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Cell_Biology_Alberts. Interaction domains enable signaling proteins to bind to one another in multiple specific combinations. Like Lego® bricks, the proteins can form linear or branching chains or three-dimensional networks, which determine the route followed by the signaling pathway. As an example, Figure 15–11 illustrates how some interaction domains mediate the formation of a large signaling complex around the receptor for the hormone insulin. Another way of bringing receptors and intracellular signaling proteins together is to concentrate them in a specific region of the cell. An important example is the primary cilium that projects like an antenna from the surface of most vertebrate cells (discussed in Chapter 16). It is usually short and nonmotile and has micro-tubules in its core, and a number of surface receptors and signaling proteins are concentrated there. We shall see later that light and smell receptors are also highly concentrated in specialized cilia.


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Cell_Biology_Alberts. The Relationship Between Signal and Response Varies in Different Signaling Pathways The function of an intracellular signaling system is to detect and measure a specific stimulus in one location of a cell and then generate an appropriately timed and measured response at another location. The system accomplishes this task by sending information in the form of molecular “signals” from the sensor to the target, often through a series of intermediaries that do not simply pass the signal along but process it in various ways. All signaling systems do not work in precisely the same way: each has evolved specialized behaviors that produce a response that is appropriate for the cell function that system controls. In the following paragraphs, we list some of these behaviors and describe how they vary in different systems, as a foundation for more detailed discussions later. 1.


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Cell_Biology_Alberts. 1. Response timing varies dramatically in different signaling systems, according to the speed required for the response. In some cases, such as synaptic signaling (see Figure 15–2C), the response can occur within milliseconds. In other cases, as in the control of cell fate by morphogens during development, a full response can require hours or days. 2.


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Cell_Biology_Alberts. 2. Sensitivity to extracellular signals can vary greatly. Hormones tend to act at very low concentrations on their distant target cells, which are therefore highly sensitive to low concentrations of signal. Neurotransmitters, on the other hand, operate at much higher concentrations at a synapse, reducing the need for high sensitivity in postsynaptic receptors. Sensitivity is often controlled by changes in the number or affinity of the receptors on the target cell. A particularly important mechanism for increasing the sensitivity of a signaling system is signal amplification, whereby a small number of activated cell-surface receptors evoke a large intracellular response either by producing large amounts of a second messenger or by activating many copies of a downstream signaling protein. 3. Dynamic range of a signaling system is related to its sensitivity. Some systems, like those involved in simple developmental decisions, are responsive


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Cell_Biology_Alberts. Figure 15–11 A specific signaling complex formed using modular interaction domains. This example is based on the insulin receptor, which is an enzyme-coupled receptor (a receptor tyrosine kinase, discussed later). First, the activated receptor phosphorylates itself on tyrosines, and one of the phosphotyrosines then recruits a docking protein called insulin receptor substrate-1 (IRS1) via a PTB domain of IRS1; the PH domain of IRS1 also binds to specific phosphoinositides on the inner surface of the plasma membrane. Then, the activated receptor phosphorylates IRS1 on tyrosines, and one of these phosphotyrosines binds the SH2 domain of the adaptor protein Grb2. Next, Grb2 uses one of its two SH3 domains to bind to a proline-rich region of a protein called Sos, which relays the signal downstream by acting as a GEF (see Figure 15–8) to activate a monomeric GTPase called Ras (not shown). Sos also binds to phosphoinositides in the plasma membrane via its PH domain. Grb2 uses its other SH3


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Cell_Biology_Alberts. by acting as a GEF (see Figure 15–8) to activate a monomeric GTPase called Ras (not shown). Sos also binds to phosphoinositides in the plasma membrane via its PH domain. Grb2 uses its other SH3 domain to bind to a proline-rich sequence in a scaffold protein. The scaffold protein binds several other signaling proteins, and the other phosphorylated tyrosines on IRS1 recruit additional signaling proteins that have SH2 domains (not shown).


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Cell_Biology_Alberts. over a narrow range of extracellular signal concentrations. Other systems, like those controlling vision or the metabolic response to some hormones, are highly responsive over a much broader range of signal strengths. We will see that broad dynamic range is often achieved by adaptation mechanisms that adjust the responsiveness of the system according to the prevailing amount of signal. 4. Persistence of a response can vary greatly. A transient response of less than a second is appropriate in some synaptic responses, for example, while a prolonged or even permanent response is required in cell fate decisions during development. Numerous mechanisms, including positive feedback, can be used to alter the duration and reversibility of a response. 5.


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Cell_Biology_Alberts. 5. Signal processing can convert a simple signal into a complex response. In many systems, for example, a gradual increase in an extracellular signal is converted into an abrupt, switchlike response. In other cases, a simple input signal is converted into an oscillatory response, produced by a repeating series of transient intracellular signals. Feedback usually lies at the heart of biochemical switches and oscillators, as we describe later. 6.


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Cell_Biology_Alberts. 6. Integration allows a response to be governed by multiple inputs. As discussed earlier, for example, specific combinations of extracellular signals are generally required to stimulate complex cell behaviors such as cell survival and proliferation (see Figure 15–4). The cell therefore has to integrate information coming from multiple signals, which often depends on intracellular coincidence detectors; these proteins are equivalent to AND gates in the microprocessor of a computer, in that they are only activated if they receive multiple converging signals (Figure 15–12). 7.


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Cell_Biology_Alberts. 7. Coordination of multiple responses in one cell can be achieved by a single extracellular signal. Some extracellular signal molecules, for example, stimulate a cell to both grow and divide. This coordination generally depends on mechanisms for distributing a signal to multiple effectors, by creating branches in the signaling pathway. In some cases, the branching of signaling pathways can allow one signal to modulate the strength of a response to other signals. Given the complexity that arises from behaviors like signal integration, distribution, and feedback, it is clear that signaling systems rarely depend on a simple linear sequence of steps but are often more like a network, in which information flows not just forward but in multiple directions—and sometimes even backward. A major research challenge is to understand the nature of these networks and the response behaviors they can achieve. The Speed of a Response Depends on the Turnover of Signaling Molecules


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Cell_Biology_Alberts. The Speed of a Response Depends on the Turnover of Signaling Molecules The speed of any signaling response depends on the nature of the intracellular signaling molecules that carry out the target cell’s response. When the response requires only changes in proteins already present in the cell, it can occur very rapidly: an allosteric change in a neurotransmitter-gated ion channel (discussed in Chapter 11), for example, can alter the plasma membrane electrical potential in milliseconds, and responses that depend solely on protein phosphorylation can occur within seconds. When the response involves changes in gene expression and the synthesis of new proteins, however, it usually requires many minutes or hours, regardless of the mode of signal delivery (Figure 15–13).


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Cell_Biology_Alberts. It is natural to think of intracellular signaling systems in terms of the changes produced when an extracellular signal is delivered. But it is just as important to consider what happens when the signal is withdrawn. During development, transient extracellular signals often produce lasting effects: they can trigger a change in the cell’s development that persists indefinitely through cell memory mechanisms, as we discuss later (and in Chapters 7 and 22). In most cases in adult tissues, however, the response fades when a signal ceases. Often the effect is transient Figure 15–12 Signal integration. Extracellular signals A and B activate different intracellular signaling pathways, