CHAPTER 12 BIOCHEMICAL SIGNALING key metabolites — which trigger appropriate responses in such cellular activities as metabolism, cell division, embryonic growth and development, movement, and defense. In all these cases, the signal represents information that is detected by specific receptors and converted to a cellular response, which always involves a chemical process. This conversion of information into a chemical change, signal transduction, is a universal property of living cells. In this chapter, we present examples of specific biological signaling systems from which we have acquired our current understanding of the biochemistry of signal transduction in animals. We emphasize the following principles: Cells respond to external signals through receptor- mediated processes that amplify the signal, integrate it with input from other receptors, transmit the signal to the appropriate effectors, and eventually end the response. There is a high degree of evolutionary conservation of signaling proteins and transduction mechanisms across the animal phyla. At least a billion years of evolution have passed since the plant and animal branches of the eukaryotes diverged, which is reflected in the differences in signaling mechanisms used in the two kingdoms. We focus here on the animal kingdom. In multicellular animals, GPCRs with seven transmembrane helices are the largest group of plasma membrane receptors. These G Protein–Coupled Receptors act through G proteins, which are turned on when they bind guanosine triphosphate (GTP). Animals have hundreds of different GPCRs, each able to respond to a specific signal. Plasma membrane receptors with an intracellular tyrosine kinase activity act through cascades of protein kinases to transduce signals about the metabolic state, including growth factors. Phosphorylation of intrinsically disordered regions of signaling proteins acts as a switch, toggling enzyme activity, or creating binding sites for other molecules. Signal responses are integrated by multiprotein signaling complexes with modular domains that bind phosphorylated Tyr, Ser, or Thr residues. Ion channels gated by membrane potential or ligands are central to signaling in all cells, including bacteria, plants, and animals. Some hormone signals act inside the cell, not at the plasma membrane, forming complexes with specific receptor proteins that regulate gene expression. Cells receive extracellular signals that determine progress through the cell division cycle, or trigger cell death — processes regulated by phosphorylation and dephosphorylation of key regulatory proteins. Defective signaling proteins or defective regulation of their synthesis and breakdown can disrupt cell cycle regulation and lead to tumor formation (cancer). 12.1 General Features of Signal Transduction Signal transductions are remarkably specific and exquisitely sensitive. Specificity is achieved by precise molecular complementarity between the signal and receptor molecules (Fig. 12-1a), mediated by the same kinds of weak (noncovalent) forces that mediate enzyme-substrate and antigen-antibody interactions. Multicellular organisms have an additional level of specificity, because the receptors for a given signal, or the intracellular targets of a given signal pathway, are present only in certain cell types. Thyrotropin-releasing hormone, for example, triggers responses in the cells of the anterior pituitary but not in hepatocytes, which lack receptors for this hormone. Epinephrine alters glycogen metabolism in hepatocytes but not in adipocytes; in this case, both cell types have receptors for the hormone, but whereas hepatocytes contain glycogen and the glycogen- metabolizing enzyme that is stimulated by epinephrine, adipocytes contain neither. FIGURE 12-1 Eight features of signal-transducing systems. Signal-Transducing Systems Share Common Features The extraordinary sensitivity of signal transduction results from the high affinity of signal receptors for their ligands (Fig. 12-1b). This affinity can be expressed as the dissociation constant, Kd, for the receptor-ligand interaction. Kd is commonly 10−7 M or less, meaning that the receptor detects micromolar to nanomolar concentrations of a signaling ligand. In some cases, cooperativity in receptor-ligand interactions results in relatively large changes in receptor activation with small changes in ligand concentration, further increasing the sensitivity of signal detection. Amplification results when an enzyme is activated by a signal receptor and, in turn, catalyzes the activation of many molecules of a second enzyme, each of which activates many molecules of a third enzyme and so on, in an enzyme cascade (Fig. 12-1c). Such cascades can produce amplifications of several orders of magnitude within milliseconds. The response to a signal must also be terminated, such that the downstream effects are in proportion to the strength of the original stimulus. Interacting signaling proteins are modular. Many signaling proteins have multiple domains that recognize specific features in several other proteins, or in the cytoskeleton or plasma membrane. This modularity allows a cell to mix and match a set of signaling molecules to create a wide variety of multienzyme complexes with different functions or cellular locations. One common theme in these interactions is the binding of one modular signaling protein to phosphorylated residues in another protein; the resulting interaction can be regulated by phosphorylation or dephosphorylation of the protein partner (Fig. 12-1d). Nonenzymatic scaffold proteins with affinity for several enzymes that interact in cascades bring these enzymes together, ensuring that they interact at specific cellular locations and at specific times. Many of the regions involved in protein- protein interactions are intrinsically disordered (see Fig. 4-20), capable of folding differently depending on which protein they interact with. As a result, a single protein can have multiple functions in signaling pathways. The sensitivity of receptor systems is subject to modification. When a signal is present continuously, the receptor system becomes desensitized (Fig. 12-1e), so that it no longer responds to the signal. When the stimulus falls below a certain threshold, the system again becomes sensitive. Think of what happens to your visual transduction system when you walk from bright sunlight into a darkened room or from darkness into the light. Signal integration (Fig. 12-1f) is the ability of the system to receive multiple signals and produce a unified response appropriate to the combined needs of the cell or organism. Different signaling pathways converse with each other at several levels, generating complex cross talk that maintains homeostasis in the cell and the organism. Signaling pathways are o en divergent — branched rather than linear; one stimulus acts through one receptor, which activates two or more pathways with different downstream targets and responses (Fig. 12-1g). A final noteworthy feature of signal-transducing systems is response localization within a cell (Fig. 12-1h). When the components of a signaling system are confined to a specific subcellular structure (a ra in the plasma membrane, for example), a cell can regulate a process locally, without affecting distant regions of the cell. One of the revelations of research on signaling is the remarkable degree to which signaling mechanisms have been conserved during evolution. Although the number of different biological signals to which cells respond is probably in the thousands (Table 12-1 lists a few important types), and the kinds of responses elicited by these signals are comparably numerous, the machinery for transducing all of these signals is built from about 10 basic types of protein components (Table 12-2). TABLE 12-1 Some Signals to Which Cells Respond Antigens Mechanical touch Cell surface glycoproteins, oligosaccharides Microbial, insect pathogens Developmental signals Neurotransmitters Extracellular matrix components Nutrients Growth factors Odorants Hormones Pheromones Hypoxia Tastants Light TABLE 12-2 Some Conserved Elements of Animal Signaling Systems Plasma membrane receptors with 7 transmembrane (7tm) helices G proteins that bind GTP or GDP and interface with membrane receptors Membrane enzymes with cyclic nucleotides as substrates or products Protein kinases that phosphorylate GPCR receptors Membrane protein tyrosine kinases Cyclic nucleotide–dependent protein kinases Ca2+-binding proteins Ca2+-dependent protein kinases Protein kinases that are activated during cell division Nonenzymic protein scaffolds that bring modules together The General Process of Signal Transduction in Animals Is Universal In this chapter we consider the molecular details of several representative signal-transduction systems, classified according to the type of receptor. The trigger for each system is different, but the general features of signal transduction are common to all: a signal (ligand) interacts with a receptor; the activated receptor interacts with cellular machinery, producing a second signal or a change in the activity of a cellular protein; the metabolic activity of the target cell undergoes a change; and finally, the transduction event ends. To illustrate these general features of signaling systems, we will look at examples of four basic receptor types (Fig. 12-2). 1. G protein–coupled receptors that indirectly activate (through GTP-binding proteins, or G proteins) enzymes that generate intracellular second messengers. Examples include the β - adrenergic receptor system that responds to epinephrine (Section 12.2) and the vision, olfaction, and gustation systems (Section 12.3). 2. Receptor enzymes in the plasma membrane that have an enzymatic activity on the cytoplasmic side, triggered by ligand binding on the extracellular side. Receptors with tyrosine kinase activity, for example, catalyze the phosphorylation of Tyr residues in specific intracellular target proteins. Examples include the insulin receptor (Section 12.4) and receptor guanylyl cyclases (see Box 12-2). 3. Gated ion channels of the plasma membrane that open and close (hence the term “gated”) in response to the binding of chemical ligands or changes in transmembrane potential. These are the simplest signal transducers (Section 12.6). 4. Nuclear receptors that bind specific ligands (such as the hormone estrogen) and alter the rate at which specific genes are transcribed and translated into cellular proteins. Because steroid hormones function through mechanisms intimately related to the regulation of gene expression, we consider them briefly in Section 12.7 and defer a detailed discussion of their action until Chapters 23 and 28. FIGURE 12-2 Four general types of signal transducers. As we begin this discussion of biological signaling, a word about the nomenclature of signaling proteins is in order. These proteins are typically discovered in one context and named accordingly, then prove to be involved in a broader range of biological functions for which the original name is not helpful. For example, the retinoblastoma protein, pRb, was initially identified as the product of a mutation that contributes to cancer of the retina (retinoblastoma), but it is now known to function in many pathways essential to cell division in all cells, not just those of the retina. Some genes and proteins are given noncommittal names: the tumor suppressor protein p53, for example, is a protein of 53 kDa, but its name gives no clue to its great importance in the regulation of cell division and the development of cancer. In this chapter we generally define these protein names as we encounter them, introducing the names that are commonly used by researchers in the field. SUMMARY 12.1 General Features of Signal Transduction All cells have specific and highly sensitive signal-transducing mechanisms, which have been conserved during evolution. A wide variety of stimuli act through specific protein receptors in the plasma membrane. In all types of signal transduction, receptors bind the signal ligand and initiate a process that amplifies the signal, integrates it with input from other receptors, and transmits the information throughout the cell. If the signal persists, receptor desensitization reduces or ends the response. Multicellular organisms have four general types of signaling mechanisms: plasma membrane proteins that act through G proteins, receptors with internal enzyme activity (such as tyrosine kinase), gated ion channels, and nuclear receptors that bind steroids and alter gene expression. 12.2 G Protein–Coupled Receptors and Second Messengers As their name implies, G protein–coupled receptors (GPCRs) are receptors that act through a member of the guanosine nucleotide–binding protein, or G protein, family. Three essential components define signal transduction through GPCRs: a plasma membrane receptor with seven transmembrane helical segments, a G protein that cycles between active (guanosine triphosphate (GTP)-bound) and inactive (guanosine diphosphate (GDP)-bound) forms, and an effector enzyme (or ion channel) in the plasma membrane that is regulated by the activated G protein. An extracellular signal such as a hormone, growth factor, or neurotransmitter is the “first messenger” that activates a receptor from outside the cell. Ligand binding to the receptor forces an allosteric transition that allows the receptor to interact with a G protein, causing it to exchange its bound GDP for a GTP from the cytosol. The G protein then dissociates from the activated receptor and binds to the nearby effector enzyme, altering its activity. The effector enzyme then causes a change in the cytosolic concentration of a low molecular weight metabolite (such as 3′,5′-cyclic AMP) or inorganic ion (Ca2+), which acts as a second messenger to activate or inhibit one or more downstream targets, o en protein kinases. The human genome encodes just over 800 GPCRs, about 350 for detecting hormones, growth factors, and other endogenous ligands, and up to 500 that serve as olfactory (smell) and gustatory (taste) receptors. The largest superfamily of proteins encoded in the human genome, GPCRs have been implicated in many common medical conditions, including allergies, depression, blindness, diabetes, and various cardiovascular defects. GPCR mutations are also found in 20% of all cancers. In the United States, more than a third of all pharmaceuticals on the market target a GPCR. For example, the β -adrenergic receptor, which mediates the effects of epinephrine, is the target of the “beta blockers,” prescribed for such diverse conditions as hypertension, cardiac arrhythmia, glaucoma, anxiety, and migraine headache. More than 100 of the GPCRs found in the human genome are still “orphan receptors,” meaning that their natural ligands are not yet identified, and so we know little about their biology. The β - adrenergic receptor, with well-understood biology and pharmacology, is the prototype for all GPCRs, and our discussion of signal-transducing systems begins there. The β -Adrenergic Receptor System Acts through the Second Messenger cAMP Epinephrine released from the adrenal glands sounds the alarm when a threat requires an animal to mobilize its energy- generating machinery; it signals the need to fight or flee. Epinephrine action begins when the hormone binds to a protein receptor in the plasma membrane of an epinephrine-sensitive cell (a myocyte in muscle, for example). Adrenergic receptors (“adrenergic” reflects the alternative name for epinephrine, adrenaline) are of four general types, α1, α2, β1, and β2, defined by differences in their affinities and responses to a group of agonists and antagonists. Agonists are molecules (natural ligands or their structural analogs) that bind a receptor and produce the effects of the natural ligand; antagonists are analogs that bind the receptor without triggering the normal effect and thereby block the effects of agonists, including the natural ligand. In some cases, the affinity of a synthetic agonist or antagonist for the receptor is greater than that of the natural agonist (Fig. 12-3). The four types of adrenergic receptors are found in different target tissues and mediate different responses to epinephrine. Here we focus on the β -adrenergic receptors of muscle, liver, and adipose tissue. These receptors mediate changes in fuel metabolism, as described in Chapter 23, including the increased breakdown of glycogen and fat. Adrenergic receptors of the β1 and β2 subtypes act through the same mechanism, so in our discussion, “β -adrenergic” applies to both types. FIGURE 12-3 Epinephrine and its synthetic analogs. Epinephrine regulates energy-yielding metabolism in muscle, liver, and adipose tissue. Its affinity for its receptor is expressed as a dissociation constant for the receptor- ligand complex. Isoproterenol and propranolol are synthetic analogs, one an agonist with an affinity for the receptor that is higher than that of epinephrine, and the other an antagonist with extremely high affinity. Like all GPCRs, the β -adrenergic receptor is an integral protein with seven hydrophobic, helical regions of 20 to 28 amino acid residues that span the plasma membrane seven times, thus the alternative names for GPCRs: seven-transmembrane (7tm) or heptahelical receptors. GPCRs effect signal transduction through interaction with heterotrimeric G proteins, a conserved family of signaling proteins with three subunits, α , β , and γ . The binding site for GDP or GTP is on the α subunit. When GDP is bound, the G protein is in its trimeric, inactive form. GPCRs are allosteric proteins. When epinephrine binds to the β - adrenergic receptor (Fig. 12-4a, step ), allosteric transitions in the receptor and its associated G protein favor the replacement of GDP with GTP. Thus the hormone-bound GPCR acts as a guanosine nucleotide–exchange factor (GEF). In this active form, the G protein (step ) can transmit the signal from the activated receptor to the downstream effector protein, adenylyl cyclase. Because this G protein stimulates its effector, it is referred to as a stimulatory G protein, or Gs. In the active form, the β and γ subunits of Gs dissociate from the α subunit as a βγ dimer, and Gsα, with its bound GTP, moves in the plane of the membrane from the receptor to a nearby molecule of adenylyl cyclase (step ). FIGURE 12-4 Transduction of the epinephrine signal: the β -adrenergic pathway. (a) The mechanism that couples binding of epinephrine to its receptor with activation of adenylyl cyclase; the seven steps are discussed in the text. The same adenylyl cyclase molecule in the plasma membrane may be regulated by a stimulatory G protein (Gs), as shown, or by an inhibitory G protein (Gi, not shown). Gs and Gi are under the influence of different hormones. Hormones that induce GTP binding to Gi cause inhibition of adenylyl cyclase, resulting in lower cellular [cAMP]. (b) The combined action of the enzymes that catalyze steps and , synthesis and hydrolysis of cAMP by adenylyl cyclase and cAMP phosphodiesterase, respectively. Adenylyl cyclase is an integral protein of the plasma membrane, with its active site on the cytoplasmic face. The association of active Gsα with adenylyl cyclase stimulates the cyclase to catalyze the synthesis of second messenger cAMP from ATP (Fig. 12-4a, step ; Fig. 12-4b), raising the cytosolic [cAMP]. The interaction between Gsα and adenylyl cyclase occurs only when Gsα is bound to GTP. The stimulation by Gsα is self-limiting; Gsα has intrinsic GTPase activity that switches Gsα to its inactive form by converting its bound GTP to GDP (Fig. 12-5). The inactive Gsα dissociates from adenylyl cyclase, rendering the cyclase inactive. Gsα reassociates with the βγ dimer (Gsβγ), and inactive Gs is again available to interact with a hormone-bound receptor. FIGURE 12-5 The GTPase switch. G proteins cycle between GDP-bound (off) and GTP-bound (on). The protein’s GTPase activity, in many cases stimulated by RGS proteins (regulators of G-protein signaling; see Fig. 12-8), determines how quickly bound GTP is hydrolyzed to GDP and thus how long the G protein remains active. Cyclic AMP Activates Protein Kinase A Epinephrine exerts its downstream effects through the increase in [cAMP] that results from activation of adenylyl cyclase. Cyclic AMP, the second messenger, allosterically activates cAMP- dependent protein kinase, also called protein kinase A or PKA (Fig. 12-4a, step ), which catalyzes the phosphorylation of specific Ser or Thr residues of targeted proteins, such as glycogen phosphorylase b kinase. The latter enzyme is active when phosphorylated and can begin the process of mobilizing glycogen stores in muscle and liver in anticipation of the need for energy, as signaled by epinephrine. The inactive form of PKA has two identical catalytic subunits (C) and two identical regulatory subunits (R) (Fig. 12-6a). The tetrameric R2C2 complex is catalytically inactive, because an autoinhibitory domain of each R subunit occupies the substrate- binding cle of each C subunit. The autoinhibitory domain is an intrinsically disordered region that can fold to fit any of several protein partners. Cyclic AMP is an allosteric activator of PKA. When cAMP binds to the R subunits, they undergo a conformational change that moves the autoinhibitory domain of R out of the catalytic domain of C, and the R2C2 complex dissociates to yield two free, catalytically active C subunits. This same basic mechanism — displacement of an autoinhibitory domain — mediates the allosteric activation of many types of protein kinases by their second messengers (as in Fig. 12-21 and 12-29, for example). The structure of the substrate-binding cle in PKA is the prototype for all known protein kinases (Fig. 12-6b); certain residues in this cle region have identical counterparts in all of the 544 protein kinases encoded in the human genome. The ATP-binding site of each catalytic subunit positions ATP perfectly for the transfer of its terminal (γ) phosphoryl group to the — OH in the side chain of a Ser or Thr residue in the target protein. FIGURE 12-6 Activation of cAMP-dependent protein kinase (PKA). (a) When [cAMP] is low, the two identical regulatory subunits (R; red) associate with the two identical catalytic subunits (C; blue). In this R2C2 complex, the inhibitor sequences of the R subunits lie in the substrate-binding cle of the C subunits and prevent binding of protein substrates; the complex is therefore catalytically inactive. The amino-terminal sequences of the R subunits interact to form an R2 dimer, the site of binding to an A kinase anchoring protein (AKAP; Fig. 12-11). When [cAMP] rises in response to a hormonal signal, each R subunit binds two cAMP molecules and undergoes a dramatic reorganization that pulls its inhibitory sequence away from the C subunit, opening up the substrate-binding cle and releasing each C subunit in its catalytically active form. (b) A crystal structure showing part of the R2C2 complex — one C subunit and part of one R subunit. The amino-terminal dimerization region of the R subunit is omitted for simplicity. The small lobe of C contains the ATP-binding site, and the large lobe surrounds and defines the cle where the protein substrate binds and undergoes phosphorylation at a Ser or Thr residue, with a phosphoryl group transferred from ATP. In this inactive form, the inhibitor sequence of R blocks the substrate-binding cle of C, inactivating it. [(b) Data from PDB ID 3FHI, C. Kim et al., Science 307:690, 2005.] As indicated in Figure 12-4a (step ), PKA regulates many enzymes downstream in the signaling pathway. Although these downstream targets have diverse functions, they share a region of sequence similarity around the Ser or Thr residue that undergoes phosphorylation, a sequence that marks them for regulation by PKA. The substrate-binding cle of PKA recognizes these sequences and phosphorylates their Thr or Ser residue. Comparison of the sequences of various protein substrates for PKA has yielded the short consensus sequence in which the target for phosphorylation (Ser or Thr) is most commonly embedded. For PKA, the consensus sequence is xR[RK]x[ST]B, where x can be any residue, R is Arg, [RK] can be either Arg or Lys, [ST] is either Ser or Thr (which is the residue phosphorylated), and B is any basic residue. The consensus sequences of a number of protein kinases are shown in Table 6- 10; they define the targets of the hundreds of protein kinases in the eukaryotic cell. How do we know that two proteins interact, or where in the cell they interact? Changes in the state of association of two proteins (such as the R and C subunits of PKA) can be seen by measuring the nonradiative transfer of energy between fluorescent probes attached to each protein, a technique called fluorescence resonance energy transfer (FRET), which we describe in Box 12-1. BOX 12-1 FRET: Biochemistry in a Living Cell Fluorescent probes are commonly used to detect biochemical changes in single living cells on a nanosecond time scale. In one widely used procedure, green fluorescent protein (GFP) and variants with different fluorescence spectra, described in Chapter 9 (see Fig. 9-16), are genetically fused with another protein. These fluorescent hybrid proteins act as spectroscopic rulers for measuring distances between interacting proteins within a cell. They can be used indirectly as measures of local concentrations of compounds that change the distance between two proteins. An excited fluorescent molecule such as GFP or YFP (yellow fluorescent protein) can dispose of the energy from the absorbed photon in either of two ways: (1) by fluorescence, emitting a photon of slightly longer wavelength (lower energy) than the exciting light, or (2) by nonradiative fluorescence resonance energy transfer (FRET), in which the energy of the excited molecule (the donor) passes directly to a nearby molecule (the acceptor) without emission of a photon, exciting the acceptor (Fig. 1). The acceptor can now decay to its ground state by fluorescence; the emitted photon has a longer wavelength (lower energy) than both the original exciting light and the fluorescence emission of the donor. This second mode of decay (FRET) is possible only when donor and acceptor are close to each other (within 1 to 50 Å); the efficiency of FRET is inversely proportional to the sixth power of the distance between donor and acceptor. Thus, very small changes in the distance between donor and acceptor register as very large changes in FRET, measured as the fluorescence of the acceptor molecule when the donor is excited. With sufficiently sensitive light detectors, this fluorescence signal can be located to specific regions of a single, living cell. FIGURE 1 When the donor protein (CFP) is excited with monochromatic light of wavelength 433 nm, it emits fluorescent light at 476 nm (le ). When the (red) protein fused with CFP interacts with the (purple) protein fused with YFP, that interaction brings CFP and YFP close enough to allow fluorescence resonance energy transfer (FRET) between them. Now, when CFP absorbs light of 433 nm, instead of fluorescing at 476 nm, it transfers energy directly to YFP, which then fluoresces at its characteristic emission wavelength, 527 nm. The ratio of light emission at 527 nm and 476 nm is therefore a measure of the extent of interaction between the red and purple proteins. FRET has been used to measure [cAMP] in living cells. The gene for BFP (blue fluorescent protein) is fused with that for the regulatory subunit (R) of cAMP- dependent protein kinase (PKA), and the gene for GFP is fused with that for the catalytic subunit (C) (Fig. 2). When these two hybrid proteins are expressed in a cell, BFP (donor) and GFP (acceptor) in the inactive PKA (R2C2 tetramer) are close enough to undergo FRET. Wherever in the cell [cAMP] increases, the R2C2 complex dissociates into R2 and 2 C and the FRET signal is lost, because donor and acceptor are now too far apart for efficient FRET. Viewed in the fluorescence microscope, the region of higher [cAMP] has a minimal GFP signal and higher BFP signal. Measuring the ratio of emission at 460 nm and 545 nm gives a sensitive measure of the change in [cAMP]. By determining this ratio for all regions of the cell, the investigator can generate a false color image of the cell in which the ratio, or relative [cAMP], is represented by the intensity of the color. Images recorded at timed intervals reveal changes in [cAMP] over time. FIGURE 2 Measuring [cAMP] with FRET. Gene fusion creates hybrid proteins that exhibit FRET when the PKA regulatory (R) and catalytic (C) subunits are associated (low [cAMP]). When [cAMP] rises, the subunits dissociate and FRET ceases. The ratio of emission at 460 nm (dissociated) and 545 nm (complexed) thus offers a sensitive measure of [cAMP]. A variation of this technology has been used to measure the activity of PKA in a living cell (Fig. 3). Researchers create a phosphorylation target for PKA by producing a hybrid protein containing four elements: YFP (acceptor); a short peptide with a Ser residue surrounded by the consensus sequence for PKA; – Ser-binding domain (called 14-3-3); and CFP (donor). When the Ser residue is not phosphorylated, 14-3-3 has no affinity for the Ser residue and the hybrid protein exists in an extended form, with the donor and acceptor too far apart to generate a FRET signal. Wherever PKA is active in the cell, it phosphorylates the Ser residue of the hybrid protein, and 14-3-3 binds to the –Ser. In doing so, it draws YFP and CFP together and a FRET signal is detected with the fluorescence microscope, revealing the presence of active PKA. FIGURE 3 Measuring the activity of PKA with FRET. An engineered protein links YFP and CFP via a peptide that contains (1) a Ser residue surrounded by the consensus sequence for phosphorylation by PKA and (2) the 14-3-3 –Ser-binding domain. Active PKA phosphorylates the Ser residue, which docks with the 14-3-3 binding domain, bringing the fluorescence proteins close enough to allow FRET, revealing the presence of active PKA. With more sophisticated microscopic equipment, the FRET signal from a single protein molecule can be detected with much higher spatial and temporal resolution than is available in FRET with an ensemble of molecules. As in many signaling pathways, signal transduction by the β -adrenergic receptor and adenylyl cyclase entails several steps that amplify the original hormone signal (Fig. 12-7). First, the binding of one hormone molecule to one receptor molecule catalytically activates many Gs molecules that associate with the activated receptor, one a er the other. Next, by activating one molecule of adenylyl cyclase, each active Gsα molecule stimulates the catalytic synthesis of many molecules of cAMP. The second messenger cAMP now activates PKA, each molecule of which catalyzes the phosphorylation of many molecules of the target protein — phosphorylase b kinase in Figure 12-7. This kinase activates glycogen phosphorylase b, which leads to the rapid mobilization of glucose from glycogen. The net effect of the cascade is amplification of the hormonal signal by orders of magnitude, which accounts for the very low concentration of epinephrine (or any other hormone) required for hormone activity. This signaling pathway is also rapid: the signal leads to intracellular changes within fractions of a second.
FIGURE 12-7 Epinephrine cascade. Epinephrine triggers a series of reactions in hepatocytes in which catalysts activate catalysts, resulting in great amplification of the original hormone signal. The numbers of molecules shown are simply to illustrate amplification and are almost certainly gross underestimates. Binding of one molecule of epinephrine to one β -adrenergic receptor on the cell surface activates many (possibly hundreds of) G proteins, one a er another, each of which goes on to activate a molecule of the enzyme adenylyl cyclase. Adenylyl cyclase acts catalytically, producing many molecules of cAMP for each activated adenylyl cyclase. (Because two molecules of cAMP are required to activate one PKA catalytic subunit, this step does not amplify the signal.) Several Mechanisms Cause Termination of the β -Adrenergic Response To be useful, a signal-transducing system has to turn off a er the hormonal or other stimulus has ended, and mechanisms for shutting off the signal are part of all signaling systems. Most systems also adapt to the continued presence of the signal by becoming less sensitive to it, by desensitizing. The β -adrenergic system illustrates both. Here, our focus is on termination. The response to β -adrenergic stimulation will end when the concentration of the ligand (epinephrine) in the blood drops below the Kd for its receptor. The epinephrine then dissociates from the receptor, and the latter reassumes its inactive conformation, in which it can no longer activate Gs. A second means of ending the response is the hydrolysis of GTP bound to the Gα subunit, catalyzed by the GTPase activity of the G protein. Conversion of bound GTP to GDP favors the return of Gα to the conformation in which it binds the Gβγ subunits — the conformation in which the G protein is unable to interact with or stimulate adenylyl cyclase. This ends the production of cAMP. The rate of inactivation of Gs depends on the GTPase activity, which for Gα alone is very feeble. However, GTPase activator proteins (GAPs) strongly stimulate this GTPase activity, causing more-rapid inactivation of the G protein (Fig. 12-8). GAPs can themselves be regulated by other factors, providing a fine-tuning of the response to β -adrenergic stimulation. A third mechanism for terminating the response is to remove the second messenger: cAMP is hydrolyzed to 5′-AMP (which is not active as a second messenger) by cyclic nucleotide phosphodiesterase (Fig. 12-4a, step ; 12- 4b).
FIGURE 12-8 Factors that regulate G-protein activity. Inactive G proteins, both small G proteins such as Ras and heterotrimeric G proteins such as Gs, interact with upstream GTP-GDP exchange factors (red). O en these exchange factors are activated (*) receptors such as rhodopsin (Rh) and β - adrenergic receptors (AR). The G proteins are activated by GTP binding, and in the GTP-bound form, activate downstream effector enzymes (blue), such as cGMP phosphodiesterase (PDE), adenylyl cyclase (AC), and Raf. By modulating the GTPase activity of G proteins, GTPase activator proteins (GAPs, in the case of small G proteins) and regulators of G-protein signaling (RGSs) (yellow), determine how long the G protein will remain active. Finally, at the end of the signaling pathway, the metabolic effects that result from phosphorylation of target enzymes by PKA are reversed by the action of phosphoprotein phosphatases, which hydrolyze phosphorylated Tyr, Ser, or Thr residues, releasing inorganic phosphate (Pi). About 190 genes in the human genome encode phosphoprotein phosphatases, fewer than the number (about 540) that encode protein kinases, reflecting the relative promiscuity of the phosphoprotein phosphatases. A single phosphoprotein phosphatase (PP1) dephosphorylates some 200 different phosphoprotein targets, including the β -adrenergic receptor and other GPCRs. Some phosphatases are known to be regulated; others may be constantly active. When [cAMP] drops and PKA returns to its inactive form (step in Fig. 12-4a), the balance between phosphorylation and dephosphorylation is tipped toward dephosphorylation by these phosphatases. The β -Adrenergic Receptor Is Desensitized by Phosphorylation and by Association with Arrestin The mechanisms for signal termination described above take effect when the stimulus ends. A different mechanism, desensitization, damps the response even while the signal persists. When the receptor remains occupied with epinephrine, β - adrenergic receptor kinase, or β ARK, phosphorylates several Ser residues near the receptor’s carboxyl terminus, which is on the cytoplasmic side of the plasma membrane (Fig. 12-9). β ARK is drawn to the plasma membrane by its association with the Gsβγ subunits and is thus positioned to phosphorylate the receptor. Receptor phosphorylation creates a binding site for the protein β -arrestin, or β arr (also called arrestin 2), and binding of β - arrestin blocks the sites in the receptor that interact with the G protein (Fig. 12-10). The binding of β -arrestin also facilitates the sequestration of receptor molecules and their removal from the plasma membrane by endocytosis into small intracellular vesicles (endosomes). The arrestin-receptor complex recruits clathrin and other proteins involved in vesicle formation, which initiate membrane invagination, leading to the formation of endosomes containing the adrenergic receptor. In this state, the receptors are inaccessible to epinephrine and therefore inactive. These receptor molecules are eventually dephosphorylated and returned to the plasma membrane, completing the circuit and resensitizing the system to epinephrine. FIGURE 12-9 Desensitization of the β -adrenergic receptor in the continued presence of epinephrine. This process is mediated by two proteins: β -adrenergic protein kinase (β ARK) and β -arrestin (β arr). Not shown here is the phosphorylation and activation of β ARK by PKA. PKA is activated by the rise in [cAMP] in response to the initial signal (epinephrine). FIGURE 12-10 Mutual exclusion of trimeric G protein and arrestin in their interaction with a GPCR. (a) The β -adrenergic receptor complexed with its trimeric G protein, Gs. (b) Another GPCR, the rhodopsin receptor, phosphorylated near its carboxyl terminus, and bound to arrestin. Binding of arrestin blocks binding and further activation of the G protein and ends the response to the initial signal. [Data from (a) PDB ID 3SN6, S. G. F. Rasmussen et al., Nature 477:549, 2011; (b) PDB ID 4ZWJ, Y. Kang et al., Nature 523:561, 2015.] β -Adrenergic receptor kinase is a member of a family of G protein–coupled receptor kinases (GRKs), all of which phosphorylate GPCRs on their carboxyl-terminal cytoplasmic domains and play roles similar to that of β ARK in desensitization and resensitization of their receptors. Seven GRKs and four different arrestins are encoded in the human genome; each GRK is capable of desensitizing a particular subset of GPCRs, and each arrestin can interact with many different types of phosphorylated receptors. The receptor-arrestin complex has another important role: it initiates signaling by a second pathway, the MAPK cascade described below. Thus, acting through a single GPCR, epinephrine triggers two divergent signaling pathways. The two pathways, one initiated by the receptor’s interaction with a G protein and the other initiated by its interaction with arrestin, can be differentially affected by the agonist; in some cases, one agonist favors the G-protein pathway and another favors the arrestin pathway. This bias is an important consideration in the development of a medication that acts through a GPCR. For example, the most addictive of the opioid drugs of abuse act more strongly through G-protein signaling than through arrestin. An ideal opioid pain medication would act through the branch of the pathway that has therapeutic effects and not through the pathway that leads to addiction. Cyclic AMP Acts as a Second Messenger for Many Regulatory Molecules Epinephrine is just one of many hormones, growth factors, and other regulatory molecules that act by changing the intracellular [cAMP] and thus the activity of PKA. Table 12-3 lists a few examples. Glucagon binds to its receptors in the plasma membrane of adipocytes, activating (via a Gs protein) adenylyl cyclase. PKA, stimulated by the resulting rise in [cAMP], phosphorylates and activates two proteins critical to the mobilization of the fatty acids of stored fats (see Fig. 17-2). Similarly, the peptide hormone ACTH (adrenocorticotropic hormone, also called corticotropin), produced by the anterior pituitary, binds to specific receptors in the adrenal cortex, activating adenylyl cyclase and raising the intracellular [cAMP]. PKA then phosphorylates and activates several of the enzymes required for the synthesis of cortisol and other steroid hormones. In many cell types, the catalytic subunit of PKA can also move into the nucleus, where it phosphorylates the cAMP response element binding protein (CREB), which alters the expression of specific genes regulated by cAMP. TABLE 12-3 Some Signals That Use cAMP as Second Messenger Corticotropin (ACTH) Corticotropin-releasing hormone (CRH) Dopamine [D1, D2] Epinephrine (β -adrenergic) Follicle-stimulating hormone (FSH) Glucagon Histamine [H2] Luteinizing hormone (LH) Melanocyte-stimulating hormone (MSH) Odorants (many) Parathyroid hormone Prostaglandins E1, E2 (PGE1,PGE2) Serotonin [5-HT 1,5-HT 4] Somatostatin Tastants (sweet, bitter) Thyroid-stimulating hormone (TSH) Note: Receptor subtypes in square brackets. Subtypes may have different transduction mechanisms. For example, serotonin is detected in some tissues by receptor subtypes 5-HT 1 and 5-HT 4, which act through adenylyl cyclase and cAMP, and in other tissues by receptor subtype 5-HT 2, acting through the phospholipase C-IP3 mechanism (see Table 12-4). Some hormones act by inhibiting adenylyl cyclase, thus lowering [cAMP] and suppressing protein phosphorylation by PKA. For example, the binding of somatostatin to its receptor in the pancreas leads to activation of an inhibitory G protein, or Gi, structurally homologous to Gs, that inhibits adenylyl cyclase and lowers [cAMP]. In this way, somatostatin inhibits the secretion of several hormones, including glucagon. In adipose tissue, prostaglandin E2 (PGE2; see Fig. 10-17) inhibits adenylyl cyclase, thus lowering [cAMP] and slowing the mobilization of lipid reserves triggered by epinephrine and glucagon. In certain other tissues, PGE2 stimulates cAMP synthesis: its receptors are coupled to adenylyl cyclase through a stimulatory G protein, Gs. In tissues with α2-adrenergic receptors, epinephrine lowers [cAMP]; in this case, the receptors are coupled to adenylyl cyclase through an inhibitory G protein, Gi. In short, an extracellular signal such as epinephrine or PGE2 can have different effects on different tissues or cell types, depending on three factors: the type of receptor in the tissue, the type of G protein (Gs or Gi) with which the receptor is coupled, and the set of PKA target enzymes in the cell. By summing the influences that tend to increase and decrease [cAMP], a cell achieves the integration of signals that is a general feature of signal-transducing mechanisms (Fig. 12-1f). Another factor that explains how so many types of signals can be mediated by a single second messenger (cAMP) is the confinement of the signaling process to a specific region of the cell by adaptor proteins — noncatalytic proteins that hold together other protein molecules that function in concert (further described below). AKAPs (A kinase anchoring proteins) have multiple, distinct protein-binding domains, o en intrinsically disordered regions; they are multivalent adaptor proteins. One domain binds to the R subunits of PKA and another binds to a specific structure in the cell, confining the PKA to the vicinity of that structure. For example, specific AKAPs bind PKA to microtubules, actin filaments, ion channels, mitochondria, or the nucleus. Different types of cells have different complements of AKAPs, so cAMP might stimulate phosphorylation of mitochondrial proteins in one cell and stimulate phosphorylation of actin filaments in another. In some cases, an AKAP connects PKA with the enzyme that triggers PKA activation (adenylyl cyclase) or terminates PKA action (cAMP phosphodiesterase or phosphoprotein phosphatase) (Fig. 12-11). The very close proximity of these activating and inactivating enzymes presumably achieves a highly localized, and very brief, response. FIGURE 12-11 Nucleation of supramolecular complexes by A kinase anchoring proteins (AKAPs). AKAP5 is one of a family of proteins that act as multivalent scaffolds, holding PKA catalytic subunits — through interaction of the AKAP with the PKA regulatory subunits — in proximity to a particular region or structure in the cell. AKAP5 is targeted to ra s in the cytoplasmic face of the plasma membrane by two covalently attached palmitoyl groups and a site that binds phosphatidylinositol 3,4,5-trisphosphate (PIP3) in the membrane. AKAP5 also has binding sites for the β -adrenergic receptor, adenylyl cyclase, PKA, and a phosphoprotein phosphatase (PP2A), bringing them all together in the plane of the membrane. When epinephrine binds to the β -adrenergic receptor, adenylyl cyclase produces cAMP, which reaches the nearby PKA quickly and with very little dilution. PKA phosphorylates its target protein, altering its activity, until the phosphoprotein phosphatase removes the phosphoryl group and returns the target protein to its prestimulus state. The AKAPs in this and other cases bring about a high local concentration of enzymes and second messengers, so that the signaling circuit remains highly localized and the duration of the signal is limited. In a process analogous with the cAMP-PKA pathway, a cyclic derivative of GTP (3′,5′-cyclic GMP; cGMP) is generated in response to an extracellular signal. The cGMP activates a cGMP- dependent protein kinase (PKG), which phosphorylates specific protein substrates, changing their activities in response to the initial signal (Box 12-2). BOX 12-2 MEDICINE Receptor Guanylyl Cyclases, cGMP, and Protein Kinase G Guanylyl cyclases (Fig. 1) are receptor enzymes that, when activated, convert GTP to the second messenger guanosine 3′,5′-cyclic monophosphate (cGMP) (Fig. 2). Many of the actions of cGMP in animals are mediated by cGMP- dependent protein kinase, also called protein kinase G (PKG). On activation by cGMP, PKG phosphorylates Ser and Thr residues in target proteins. The catalytic and regulatory domains of this enzyme are in a single polypeptide (Mr ~80,000). Part of the regulatory domain fits snugly in the substrate-binding cle . Binding of cGMP forces this activation loop out of the binding site, opening the site to PKG target proteins. FIGURE 1 Two types of signal-transducing guanylyl cyclase. (a) Membrane- spanning guanylyl cyclases such as the ANF and guanylin receptors are homodimers with an extracellular ligand-binding domain and an intracellular guanylyl cyclase domain. (b) A soluble heme-containing guanylyl cyclase is activated by intracellular NO. FIGURE 2 Synthesis of cGMP by guanylyl cyclase and its hydrolysis by cGMP phosphodiesterase. Cyclic GMP carries different messages in different tissues. In cardiac muscle (a type of smooth muscle), it signals relaxation. In the kidney and the intestine, it triggers changes in ion transport and water retention. Guanylyl cyclase in the kidney is activated by the peptide hormone atrial natriuretic factor (ANF; Fig. 1a), which is released by cells in the cardiac atrium when the heart is stretched by increased blood volume. Carried in the blood to the kidney, ANF activates guanylyl cyclase in cells of the collecting ducts. The resulting rise in [cGMP] triggers increased renal excretion of Na+ and consequently of water, driven by the change in osmotic pressure. Water loss reduces the blood volume, countering the stimulus that initially led to ANF secretion. Vascular smooth muscle also has an ANF receptor–guanylyl cyclase; on binding to this receptor, ANF causes relaxation (vasodilation) of the blood vessels, which increases blood flow while decreasing blood pressure. A similar receptor guanylyl cyclase in the plasma membrane of epithelial cells lining the intestine is activated by the peptide guanylin (Fig. 1a), which regulates Cl− secretion in the intestine. This receptor is also the target of a heat-stable peptide endotoxin produced by Escherichia coli and other gram- negative bacteria. The elevation in [cGMP] caused by the endotoxin increases CI− secretion and consequently decreases reabsorption of water by the intestinal epithelium, producing diarrhea. A distinctly different type of guanylyl cyclase is a soluble cytosolic protein with a tightly associated heme group (Fig. 1b), an enzyme activated by nitric oxide (NO). Nitric oxide is produced from arginine by Ca2+-dependent NO synthase, present in many mammalian tissues, and diffuses from its cell of origin into nearby cells (see Chapter 22). In the target cell, NO binds to the heme group of guanylyl cyclase and activates cGMP production. In the heart, cGMP-dependent protein kinase reduces the forcefulness of contractions by stimulating the ion pump(s) that remove Ca2+ from the cytosol. NO-induced relaxation of cardiac muscle is the same response brought about by nitroglycerin and other nitrovasodilators taken to relieve angina pectoris, the pain caused by contraction of a heart deprived of O2 because of blocked coronary arteries. Nitric oxide is unstable and its action is brief; within seconds of its formation, it undergoes oxidation to nitrite or nitrate. Nitrovasodilator drugs produce long-lasting relaxation of cardiac muscle because they break down over several hours, yielding a steady stream of NO. The value of nitroglycerin as a treatment for angina was discovered serendipitously in factories producing nitroglycerin as an explosive in the 1860s. Workers with angina reported that their condition was much improved during the workweek but worsened on weekends. The physicians treating these workers heard this story so o en that they made the connection, and a drug was born. The effects of increased cGMP synthesis diminish a er the stimulus ceases, because a specific phosphodiesterase (cGMP PDE) converts cGMP to the inactive 5′-GMP (see Fig. 2). Humans have several isoforms of cGMP PDE, with different tissue distributions. The isoform in the blood vessels of the penis is inhibited by the drugs sildenafil (Viagra) and tadalafil (Cialis), which therefore cause [cGMP] to remain elevated once raised by an appropriate stimulus, accounting for the usefulness of this drug in the treatment of erectile dysfunction.
G Proteins Act as Self-Limiting Switches in Many Processes Proteins sensitive to the binding of either GTP or GDP play critical roles in many cellular processes, including sensory perception, signaling for cell division, growth and differentiation, intracellular movements of proteins and membrane vesicles, and protein synthesis. The human genome encodes nearly 200 of these proteins, which differ in size and subunit structure, intracellular location, and function. But all G proteins share a common feature: they can become activated by binding GTP and then, a er a brief period, can inactivate themselves with their GTPase activity, thereby serving as molecular binary switches with built-in timers. This superfamily of proteins includes the trimeric G proteins involved in β -adrenergic signaling (Gs or Gi) and vision (transducin); small, monomeric G proteins such as that involved in insulin signaling (Ras; see below) and others that function in vesicle trafficking (ARF, RAC1, and Rab), transport into and out of the nucleus (Ran), and timing of the cell cycle (Rho); and several proteins involved in protein synthesis (initiation factor IF2 and elongation factors EF-Tu and EF-G; see Chapter 27). Among the trimeric G proteins, Gα subunits have covalently linked lipids: the amino terminus is palmitoylated, and some are also myristoylated. Gγ and the monomeric Ras protein have an isoprenyl lipid at their carboxyl termini. The attached lipids keep them anchored to the plasma membrane, restricting their action to that two-dimensional plane. All G proteins have the same core structure and use the same mechanism for switching between an inactive conformation, favored when GDP is bound, and an active conformation, favored when GTP is bound. We can use Ras (~20 kDa, a minimal signaling unit) as a prototype for all members of this superfamily (Fig. 12-12). FIGURE 12-12 Ras, the G-protein prototype. M g2+-GTP is held by critical residues in the phosphate-binding P loop (blue) and by Thr35 in the switch I region (red) and Gly60 in the switch II region (green). Ala146 gives specificity for GTP over ATP. In the structure shown here, the nonhydrolyzable GTP analog Gpp(NH)p is in the GTP-binding site. [Data from PDB ID 5P21, E. F. Pai et al., EMBO J. 9:2351, 1990.] In the nucleotide-binding site of Ras, Ala146 hydrogen bonds to the guanine oxygen, allowing GTP, but not ATP, to bind. In the GTP-bound conformation, the G protein exposes previously buried regions (called switch I and switch II) that interact with proteins downstream in the signaling pathway until the G protein inactivates itself by hydrolyzing its bound GTP to GDP. The critical determinant of G-protein conformation is the γ phosphate of GTP, which interacts with a region called the P loop (phosphate- binding). In Ras, the γ phosphate of GTP binds to a Lys residue in the P loop and to two critical residues, T hr35 in switch I and Gly60 in switch II, that hydrogen-bond with the oxygens of the γ phosphate of GTP. These hydrogen bonds act like a pair of springs holding the protein in its active conformation (Fig. 12-13). When GTP is cleaved to GDP, and Pi is released, these hydrogen bonds are lost; the protein then relaxes into its inactive conformation, burying switch I and II so they are no longer available to interact with other partners. FIGURE 12-13 GTP hydrolysis flips the switches in Ras. When bound GTP is hydrolyzed by the GTPase activities of Ras and its GTPase activator protein (GAP), loss of hydrogen bonds to Thr35 and Gly60 allows the switch I and switch II regions to relax into a conformation in which they are no longer available to interact with downstream targets. [Information from I. R. Vetter and A. Wittinghofer, Science 294:1299, 2001, Fig. 3.] The GTPase activity of most G proteins is very weak, but it is increased up to 105-fold by GTPase activator proteins (GAPs), also called, in the case of heterotrimeric G proteins, regulators of G-protein signaling (RGSs; Fig. 12-8). GAPs and RGSs thus determine how long the G-protein switch remains on. There are about 40 different RGS proteins associated with a variety of processes and expressed in most tissues. They have a critical Arg residue that reaches into the G-protein GTPase active site and contributes to catalysis. The intrinsically slow process of replacing bound GDP with GTP, switching the protein on, is catalyzed by guanosine nucleotide–exchange factors (GEFs, such as the β -adrenergic receptor) associated with the G protein. The ligand-bound β -adrenergic receptor is one of many GEFs, and a broad range of proteins act as GAPs. Their combined effects set the level of GTP-bound G proteins, and thus the strength of the response to signals that arrive at the receptors.
Because G proteins play crucial roles in so many signaling processes, it is not surprising that defects in G proteins lead to a variety of diseases. In about 25% of all human cancers (and in a much higher proportion of certain types of cancer), a mutation in a Ras protein — typically in one of the critical residues around the GTP-binding site or in the P loop — virtually eliminates its GTPase activity. Once activated by GTP binding, this Ras protein remains constantly active, promoting cell division in cells that should not divide. The tumor suppressor gene NF1 encodes a GAP that enhances the GTPase activity of normal Ras. Mutations in NF1 that result in a nonfunctioning GAP leave Ras with only its GTPase activity, which is very weak (that is, has a very low turnover number); once activated by GTP binding, Ras stays active for an extended period, continuing to send the signal: divide. Defective heterotrimeric G proteins can also lead to disease. Mutations in the gene that encodes the α subunit of Gs (which mediates changes in [cAMP] in response to hormonal stimuli) may result in a Gα that is permanently active or permanently inactive. “Activating” mutations generally occur in residues crucial to GTPase activity; they lead to a continuously elevated [cAMP], with significant downstream consequences, including undesirable cell proliferation. Such mutations are found in about 40% of pituitary tumors (adenomas). Individuals with “inactivating” mutations in Gα are unresponsive to hormones (such as thyroid hormone) that act through cAMP. Mutation in the gene for the transducin α subunit (T α), which is involved in visual signaling, leads to a type of night blindness, apparently due to defective interaction between the activated T α subunit and the phosphodiesterase of the rod outer segment (see Fig. 12-19). A sequence variation in the gene encoding the β subunit of a heterotrimeric G protein is commonly found in individuals with hypertension (high blood pressure), and this variant gene is suspected of involvement in obesity and atherosclerosis. The pathogenic bacterium that causes cholera produces a toxin that targets a G protein, interfering with normal signaling in host cells. Cholera toxin, secreted by Vibrio cholerae in the intestine of an infected person, is a heterodimeric protein. Subunit B recognizes and binds to specific gangliosides on the surface of intestinal epithelial cells and provides a route for subunit A to enter these cells. A er entry, subunit A is broken into two fragments, A1 and A2. A1 associates with the host cell’s ADP- ribosylation factor ARF6, a small G protein, through residues in its switch I and switch II regions — which are accessible only when ARF6 is in its active (GTP-bound) form. This association with ARF6 activates A1, which catalyzes the transfer of ADP- ribose from NAD+ to the critical Arg residue in the P loop of the α subunit of Gs (Fig. 12-14). ADP-ribosylation blocks the GTPase activity of Gs and thereby renders Gs permanently active. This results in continuous activation of the adenylyl cyclase of intestinal epithelial cells, chronically high [cAMP], and chronically active PKA. PKA phosphorylates the CFTR Cl− channel (see Box 11-2) and a Na+-H+ exchanger in the intestinal epithelial cells. The resultant efflux of NaCl triggers massive water loss through the intestine as cells respond to the ensuing osmotic imbalance. Severe dehydration and electrolyte loss are the major pathologies in cholera. These can be fatal in the absence of prompt rehydration therapy. FIGURE 12-14 ADP-ribosylation locks Gsα in the active conformation. The bacterial toxin that causes cholera is an enzyme that catalyzes transfer of the ADP-ribose moiety of NAD+ (nicotinamide adenine dinucleotide) to an Arg residue of Gsα. The G proteins thus modified fail to respond to normal hormonal stimuli. The pathology of cholera results from defective regulation of adenylyl cyclase and overproduction of cAMP. Diacylglycerol, Inositol Trisphosphate, and Ca2+ Have Related Roles as Second Messengers A second broad class of GPCRs is coupled through a G protein to a plasma membrane phospholipase C (PLC) that catalyzes cleavage of the membrane phospholipid phosphatidylinositol 4,5- bisphosphate, or PIP2 (see Fig. 10-14). When one of the agonists (hormone, neurotransmitter, growth factor; Table 12-4) that act by this mechanism binds its specific receptor in the plasma membrane (Fig. 12-15, step ), the receptor-hormone complex catalyzes GTP-GDP exchange on an associated trimeric G protein, Gq (step ). This activates the Gq proteins in much the same way that the β -adrenergic receptor activates Gs (Fig. 12-4). The activated Gq activates the PIP2-specific PLC (Fig. 12-15, step ), which catalyzes the production of two potent second messengers (step ), diacylglycerol and inositol 1,4,5-trisphosphate, or IP3 (not to be confused with PIP3, p. 435 or Fig. 12-24). TABLE 12-4 Some Signals That Act through Phospholipase C, IP3 , and Ca2+ Acetylcholine [muscarinic M 1] Gastrin-releasing peptide Platelet-derived growth factor (PDGF) α1-Adrenergic agonists Glutamate Serotonin [5-HT 2] Angiogenin Gonadotropin-releasing hormone (GRH) Thyrotropin-releasing hormone (TRH) Angiotensin II Vasopressin Histamine [H1] ATP [P2x,P2y] Light (Drosophila) Auxin Oxytocin Note: Receptor subtypes are in square brackets; see footnote to Table 12-3. FIGURE 12-15 Hormone-activated phospholipase C and IP3. Two intracellular second messengers are produced in the hormone-sensitive phosphatidylinositol system: inositol 1,4,5-trisphosphate (IP3) and diacylglycerol are cleaved from phosphatidylinositol 4,5-bisphosphate (PIP2). Both contribute to the activation of protein kinase C. By raising cytosolic [Ca2+], IP3 also activates other Ca2+-dependent enzymes; thus Ca2+ also acts as a second messenger. Inositol trisphosphate, a water-soluble compound, diffuses from the plasma membrane to the endoplasmic reticulum (ER), where it binds to the IP3-gated Ca2+ channel, causing the channel to open. The action of the SERCA pump (pp. 393–394) ensures that [Ca2+] in the ER is orders of magnitude higher than that in the cytosol, so when these gated Ca2+ channels open, Ca2+ rushes into the cytosol (Fig. 12-15, step ), and the cytosolic [Ca2+] rises sharply to about 10−6 M . One effect of elevated [Ca2+] is the activation of protein kinase C (PKC; C for Ca2+). Diacylglycerol cooperates with Ca2+ in activating PKC, thus also acting as a second messenger (step ). Activation involves the movement of a PKC domain (the pseudosubstrate domain) away from its location in the substrate-binding region of the enzyme, allowing the enzyme to bind and phosphorylate proteins that contain a PKC consensus sequence — Ser or Thr residues embedded in an amino acid sequence recognized by PKC (step ). Figure 12-16 shows the structure of the IP3 receptor and a proposed mechanism of its action as a gated Ca2+ channel. FIGURE 12-16 Proposed mechanism of action of the IP3 -gated Ca2+ channel. (a) The IP3 receptor in its closed conformation, determined by cryo-EM. The 1.3 MDa tetramer has 24 transmembrane helices that surround a central pore. The bulk of the protein protrudes out of the ER into the cytosol and contains the IP3-binding sites. The central pore is the channel for Ca2+ movement, which does not conduct Ca2+ in the absence of IP3. (b, c) Model for receptor activation by IP3, showing only two of the four identical subunits (b) in the absence of IP3, and (c) with one IP3 bound to each subunit. According to this model, IP3 binding near the amino-terminal end of a subunit causes a major rearrangement of the α helix at the carboxyl-terminal end, opening the Ca2+ channel. [(a) Data from PDB ID 6MU2, G. Fan et al., Cell Res. 28:1158, 2018. (b, c) Information from M. J. Berridge, Physiol. Rev. 96:1261, 2016, Fig. 3.] Several isozymes of PKC are known, each with a characteristic tissue distribution, target protein specificity, and role. PLCβ is activated by GPCRs; another isoform, PLCγ , is activated by receptor tyrosine kinases, as described below. The ultimate PKC targets include cytoskeletal proteins, enzymes, and nuclear proteins that regulate gene expression. Taken together, this family of enzymes has a wide range of cellular actions, affecting neuronal and immune function and the regulation of cell division. Compounds that lead to overexpression of PKC or that increase its activity to abnormal levels act as tumor promoters; animals exposed to these substances have increased rates of cancer. Calcium Is a Second Messenger That Is Limited in Space and Time There are many variations on this basic scheme for Ca2+ signaling. In many cell types that respond to extracellular signals, Ca2+ serves as a second messenger that triggers intracellular responses, such as exocytosis in neurons and endocrine cells, contraction in skeletal muscle, and cytoskeletal rearrangements during amoeboid movement. In unstimulated cells, cytosolic [Ca2+] is kept very low (< 10−7 M ) by the action of Ca2+ pumps in the ER, mitochondria, and plasma membrane (as further discussed below). Hormonal, neural, or other stimuli cause either an influx of Ca2+ into the cell through specific Ca2+ channels in the plasma membrane or the release of sequestered Ca2+ from the ER or mitochondria, in either case raising the cytosolic [Ca2+] and triggering a cellular response. Changes in intracellular [Ca2+] are detected by Ca2+-binding proteins that regulate a variety of Ca2+-dependent enzymes. Calmodulin (CaM; Mr 17,000) is an acidic protein with four high- affinity Ca2+-binding sites (Kd ≈ 0.1 to 1 μ M) (Fig. 12-17). When intracellular [Ca2+] rises to about 10−6 M (1 μ M), the binding of Ca2+ to calmodulin drives a conformational change in the protein. Calmodulin associates with a variety of proteins and, in its Ca2+-bound state, modulates their activities. It is a member of a family of Ca2+-binding proteins that also includes troponin (see Fig. 5-30), which triggers skeletal muscle contraction in response to increased [Ca2+]. Members of this family share a characteristic Ca2+-binding structure, the EF hand. FIGURE 12-17 Calmodulin, the protein mediator of many Ca2+- stimulated enzymatic reactions. (a) In this ribbon model of the crystal structure of calmodulin, the four high-affinity Ca2+-binding sites are occupied by Ca2+ (purple). The amino-terminal domain is on the le ; the carboxyl-terminal domain on the right. (b) Calmodulin associated with a helical domain of one of the many enzymes it regulates, calmodulin- dependent protein kinase II. Notice that the long central α helix of calmodulin visible in (a) has bent back on itself in binding to the helical substrate domain. The central helix of calmodulin is clearly more flexible in solution than in the crystal. (c) Each of the four Ca2+-binding sites occurs in a helix-loop-helix motif called the EF hand, found in many Ca2+-binding proteins. [Data from (a) PDB ID 1CLL, R. Chattopadhyaya et al., J. Mol. Biol. 228:1177, 1992; (b, c) PDB ID 1CDL, W. E. Meador et al., Science 257:1251, 1992.] Calmodulin is a subunit of the Ca2+/calmodulin-dependent protein kinases (CaM kinases, types I through IV). When intracellular [Ca2+] increases in response to a stimulus, calmodulin binds Ca2+, undergoes a change in conformation, and activates the CaM kinase. The kinase then phosphorylates target enzymes, regulating their activities. Calmodulin is also a regulatory subunit of phosphorylase b kinase of muscle, which is activated by Ca2+. Thus Ca2+ triggers ATP-requiring muscle contractions while also activating glycogen breakdown, providing fuel for ATP synthesis. Many other enzymes are known to be modulated by Ca2+ through calmodulin (Table 12-5). The activity of the second messenger Ca2+, like that of cAMP, can be spatially restricted; a er its release triggers a local response, Ca2+ is generally removed before it can diffuse to distant parts of the cell. TABLE 12-5 Some Proteins Regulated by Ca2+ and Calmodulin Adenylyl cyclase (brain) Ca2+/calmodulin-dependent protein kinases (CaM kinases I to IV) Ca2+-dependent Na+ channel (Paramecium) Ca2+-release channel of sarcoplasmic reticulum Calcineurin (phosphoprotein phosphatase 2B) cAMP phosphodiesterase cAMP-gated olfactory channel cGMP-gated Na+, Ca2+ channels (rod and cone cells) Glutamate decarboxylase Myosin light-chain kinases NAD+ kinase Nitric oxide synthase Phosphatidylinositol 3-kinase Plasma membrane Ca2+ ATPase (Ca2+ pump) RNA helicase (p68) Commonly, Ca2+ level does not simply rise and then fall, but rather oscillates with a period of a few seconds (Fig. 12-18) — even when the extracellular concentration of the triggering hormone remains constant. The mechanism underlying [Ca2+] oscillations presumably entails feedback regulation by Ca2+ on some part of the Ca2+-release process. Whatever the mechanism, the effect is that one kind of signal (hormone concentration, for example) is converted into another (frequency and amplitude of intracellular [Ca2+] “spikes”). The Ca2+ signal diminishes as Ca2+ diffuses away from the initial source (the Ca2+ channel), is sequestered in the ER, or is pumped out of the cell. FIGURE 12-18 Triggering of oscillations in intracellular [Ca2+] by extracellular signals. (a) The dye fura, which undergoes fluorescence changes when it binds Ca2+, can be used with fluorescence microscopy to measure the instantaneous Ca2+ in cells. The color scale relates fluorescence intensity to [Ca2+]. Here, thymus cells have been stimulated with extracellular ATP, which raises their internal [Ca2+]. The cells are heterogeneous in their responses: some have high intracellular [Ca2+] (red), others have much lower [Ca2+] (blue). (b) When such a probe is used in a single hepatocyte, the agonist norepinephrine (added at the arrow) causes oscillations of [Ca2+] from 200 to 500 n . Similar oscillations are induced in other cell types by other extracellular signals. [(a) Courtesy Michael D. Cahalan, University of California, Irvine, Department of Physiology and Biophysics. (b) Data from T. A. Rooney et al., J. Biol. Chem. 264:17,131, 1989.] There is significant cross talk between the Ca2+ and cAMP signaling systems. In some tissues, both the enzyme that produces cAMP (adenylyl cyclase) and the enzyme that degrades cAMP (phosphodiesterase) are stimulated by Ca2+. Temporal and spatial changes in [Ca2+] can therefore produce transient, localized changes in [cAMP]. We have noted already that PKA, the enzyme that responds to cAMP, is o en part of a highly localized supramolecular complex assembled on scaffold proteins such as AKAPs. This subcellular localization of target enzymes, combined with temporal and spatial gradients in [Ca2+] and [cAMP], allows a cell to respond to one or several signals with subtly nuanced metabolic changes. SUMMARY 12.2 G Protein–Coupled Receptors and Second Messengers G protein–coupled receptors (GPCRs) have seven transmembrane helices and act through heterotrimeric G proteins. Ligand binding activates the G protein, which then stimulates or inhibits the activity of an effector enzyme, changing the local concentration of its second-messenger product cAMP. Epinephrine, acting through its GPCR and the Gs protein, stimulates adenylyl cyclase, which produces cAMP. Cyclic AMP activates protein kinase A (PKA), which then phosphorylates target proteins on a Ser or Thr residue, changing their biological activity. To end the response to epinephrine, a phosphodiesterase breaks down cAMP, and the G protein is inactivated by its own GTPase activity. Phosphoprotein phosphatases reverse the effects of PKA. When the epinephrine signal persists, β -adrenergic receptor– specific protein kinase phosphorylates the GPCR, creating a binding site for the protein β -arrestin, which prevents interaction between the GPCR and its G protein. Arrestin triggers desensitization by movement of the receptor into intracellular vesicles. GPCRs typically have their effects through the G protein-cAMP- PKA pathway; some act through Gs, raising [cAMP], others through Gi, lowering [cAMP]. Adaptor proteins such as AKAPs tether PKA and limit its area of action and its target proteins. Trimeric G proteins coupled to GPCRs are activated when their bound GDP is exchanged for GTP, and they remain active until their GTPase converts bound GTP to GDP. Their GTPase activity is modulated by GTPase activator proteins (GAPs) and regulators of G-protein signaling (RGSs). Monomeric (small) G proteins such as Ras, Rab, and Ran also serve as self-limiting switches. Defective G-protein signaling is common in individuals with some types of cancer. Some GPCRs are coupled to a G protein (Gq) that acts through a plasma membrane phospholipase C that cleaves PIP2 to diacylglycerol and IP3. By opening Ca2+ channels in the endoplasmic reticulum, IP3 raises cytosolic [Ca2+]. Diacylglycerol and Ca2+ act together to activate protein kinase C, which phosphorylates and changes the activity of specific cellular proteins. Cellular [Ca2+] also regulates (o en through calmodulin) many other enzymes and proteins involved in secretion, cytoskeletal rearrangements, or contraction. Many of the target enzymes are in the family of Ca2+-activated protein kinases (PKCs). 12.3 GPCRs in Vision, Olfaction, and Gustation The detection of light, odors, and tastes (vision, olfaction, and gustation, respectively) in animals is accomplished by specialized sensory neurons that use signal-transduction mechanisms fundamentally similar to those that detect hormones, neurotransmitters, and growth factors. An initial sensory signal is greatly amplified by mechanisms that include gated ion channels and intracellular second messengers; the system adapts to continued stimulation by changing its sensitivity to the stimulus (desensitization); and sensory input from several receptors is integrated before the final signal goes to the brain. The Vertebrate Eye Uses Classic GPCR Mechanisms Visual transduction (Fig. 12-19) begins when light falls on rhodopsin, a GPCR in the disk membranes of rod cells of the vertebrate eye. (Rod cells do not detect colors; cone cells do, as we shall see in Box 12-3.) The light-absorbing pigment (chromophore) 11-cis-retinal is covalently attached to opsin, the protein component of rhodopsin, which lies near the middle of the disk membrane bilayer. When a photon is absorbed by the retinal component of rhodopsin (step ), the energy causes a photochemical change; 11-cis-retinal is converted to all-trans- retinal (see Fig. 10-20). This change in the structure of the chromophore forces conformational changes in the rhodopsin molecule, allowing it to interact with and thus activate its trimeric G protein, transducin. Rhodopsin now stimulates the exchange of bound GDP on transducin for GTP from the cytosol (Fig. 12-19, step ), and activated transducin stimulates the membrane protein cyclic GMP (cGMP) phosphodiesterase (PDE) by removing an inhibitory subunit (step ). The activated cGMP PDE degrades the second messenger 3′,5′-cGMP to 5′-GMP, lowering [cGMP] (step ). A cGMP-dependent Na+ or Ca2+ channel in the plasma membrane closes (step ), while a Na+-Ca2+ active antiporter continues to pump Ca2+ outward across the plasma membrane (step ), making the transmembrane electrical potential more negative inside (that is, hyperpolarizing the rod cell). This electrical change passes through a series of specialized nerve cells to the visual cortex of the brain. FIGURE 12-19 Molecular consequences of photon absorption by rhodopsin in the rod outer segment. The top half of the figure (steps to ) describes excitation; the bottom shows post-illumination steps: recovery (steps and ) and adaptation (steps and ). BOX 12-3 MEDICINE Color Blindness: John Dalton’s Experiment from the Grave Color vision involves a path of sensory transduction in specialized cells in the retina. Three types of cone cells are specialized to detect light from different regions of the spectrum, using three related photoreceptor proteins (opsins). Each cone cell expresses only one kind of opsin, but each type is closely related to rhodopsin in size, amino acid sequence, and, presumably, three- dimensional structure. The differences among the opsins, however, are great enough to place the chromophore, 11-cis-retinal, in three slightly different environments, with the result that the three photoreceptors have different absorption spectra (Fig. 1). We discriminate colors and hues by integrating the output from the three types of cone cells, each containing one of the three types of photoreceptors. FIGURE 1 Absorption spectra of purified rhodopsin and the red, green, and blue receptors of cone cells. The receptor spectra, obtained from individual cone cells isolated from cadavers, peak at about 420, 530, and 560 nm, and the maximum absorption for rhodopsin is at about 500 nm. For reference, the visible spectrum for humans is about 380 to 750 nm. [Data from J. Nathans, Sci. Am. 260 (February):42, 1989.] Color blindness, such as the inability to distinguish red from green, is a fairly common, genetically inherited trait in humans. The various types of color blindness result from different opsin mutations. One form is due to loss of the red photoreceptor; affected individuals are red− dichromats (they see only two primary colors). Others lack the green pigment and are green− dichromats. In some cases, the red and green photoreceptors are present but have a changed amino acid sequence that causes a change in their absorption spectra, resulting in abnormal color vision. Depending on which pigment is altered, these individuals are red-anomalous trichromats or green- anomalous trichromats. Examination of the genes for the visual receptors has allowed the diagnosis of color blindness in the chemist John Dalton more than a century a er his death. Dalton (of atomic theory fame) was color-blind. He thought it probable that the vitreous humor of his eyes (the fluid that fills the eyeball behind the lens) was tinted blue, unlike the colorless fluid of normal eyes. He proposed that a er his death, his eyes should be dissected and the color of the vitreous humor determined. His wish was honored. The day a er Dalton’s death in July 1844, Joseph Ransome dissected his eyes and found the vitreous humor to be perfectly colorless. Ransome, like many scientists, was reluctant to throw samples away. He placed Dalton’s eyes in a jar of preservative, where they stayed for a century and a half (Fig. 2). FIGURE 2 Dalton’s eyes. Then, in the mid-1990s, molecular biologists in England took small samples of Dalton’s retinas and extracted DNA. Using the known gene sequences for the opsins of the red and green light receptors, they amplified the relevant sequences using PCR and determined that Dalton had the opsin gene for the red photopigment but lacked the opsin gene for the green photopigment. Dalton was a green− dichromat. So, 150 years a er his death, the experiment Dalton started — by hypothesizing about the cause of his color blindness — was finally finished. Several steps in the visual-transduction process result in a huge amplification of the signal. Each excited rhodopsin molecule activates at least 500 molecules of transducin, and each transducin molecule can activate a molecule of cGMP PDE. This phosphodiesterase has a remarkably high turnover number: each activated molecule hydrolyzes 4,200 molecules of cGMP per second. The binding of cGMP to cGMP-gated ion channels is cooperative, and a relatively small change in [cGMP] therefore registers as a large change in ion conductance. The result of these amplifications is exquisite sensitivity to light. Absorption of a single photon closes 1,000 or more ion channels for Na+ and Ca2+, hyperpolarizing the cell’s membrane by about 1 mV. As your eyes move across this line of type, the retinal images of the first words disappear rapidly — before you see the next series of words. In that short interval, a great deal of biochemistry has taken place. Very soon a er illumination of the rod or cone cells stops, the photosensory system shuts off. The α subunit of transducin (T α, with bound GTP) has GTPase activity. Within milliseconds a er the decrease in light intensity, GTP is hydrolyzed and T α reassociates with T βγ. The inhibitory subunit of PDE, which had been bound to T α-GTP, is released and reassociates with PDE, strongly inhibiting its activity and thus slowing cGMP breakdown. At the same time, a second factor that helps to end the response to light is the reduction of intracellular [Ca2+] that results from continued Ca2+ efflux through the Na+-Ca2+ exchanger (Fig. 12- 19, step ). High [Ca2+] inhibits the enzyme that makes cGMP (guanylyl cyclase; step ), so cGMP production rises when [Ca2+] falls, quickly reaching its prestimulus level. In response to prolonged illumination, rhodopsin itself undergoes changes that limit the duration of its signaling activity. The conformational change induced in rhodopsin by light absorption exposes several Thr and Ser residues in its carboxyl- terminal domain, and these residues are phosphorylated by rhodopsin kinase (step ), which is functionally and structurally homologous to the β -adrenergic kinase (β ARK) that desensitizes the β -adrenergic receptor. The phosphorylated carboxyl- terminal domain of rhodopsin is bound by the protein arrestin 1, preventing further interaction between activated rhodopsin and transducin (see Fig. 12-10b). Arrestin 1 is a close homolog of arrestin 2 (β arr) of the β -adrenergic system. On a much longer time scale (step ), the all-trans-retinal bound to light-bleached rhodopsin is removed and replaced with 11-cis-retinal, making rhodopsin ready to detect another photon. Vertebrate Olfaction and Gustation Use Mechanisms Similar to the Visual System The sensory cells that detect odors and tastes have much in common with the visual receptor system. Binding of an odorant molecule to one of its specific GPCRs (humans have about 800 different GPCRs; rodents have about 1,200) triggers a change in receptor conformation, activating a G protein, G olf, analogous to transducin and to G s of the β -adrenergic system. The activated G olf activates adenylyl cyclase, raising the local [cAMP]. The cAMP-gated Na+ and Ca2+ channels of the plasma membrane open, and the influx of Na+ and Ca2+ produces a small depolarization called the receptor potential. If a sufficient number of odorant molecules encounter receptors, the receptor potential is strong enough to cause the neuron to fire an action potential. This signal is relayed to the brain in several stages and registers as a specific smell. All these events occur within 100 to 200 ms. When the olfactory stimulus is no longer present, the transducing machinery shuts itself off in several ways. A cAMP phosphodiesterase returns [cAMP] to the prestimulus level. G olf hydrolyzes its bound GTP to GDP, thereby inactivating itself. Phosphorylation of the receptor by a specific kinase prevents its interaction with G olf, by a mechanism analogous to that used to desensitize the β -adrenergic receptor and rhodopsin. Some odorants are detected by another mechanism we have seen in other signal transductions: activation of a phospholipase and production of IP3, leading to a rise in intracellular [Ca2+]. The sense of taste in vertebrates reflects the activity of gustatory neurons clustered in taste buds on the surface of the tongue. In taste sensory neurons, GPCRs are coupled to the heterotrimeric G protein gustducin. When the tastant molecule binds its receptor, gustducin is activated and stimulates cAMP production by adenylyl cyclase. The resulting elevation of [cAMP] activates PKA, which phosphorylates K+ channels in the plasma membrane, causing them to close and sending an electrical signal to the brain. Other taste buds specialize in detecting bitter, sour, salty, or umami (savory) tastants, using various combinations of second messengers and ion channels in the transduction mechanisms. All GPCR Systems Share Universal Features We have now looked at several types of signaling systems (hormone signaling, vision, olfaction, and gustation) in which membrane receptors are coupled to second messenger– generating enzymes through G proteins. As we have intimated, signaling mechanisms must have arisen early in evolution; genomic studies have revealed hundreds of genes encoding GPCRs in vertebrates, arthropods (Drosophila and mosquito), and the roundworm Caenorhabditis elegans. Even the common budding yeast Saccharomyces uses GPCRs and G proteins to detect the opposite mating type. Overall patterns have been conserved, and the introduction of variety has given modern organisms the ability to respond to a wide range of stimuli (Table 12-6). Of the approximately 20,000 genes in the human genome, as many as 800 encode GPCRs, including hundreds for olfactory stimuli and many orphan receptors, for which the natural ligand is not yet known. TABLE 12-6 Some Signals That Act through GPCRs Amines Acetylcholine (muscarinic) Dopamine Epinephrine Histamine Serotonin Peptides Angiotensin Bombesin Bradykinin Chemokine Colecystokinin (CCK) Endothelin Gonadotropin-releasing hormone Interleukin-8 Melanocortin Neuropeptide Y Neurotensin Orexin Somatostatin Tachykinin Thyrotropin-releasing hormone Vasopressin Protein hormones Follicle-stimulating hormone Gonadotropin Lutropin-choriogonadotropic hormone Thyrotropin Prostanoids Prostacyclin Prostaglandin Thromboxane Others Cannabinoids Lysosphingolipids Melatonin Olfactory stimuli Opioids Rhodopsin All well-studied signal-transducing systems that act through heterotrimeric G proteins share some common features that reflect their evolutionary relatedness (Fig. 12-20). The receptors have seven transmembrane segments, a domain (generally the loop between transmembrane helices 6 and 7) that interacts with a G protein, and a carboxyl-terminal cytoplasmic domain that undergoes reversible phosphorylation on several Ser or Thr residues. The ligand-binding site (or, in the case of light reception, the light receptor) is buried deep in the membrane and includes residues from several of the transmembrane segments. Ligand binding (or light) induces a conformational change in the receptor, exposing a domain that can interact with a G protein. Heterotrimeric G proteins activate or inhibit effector enzymes (adenylyl cyclase, PDE, or PLC), which change the concentration of a second messenger (cAMP, cGMP, IP3, or Ca2+). In the hormone-detecting systems, the final output is an activated protein kinase that regulates some cellular process by phosphorylating a protein critical to that process. In sensory neurons, the output is a change in membrane potential and a consequent electrical signal that passes to another neuron in the pathway connecting the sensory cell to the brain. FIGURE 12-20 Common features of signaling systems that detect hormones, light, smells, and tastes. GPCRs provide signal specificity, and their interaction with G proteins provides signal amplification. Heterotrimeric G proteins activate or inhibit effector enzymes: adenylyl cyclase (AC) and phosphodiesterases (PDEs) that degrade cAMP or cGMP. Changes in concentration of the second messengers (cAMP, cGMP) result in alterations in enzymatic activities via phosphorylation or alterations in the permeability (P) of surface membranes to Ca2+, Na+, and K+. The resulting depolarization or hyperpolarization of the sensory cell (the signal) passes through relay neurons to sensory centers in the brain. In the best- studied cases, desensitization includes phosphorylation of the receptor and binding of a protein (arrestin) that interrupts receptor–G protein interactions. (The path of odorant detection by production of IP3 and increase in intracellular [Ca2+], mentioned in the text, is not shown here.) VR is the vasopressin receptor; β -AR, the β -adrenergic receptor; Rh, rhodopsin; OR, olfactory receptor; SR, sweet-taste receptor. All these systems self-inactivate. Bound GTP is converted to GDP by the GTPase activity of G proteins, o en augmented by GTPase- activating proteins (GAPs) or regulators of G-protein signaling (RGS). In some cases, the effector enzymes that are the targets of modulation by G proteins also serve as GAPs. The desensitization mechanism involving phosphorylation of the carboxyl-terminal region followed by arrestin binding is widespread and may be universal. SUMMARY 12.3 GPCRs in Vision, Olfaction, and Gustation Vision, olfaction, and gustation in vertebrates employ GPCRs, which act through heterotrimeric G proteins to change the membrane potential (Vm ) of a sensory neuron. Light activates the GPCR rhodopsin, which allosterically activates the trimeric G protein transducin. T α activates a cGMP phosphodiesterase, lowering [cGMP] and closing cGMP- dependent ion channels. The resulting electrical impulse carries the signal to the brain. In olfactory neurons, olfactory stimuli, acting through GPCRs and G proteins, trigger an increase in [cAMP] (by activating adenylyl cyclase) or [Ca2+] (by activating PLC). These second messengers affect ion channels and thus the Vm. Gustatory neurons have GPCRs that respond to tastants by altering levels of cAMP, which changes Vm by gating ion channels. There is a high degree of conservation of signaling proteins and transduction mechanisms across signaling systems and across species. GPCRs with seven transmembrane helices, G proteins with intrinsic GTPase activities, cyclic nucleotides, and protein kinases are central to signaling. 12.4 Receptor Tyrosine Kinases The receptor tyrosine kinases (RTKs), a family of plasma membrane receptors with protein kinase activity, transduce extracellular signals by a mechanism fundamentally different from that of GPCRs. RTKs have a ligand-binding domain on the extracellular face of the plasma membrane and an enzyme active site on the cytoplasmic face, connected by a single transmembrane segment, as seen for insulin in Figure 12-21a. The cytoplasmic domain is a Tyr kinase, a protein kinase that phosphorylates specific Tyr residues in target proteins. The receptors for insulin and epidermal growth factor are prototypes for the 58 RTKs in humans (see Fig. 12-26). FIGURE 12-21 Activation of the insulin-receptor tyrosine kinase by autophosphorylation. (a) A structural model assembled from crystal structures of the extracellular and tyrosine kinase domains and NMR solution structure of the transmembrane domain. The insulin-binding region of the insulin receptor lies outside the cell and comprises two intertwined α subunits. (b) The binding of a single molecule of insulin is communicated through the single transmembrane helix of each β subunit to the paired Tyr kinase domains inside the cell, moving them toward each other and activating them to phosphorylate each other on three Tyr residues. (c) In the inactive form of the Tyr kinase domain, the activation loop (backbone shown in teal) sits in the active site, and none of the critical Tyr residues (stick structures) are phosphorylated. This conformation is stabilized by hydrogen bonding between Tyr1162 and Asp1132. (d) Activation of the Tyr kinase allows each β subunit to phosphorylate three Tyr residues (Tyr1158,Tyr1162, Tyr1163) on the other β subunit. (Phosphoryl groups are depicted in red and orange.) The introduction of three highly charged –Tyr residues forces a 30 Å change in the position of the activation loop, away from the substrate-binding site, which thus becomes available to bind and phosphorylate a target protein. [(a) Data and information from T. Gutmann et al., J. Cell Biol. 21:1643, 2018, Fig. 1. Data from (c) PDB ID 1IRK, S. R. Hubbard et al., Nature 372:746, 1994; (d) PDB ID 1IR3, S. R. Hubbard, EMBO J. 16:5572, 1997.] Stimulation of the Insulin Receptor Initiates a Cascade of Protein Phosphorylation Reactions Insulin regulates both metabolic enzymes and gene expression. It initiates a signal that travels divergent pathways from the plasma membrane receptor to insulin-sensitive enzymes in the cytosol, and to enzymes that act in the nucleus by stimulating the transcription of specific genes. The active insulin receptor protein (INSR) consists of two identical α subunits protruding from the outer face of the plasma membrane and two transmembrane β subunits with their carboxyl termini protruding into the cytosol — a dimer of αβ monomers (Fig. 12-21). The α subunits contain the insulin-binding domain, and the intracellular domains of the β subunits contain the protein kinase activity that transfers a phosphoryl group from ATP to the hydroxyl group of Tyr residues in specific target proteins. Signaling through INSR begins with the binding of one insulin molecule between the two subunits of the receptor on the extracellular side, causing movement of the Tyr kinase domains together and stimulating their activity. Each β subunit phosphorylates three essential Tyr residues near the carboxyl terminus of the other β subunit. This autophosphorylation opens the active site so that the enzyme can phosphorylate Tyr residues of other target proteins. The mechanism of activation of the INSR protein kinase is similar to that described for PKA and PKC: a region of the cytoplasmic domain called the activation loop that usually occludes the active site moves out of the active site a er being phosphorylated, opening the site for the binding of target proteins (Fig. 12-21c, d). When INSR is autophosphorylated (Fig. 12-22, step ) and becomes an active Tyr kinase, one of its targets is the protein insulin receptor substrate 1 (IRS1; step ). Once phosphorylated on several of its Tyr residues, IRS1 becomes the point of nucleation for a complex of proteins (step ) that carry the message from the insulin receptor to end targets in the cytosol and nucleus, through a long series of intermediate proteins. First, a –Tyr residue of IRS1 binds to the SH2 domain of the protein Grb2. Many signaling proteins contain SH2 (Src homology 2) domains, all of which bind –Tyr residues in a protein partner. Grb2 (growth factor receptor-bound protein 2) is an adaptor protein, with no intrinsic enzymatic activity. Its function is to bring together two proteins (in this case, IRS1 and the protein Sos) that must interact to enable signal transduction. In addition to its SH2 ( –Tyr-binding) domain, Grb2 contains a second protein-binding domain, SH3, that binds to a proline-rich region of Sos (son of sevenless), recruiting Sos to the growing receptor complex. When bound to Grb2, Sos acts as a guanosine nucleotide–exchange factor (GEF), catalyzing the replacement of bound GDP with GTP on the small G protein Ras. FIGURE 12-22 Regulation of gene expression by insulin through a MAP kinase cascade. The signaling pathway by which insulin regulates the expression of specific genes consists of a cascade of protein kinases, each of which activates the next. The insulin receptor is a Tyr-specific kinase; the other kinases (all shown in blue) phosphorylate Ser or Thr residues. MEK is a dual-specificity kinase that phosphorylates both a Thr residue and a Tyr residue in ERK (extracellular regulated kinase). MEK is mitogen-activated, ERK-activating kinase; SRF is serum response factor. In its GTP-bound active form, Ras can activate a protein kinase, Raf-1 (Fig. 12-22, step ), the first of three protein kinases — Raf- 1, MEK, and ERK — that form a cascade in which each kinase activates the next by phosphorylation (step ). The protein kinases MEK and ERK are activated by phosphorylation of both a Thr residue and a Tyr residue. When activated, ERK mediates some of the biological effects of insulin by entering the nucleus and phosphorylating transcription factors, including Elk1 (step ), that modulate the transcription of about 100 insulin-regulated genes (step ), some of which encode proteins essential for cell division. Thus, insulin acts as a growth factor. The proteins Raf-1, MEK, and ERK are members of three larger families, for which several nomenclatures are used. ERK is in the MAPK family (mitogen-activated protein kinases; mitogens are extracellular signals that induce mitosis and cell division). Soon a er discovery of the first MAPK enzyme, that enzyme was found to be activated by another protein kinase, which was named MAP kinase kinase (MEK belongs to this family), and when a third kinase that activated MAP kinase kinase was discovered, it was given the slightly ludicrous family name MAP kinase kinase kinase (Raf-1 is in this family). Somewhat less cumbersome are the abbreviations for these three families: MAPK, MAPKK, and MAPKKK. Kinases in the MAPK and MAPKKK families are specific for Ser residues or Thr residues, and MAPKKs (here, MEK) phosphorylate both a Ser residue and a Tyr residue in their substrate, a MAPK (here, ERK). This insulin pathway is but one instance of a more general scheme in which hormone signals, via pathways similar to that shown in Figure 12-22, result in a change in the phosphorylation of target enzymes by protein kinases or phosphoprotein phosphatases. The target of phosphorylation is o en another protein kinase, which then phosphorylates a third protein kinase, and so on. The result is a cascade of reactions that amplifies the initial signal by many orders of magnitude (see Fig. 12-7). MAPK cascades (such as the Raf-MEK-ERK sequence in Fig. 12-22) mediate signaling initiated by a variety of growth factors, such as platelet-derived growth factor (PDGF) and epidermal growth factor (EGF), both of which are receptor tyrosine kinases like the insulin receptor. Another general scheme exemplified by the insulin receptor pathway is the use of nonenzymatic adaptor proteins, o en with intrinsically disordered regions, to bring together the components of divergent signaling pathways, to which we now turn. The Membrane Phospholipid PIP3 Functions at a Branch in Insulin Signaling The signaling pathway from insulin branches at IRS1 (step in Fig. 12-22 and Fig. 12-23). Grb2 is not the only protein that associates with phosphorylated IRS1. The enzyme phosphoinositide 3-kinase (PI3K) binds IRS1 through PI3K’s SH2 domain. Thus activated, PI3K converts the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) to phosphatidylinositol 3,4,5-trisphosphate (PIP3) by the transfer of a phosphoryl group from ATP (Fig. 12-24). The multiply (negatively) charged head group of PIP3, protruding on the cytoplasmic side of the plasma membrane, is the starting point for a second signaling branch involving another cascade of protein kinases. When bound to PIP3, protein kinase B (PKB; also called Akt) is phosphorylated and activated by yet another protein kinase, PDK1 (not shown in Fig. 12-23). The activated PKB then phosphorylates Ser or Thr residues in its target proteins, one of which is glycogen synthase kinase 3 (GSK3). In its active, nonphosphorylated form, GSK3 phosphorylates glycogen synthase, inactivating it and thereby contributing to the slowing of glycogen synthesis. (This mechanism is only part of the explanation for the effects of insulin on glycogen metabolism; see Fig. 15-16.) When phosphorylated by PKB, GSK3 is inactivated. By thus preventing inactivation of glycogen synthase in liver and muscle, the cascade of protein phosphorylations initiated by insulin ultimately stimulates glycogen synthesis (Fig. 12-23). FIGURE 12-23 Insulin action on glycogen synthesis and GLUT4 movement to the plasma membrane. The activation of PI3 kinase (PI3K) by phosphorylated IRS1 initiates (through protein kinase B, PKB) movement of the glucose transporter GLUT4 to the plasma membrane, and the activation of glycogen synthase.
FIGURE 12-24 Regulation of PIP3 formation and breakdown. Phosphoinositide 3- kinase (PI3K) responds to the insulin signal by catalyzing the transfer of a phosphoryl group from ATP to C-3 of the inositol ring of phosphatidylinositol 4,5-bisphosphate (PIP2) to form phosphatidylinositol 3,4,5-trisphosphate (PIP3), which serves as a nucleation point for proteins involved in the MAPK cascade. The enzyme PTEN (phosphatase and tensin homolog) ends the response by catalyzing removal of the same phosphoryl group, which is released as Pi. In a third signaling branch important in muscle and fat tissue, PKB, acting through two small G proteins (RAC1 and Rab) triggers the clathrin-aided movement of glucose transporters (GLUT4) from intracellular vesicles to the plasma membrane, stimulating glucose uptake from the blood (Fig. 12-23, step ; see also Box 11- 1). This increase in glucose uptake triggered by insulin has profound metabolic and medical consequences, as we shall see in Chapter 23. How fast is the response to insulin? Phosphoproteomics uses high-resolution, high-throughput mass spectrometry to determine, for thousands of proteins in a cell type, which residues in which proteins are phosphorylated in response to a stimulus such as insulin in the living animal (Fig. 12-25). Autophosphorylation of the insulin receptor and IRS1 occurs within a few seconds of insulin addition; protein kinase Cβ is phosphorylated slightly later (within 15 s), and Sos and Gab are phosphorylated within 0.5 and 1 min. ERK1, the end target in this signaling pathway (Fig. 12-22), is maximally phosphorylated within 3 min. The movement of glucose transporter GLUT4 into the plasma membrane takes about 15 min; and the many changes in gene expression in response to insulin occur over several hours. A recently discovered action of insulin has a different mechanism and a slower time course. In some cases, insulin enters the cell and the nucleus, where, with the help of several other nuclear proteins, it regulates gene expression by binding to promoter regions on the DNA. FIGURE 12-25 Time course of phosphorylations triggered by insulin. Mouse liver proteins were examined by mass spectrometry at intervals a er injection of insulin to determine quantitatively for each protein which amino acid residues became phosphorylated and when phosphorylation occurred. Each graph represents the phosphorylation of a single residue in the protein, designated by its one-letter abbreviation and position in the primary sequence. Proteins are INSR, insulin receptor; IRS2, insulin receptor substrate 2; PKCβ , protein kinase Cβ; Sos and ERK, as in Fig. 12- 22. Gab2 is an adaptor protein involved in the MAPK and PI3K signaling pathways. [Data from S. J. Humphrey et al., Nature Biotechnol. 33:990, 2015, Fig. 4.] As in all signaling pathways, there is a mechanism for terminating the activity of the PI3K-PKB pathway. A PIP3-specific phosphatase (in humans, PTEN) removes the phosphoryl group at the 3 position of PIP3 to produce PIP2 (see Fig. 12-24), which no longer serves as a binding site for PKB, and the signaling chain is broken. In several types of cancer, the PTEN gene has o en undergone mutation, resulting in a defective regulatory circuit and abnormally high levels of PIP3 activity. The result is a continuing signal for cell division and thus tumor growth. The unmutated PTEN gene is a tumor suppressor, the subject of Section 12.9. The insulin receptor is the prototype for several receptor enzymes with a similar structure and RTK activity (Fig. 12-26). The receptors for EGF and PDGF, for example, have structural and sequence similarities to INSR, and both have a protein Tyr kinase activity that phosphorylates IRS1. Many of these receptors dimerize a er binding ligand and before autophosphorylation. INSR is the exception, as it is already an (αβ)2 dimer before insulin binds. (The protomer of the insulin receptor is one αβ unit.) The binding of adaptor proteins such as Grb2 to –Tyr residues is a common mechanism for promoting protein-protein interactions initiated by RTKs, a subject to which we return in Section 12.5. FIGURE 12-26 Receptor tyrosine kinases. Growth factor receptors that initiate signals through Tyr kinase activity include those for insulin (INSR), vascular endothelial growth factor (VEGFR), platelet-derived growth factor (PDGFR), epidermal growth factor (EGFR), high-affinity nerve growth factor (TrkA), and fibroblast growth factor (FGFR). All these receptors have a Tyr kinase domain (blue) on the cytoplasmic side of the plasma membrane. The extracellular domain is unique to each type of receptor, reflecting the different growth factor specificities. These extracellular domains are typically combinations of structural motifs such as Cys- or Leu-rich segments and segments containing one of several motifs common to immunoglobulins (Ig). Many other Tyr kinase receptors are encoded in the human genome, each with a different extracellular domain and ligand specificity. All of the receptors except INSR are monomeric and their Tyr kinase activity is silent until ligand binding triggers receptor dimerization and Tyr kinase activation. Only INSR is always dimeric, but its Tyr kinase is active only when insulin is bound. What spurred the evolution of such complicated regulatory machinery? This system allows one activated receptor to activate several IRS1 molecules, amplifying the insulin signal, and it provides for the integration of signals from different receptors such as EGFR and PDGFR, each of which can phosphorylate IRS1. Furthermore, because IRS1 can activate any of several proteins that contain SH2 domains, a single receptor acting through IRS1 can trigger two or more signaling pathways; insulin affects gene expression through the mitogenic Grb2-Sos-Ras-MAPK pathway and affects glycogen metabolism and glucose transport through the metabolic PI3K-PKB pathway. Finally, there are several closely related IRS proteins (IRS2, IRS3), each with its own characteristic tissue distribution and function, further enriching the signaling possibilities in pathways initiated by RTKs. Cross Talk among Signaling Systems Is Common and Complex For simplicity, we have treated individual signaling pathways as separate sequences of events leading to separate metabolic consequences, but there is, in fact, extensive cross talk among signaling systems. The regulatory circuitry that governs metabolism is richly interwoven and multilayered. We have discussed the signaling pathways for insulin and epinephrine separately, but they do not operate independently. Insulin opposes the metabolic effects of epinephrine in most tissues, and activation of the insulin signaling pathway directly reduces signaling through the β -adrenergic signaling system. For example, the INSR kinase directly phosphorylates two Tyr residues in the cytoplasmic tail of a β2-adrenergic receptor, and PKB, activated by insulin, phosphorylates two Ser residues in the same region (Fig. 12-27). Phosphorylation of these four residues triggers clathrin-aided internalization of the β2-adrenergic receptor, lowering the cell’s sensitivity to epinephrine. FIGURE 12-27 Cross talk between the insulin receptor and the β - adrenergic receptor (or other GPCR). When INSR is activated by insulin binding, its Tyr kinase directly phosphorylates the β -adrenergic receptor (right side) on two Tyr residues (Tyr350 and Tyr364) near its carboxyl terminus, and indirectly causes phosphorylation of two Ser residues in the same region (through activation of protein kinase B (PKB). The effect of these phosphorylations is internalization of the adrenergic receptor, reducing the response to the adrenergic stimulus. Alternatively (le side), INSR-catalyzed phosphorylation of a GPCR (an adrenergic or other receptor) on a carboxyl-terminal Tyr creates the point of nucleation for activating the MAPK cascade (see Fig. 12-22), with Grb2 serving as the adaptor protein. In this case, INSR has used the GPCR to enhance its own signaling. A second type of cross talk between these receptors occurs when –Tyr residues on the β2-adrenergic receptor, phosphorylated by INSR, serve as nucleation points for SH2 domain–containing proteins such as Grb2 (Fig. 12-27, le side). Activation of the MAPK ERK by insulin is 5- to 10-fold greater in the presence of the β2-adrenergic receptor, presumably because of this cross talk. Signaling systems that use cAMP and Ca2+ also show extensive interaction; each second messenger affects the generation and concentration of the other. Yet another factor that further complicates the signaling picture is that certain metabolic intermediates, such as fatty acids and ceramides, amino acids, and bile acids, can influence insulin signaling. One of the major challenges of systems biology is to sort out the effects of such interactions on the overall metabolic patterns in each tissue — a daunting task. SUMMARY 12.4 Receptor Tyrosine Kinases The insulin receptor, INSR, is the prototype of receptor enzymes with Tyr kinase activity. When insulin binds, the receptor Tyr kinase is activated, and phosphorylates Tyr residues on other proteins, such as IRS. Phosphotyrosine residues of IRS1 serve as binding sites for proteins with SH2 domains. These multivalent proteins can serve as adaptors that bring two proteins into proximity. Sos bound to Grb2 activates Ras, which in turn activates a MAPK cascade that ends with the phosphorylation of target proteins in the cytosol and nucleus. The result is specific metabolic changes and altered gene expression. The enzyme PI3K, activated by interaction with IRS1, phosphorylates the membrane lipid PIP2 to PIP3, which becomes the point of nucleation for proteins in a second and third branch of insulin signaling. Extensive interconnections among signaling pathways allow integration and fine-tuning of multiple hormonal effects. 12.5 Multivalent Adaptor Proteins and Membrane Ra s Two generalizations have emerged from studies of signaling systems such as those we have discussed so far. First, protein kinases that phosphorylate Tyr, Ser, and Thr residues — and phosphatases that dephosphorylate them — are central to signaling, directly affecting the activities of a large number of protein substrates. Second, protein-protein interactions brought about by the reversible phosphorylation of Tyr, Ser, and Thr residues in signaling proteins create docking sites for other proteins that bring about indirect effects on proteins downstream in the signaling pathway. In fact, many signaling proteins are multivalent: they can interact with several different proteins simultaneously to form multiprotein signaling complexes. In this section we present a few examples to illustrate the general principles of phosphorylation-dependent protein interactions in signaling pathways. Many of the proteins involved in signaling have intrinsically disordered regions (IDRs) that are flexible enough to allow specific interaction with more than one protein — and perhaps with many other proteins. Protein kinases, for example, have a well-conserved structure containing the substrate-binding and catalytic sites, but through evolution they have acquired additional sequences, typically 20–30 residues long, that are partially disordered and can fold to fit into various multienzyme regulatory networks. The activation loop found in most protein kinases is an IDR and is the universal regulator of kinase activity; its position changes when one or several residues become phosphorylated, switching on the kinase activity. Protein kinases of the PKA, PKB, and PKC families have a disordered carboxyl- terminal tail containing critical residues whose phosphorylation state switches between active and inactive kinase structures. In the MAPK cascade, an amino-terminal IDR plays a similar role as a docking site in the multienzyme complex. Given the ~1,000 protein kinase genes in humans, the many noncatalytic scaffold proteins, and the multiple interactions possible for IDRs in many of these proteins, the number of possible permutations and combinations available for switching and regulating metabolic processes by protein phosphorylation is impressively large. Protein Modules Bind Phosphorylated Tyr, Ser, or Thr Residues in Partner Proteins The protein Grb2 in the insulin signaling pathway (Figs. 12-22 and 12-27) binds through its SH2 domain to other proteins that have exposed –Tyr residues. The human genome encodes at least 87 SH2-containing proteins, many known to participate in signaling. The –Tyr residue is bound in a deep pocket in an SH2 domain, with each of its phosphate oxygens participating in hydrogen bonding or electrostatic interactions; the positive charges on two Arg residues figure prominently in the binding. Subtle differences in the structure of SH2 domains account for the specificities of the interactions of SH2-containing proteins with various –Tyr- containing proteins. The SH2 domain typically interacts with a –Tyr (which is assigned the index position 0) and the next three residues toward the carboxyl terminus (designated +1,+2,+3). Some proteins with SH2 domains (Src, Fyn, Hck, Nck) favor negatively charged residues in the +1 and +2 positions; others (PLCγ 1, SHP2) have a long hydrophobic groove that binds to aliphatic residues in positions +1 to +5. These differences define subclasses of SH2 domains that have different partner specificities. Phosphotyrosine-binding domains, or PTB domains, are another binding partner for –Tyr proteins (Fig. 12-28), but their critical sequences and three-dimensional structure distinguish them from SH2 domains. The human genome encodes at least 24 proteins that contain PTB domains, including IRS1, which we have already encountered in its role as an adaptor protein in insulin-signal transduction (Fig. 12-23). The –Tyr-binding sites for SH2 and PTB domains on partner proteins are created by Tyr kinases and eliminated by protein tyrosine phosphatases (PTPs). FIGURE 12-28 Interaction of a PTB domain with –Tyr residue in a partner protein. The PTB domain is represented as a blue surface contour. The partner protein is held to the kinase by multiple noncovalent interactions, which confer specificity on the interaction and position the –Tyr residue in a binding pocket at the enzyme’s active site. [Data from PDB ID 1SHC, M. M. Zhou et al., Nature 378:584, 1995.] As we have seen, other signaling protein kinases, including PKA, PKC, PKG, and members of the MAPK cascade, phosphorylate Ser or Thr residues in their target proteins. In some cases, these proteins acquire the ability to interact with partner proteins through the phosphorylated residue, triggering a downstream process. An alphabet soup of domains that bind –Ser or –Thr residues has been identified, and more are sure to be found. Each domain favors a certain sequence around the phosphorylated residue, so proteins with that domain bind to and interact with a specific subset of phosphorylated proteins. In some cases, the region on a protein that binds –Tyr of a substrate protein is masked by the region’s interaction with a – Tyr in the same protein. For example, the soluble protein Tyr kinase Src, when phosphorylated on a specific Tyr residue, is rendered inactive; an SH2 domain needed to bind the substrate protein instead binds the internal –Tyr. Removal of this –Tyr residue by a phosphoprotein phosphatase turns on the Tyr kinase activity of Src (Fig. 12-29a). Similarly, glycogen synthase kinase 3 (GSK3) is inactive when phosphorylated on a Ser residue in its autoinhibitory domain (Fig. 12-29b). Dephosphorylation of that domain frees the enzyme to bind (and then phosphorylate) its target proteins. FIGURE 12-29 Mechanism of autoinhibition of Src and GSK3. (a) In the active form of the Tyr kinase Src, an SH2 domain binds a –Tyr in the protein substrate, and an SH3 domain binds a proline-rich region of the substrate, thus lining up the active site of the kinase with several target Tyr residues in the substrate (top). When Src is phosphorylated on a specific Tyr residue (bottom), the SH2 domain binds the internal –Tyr instead of the –Tyr of the substrate, and the SH3 domain binds an internal proline-rich region, preventing productive enzyme-substrate binding; the enzyme is thus autoinhibited. (b) In the active form of glycogen synthase kinase 3 (GSK3), an internal –Ser-binding domain is available to bind –Ser in its substrate (glycogen synthase) and to position the kinase to phosphorylate neighboring Ser residues (top). Phosphorylation of an internal Ser residue allows this internal kinase segment to occupy the – Ser-binding site, blocking substrate binding (bottom). In addition to the three commonly phosphorylated residues in proteins (Tyr, Ser, Thr), there is a fourth phosphorylated structure that nucleates the formation of supramolecular complexes of signaling proteins: the phosphorylated head group of the membrane phosphatidylinositols. Many signaling proteins contain domains such as SH3 and PH (pleckstrin homology) that bind tightly to PIP3 protruding on the cytoplasmic side of the plasma membrane. Wherever the enzyme PI3K creates this head group (as it does in response to the insulin signal), proteins that bind PIP3 will cluster at the membrane surface. Most of the proteins involved in signaling at the plasma membrane have one or more phosphoprotein- or phospholipid- binding domain; many have three or more, and thus are multivalent in their interactions with other signaling proteins. Figure 12-30 shows just a few of the multivalent proteins known to participate in signaling. Many of the complexes include components with membrane-binding domains. Given the location of so many signaling processes at the inner surface of the plasma membrane, the molecules that must collide to produce the signaling response are effectively confined to two- dimensional space — the membrane surface; collisions are far more likely here than in the three-dimensional space of the cytosol. FIGURE 12-30 Some binding modules of signaling proteins. These signaling proteins interact with phosphorylated proteins or phospholipids in many permutations and combinations to form integrated signaling complexes. Each protein is represented by a line (with the amino terminus to the le ); symbols indicate the location of conserved binding domains (with specificities as listed in the key; abbreviations are explained in the text); green boxes indicate catalytic activities. The name of each protein is given at its carboxyl-terminal end. [Information from T. Pawson et al., Trends Cell Biol. 11:504, 2001, Fig. 5.] In summary, a remarkable picture of signaling pathways has emerged from studies of many signaling proteins and their multiple binding domains. An initial signal results in phosphorylation of the receptor or a target protein, triggering the assembly of large multiprotein complexes, held together on scaffolds with multivalent binding capacities. Some of these complexes contain several protein kinases that activate each other in turn, producing a cascade of phosphorylation and a great amplification of the initial signal. The interactions between cascade kinases are not le to the vagaries of random collisions in three-dimensional space. In the MAPK cascade, for example, a scaffold protein, KSR, binds all three kinases (MAPK, MAPKK, and MAPKKK), ensuring their proximity and correct orientation and even conferring allosteric properties on the interactions among the kinases, which makes their serial phosphorylation sensitive to very small stimuli (Fig. 12-31). FIGURE 12-31 A scaffold protein from yeast that organizes and regulates a protein kinase cascade. (a) The scaffold protein KSR has binding sites for all three of the kinases in the Raf-MEK-ERK cascade. With the binding of all three in appropriate orientations, interactions among the proteins are rapid and efficient. When ERK has been activated (le ), it phosphorylates the binding site for Raf (right), forcing a conformational change that displaces Raf and thereby prevents the phosphorylation of MEK. The result of this feedback regulation is that MEK phosphorylation is temporary. (b) In yeast cells with mutant KSR lacking the phosphorylation sites (red curve), no feedback occurs, producing a different time course of signaling. [Information from M. C. Good et al., Science 332:680, 2011, Fig. 2E.] Phosphotyrosine phosphatases remove the phosphate from – Tyr residues, reversing the effect of phosphorylation. At least 37 genes in the human genome encode protein tyrosine phosphatases (PTPs). About half of these are receptorlike integral proteins with a single transmembrane domain; they are presumably controlled by extracellular factors not yet identified. Other PTPs are soluble and contain SH2 domains that determine their molecular partners and intracellular location. In addition, animal cells have protein –Ser and –Thr phosphatases such as PP1 that reverse the effects of Ser- and Thr-specific protein kinases. We can see, then, that signaling occurs in protein circuits, which are effectively hardwired from signal receptor to response effector and can be switched off instantly by the hydrolysis of a single upstream phosphate ester bond. In these circuits, protein kinases are the writers, domains such as SH2 are the readers, and PTPs and other phosphatases are the erasers. The multivalency of signaling proteins allows the assembly of many different combinations of Lego-like signaling modules, each combination suited to particular signals, cell types, and metabolic circumstances, yielding diverse signaling circuits of extraordinary complexity. Membrane Ra s and Caveolae Segregate Signaling Proteins Membrane ra s (Chapter 11) are regions of the membrane bilayer enriched in sphingolipids, sterols, and certain proteins, including many proteins attached to the bilayer by GPI (glycosylated derivatives of phosphatidylinositol) anchors. The β -adrenergic receptor is segregated in ra s that also contain G proteins, adenylyl cyclase, PKA, and the protein phosphatase PP2, which together provide a highly integrated signaling unit. By segregating in a small region of the plasma membrane all of the elements required for responding to and ending the signal, the cell is able to produce a highly localized and brief “puff” of second messenger. Some RTKs (EGFR and PDGFR) are also localized in ra s, and this sequestration very likely has functional significance. In isolated fibroblasts, EGFR is usually concentrated in specialized ra s called caveolae (see Fig. 11-23). When the cells are treated with EGF, the receptor leaves the ra , separating it from the other components of the EGF signaling pathway. This migration depends on the receptor’s protein kinase activity; mutant receptors lacking this activity remain in the ra during treatment with EGF. Such experiments suggest that spatial segregation of signaling proteins in ra s is yet another dimension of the already complex processes initiated by extracellular signals. SUMMARY 12.5 Multivalent Adaptor Proteins and Membrane Ra s Many signaling proteins have domains that bind phosphorylated Tyr, Ser, or Thr residues in other proteins; the binding specificity for each domain is determined by sequences that adjoin the phosphorylated residue in the substrate. SH2 and PTB domains bind to proteins containing –Tyr residues; other domains bind –Ser and –Thr residues in various contexts. SH3 and PH domains bind the membrane phospholipid PIP3. Many signaling proteins are multivalent, with several different binding modules. By combining the substrate specificities of various protein kinases with the specificities of domains that bind phosphorylated Tyr, Ser, or Thr residues, and with phosphatases that can rapidly inactivate a signaling pathway, cells create a large number of multiprotein signaling complexes. Membrane ra s and caveolae sequester groups of signaling proteins in small regions of the plasma membrane, effectively raising their local concentrations and making signaling more efficient. 12.6 Gated Ion Channels Certain cells in multicellular organisms are “excitable”: they can detect an external signal, convert it into an electrical signal (specifically, a change in plasma membrane potential), and pass it on. Changes in membrane potential are effected by gated ion channels. Excitable cells play central roles in nerve conduction, muscle contraction, hormone secretion, sensory processes, and learning and memory. Ion Channels Underlie Rapid Electrical Signaling in Excitable Cells The excitability of sensory cells, neurons, and myocytes depends on ion channels, signal transducers that provide a regulated path for the movement of inorganic ions such as Na+, K+, Ca2+, and Cl− across the plasma membrane in response to various stimuli. Recall from Chapter 11 that these ion channels are gated: they may be open or closed, depending on whether the associated receptor has been activated by the binding of its specific ligand (a neurotransmitter, for example) or by a change in the transmembrane electrical potential (voltage- gated ion channels). The Na+K+ ATPase is electrogenic; it creates a charge imbalance across the plasma membrane by carrying 3 Na+ out of the cell for every 2 K+ carried in (Fig. 12- 32a). The action of the Na+K+ ATPase makes the inside of the cell negative relative to the outside. Inside the cell, [K+] is much higher and [Na+] is much lower than outside the cell (Fig. 12-32b). The direction of spontaneous ion flow across a polarized membrane is dictated by the electrochemical potential of that ion across the membrane, which has two components: the difference in concentration of the ion on the two sides of the membrane, and the difference in electrical potential (Vm), typically expressed in millivolts (see Eqn 11-4, p. 392). Given the ion concentration differences and a Vm of about −60 mV (inside negative), opening of a Na+ or Ca2+ channel will result in a spontaneous inward flow of Na+ or Ca2+ (and depolarization), whereas opening of a K+ channel will result in a spontaneous outward flow of K+ (and hyperpolarization) (Fig. 12-32b). In this case, K+ moves out of the cell against the electrical gradient, because the large concentration difference exerts a stronger effect than the Vm. For Cl−, the membrane potential predominates, so when a Cl− channel opens, Cl− flows outward. FIGURE 12-32 Transmembrane electrical potential. (a) The electrogenic Na+K+ ATPase produces a transmembrane electrical potential of about −60 mV (inside negative). (b) Blue arrows show the direction in which ions tend to move spontaneously across the plasma membrane in an animal cell, driven by the combination of chemical and electrical gradients. The chemical gradient drives Na+ and Ca2+ inward (producing depolarization) and K+ outward, against its electrical gradient (producing hyperpolarization). The electrical gradient drives Cl− outward, against its concentration gradient (producing depolarization). The number of ions that must flow to produce a physiologically significant change in the membrane potential is negligible relative to the concentrations of Na+, K+, and Cl− in cells and extracellular fluid, so the ion fluxes that occur during signaling in excitable cells have essentially no effect on the concentrations of these ions. With Ca2+, the situation is different; because the intracellular [Ca2+] is generally very low (~10−7 M ), inward flow of Ca2+ can significantly alter the cytosolic [Ca2+], allowing it to serve as a second messenger. The membrane potential of a cell at a given time is the result of the types and numbers of ion channels open at that instant. The precisely timed opening and closing of ion channels and the resulting transient changes in membrane potential underlie the electrical signaling by which the nervous system stimulates the skeletal muscles to contract, the heart to beat, or secretory cells to release their contents. Moreover, many hormones exert their effects by altering the membrane potential of their target cells. These mechanisms are not limited to animals; ion channels play important roles in the responses of bacteria, protists, and plants to environmental signals. To illustrate the action of ion channels in cell-to-cell signaling, we describe the mechanisms by which a neuron passes a signal along its length and across a synapse to the next neuron (or to a myocyte) in a cellular circuit, using acetylcholine as the neurotransmitter. Voltage-Gated Ion Channels Produce Neuronal Action Potentials Signaling in the nervous system is accomplished by networks of neurons, specialized cells that carry an electrical impulse (action potential) from one end of the cell (the cell body) through an elongated cytoplasmic extension (the axon), to the synapse with the next neuron. The electrical signal triggers release of the neurotransmitter acetylcholine into the synaptic cle , carrying the signal to the next cell (neuron) in the circuit. Initially, the plasma membrane of the presynaptic neuron is polarized (inside negative) through the action of the electrogenic Na+K+ ATPase, which pumps out three Na+ for every two K+ pumped in (Fig. 12- 32). A rapid sequence of opening and closing of several types of ion channels (Fig. 12-33) produces a wave of depolarization (an action potential) that sweeps the neuron from the cell body to the end of an axon. First, opening of a voltage-gated Na+ channel allows Na+ entry, and the resulting local depolarization causes the adjacent Na+ channel to open, and so on (Fig. 12-33, step ). The directionality of movement of the action potential is ensured by the brief refractory period that follows the opening of each voltage-gated Na+ channel. A split second a er the action potential passes a point in the axon, voltage-gated K+ channels open (step ), allowing K+ exit, which brings about repolarization of the membrane to make it ready for the next action potential (step ).
FIGURE 12-33 Role of voltage-gated and ligand-gated ion channels in neural transmission. The steps are described in the text. Stimulation of the presynaptic neuron (top) triggers a wave of depolarization (blue arrow) followed by repolarization (red arrow), producing an action potential that sweeps along the axon. At the synapse, depolarization opens Ca2+ channels, and Ca2+ entry triggers acetylcholine release into the synaptic cle . Acetylcholine diffuses across the cle , opening receptor channels, depolarizing the postsynaptic cell, and starting an action potential in the postsynaptic cell. Note that, for clarity, Na+ channels and K+ channels are drawn on opposite sides of the axon, but both types are uniformly distributed in the axonal membrane. Also, positive and negative charges are shown only on the le , but as the wave of potential sweeps the axon, the membrane potential is the same at any given point along the axon. When the wave of depolarization reaches the axon tip, voltage- gated Ca2+ channels open, allowing Ca2+ entry (step ). The resulting increase in internal [Ca2+] triggers exocytotic release of the neurotransmitter acetylcholine into the synaptic cle (step ). Acetylcholine binds to a receptor on the postsynaptic neuron or myocyte, causing its ligand-gated ion channel to open (step ). Extracellular Na+ and Ca2+ enter through this channel, depolarizing the postsynaptic cell. The electrical signal has thus passed to the cell body of the postsynaptic neuron and will move along its axon to a third neuron (or a myocyte) by this same sequence of events. We see, then, that gated ion channels convey signals in either of two ways: by changing the cytoplasmic concentration of an ion (such as Ca2+), which then serves as an intracellular second messenger, or by changing Vm and affecting other membrane proteins that are sensitive to Vm. The passage of an electrical signal through one neuron and on to the next illustrates both types of mechanism. Neurons Have Receptor Channels That Respond to Different Neurotransmitters Animal cells, especially those of the nervous system, contain a variety of ion channels gated by ligands, voltage, or both. Receptors that are themselves ion channels are classified as ionotropic, to distinguish them from receptors that generate a second messenger (metabotropic receptors). Acetylcholine acts on an ionotropic receptor in the postsynaptic cell. The acetylcholine receptor is a cation channel. When occupied by acetylcholine, the receptor opens to the passage of cations (Na+, K+, and Ca2+), triggering depolarization of the cell. The neurotransmitters serotonin, glutamate, and glycine all can act through ionotropic receptors that are structurally related to the acetylcholine receptor. Serotonin and glutamate trigger the opening of cation (Na+, K+, Ca2+) channels, whereas glycine opens Cl−-specific channels. Depending on which ion passes through a channel, binding of the ligand (neurotransmitter) for that channel results in either depolarization or hyperpolarization of the target cell. A single neuron normally receives input from many other neurons, each releasing its own characteristic neurotransmitter with its characteristic depolarizing or hyperpolarizing effect. The target cell’s Vm therefore reflects the integrated input (see Fig. 12-1f) from multiple neurons. The cell responds with an action potential only if the integrated input adds up to a net depolarization of sufficient magnitude. The receptor channels for acetylcholine, glycine, glutamate, and γ -aminobutyric acid (GABA) are gated by extracellular ligands. Intracellular second messengers — such as cAMP, cGMP, IP3, Ca2+, and ATP — regulate ion channels of the type we saw in the sensory transductions of vision, olfaction, and gustation. Toxins Target Ion Channels Many of the most potent toxins found in nature act on ion channels. For example, dendrotoxin (from the black mamba snake) blocks the action of voltage-gated K+ channels, tetrodotoxin (produced by puffer fish) acts on voltage-gated Na+ channels, and cobrotoxin (from cobras) disables acetylcholine receptor ion channels. Why, in the course of evolution, have ion channels become the preferred target of toxins, rather than some critical metabolic target such as an enzyme essential in energy metabolism? Ion channels are extraordinary amplifiers; opening of a single channel can allow the flow of 10 million ions per second. Consequently, relatively few molecules of an ion-channel protein are needed per neuron for signaling functions. This means that a relatively small number of toxin molecules with high affinity for ion channels, acting from outside the cell, can have a pronounced effect on neurosignaling throughout the body. A comparable effect by way of a metabolic enzyme, typically present in cells at much higher concentrations than ion channels, would require far greater numbers of the toxin molecule. SUMMARY 12.6 Gated Ion Channels Ion channels gated by membrane potential or ligands are central to signaling in neurons and other cells. The voltage-gated Na+ and K+ channels of neuronal membranes carry the action potential along the axon as a wave of depolarization (Na+ influx) followed by repolarization (K+ efflux). Arrival of an action potential at the distal end of a presynaptic neuron triggers neurotransmitter release. The neurotransmitter (acetylcholine, for example) diffuses to the postsynaptic neuron (or the myocyte, at a neuromuscular junction), binds to specific receptors in the plasma membrane, and triggers a change in Vm. The cell body of the neuron has receptors for a variety of neurotransmitters or extracellular signals. The neuron’s Vm is the sum of the effects of all ion- channel contributions. Neurotoxins, produced by many organisms, attack neuronal ion channels and are therefore fast-acting and deadly. 12.7 Regulation of Transcription by Nuclear Hormone Receptors The steroid, retinoic acid (retinoid), and thyroid hormones form a large group of receptor ligands that exert at least part of their effects by a mechanism fundamentally different from that of other hormones: they act directly in the nucleus to alter gene expression. We discuss their mode of action in detail in Chapter 28, along with other mechanisms for regulating gene expression. Here we give a brief overview. Steroid hormones (estrogen, progesterone, vitamin D, and cortisol, for example), too hydrophobic to dissolve readily in the blood, are transported on specific carrier proteins from their point of release to their target tissues. In target cells, these hormones pass through the plasma membrane and nuclear membrane by simple diffusion and bind to specific receptor proteins in the nucleus (Fig. 12-34). Hormone binding triggers changes in the conformation of a receptor protein so that it becomes capable of interacting with specific regulatory sequences in DNA called hormone response elements (HREs), thus altering gene expression (see Fig. 28-34). The bound receptor-hormone complex enhances the expression of specific genes adjacent to HREs, with the help of several other proteins essential for transcription. Hours or days are required for these regulators to have their full effect — the time required for the changes in RNA synthesis and subsequent protein synthesis to become evident in altered metabolism. FIGURE 12-34 General mechanism by which steroid and thyroid hormones, retinoids, and vitamin D regulate gene expression. The details of transcription and protein synthesis are discussed in Chapters 26 and 27. Some steroids also act through plasma membrane receptors by a completely different mechanism. The specificity of the steroid-receptor interaction is exploited in the use of the drug tamoxifen to treat breast cancer. In some types of breast cancer, division of the cancerous cells depends on the continued presence of estrogen. Tamoxifen is an estrogen antagonist; it competes with estrogen for binding to the estrogen receptor, but the tamoxifen-receptor complex has little or no effect on gene expression. Consequently, tamoxifen administered a er surgery or during chemotherapy for hormone-dependent breast cancer slows or stops the growth of remaining cancerous cells. Another steroid analog, the drug mifepristone (RU486), binds to the progesterone receptor and blocks hormone actions essential to implantation of the fertilized ovum in the uterus, and thus functions as a contraceptive.
SUMMARY 12.7 Regulation of Transcription by Nuclear Hormone Receptors Steroid hormones enter cells by simple diffusion and bind to specific receptor proteins, inducing a structural change that exposes a specific binding site on the DNA. The hormone-receptor complex binds specific regions of DNA at the hormone response elements, and interacts with other proteins to regulate the expression of nearby genes. 12.8 Regulation of the Cell Cycle by Protein Kinases One of the most dramatic manifestations of signaling pathways is the regulation of the eukaryotic cell cycle. During embryonic growth and later development, cell division occurs in virtually every tissue. In the adult organism, cells of most tissues stop dividing, becoming quiescent. A cell’s “decision” to divide or not is of crucial importance to the organism. When the regulatory mechanisms that limit cell division are defective and cells undergo unregulated division, the result is catastrophic — cancer. Proper cell division requires a precisely ordered sequence of biochemical events that assures every daughter cell a full complement of the molecules required for life. Investigations into the control of cell division in diverse eukaryotic cells have revealed universal regulatory mechanisms. Signaling mechanisms much like those discussed above are central in determining whether and when a cell undergoes cell division, and they also ensure orderly passage through the stages of the cell cycle. The Cell Cycle Has Four Stages Cell division accompanying mitosis in eukaryotes occurs in four well-defined stages (Fig. 12-35). In the S (synthesis) phase, the DNA is replicated to produce copies for both daughter cells. In the G2 phase (G indicates the gap between divisions), new proteins are synthesized and the cell approximately doubles in size. In the M phase (mitosis), the maternal nuclear envelope breaks down, paired chromosomes are pulled to opposite poles of the cell, each set of daughter chromosomes is surrounded by a newly formed nuclear envelope, and cytokinesis pinches the cell in half, producing two daughter cells (see Fig. 24-22). In embryonic or rapidly proliferating tissue, each daughter cell divides again, but only a er a waiting period (G1). In animal cells in the laboratory, the entire process takes about 24 hours. FIGURE 12-35 The eukaryotic cell cycle. The durations (in hours) of the four stages vary, but those shown are typical. A er passing through mitosis and into G1, a cell either continues through another division or ceases to divide, entering a quiescent phase (G0) that may last hours, days, or the lifetime of the cell. When a cell in G0 begins to divide again, it reenters the division cycle through the G1 phase. Differentiated cells such as hepatocytes or adipocytes have acquired their specialized function and form; they remain in the G0 phase. Stem cells retain their potential to divide and to differentiate into any of a number of cell types. Levels of Cyclin-Dependent Protein Kinases Oscillate The timing of the cell cycle is controlled by a family of protein kinases with activities that change in response to cellular signals. By phosphorylating specific proteins at precisely timed intervals, these protein kinases orchestrate the metabolic activities of the cell to produce orderly cell division. The kinases are heterodimers with a regulatory subunit, a cyclin, and a catalytic subunit, a cyclin-dependent protein kinase (CDK). In the absence of the cyclin, the catalytic subunit is virtually inactive. When the cyclin binds, the catalytic site opens up, a residue essential to catalysis becomes accessible, and the protein kinase activity of the catalytic subunit increases 10,000-fold. Animal cells have at least 10 different cyclins (designated A, B, and so forth) and at least 8 CDKs (CDK1 through CDK8), which act in various combinations at specific points in the cell cycle. In a population of animal cells undergoing synchronous division, some CDK activities show striking oscillations (Fig. 12-36). These oscillations are the result of four mechanisms for regulating CDK activity: phosphorylation or dephosphorylation of the CDK, controlled degradation of the cyclin subunit, periodic synthesis of CDKs and cyclins, and the action of specific CDK-inhibiting proteins. The precisely timed activation and inactivation of a series of CDKs produces signals serving as a master clock that orchestrates the events in normal cell division and ensures that one stage is completed before the next begins. FIGURE 12-36 Variations in the activities of specific CDKs during the cell cycle in animals. Early in G1, the activity of cyclin D–CDK4-6 rises slowly, then drops sharply as G1 ends. Near the end of G1, cyclin E–CDK2 activity rises and peaks near the G1 phase–S phase boundary, when the active enzyme triggers synthesis of enzymes required for DNA synthesis (see Fig. 12-40). Cyclin A–CDK2 activity rises during the S and G2 phases, then drops sharply in the M phase, as cyclin B–CDK1 peaks. [Data from P. Icard et al., Trends Biochem. Sci. 44:490, 2019, Fig. 3.] CDKs Are Regulated by Phosphorylation, Cyclin Degradation, Growth Factors, and Specific Inhibitors The activity of a CDK is strikingly affected by phosphorylation and dephosphorylation of two specific residues in the protein (Fig. 12-37). Phosphorylation of Thr160 of CDK2 stabilizes a conformation in which an autoinhibitory “T loop” is moved away from the substrate-binding cle in the kinase, opening it to bind protein substrates. Dephosphorylation of -Tyr15 of CDK2 removes a negative charge that blocks ATP from approaching its binding site. This mechanism for activating a CDK is self- reinforcing; the enzyme (PTP) that dephosphorylates -Tyr15 is itself a substrate for the CDK and is activated by phosphorylation. The combination of these factors activates the CDK manyfold, allowing it to phosphorylate downstream protein targets required for progression of the cell cycle (Fig. 12-38a). FIGURE 12-37 Activation of cyclin-dependent protein kinases (CDKs) by cyclin and phosphorylation. CDKs are active only when associated with a cyclin. The crystal structure of CDK2 with and without a cyclin reveals the basis for this activation. (a) Without the cyclin, CDK2 folds so that one segment, the T loop, obstructs the binding site for protein substrates. The binding site for ATP is also near the T loop and is blocked when Tyr15 is phosphorylated (not shown). (b) When the cyclin binds, it forces conformational changes that move the T loop away from the active site and reorient an amino-terminal helix, bringing a residue critical to catalysis (Glu51) into the active site. (c) When a Thr residue in the T loop is phosphorylated, its negative charges are stabilized by interaction with three Arg residues, holding the T loop away from the substrate-binding site. Removal of the phosphoryl group on Tyr15 gives ATP access to its binding site, fully activating CDK2 (see Fig. 12-38). [Data from (a) PDB ID 1HCK, U. Schulze-Gahmen et al., J. Med. Chem. 39:4540, 1996; (b) PDB ID 1FIN, P. D. Jeffrey et al., Nature 376:313, 1995; (c) PDB ID 1JST, A. A. Russo et al., Nature Struct. Biol. 3:696, 1996.] FIGURE 12-38 Regulation of CDK by phosphorylation and proteolysis. (a) The series of events that leads to activation of a cyclin-dependent protein kinase. (b) The periodic proteolytic degradation of cyclin, inactivating the cyclin-dependent protein kinase. The steps are described in the text. The presence of a single-strand break in DNA signals arrest of the cell cycle in G2 by activating two proteins (ATM and ATR; see Fig. 12-40). These proteins trigger a cascade of responses that include inactivation of the PTP that dephosphorylates Tyr15 of the CDK. With the CDK inactivated, the cell is arrested in G2, unable to divide until the DNA is repaired and the effects of the cascade are reversed. Highly specific and precisely timed proteolytic breakdown of mitotic cyclins regulates CDK activity throughout the cell cycle (Fig. 12-38b). How is the timing of cyclin breakdown controlled? A feedback loop occurs in the overall process shown in Figure 12- 38. As a cell enters mitosis, the M-phase CDK is inactive (step ). As cyclin is synthesized (step ), the cyclin-CDK complex forms (step ). The T loop lies in the substrate-binding site of CDK, and -Tyr15 blocks its ATP-binding site, keeping the complex inactive. When Thr160 in the T loop is phosphorylated, the loop moves out of the substrate-binding site, and when Tyr15 is dephosphorylated, ATP can bind. These two changes make the cyclin-CDK complex many times more active (step ). Further activation is achieved as CDK also phosphorylates and activates the enzyme that dephosphorylates -Tyr15 (step ). The active cyclin-CDK complex triggers its own inactivation by phosphorylation of DBRP (destruction box recognizing protein; step ). DBRP and ubiquitin ligase then attach several molecules of ubiquitin (U) to the cyclin (step ), targeting it for destruction by proteolytic enzyme complexes called proteasomes (step ). The role of ubiquitin and proteasomes is not limited to the regulation of cyclins; as we shall see in Chapter 27, both also take part in the turnover of cellular proteins, a process fundamental to cellular housekeeping. The third mechanism for changing CDK activity is regulation of the rate of synthesis of the cyclin or CDK or both. Extracellular signals such as growth factors and cytokines (developmental signals that trigger cell division) activate, by phosphorylation, the nuclear transcription factors Jun and Fos, which promote the synthesis of many gene products, including cyclins, CDKs, and the transcription factor E2F. In turn, E2F stimulates production of several enzymes essential for the synthesis of deoxynucleotides and DNA, and the CDK and cyclin allow the cell to enter the S phase (Fig. 12-39). FIGURE 12-39 Regulation of cell division by growth factors. Finally, specific protein inhibitors bind to and inactivate specific CDKs. One such protein is p21, which we discuss below. These four control mechanisms modulate the activity of specific CDKs that, in turn, control whether a cell will divide, differentiate, become permanently quiescent, or begin a new cycle of division a er a period of quiescence. The details of cell cycle regulation, such as the number of different cyclins and kinases and the combinations in which they act, differ from species to species, but the basic mechanism has been conserved in the evolution of all eukaryotic cells. CDKs Regulate Cell Division by Phosphorylating Critical Proteins We have examined how cells maintain close control of CDK activity, but how does the activity of CDKs control the cell cycle? There are scores of known CDK targets, and much remains to be learned. But we can see a general pattern behind CDK regulation by inspecting the effect of CDKs on the structures of lamin and myosin and on the activity of retinoblastoma protein. The structure of the nuclear envelope is maintained in part by highly organized meshworks of intermediate filaments composed of the protein lamin. Breakdown of the nuclear envelope before segregation of the sister chromatids in mitosis is partly due to the phosphorylation of lamin by a CDK, which causes lamin filaments to depolymerize. A second kinase target is the ATP-driven contractile machinery (actin and myosin) that pinches a dividing cell into two equal parts during cytokinesis. A er the division, a CDK phosphorylates a small regulatory subunit of myosin, causing dissociation of myosin from actin filaments and inactivating the contractile machinery. Subsequent dephosphorylation allows reassembly of the contractile apparatus for the next round of cytokinesis. A third and very important CDK substrate is the retinoblastoma protein, pRb; when DNA damage is detected, this protein participates in a mechanism that arrests cell division in G1 (Fig. 12-40). Named for the retinal tumor cell line in which it was discovered, pRb functions in most, perhaps all, cell types to regulate cell division in response to a variety of stimuli. Unphosphorylated pRb binds the transcription factor E2F; while bound to pRb, E2F cannot promote transcription of a group of genes necessary for DNA synthesis (the genes for DNA polymerase α , ribonucleotide reductase, and other proteins; see Chapter 25). In this state, the cell cycle cannot proceed from the G1 phase to the S phase, the step that commits a cell to mitosis and cell division. The pRb-E2F blocking mechanism is relieved when pRb is phosphorylated by cyclin E–CDK2, which occurs in response to a signal for cell division to proceed. FIGURE 12-40 Regulation of passage from G1 to S by phosphorylation of pRb. Transcription factor E2F promotes transcription of genes for certain enzymes essential to DNA synthesis. The retinoblastoma protein, pRb, can bind E2F (lower le ), inactivating it and preventing transcription of these genes. Phosphorylation of pRb by CDK2 prevents it from binding and inactivating E2F, and the genes are transcribed, allowing cell division. Damage to the cell’s DNA (upper le ) triggers a series of events that inactivate CDK2, blocking cell division. When the protein MRN detects damage to the DNA, it activates two protein kinases, ATM and ATR, and they phosphorylate and activate the transcription factor p53. Active p53 promotes the synthesis of another protein, p21, an inhibitor of CDK2. Inhibition of CDK2 stops the phosphorylation of pRb, which therefore continues to bind and inhibit E2F. With E2F inactivated, genes essential to cell division are not transcribed and cell division is blocked. When DNA has been repaired, this inhibition is released, and the cell divides. When the protein kinases ATM and ATR detect damage to DNA (signaled by the presence of the protein MRN at a double-strand break site), they phosphorylate p53, activating it to serve as a transcription factor that stimulates the synthesis of the protein p21 (Fig. 12-40). This protein inhibits the protein kinase activity of cyclin E–CDK2. In the presence of p21, pRb remains unphosphorylated and bound to E2F, blocking the activity of this transcription factor, and the cell cycle is arrested in G1. This gives the cell time to repair its DNA before entering the S phase, thereby avoiding the potentially disastrous transfer of a defective genome to one or both daughter cells. When the damage is too severe to allow effective repair, this same machinery triggers apoptosis (described below), a process that leads to the death of the cell, preventing the possible development of a cancer. SUMMARY 12.8 Regulation of the Cell Cycle by Protein Kinases Progression through the cell cycle is regulated by the cyclin- dependent protein kinases (CDKs), which act at specific points in the cycle, phosphorylating key proteins and modulating their activities. The catalytic subunit of CDKs is inactive unless associated with the regulatory cyclin subunit. The activity of a cyclin-CDK complex changes during the cell cycle through differential synthesis of CDKs, specific degradation of the cyclin, phosphorylation and dephosphorylation of critical residues in CDKs, and binding of inhibitory proteins to specific cyclin-CDKs. A cyclin sequence (the destruction box) marks cyclin for tagging with ubiquitin and degradation in proteasomes. The rise of cyclin concentration by its synthesis ultimately triggers its degradation, yielding oscillations in cyclin level keyed to the cell cycle. Cells receive extracellular signals that determine the timing of their division. Scores of proteins are known targets of CDKs, many with unknown functions. Among the targets phosphorylated by cyclin-CDKs are proteins of the nuclear envelope and proteins required for cytokinesis and DNA repair. 12.9 Oncogenes, Tumor Suppressor Genes, and Programmed Cell Death Tumors and cancer are the result of uncontrolled cell division. Normally, cell division is regulated by a family of extracellular growth factors, proteins that cause resting cells to divide and, in some cases, differentiate. The result is a precise balance between the formation of new cells and cell destruction. Regulation of cell division ensures that skin cells are replaced every few weeks and white blood cells are replaced every few days. This is homeostasis at the organismal level. When this balance is disturbed by defects in regulatory proteins, the result is sometimes the formation of a clone of cells that divide repeatedly and without regulation (a tumor) until their presence interferes with the function of normal tissues — cancer. The direct cause is almost always a genetic defect in one or more of the proteins that regulate cell division. In some cases, a defective gene is inherited from one parent; in other cases, the mutation occurs when a toxic compound from the environment (a mutagen or a carcinogen) or high-energy radiation interacts with the DNA of a single cell to damage it and introduce a mutation. In most cases there is both an inherited contribution and an environmental contribution, and in most cases, more than one mutation is required in order to cause completely unregulated division and full-blown cancer. Oncogenes Are Mutant Forms of the Genes for Proteins That Regulate the Cell Cycle
Oncogenes are mutated versions of genes encoding signaling proteins involved in cell cycle regulation. Oncogenes were originally discovered in tumor-causing viruses, then later found to be derived from genes in animal host cells, proto- oncogenes, which encode growth-regulating proteins. During a viral infection, the host DNA sequence of a proto-oncogene is sometimes copied into the viral genome, where it proliferates with the virus. In subsequent viral infection cycles, the proto- oncogenes can become defective by truncation or mutation. Viruses, unlike animal cells, do not have effective mechanisms for correcting mistakes during DNA replication, so they accumulate mutations rapidly. When a virus carrying an oncogene infects a new host cell, the viral DNA (and oncogene) can be incorporated into the host cell’s DNA, where it can now interfere with the regulation of cell division in the host cell. In an alternative, nonviral mechanism, a single cell in a tissue exposed to carcinogens may suffer DNA damage that renders one of its regulatory proteins defective, with the same effect as the viral oncogenic mechanism: failed regulation of cell division. The mutations that produce oncogenes are genetically dominant; if either of a pair of chromosomes contains a defective gene, that gene product sends the signal “divide,” and a tumor may result. The oncogenic defect can be in any of the proteins involved in communicating the “divide” signal. Oncogenes discovered thus far include those that encode secreted proteins that act as signaling molecules, growth factors, transmembrane proteins (receptors), cytoplasmic proteins (G proteins and protein kinases), and the nuclear transcription factors that control the expression of genes essential for cell division (Jun, Fos). Some oncogenes encode growth factor receptors with unregulated Tyr kinase activity; they signal continued cell division even when the growth factor is absent, leading to tumor formation. Tumor-producing mutations have been found in many of the signaling protein kinases we have discussed here, all of which use ATP as their substrate for phosphoryl transfer to another element in the signaling cascade. The development of drugs that inhibit the protein kinase activity is an obvious approach to treating cancers that result from unregulated kinase activity. However, most known protein kinase inhibitors act by blocking the binding site for ATP, which is similar in all protein kinases. Inhibitors that are effective against one protein kinase are likely to have intolerable side effects due to their inhibition of other, essential kinases. Nonetheless, the prominent role played by protein kinases in signaling processes related to normal and abnormal cell division has made these enzymes a prime target in the development of drugs for the treatment of cancer (Box 12-4). BOX 12-4 MEDICINE Development of Protein Kinase Inhibitors for Cancer Treatment When a single cell divides without any regulatory limitation, it eventually gives rise to a clone of cells so large that it interferes with normal physiological functions (Fig. 1). This is cancer, a leading cause of death in the developed world, and increasingly so in the developing world. In all types of cancer, the normal regulation of cell division has become dysfunctional due to defects in one or more genes. For example, genes encoding proteins that normally send intermittent signals for cell division become oncogenes, producing constitutively active signaling proteins, or genes encoding proteins that normally restrain cell division (tumor suppressor genes) mutate to produce proteins that lack this braking function. In many tumors, both kinds of mutation have occurred. FIGURE 1 Unregulated division of a single cell in the colon led to a primary cancer that metastasized to the liver. Secondary cancers are seen as white patches in this liver obtained at autopsy. Many oncogenes and tumor suppressor genes encode protein kinases or proteins that act in pathways upstream from protein kinases. It is therefore reasonable to hope that specific inhibitors of protein kinases could prove valuable in the treatment of cancer. For example, a mutant form of the EGF (epidermal growth factor) receptor is a constantly active receptor Tyr kinase (RTK), signaling cell division whether EGF is present or not. In about 30% of all women with invasive breast cancer, the gene for the receptor ErbB2 (also called HER2/neu) is overexpressed, sometimes by as much as 100-fold. Another RTK, vascular endothelial growth factor receptor (VEGFR), must be activated for the formation of new blood vessels (angiogenesis) to provide a solid tumor with its own blood supply, and inhibition of VEGFR might starve a tumor of essential nutrients. Nonreceptor Tyr kinases can also mutate, resulting in constant signaling and unregulated cell division. For example, the oncogene Abl (from the Abelson leukemia virus) is associated with acute myeloid leukemia, a relatively rare blood disease (~5,000 cases a year in the United States). Another group of oncogenes encode unregulated cyclin-dependent protein kinases. In each of these cases, specific protein kinase inhibitors might be valuable chemotherapeutic agents in the treatment of disease. Not surprisingly, huge efforts are under way to develop such inhibitors. How should one approach this challenge? Protein kinases of all types show striking conservation of structure at the active site. All share with the prototypical PKA structure the features shown in Figure 2: two lobes that enclose the active site, with a P loop that helps to align and bind the phosphoryl groups of ATP, an activation loop that moves to open the active site to the protein substrate, and a C helix that changes position as the enzyme is activated, bringing the residues in the substrate-binding cle into their binding positions. Detailed knowledge of the structure around the ATP- binding site makes it possible to design drugs that inhibit a specific protein kinase by (1) blocking the critical ATP-binding site, while (2) interacting with residues around that site that are unique to that particular protein kinase. FIGURE 2 Conserved features of the active site of protein kinases. The amino- terminal and carboxyl-terminal lobes surround the active site of the enzyme, near the catalytic loop and the site where ATP binds. The activation loop of this and many other kinases undergoes phosphorylation, then moves away from the active site to expose the substrate-binding cle , which in this image is occupied by a specific inhibitor of this enzyme, PD318088. The P loop is essential in the binding of ATP, and the C helix must also be correctly aligned for ATP binding and kinase activity. [Data from PDB ID 1S9I, J. F. Ohren et al., Nature Struct. Mol. Biol. 11:1192, 2004.] The simplest protein kinase inhibitors are ATP analogs that occupy the ATP- binding site but cannot serve as phosphoryl group donors. Many such compounds are known, but their clinical usefulness is limited by their lack of selectivity — they inhibit virtually all protein kinases and would produce unacceptable side effects. More selectivity is seen with compounds that fill part of the ATP-binding site but also interact outside this site with parts of the protein unique to the target protein kinase. A third possible strategy is based on the fact that although the active conformations of all protein kinases are similar, their inactive conformations are not. Drugs that target the inactive conformation of a specific protein kinase and prevent its conversion to the active form may have a higher specificity of action. A fourth approach employs the great specificity of antibodies. For example, monoclonal antibodies (p. 167) that bind the extracellular portions of specific RTKs could eliminate the receptors’ kinase activity by preventing dimerization or by causing their removal from the cell surface. In some cases, an antibody selectively binding to the surface of cancer cells could cause the immune system to attack those cells. The search for drugs active against specific protein kinases has yielded encouraging results. For example, imatinib mesylate (Gleevec; Fig. 3a), a small- molecule inhibitor, has proved nearly 100% effective in bringing about remission in patients with early-stage chronic myeloid leukemia. Erlotinib (Tarceva; Fig. 3b), which targets EGFR, is effective against advanced non-small- cell lung cancer (NSCLC). Because many cell-division signaling systems involve more than one protein kinase, inhibitors that act on several protein kinases may be useful in the treatment of cancer. Sunitinib (Sutent) and sorafenib (Nexavar) target several protein kinases, including VEGFR and PDGFR. These two drugs are in clinical use for patients with gastrointestinal stromal tumors and advanced renal cell carcinoma, respectively. Trastuzumab (Herceptin), cetuximab (Erbitux), and bevacizumab (Avastin) are monoclonal antibodies that target ErbB2/HER2/neu, EGFR, and VEGFR, respectively; all three drugs are in clinical use for certain types of cancer. FIGURE 3 Some protein kinase inhibitors now in clinical trials or clinical use, showing their binding to the target protein. (a) Imatinib binds to the Abl kinase (an oncogene product) active site; it occupies both the ATP-binding site and a region adjacent to that site. (b) Erlotinib binds to the active site of EGFR. (c), (d) Roscovitine is an inhibitor of the cyclin-dependent kinases CDK2, CDK7, and CDK9; shown here are normal M g2+-ATP binding at the active site (c) and roscovitine binding (d), which prevents the binding of ATP. [Data from (a) PDB ID 1IEP, B. Nagar et al., Cancer Res. 62:4236, 2002; (b) PDB ID 1M17, J. Stamos et al., J. Biol. Chem. 277:46,265, 2002; (c) PDB ID 1S9I, J. F. Ohren et al., Nature Struct. Mol. Biol. 11:1192, 2004; (d) PDB ID 2A4L, W. F. De Azevedo et al., Eur. J. Biochem. 243:518, 1997.] At least a hundred more compounds are in preclinical trials. Among the drugs being evaluated are some obtained from natural sources and some produced by synthetic chemistry. Indirubin is a component of a Chinese herbal preparation traditionally used to treat certain leukemias; it inhibits CDK2 and CDK5. Roscovitine (Fig. 3d), a substituted adenine, has a benzyl ring that makes it highly specific as an inhibitor of CDK2. With several hundred potential anticancer drugs heading toward clinical testing, it is realistic to hope that some will prove more effective or more target-specific than those now in use. Defects in Certain Genes Remove Normal Restraints on Cell Division Tumor suppressor genes encode proteins that normally restrain cell division. Mutation in one or more of these genes can lead to tumor formation. Unregulated growth due to defective tumor suppressor genes, unlike that due to oncogenes, is genetically recessive; tumors form only if both chromosomes contain a defective gene. This is because the function of these genes is to prevent cell division, and if either copy of the gene is normal, it will produce a normal protein and normal inhibition of division. In a person who inherits one correct copy and one defective copy, every cell begins with one defective copy of the gene. If any one of the individual’s 1012 somatic cells undergoes mutation in the one good copy, a tumor may grow from that doubly mutant cell. Mutations in both copies of the genes for pRb, p53, or p21 yield cells in which the normal restraint on cell division is lost and a tumor forms. Retinoblastoma occurs in children and causes blindness if not surgically treated. The cells of a retinoblastoma have two defective versions of the Rb gene (two defective alleles). Very young children who develop retinoblastoma commonly have multiple tumors in both eyes. These children have inherited one defective copy of the Rb gene, which is present in every cell; each tumor is derived from a single retinal cell that has undergone a mutation in its remaining good copy of the Rb gene. (A fetus with two mutant alleles in every cell is nonviable.) People with retinoblastoma who survive childhood also have a high incidence of cancers of the lung, prostate, and breast later in life. A far less likely event is that a person born with two good copies of the Rb gene will have independent mutations in both copies in the same cell. Some individuals do develop retinoblastomas later in childhood, usually with only one tumor in one eye. These individuals, presumably, were born with two good copies (alleles) of Rb in every cell, but both Rb alleles in a single retinal cell have undergone mutation, leading to a tumor. A er the child reaches about age 3, retinal cells stop dividing, and retinoblastomas at later ages are quite rare. Stability genes (also called caretaker genes) encode proteins that function in the repair of major genetic defects that result from aberrant DNA replication, ionizing radiation, or environmental carcinogens. Mutations in these genes lead to a high frequency of unrepaired damage (mutations) in other genes, including proto- oncogenes and tumor suppressor genes, and thus to cancer. Among the stability genes are ATM (see Fig. 12-40); the XP gene family, in which mutations lead to xeroderma pigmentosum; and the BRCA1 genes associated with some types of breast cancer (see Box 25-1). Mutations in the gene for p53 also cause tumors; in more than 90% of human cutaneous squamous cell carcinomas (skin cancers) and in about 50% of all other human cancers, p53 is defective. Those very rare individuals who inherit one defective copy of p53 commonly have the Li-Fraumeni cancer syndrome, with multiple cancers (of the breast, brain, bone, blood, lung, and skin) occurring at high frequency and at an early age. The explanation for multiple tumors in this case is the same as that for Rb mutations: an individual born with one defective copy of p53 in every somatic cell is likely to suffer a second p53 mutation in more than one cell during his or her lifetime. In summary, then, three classes of defects can contribute to the development of cancer: (1) oncogenes, in which the defect is the equivalent of a car’s accelerator pedal being stuck down, with the engine racing; (2) mutated tumor suppressor genes, in which the defect leads to the equivalent of brake failure; and (3) mutated stability genes, with the defect leading to unrepaired damage to the cell’s replication machinery — the equivalent of an unskilled car mechanic. Mutations in oncogenes and tumor suppressor genes do not have an all-or-none effect. In some cancers, perhaps in all, the progression from a normal cell to a malignant tumor requires an accumulation of mutations (sometimes over several decades), none of which, alone, is responsible for the end effect. For example, the development of colorectal cancer has several recognizable stages, each associated with a mutation (Fig. 12-41). If an epithelial cell in the colon undergoes mutation of both copies of the tumor suppressor gene APC (adenomatous polyposis coli), it begins to divide faster than normal and produces a clone of itself, a benign polyp (early adenoma). For reasons not yet known, the APC mutation results in chromosomal instability, and whole regions of a chromosome are lost or rearranged during cell division. This instability can lead to another mutation, commonly in ras, that converts the clone into an intermediate (precancerous) adenoma. A third mutation (o en in the tumor suppressor gene DCC) leads to a late adenoma. Only when both copies of p53 become defective does this cell mass become a carcinoma — a malignant, life-threatening tumor. The full sequence therefore requires at least seven genetic “hits”: two on each of three tumor suppressor genes (APC, DCCΡ , and p53) and one on the proto-oncogene ras. There are probably several other routes to colorectal cancer as well, but the principle that full malignancy results only from multiple mutations is likely to hold true for all of them. Because mutations accumulate over time, the chances of developing full- blown metastatic cancer rise with age. FIGURE 12-41 Multistep transition from normal epithelial cell to colorectal cancer. Serial mutations in oncogenes (green) or tumor suppressor genes (red) lead to progressively less control of cell division, until finally an active tumor forms, which can sometimes metastasize (spread from the initial site to other regions of the body). Mutation of the MMR gene leads to defective DNA repair and consequently to a higher rate of mutation. Mutations in both copies of the tumor suppressor gene APC lead to benign clusters of epithelial cells that multiply too rapidly (early adenoma). The CDC4 oncogene results in defective ubiquitination, which is essential to the regulation of cyclin-dependent kinases (see Fig. 12-38). The oncogenes KRAS and BRAF encode Ras and Raf proteins (see Fig. 12-22), and this further disruption of signaling leads to the formation of a large adenoma, which may be detected by colonoscopy as a benign polyp. Oncogenic mutations in the PI3K gene, which encodes the enzyme phosphoinositide-3 kinase, or in PTEN, which regulates the synthesis of this enzyme, lead to a further strengthening of the signal: divide now. When a cell in one of the polyps undergoes further mutations, such as in the tumor suppressor genes DCC and p53 (see Fig. 12-40), increasingly aggressive tumors form. Finally, mutations in other tumor suppressor genes such as SMAD4 lead to a malignant tumor and sometimes to a metastatic tumor that can spread to other tissues. [Information from S. D. Markowitz and M. M. Bertagnolli, N. Engl. J. Med. 361:2449, 2009, Fig. 2.] When a polyp is detected in the early adenoma stage and the cells containing the first mutations are removed surgically, late adenomas and carcinomas will not develop; hence the importance of early detection. Cells and organisms, too, have their early detection systems. For example, the ATM and ATR proteins can detect DNA damage too extensive to be repaired effectively. They then trigger, through a pathway that includes p53, the process of apoptosis, in which a cell that has become dangerous to the organism kills itself. The development of fast and inexpensive sequencing methods has opened a new window on the process by which cancer develops. In a typical study of cancers in humans, the sequences of all 20,000 genes were determined in about 3,300 different tumors, and then compared with the gene sequences in noncancerous tissue from the same patient. Almost 300,000 mutations were detected in all. Only a small fraction of these mutations, the driver mutations, were the cause of unregulated cell division; the vast majority (>99.9%) were “passenger mutations,” which occurred randomly and did not confer a selective growth advantage on the tissue in which they occurred. Among the driver mutations were those in about 75 tumor suppressor genes and about 65 oncogenes. These 140 driver mutations fell in three general categories: those that affect cell survival (in genes encoding Ras, PI3K, MAPK, for example), those that affect cells’ ability to maintain an intact genome (ATM, ATR), and those that affect cell fate, causing cells to divide, differentiate, or become quiescent (APC is one example). A relatively small number of mutations were very common in multiple types of cancer, in the genes for Ras, p53, and pRb, for example. Apoptosis Is Programmed Cell Suicide Many cells can precisely control the time of their own death by the process of programmed cell death, or apoptosis (pronounced app′-a-toe′-sis; from the Greek for “dropping off,” as in leaves dropping in the fall). One trigger for apoptosis is irreparable damage to DNA. Programmed cell death also occurs during the normal development of an embryo, when some cells must die to give a tissue or an organ its final shape. Carving fingers from stubby limb buds requires the precisely timed death of cells between developing finger bones. During development of the nematode C. elegans from a fertilized egg, exactly 131 cells (of a total of 1,090 somatic cells in the embryo) must undergo programmed death in order to construct the adult body. Apoptosis also has roles in processes other than development. If a developing antibody-producing cell generates antibodies against a protein or glycoprotein that is normally present in the body, that cell undergoes programmed death in the thymus gland — an essential mechanism for eliminating anti-self antibodies (the cause of many autoimmune diseases). The monthly sloughing of cells of the uterine wall (menstruation) is another case of apoptosis mediating normal cell death. The dropping of leaves in the fall is the result of apoptosis in specific cells of the stem of a plant. Sometimes cell suicide is not programmed but occurs in response to biological circumstances that threaten the rest of the organism. For example, a virus-infected cell that dies before completion of the infection cycle prevents spread of the virus to nearby cells. Severe stresses such as heat, hyperosmolarity, UV light, and gamma irradiation also trigger cell suicide; presumably the organism is better off with any aberrant, potentially mutated cells dead. The regulatory mechanisms that trigger apoptosis involve some of the same proteins that regulate the cell cycle. The signal for suicide o en comes from outside, through a surface receptor. Tumor necrosis factor (TNF), produced by cells of the immune system, interacts with cells through specific TNF receptors. These receptors have TNF-binding sites on the outer face of the plasma membrane and a “death domain” (~80 amino acid residues) that carries the self-destruct signal through the membrane to cytosolic proteins such as TRADD (TNF receptor–associated death domain) (Fig. 12-42).
FIGURE 12-42 Initial events of apoptosis. An apoptosis-triggering signal from outside the cell (TNFα ) binds to its specific receptor in the plasma membrane. The occupied receptor interacts with the cytosolic protein TRADD through “death domains” (80-residue domains on both TNFα receptor and TRADD), activating TRADD. Activated TRADD initiates a proteolytic cascade that leads to apoptosis: TRADD activates caspase-8, which acts to release cytochrome c from mitochondria, which, in concert with protein Apaf-1, activates caspase-9, triggering apoptosis. When caspase-8, an “initiator” caspase, is activated by an apoptotic signal carried through TRADD, it further self-activates by cleaving its own proenzyme form. Mitochondria are one target of active caspase-8. The protease causes the release of certain proteins contained between the inner and outer mitochondrial membranes: cytochrome c and several “effector” caspases (see Fig. 19-39). Cytochrome c binds to the proenzyme form of the effector enzyme caspase-9 and stimulates its proteolytic activation. The activated caspase-9, in turn, catalyzes wholesale destruction of cellular proteins — a major cause of apoptotic cell death. One specific target of caspase action is a caspase-activated deoxyribonuclease. In apoptosis, the monomeric products of protein and DNA degradation (amino acids and nucleotides) are released in a controlled process that allows them to be taken up and reused by neighboring cells. Apoptosis thus allows the organism to eliminate a cell that is unneeded or potentially dangerous without wasting its components. SUMMARY 12.9 Oncogenes, Tumor Suppressor Genes, and Programmed Cell Death Oncogenes encode defective signaling proteins. By continually giving the signal for cell division, they lead to tumor formation. Oncogenes are genetically dominant and may encode defective growth factors, receptors, G proteins, protein kinases, or nuclear regulators of transcription. Tumor suppressor genes encode regulatory proteins that normally inhibit cell division; mutations in these genes are genetically recessive but can lead to tumor formation. Cancer is generally the result of an accumulation of mutations in oncogenes and tumor suppressor genes. When stability genes, which encode proteins necessary for the repair of genetic damage, are mutated, other mutations go unrepaired, including mutations in proto-oncogenes and tumor suppressor genes that can lead to cancer. Apoptosis is programmed and controlled cell death that functions during normal development and adulthood to destroy and recycle unnecessary, damaged, or infected cells. Apoptosis can be triggered by extracellular signals such as TNF, acting through plasma membrane receptors. Chapter Review KEY TERMS Terms in bold are defined in the glossary. signal transduction specificity sensitivity amplification enzyme cascade modularity scaffold proteins desensitization integration divergence response localization G protein–coupled receptors (GPCRs) guanosine nucleotide–binding proteins G proteins second messenger agonist antagonist β -adrenergic receptors seven-transmembrane (7tm) receptors guanosine nucleotide–exchange factor (GEF) stimulatory G protein (Gs) adenylyl cyclase cAMP-dependent protein kinase (protein kinase A; PKA) consensus sequence green fluorescent protein (GFP) fluorescence resonance energy transfer (FRET) β -adrenergic receptor kinase (β ARK) β -arrestin (β arr) G protein–coupled receptor kinases (GRKs) cAMP response element binding protein (CREB) inhibitory G protein (Gi) adaptor proteins AKAPs (A kinase anchoring proteins) Ras guanosine 3′,5′-cyclic monophosphate (cyclic GMP; cGMP) cGMP-dependent protein kinase (protein kinase G; PKG) GTPase activator protein (GAP) regulator of G-protein signaling (RGS) NO synthase phospholipase C (PLC) inositol 1,4,5-trisphosphate (IP3) IP3-gated Ca2+ channel protein kinase C (PKC) calmodulin (CaM) Ca2+/calmodulin-dependent protein kinases (CaM kinases) rhodopsin rhodopsin kinase receptor potential receptor Tyr kinase (RTK) autophosphorylation SH2 domain MAPKs voltage-gated ion channels ionotropic metabotropic hormone response element (HRE) cyclin cyclin-dependent protein kinase (CDK) ubiquitin proteasome growth factors retinoblastoma protein (pRb) oncogene proto-oncogene tumor suppressor gene programmed cell death apoptosis PROBLEMS 1. Hormone Experiments in Cell-Free Systems In the 1950s, Earl W. Sutherland, Jr., and his colleagues carried out pioneering experiments to elucidate the mechanism of action of epinephrine and glucagon. Given what you have learned in this chapter about hormone action, interpret each of the experiments described below. Identify substance X and indicate the significance of the results. a. Addition of epinephrine to a homogenate of normal liver resulted in an increase in the activity of glycogen phosphorylase. However, when the homogenate was first centrifuged at a high speed and epinephrine or glucagon was added to the clear supernatant fraction that contains phosphorylase, no increase in the phosphorylase activity occurred. b. When the particulate fraction from the centrifugation in (a) was treated with epinephrine, substance X was produced. The substance was isolated and purified. Unlike epinephrine, substance X activated glycogen phosphorylase when added to the clear supernatant fraction of the centrifuged homogenate. c. Substance X was heat-stable; that is, heat treatment did not affect its capacity to activate phosphorylase. (Hint: Would this be the case if substance X were a protein?) Substance X was nearly identical to a compound obtained when pure ATP was treated with barium hydroxide. (Fig. 8-6 will be helpful.) 2. Effect of Dibutyryl cAMP versus cAMP on Intact Cells In principle, the physiological effects of epinephrine should be mimicked by addition of cAMP to the target cells. In practice, addition of cAMP to intact target cells elicits only a minimal physiological response. Why? When the structurally related derivative dibutyryl cAMP (shown) is added to intact cells, the expected physiological response is readily apparent. Explain the basis for the difference in cellular response to these two substances. Dibutyryl cAMP is widely used in studies of cAMP function. 3. Effect of Cholera Toxin on Adenylyl Cyclase The gram-negative bacterium Vibrio cholerae produces a protein, cholera toxin (Mr 90,000), that is responsible for the characteristic symptoms of cholera: extensive loss of body water and Na+ through continuous, debilitating diarrhea. If body fluids and Na+ are not replaced, severe dehydration results; untreated, the disease is o en fatal. When the cholera toxin gains access to the human intestinal tract, it binds tightly to specific sites in the plasma membrane of the epithelial cells lining the small intestine, causing adenylyl cyclase to undergo prolonged activation (hours or days). a. What is the expected effect of cholera toxin on [cAMP] in the intestinal cells? b. Based on the information above, suggest how cAMP normally functions in intestinal epithelial cells. c. Suggest a possible treatment for cholera. 4. Mutations in PKA Explain how mutations in the R or C subunit of cAMP-dependent protein kinase (PKA) might lead to (a) a constantly active PKA or (b) a constantly inactive PKA. 5. Therapeutic Effects of Albuterol The respiratory symptoms of asthma result from constriction of the bronchi and bronchioles of the lungs, caused by contraction of the smooth muscle of their walls. Raising [cAMP] in the smooth muscle reverses the constriction of the bronchi and bronchioles. Explain the therapeutic effects of albuterol, an inhaled β -adrenergic agonist, in treating asthma. Would you expect this drug to have any side effects? If so, what design change could you make to the drug to minimize side effects? 6. Termination of Hormonal Signals Signals carried by hormones must eventually be terminated. Describe several mechanisms for signal termination. 7. Using FRET to Explore Protein-Protein Interactions In Vivo Figure 12-9 shows the interaction between β -arrestin and the β -adrenergic receptor. How would you use FRET (see Box 12-1) to demonstrate this interaction in living cells? Which proteins would you fuse? Which wavelengths would you use to illuminate the cells, and which wavelengths would you monitor? What would you expect to observe if the interaction occurred? If it did not occur? How might you explain the failure of this approach to demonstrate this interaction? 8. EGTA Injection EGTA (ethylene glycol-bis(β -amino ethyl ether)-N,N,N ′,N ′-tetraacetic acid) is a chelating agent with high affinity and specificity for Ca2+. By microinjecting a cell with an appropriate Ca2+-EGTA solution, an experimenter can prevent cytosolic [Ca2+] from rising above 10−7 M . How would EGTA microinjection affect a cell’s response to vasopressin (see Table 12-4)? To glucagon? 9. Amplification and Termination of Hormonal Signals In the β -adrenergic system, which of these contributes to the amplification of the signal (epinephrine) and which to the termination of the signal? Do any contribute to both amplification and termination of the signal? a. One Gα activates many adenylyl cyclase molecules. b. One protein kinase A (PKA) phosphorylates many target proteins. c. The intrinsic GTPase of G protein converts bound GTP to GDP. d. A phosphodiesterase acts on many molecules of cAMP. e. One epinephrine molecule activates many adrenergic receptors. f. One protein kinase phosphorylates many molecules of another protein kinase. 10. The Insulin Signaling System Place these components of the insulin receptor system in the order in which they occur in the sequence of events triggered by insulin: MEK, Ras, ERK, GRK, Raf, Sos, IRS1, PKA, Grb2. Some of these may not participate in that path. 11. Mutations in ras How would a mutation in ras that leads to formation of a Ras protein with no GTPase activity affect a cell’s response to insulin? 12. Differences among G Proteins Compare the G protein Gs, which acts in transducing the signal from β -adrenergic receptors, and the G protein Ras. What properties do they share? How do they differ? What is the functional difference between Gs and Gi? 13. Mechanisms for Regulating Protein Kinases Identify eight general types of protein kinases found in eukaryotic cells, and explain what factor is directly responsible for activating each type. 14. Nonhydrolyzable GTP Analogs Many enzymes can hydrolyze GTP between the β and γ phosphates. The GTP analog β ,γ -imidoguanosine 5′-triphosphate (Gpp(NH)p), shown here, cannot be hydrolyzed between the β and γ phosphates. Predict the effect of microinjection of Gpp(NH)p into a myocyte on the cell’s response to β -adrenergic stimulation. 15. Visual Desensitization Oguchi disease is an inherited form of night blindness. Affected individuals are slow to recover vision a er a flash of bright light against a dark background, such as the headlights of a car on the freeway. Suggest what the molecular defect(s) might be in Oguchi disease. Explain in molecular terms how this defect would account for night blindness. 16. Effect of a Permeant cGMP Analog on Rod Cells An analog of cGMP, 8-Br-cGMP, will permeate cellular membranes, is only slowly degraded by a rod cell’s PDE activity, and is as effective as cGMP in opening the gated channel in the cell’s outer segment. If you suspended rod cells in a buffer containing a relatively high [8-Br-cGMP], then illuminated the cells while measuring their membrane potential, what would you expect to see? 17. Effect of Insulin on Glycogen Synthesis Protein kinase B (PKB) inactivates glycogen synthase kinase (GSK3), and GSK3 inactivates glycogen synthase. Predict the effect of insulin on glycogen synthesis. 18. Role of Intrinsically Disordered Regions of Signaling Proteins Signaling proteins, including protein kinases, o en have intrinsically disordered regions (IDRs) that are important in signaling. Describe a case in which IDRs and their interactions with other proteins are important in signaling. 19. The Action Potential Place these events in the order in which they occur a er a presynaptic neuron releases acetylcholine into the synaptic cle . a. Vesicles containing a neurotransmitter fuse with the cell membrane. b. Ligand-gated Na+ channels open, causing an influx of Na+ ions. c. Voltage-gated Na+ channels open in the axon. d. Membrane depolarization triggers voltage-gated Ca2+ channels to open. e. Local membrane depolarization in the axon triggers an efflux of K+. 20. Hot and Cool Taste Sensations The sensations of heat and cold are transduced by a group of temperature-gated cation channels. For example, TRPV1, TRPV3, and TRPM8 are usually closed, but they open at different temperatures. TRPV1 opens at ≥43 °C, TRPV3 opens at ≥33 °C, and TRPM8 opens at < 25 °C. These channel proteins are expressed in sensory neurons known to be responsible for temperature sensation. a. Propose a reasonable model to explain how exposing a sensory neuron containing TRPV1 to high temperature leads to a sensation of heat. b. Capsaicin, one of the active ingredients in “hot” peppers, is an agonist of TRPV1. Capsaicin shows 50% activation of the TRPV1 response at a concentration of 32 nM — a property known as EC50. Explain why even a very few drops of hot pepper sauce can taste very “hot” without actually burning you. c. Menthol, one of the active ingredients in mint, is an agonist of TRPM8 (EC50 = 30 μM ) and TRPV3 (EC50 = 20 mM ). What sensation would you expect from contact with low levels of menthol? With high levels? 21. Oncogenes, Tumor Suppressor Genes, and Tumors For each of the situations listed, provide a plausible explanation for how it could lead to unrestricted cell division. a. Colon cancer cells o en contain mutations in the gene encoding the prostaglandin E2 receptor. PGE2 is a growth factor required for the division of cells in the gastrointestinal tract. b. Kaposi sarcoma, a common tumor in people with untreated AIDS, is caused by a virus carrying a gene for a protein similar to the chemokine receptors CXCR1 and CXCR2. Chemokines are cell-specific growth factors. c. Adenovirus, a tumor virus, carries a gene for the protein E1A, which binds to the retinoblastoma protein, pRb. (Hint: See Fig. 12-40.) d. An important feature of many oncogenes and tumor suppressor genes is their cell-type specificity. For example, mutations in the PGE2 receptor are not typically found in lung tumors. Explain this observation. (Note that PGE2 acts through a GPCR in the plasma membrane.) 22. Mutations in Tumor Suppressor Genes and Oncogenes Explain why mutations in tumor suppressor genes are recessive (both copies of the gene must be defective for the regulation of cell division to be defective), whereas mutations in oncogenes are dominant. 23. Retinoblastoma in Children Explain why some children with retinoblastoma develop multiple tumors of the retina in both eyes, whereas others have a single tumor in only one eye. 24. Specificity of a Signal for a Single Cell Type Discuss the validity of the proposition that a signaling molecule (hormone, growth factor, or neurotransmitter) elicits identical responses in different types of target cells if those cells contain identical receptors. DATA ANALYSIS PROBLEM 25. Exploring Taste Sensation in Mice Pleasing tastes are an evolutionary adaptation to encourage animals to consume nutritious foods. Zhao and coauthors (2003) examined the two major pleasurable taste sensations: sweet and umami. Umami is a “distinct savory taste” triggered by amino acids, especially aspartate and glutamate, and it probably encourages animals to consume protein-rich foods. Monosodium glutamate (MSG) is a flavor-enhancer that exploits this sensitivity. At the time the article was published, three taste receptor proteins for sweet and umami had been tentatively characterized: T1R1, T1R2, and T1R3. These proteins function as heterodimeric receptor complexes: T1R1-T1R3 was tentatively identified as the umami receptor, and T1R2- T1R3 as the sweet receptor. It was not clear how taste sensation was encoded and sent to the brain, and two possible models had been suggested. In the cell-based model, individual taste-sensing cells express only one kind of receptor; that is, there are “sweet cells,” “bitter cells,” “umami cells,” and so on, and each type of cell sends its information to the brain via a different nerve. The brain “knows” which taste is detected by the identity of the nerve fiber that transmits the message. In the receptor-based model, individual taste-sensing cells have several kinds of receptors and send different messages along the same nerve fiber to the brain, the message depending on which receptor is activated. Also unclear at the time was whether there was any interaction between the different taste sensations, or whether parts of one taste-sensing system were required for other taste sensations. a. Previous work had shown that different taste receptor proteins are expressed in nonoverlapping sets of taste receptor cells. Which model does this support? Explain your reasoning. Zhao and colleagues constructed a set of “knockout mice” — mice homozygous for loss-of-function alleles for one of the three receptor proteins, T1R1, T1R2, or T1R3 — and double-knockout mice with nonfunctioning T1R2 and T1R3. The researchers measured the taste perception of these mice by measuring their “lick rate” of solutions containing different taste molecules. Mice will lick the spout of a feeding bottle with a pleasant-tasting solution more o en than one with an unpleasant-tasting solution. The researchers measured relative lick rates: how o en the mice licked a sample solution compared with water. A relative lick rate of 1 indicated no preference; < 1, an aversion; and > 1, a preference. b. All four types of knockout strains had the same responses to salt and bitter tastes as did wild-type mice. Which of the above issues did this experiment address? What do you conclude from these results? The researchers then studied umami taste reception by measuring the relative lick rates of the different mouse strains with different quantities of MSG in the feeding solution. Note that the solutions also contained inosine monophosphate (IMP), a strong potentiator of umami taste reception (and a common ingredient in ramen soups, along with MSG), and amiloride, which suppresses the pleasant salty taste imparted by the sodium of MSG. The results are shown in the graph. c. Are these data consistent with the umami taste receptor consisting of a heterodimer of T1R1 and T1R3? Why or why not? d. Which model(s) of taste encoding does this result support? Explain your reasoning. Zhao and coworkers then performed a series of similar experiments using sucrose as a sweet taste. These results are shown below. e. Are these data consistent with the sweet taste receptor consisting of a heterodimer of T1R2 and T1R3? Why or why not? f. There were some unexpected responses at very high sucrose concentrations. How do these complicate the idea of a heterodimeric system as presented above? In addition to sugars, humans also taste other compounds (e.g., saccharin and the peptides monellin and aspartame) as sweet; mice do not taste these as sweet. Zhao and coworkers inserted into TIR2- knockout mice a copy of the human T1R2 gene under the control of the mouse T1R2 promoter. These modified mice now tasted monellin and saccharin as sweet. The researchers then went further, adding to T1R1-knockout mice the RASSL protein — a G protein– linked receptor for the synthetic opiate spiradoline; the RASSL gene was under the control of a promoter that could be induced by feeding the mice tetracycline. These mice did not prefer spiradoline in the absence of tetracycline; in the presence of tetracycline, they showed a strong preference for nanomolar concentrations of spiradoline. g. Do these results strengthen your conclusions about the mechanism of taste sensation? Reference Zhao, G.Q., Y. Zhang, M.A. Hoon, J. Chandrashekar, I. Erlenbach, N.J.P. Ryba, and C. Zuker. 2003. The receptors for mammalian sweet and umami taste. Cell 115:255–266. PART I I BIOENERGETICS AND METABOLISM PART OUTLINE 13 Introduction to Metabolism 14 Glycolysis, Gluconeogenesis, and the Pentose Phosphate Pathway 15 The Metabolism of Glycogen in Animals 16 The Citric Acid Cycle 17 Fatty Acid Catabolism 18 Amino Acid Oxidation and the Production of Urea 19 Oxidative Phosphorylation 20 Photosynthesis and Carbohydrate Synthesis in Plants 21 Lipid Biosynthesis 22 Biosynthesis of Amino Acids, Nucleotides, and Related Molecules 23 Hormonal Regulation and Integration of Mammalian Metabolism Every enzyme-catalyzed reaction and reaction sequence serves an important role in an organism’s physiology: to (1) obtain chemical energy by capturing solar energy or degrading energy-rich nutrients from the environment; (2) convert nutrient molecules into the cell’s own characteristic molecules, including precursors of macromolecules; (3) polymerize monomeric precursors into macromolecules: proteins, nucleic acids, and polysaccharides; and (4) synthesize and degrade biomolecules required for specialized cellular functions, such as membrane lipids, intracellular messengers, and pigments. Although metabolism embraces many thousands of different enzyme-catalyzed reactions, our major concern in Part II is the central metabolic pathways, which are few in number and remarkably similar in all forms of life. Living organisms can be divided into two large groups according to the chemical form in which they obtain carbon from the environment. Autotrophs (such as photosynthetic bacteria, green algae, and vascular plants) can use carbon dioxide from the atmosphere as their sole source of carbon, from which they construct all their carbon- containing biomolecules (see Fig. 1). Heterotrophs cannot use atmospheric carbon dioxide and must obtain carbon from their environment in the form of relatively complex organic molecules such as glucose. Multicellular animals and most microorganisms are heterotrophic. Autotrophic cells and organisms are relatively self-sufficient, whereas heterotrophic cells and organisms, with their requirements for carbon in more complex forms, must subsist on the products of other organisms. FIGURE 1 Cycling of carbon dioxide and oxygen between the autotrophic (photosynthetic) and heterotrophic domains in the biosphere. The flow of mass through this cycle is enormous; about 4× 1011 metric tons of carbon are turned over in the biosphere annually. Many autotrophic organisms are photosynthetic and obtain their energy from sunlight, whereas heterotrophic organisms obtain their energy from the degradation of organic nutrients produced by autotrophs. In our biosphere, autotrophs and heterotrophs live together in a vast, interdependent cycle in which autotrophic organisms use atmospheric carbon dioxide to build their organic biomolecules, some of them generating oxygen from water in the process. Heterotrophs, in turn, use the organic products of autotrophs as nutrients, which they oxidize, releasing to the atmosphere the CO2 produced by oxidation. Thus carbon, oxygen, and water are constantly cycled between the heterotrophic and autotrophic worlds, with solar energy as the driving force for this global process. A similarly complex global web connects molecular nitrogen (N2) with nitrogen in its other oxidation states, including NH3 (the form in which nitrogen enters metabolism). The cycling of carbon, oxygen, and nitrogen, which ultimately involves all species, depends on a proper balance between the activities of the producers (autotrophs) and consumers (heterotrophs) in our biosphere. Global warming by the greenhouse effect (the result of rising CO2 concentrations in our atmosphere) is a biochemical phenomenon, occurring on a very large scale. These cycles of matter are driven by an enormous flow of energy into and through the biosphere, beginning with the capture of solar energy by photosynthetic organisms and use of this energy to generate energy-rich carbohydrates and other organic nutrients; these nutrients are then used as energy sources by heterotrophic organisms. Precursors are converted into products through a series of intermediates called metabolites. The term intermediary metabolism is o en applied to the combined activities of all the metabolic pathways that interconvert precursors, metabolites, and products of low molecular weight (generally, Mr< 1,000). Catabolism is the degradative phase of metabolism in which organic nutrient molecules (carbohydrates, fats, and proteins) are converted into smaller, simpler end products (such as lactic acid, CO2, and NH3). Catabolic pathways release energy, some of which is conserved in the formation of ATP and reduced electron carriers (NADH or NADPH); the rest is lost as heat. In anabolism, also called biosynthesis, small, simple precursors are built up into larger and more complex molecules, including lipids, polysaccharides, proteins, and nucleic acids. Anabolic reactions require an input of energy, generally in the form of the phosphoryl group transfer potential of ATP and the reducing power of NADH and NADPH (Fig. 2). FIGURE 2 The big picture: energy relationships between catabolic and anabolic pathways. Catabolic pathways deliver chemical energy in the form of ATP, NADH, NADPH, and FADH2. These energy carriers are used in anabolic pathways to convert small precursor molecules into cellular macromolecules. Some metabolic pathways are linear, and some are branched, yielding multiple useful end products from a single precursor or converting several starting materials into a single product. In general, catabolic pathways are convergent and anabolic pathways are divergent (Fig. 3). Some pathways are cyclic: one starting component of the pathway is regenerated in a series of reactions that converts another starting component into a product. We shall see examples of each type of pathway in the following chapters. FIGURE 3 Three types of nonlinear metabolic pathways. (a) Converging, catabolic, (b) diverging, anabolic, and (c) cyclic pathways. In (c), one of the starting materials (oxaloacetate in this case) is regenerated and reenters the pathway. Acetate, a key metabolic intermediate, is the breakdown product of a variety of fuels (a), serves as the precursor for an array of products (b), and is consumed in the catabolic pathway known as the citric acid cycle (c). Most cells have the enzymes to carry out both the degradation and the synthesis of the important categories of biomolecules — fatty acids, for example. The simultaneous synthesis and degradation of fatty acids would be wasteful, however. This is prevented by reciprocally regulating the anabolic and catabolic reaction sequences: when one sequence is active, the other is suppressed. Such regulation could not occur if anabolic and catabolic pathways were catalyzed by exactly the same set of enzymes, operating in one direction for anabolism, the opposite direction for catabolism: inhibition of an enzyme involved in catabolism would also inhibit the reaction sequence in the anabolic direction. Catabolic and anabolic pathways that connect the same two end points (glucose →→ pyruvate, and pyruvate →→ glucose, for example) may employ many of the same enzymes; but, invariably, at least one of the steps is catalyzed by different enzymes in the catabolic and anabolic directions, and these enzymes are the sites of separate regulation. Moreover, for both anabolic and catabolic pathways to be essentially irreversible, the reactions unique to each direction must include at least one that is thermodynamically very favorable — in other words, a reaction for which the reverse reaction is very unfavorable. As a further contribution to the separate regulation of catabolic and anabolic reaction sequences, paired catabolic and anabolic pathways commonly take place in different cellular compartments: for example, fatty acid catabolism occurs in animal mitochondria, whereas fatty acid synthesis occurs in the cytosol. The concentrations of intermediates, enzymes, and regulators can be maintained at different levels in these different compartments. Because metabolic pathways are subject to kinetic control by substrate concentration, separate pools of anabolic and catabolic intermediates also contribute to the control of metabolic rates. Devices that separate anabolic and catabolic processes will be of particular interest in our discussions of metabolism. Metabolic pathways are regulated at several levels, from within the cell and from outside. A key enzyme in a pathway may be activated allosterically, or its amount may be changed by the rates of synthesis and breakdown of the enzyme. In multicellular organisms, the metabolic activities of different tissues are regulated and integrated by growth factors and hormones that act from outside the cell. We begin Part II with a discussion of the basic energetic principles that govern all metabolism, as well as a refresher on the reactions from organic chemistry that we will see in metabolism (Chapter 13). We then consider the major catabolic pathways by which cells obtain energy from the oxidation of various fuels (Chapters 14 through 20). Chapters 19 and 20 represent the pivotal point of our discussion of metabolism; they concern chemiosmotic energy coupling, a universal mechanism in which a transmembrane electrochemical potential, produced either by substrate oxidation or by light absorption, drives the synthesis of ATP. Chapters 20 through 22 describe the major anabolic pathways by which cells use the energy in ATP to produce carbohydrates, lipids, amino acids, and nucleotides from simpler precursors. In Chapter 23 we step back from our detailed look at the metabolic pathways — as they occur in all organisms, from E. coli to humans — and consider how they are regulated and integrated in mammals by hormonal mechanisms. As we undertake our study of intermediary metabolism, a final word. Keep in mind that the many reactions described in these pages take place in, and play crucial roles in, living organisms. As you encounter each reaction and each pathway, ask: Where does this piece fit in the big picture? What does this chemical transformation do for the organism? How does this pathway interconnect with the other pathways operating simultaneously in the same cell to produce the energy and products required for cell maintenance and growth? What would be the expected result of a defect in this enzyme or that pathway? Studied with this perspective, metabolism provides fascinating and revealing insights into life, with countless applications in medicine, agriculture, and biotechnology.
Stems are from the chapter Problems section; correct choices are drawn from Abbreviated Solutions to Problems (Appendix B) in the same edition.
1. Hormone Experiments in Cell-Free Systems In the 1950s, Earl W. Sutherland, Jr., and his colleagues carried out pioneering experiments to elucidate the mechanism of action of epinephrine and glucagon. Given what you have learned in this chapter about hormone action, interpret each of the experiments described below. Identify substance X and indicate the significance of the results. a. Addition of epinephrine to a homogenate of normal liver resulted in an increase in the activity of glycogen phosphorylase. However, when the homogenate was first centrifuged at a high speed and epinephrine or glucagon was added to the clear supernatant fraction that contains phosphorylase, no increase in the phosphorylase activity occurred. b. When the particulate fraction from the centrifugation in (a) was treated with epinephrine, substance X was produced. The substance was isolated and purified. Unlike epinephrine, substance X activated glycogen phosphorylase when added to the clear supernatant fraction of the centrifuged homogenate. c. Substance X was heat-stable; that is, heat treatment did not affect its capacity to activate phosphorylase. (Hint: Would this be the case if substance X were a protein?) Substance X was nearly identical to a compound obtained when pure ATP was treated with barium hydroxide. (Fig. 8-6 will be helpful.)
2. Effect of Dibutyryl cAMP versus cAMP on Intact Cells In principle, the physiological effects of epinephrine should be mimicked by addition of cAMP to the target cells. In practice, addition of cAMP to intact target cells elicits only a minimal physiological response. Why? When the structurally related derivative dibutyryl cAMP (shown) is added to intact cells, the expected physiological response is readily apparent. Explain the basis for the difference in cellular response to these two substances. Dibutyryl cAMP is widely used in studies of cAMP function.
3. Effect of Cholera Toxin on Adenylyl Cyclase The gram-negative bacterium Vibrio cholerae produces a protein, cholera toxin (Mr 90,000), that is responsible for the characteristic symptoms of cholera: extensive loss of body water and Na+ through continuous, debilitating diarrhea. If body fluids and Na+ are not replaced, severe dehydration results; untreated, the disease is o en fatal. When the cholera toxin gains access to the human intestinal tract, it binds tightly to specific sites in the plasma membrane of the epithelial cells lining the small intestine, causing adenylyl cyclase to undergo prolonged activation (hours or days). a. What is the expected effect of cholera toxin on [cAMP] in the intestinal cells? b. Based on the information above, suggest how cAMP normally functions in intestinal epithelial cells. c. Suggest a possible treatment for cholera.
4. Mutations in PKA Explain how mutations in the R or C subunit of cAMP-dependent protein kinase (PKA) might lead to (a) a constantly active PKA or (b) a constantly inactive PKA.
5. Therapeutic Effects of Albuterol The respiratory symptoms of asthma result from constriction of the bronchi and bronchioles of the lungs, caused by contraction of the smooth muscle of their walls. Raising [cAMP] in the smooth muscle reverses the constriction of the bronchi and bronchioles. Explain the therapeutic effects of albuterol, an inhaled β -adrenergic agonist, in treating asthma. Would you expect this drug to have any side effects? If so, what design change could you make to the drug to minimize side effects?
6. Termination of Hormonal Signals Signals carried by hormones must eventually be terminated. Describe several mechanisms for signal termination.
7. Using FRET to Explore Protein-Protein Interactions In Vivo Figure 12-9 shows the interaction between β -arrestin and the β -adrenergic receptor. How would you use FRET (see Box 12-1) to demonstrate this interaction in living cells? Which proteins would you fuse? Which wavelengths would you use to illuminate the cells, and which wavelengths would you monitor? What would you expect to observe if the interaction occurred? If it did not occur? How might you explain the failure of this approach to demonstrate this interaction?
8. EGTA Injection EGTA (ethylene glycol-bis(β -amino ethyl ether)-N,N,N ′,N ′-tetraacetic acid) is a chelating agent with high affinity and specificity for Ca2+. By microinjecting a cell with an appropriate Ca2+-EGTA solution, an experimenter can prevent cytosolic [Ca2+] from rising above 10−7 M . How would EGTA microinjection affect a cell’s response to vasopressin (see Table 12-4)? To glucagon?
9. Amplification and Termination of Hormonal Signals In the β -adrenergic system, which of these contributes to the amplification of the signal (epinephrine) and which to the termination of the signal? Do any contribute to both amplification and termination of the signal? a. One Gα activates many adenylyl cyclase molecules. b. One protein kinase A (PKA) phosphorylates many target proteins. c. The intrinsic GTPase of G protein converts bound GTP to GDP. d. A phosphodiesterase acts on many molecules of cAMP. e. One epinephrine molecule activates many adrenergic receptors. f. One protein kinase phosphorylates many molecules of another protein kinase.
10. The Insulin Signaling System Place these components of the insulin receptor system in the order in which they occur in the sequence of events triggered by insulin: MEK, Ras, ERK, GRK, Raf, Sos, IRS1, PKA, Grb2. Some of these may not participate in that path.
11. Mutations in ras How would a mutation in ras that leads to formation of a Ras protein with no GTPase activity affect a cell’s response to insulin?
12. Differences among G Proteins Compare the G protein Gs, which acts in transducing the signal from β -adrenergic receptors, and the G protein Ras. What properties do they share? How do they differ? What is the functional difference between Gs and Gi?
13. Mechanisms for Regulating Protein Kinases Identify eight general types of protein kinases found in eukaryotic cells, and explain what factor is directly responsible for activating each type.
14. Nonhydrolyzable GTP Analogs Many enzymes can hydrolyze GTP between the β and γ phosphates. The GTP analog β ,γ -imidoguanosine 5′-triphosphate (Gpp(NH)p), shown here, cannot be hydrolyzed between the β and γ phosphates. Predict the effect of microinjection of Gpp(NH)p into a myocyte on the cell’s response to β -adrenergic stimulation.
15. Visual Desensitization Oguchi disease is an inherited form of night blindness. Affected individuals are slow to recover vision a er a flash of bright light against a dark background, such as the headlights of a car on the freeway. Suggest what the molecular defect(s) might be in Oguchi disease. Explain in molecular terms how this defect would account for night blindness.
16. Effect of a Permeant cGMP Analog on Rod Cells An analog of cGMP, 8-Br-cGMP, will permeate cellular membranes, is only slowly degraded by a rod cell’s PDE activity, and is as effective as cGMP in opening the gated channel in the cell’s outer segment. If you suspended rod cells in a buffer containing a relatively high [8-Br-cGMP], then illuminated the cells while measuring their membrane potential, what would you expect to see?
17. Effect of Insulin on Glycogen Synthesis Protein kinase B (PKB) inactivates glycogen synthase kinase (GSK3), and GSK3 inactivates glycogen synthase. Predict the effect of insulin on glycogen synthesis.
18. Role of Intrinsically Disordered Regions of Signaling Proteins Signaling proteins, including protein kinases, o en have intrinsically disordered regions (IDRs) that are important in signaling. Describe a case in which IDRs and their interactions with other proteins are important in signaling.
19. The Action Potential Place these events in the order in which they occur a er a presynaptic neuron releases acetylcholine into the synaptic cle . a. Vesicles containing a neurotransmitter fuse with the cell membrane. b. Ligand-gated Na+ channels open, causing an influx of Na+ ions. c. Voltage-gated Na+ channels open in the axon. d. Membrane depolarization triggers voltage-gated Ca2+ channels to open. e. Local membrane depolarization in the axon triggers an efflux of K+.
20. Hot and Cool Taste Sensations The sensations of heat and cold are transduced by a group of temperature-gated cation channels. For example, TRPV1, TRPV3, and TRPM8 are usually closed, but they open at different temperatures. TRPV1 opens at ≥43 °C, TRPV3 opens at ≥33 °C, and TRPM8 opens at < 25 °C. These channel proteins are expressed in sensory neurons known to be responsible for temperature sensation. a. Propose a reasonable model to explain how exposing a sensory neuron containing TRPV1 to high temperature leads to a sensation of heat. b. Capsaicin, one of the active ingredients in “hot” peppers, is an agonist of TRPV1. Capsaicin shows 50% activation of the TRPV1 response at a concentration of 32 nM — a property known as EC50. Explain why even a very few drops of hot pepper sauce can taste very “hot” without actually burning you. c. Menthol, one of the active ingredients in mint, is an agonist of TRPM8 (EC50 = 30 μM ) and TRPV3 (EC50 = 20 mM ). What sensation would you expect from contact with low levels of menthol? With high levels?
21. Oncogenes, Tumor Suppressor Genes, and Tumors For each of the situations listed, provide a plausible explanation for how it could lead to unrestricted cell division. a. Colon cancer cells o en contain mutations in the gene encoding the prostaglandin E2 receptor. PGE2 is a growth factor required for the division of cells in the gastrointestinal tract. b. Kaposi sarcoma, a common tumor in people with untreated AIDS, is caused by a virus carrying a gene for a protein similar to the chemokine receptors CXCR1 and CXCR2. Chemokines are cell-specific growth factors. c. Adenovirus, a tumor virus, carries a gene for the protein E1A, which binds to the retinoblastoma protein, pRb. (Hint: See Fig. 12-40.) d. An important feature of many oncogenes and tumor suppressor genes is their cell-type specificity. For example, mutations in the PGE2 receptor are not typically found in lung tumors. Explain this observation. (Note that PGE2 acts through a GPCR in the plasma membrane.)
22. Mutations in Tumor Suppressor Genes and Oncogenes Explain why mutations in tumor suppressor genes are recessive (both copies of the gene must be defective for the regulation of cell division to be defective), whereas mutations in oncogenes are dominant.
23. Retinoblastoma in Children Explain why some children with retinoblastoma develop multiple tumors of the retina in both eyes, whereas others have a single tumor in only one eye.
24. Specificity of a Signal for a Single Cell Type Discuss the validity of the proposition that a signaling molecule (hormone, growth factor, or neurotransmitter) elicits identical responses in different types of target cells if those cells contain identical receptors. DATA ANALYSIS PROBLEM
25. Exploring Taste Sensation in Mice Pleasing tastes are an evolutionary adaptation to encourage animals to consume nutritious foods. Zhao and coauthors (2003) examined the two major pleasurable taste sensations: sweet and umami. Umami is a “distinct savory taste” triggered by amino acids, especially aspartate and glutamate, and it probably encourages animals to consume protein-rich foods. Monosodium glutamate (MSG) is a flavor-enhancer that exploits this sensitivity. At the time the article was published, three taste receptor proteins for sweet and umami had been tentatively characterized: T1R1, T1R2, and T1R3. These proteins function as heterodimeric receptor complexes: T1R1-T1R3 was tentatively identified as the umami receptor, and T1R2- T1R3 as the sweet receptor. It was not clear how taste sensation was encoded and sent to the brain, and two possible models had been suggested. In the cell-based model, individual taste-sensing cells express only one kind of receptor; that is, there are “sweet cells,” “bitter cells,” “umami cells,” and so on, and each type of cell sends its information to the brain via a different nerve. The brain “knows” which taste is detected by the identity of the nerve fiber that transmits the message. In the receptor-based model, individual taste-sensing cells have several kinds of receptors and send different messages along the same nerve fiber to the brain, the message depending on which receptor is activated. Also unclear at the time was whether there was any interaction between the different taste sensations, or whether parts of one taste-sensing system were required for other taste sensations. a. Previous work had shown that different taste receptor proteins are expressed in nonoverlapping sets of taste receptor cells. Which model does this support? Explain your reasoning. Zhao and colleagues constructed a set of “knockout mice” — mice homozygous for loss-of-function alleles for one of the three receptor proteins, T1R1, T1R2, or T1R3 — and double-knockout mice with nonfunctioning T1R2 and T1R3. The researchers measured the taste perception of these mice by measuring their “lick rate” of solutions containing different taste molecules. Mice will lick the spout of a feeding bottle with a pleasant-tasting solution more o en than one with an unpleasant-tasting solution. The researchers measured relative lick rates: how o en the mice licked a sample solution compared with water. A relative lick rate of 1 indicated no preference; < 1, an aversion; and > 1, a preference. b. All four types of knockout strains had the same responses to salt and bitter tastes as did wild-type mice. Which of the above issues did this experiment address? What do you conclude from these results? The researchers then studied umami taste reception by measuring the relative lick rates of the different mouse strains with different quantities of MSG in the feeding solution. Note that the solutions also contained inosine monophosphate (IMP), a strong potentiator of umami taste reception (and a common ingredient in ramen soups, along with MSG), and amiloride, which suppresses the pleasant salty taste imparted by the sodium of MSG. The results are shown in the graph. c. Are these data consistent with the umami taste receptor consisting of a heterodimer of T1R1 and T1R3? Why or why not? d. Which model(s) of taste encoding does this result support? Explain your reasoning. Zhao and coworkers then performed a series of similar experiments using sucrose as a sweet taste. These results are shown below. e. Are these data consistent with the sweet taste receptor consisting of a heterodimer of T1R2 and T1R3? Why or why not? f. There were some unexpected responses at very high sucrose concentrations. How do these complicate the idea of a heterodimeric system as presented above? In addition to sugars, humans also taste other compounds (e.g., saccharin and the peptides monellin and aspartame) as sweet; mice do not taste these as sweet. Zhao and coworker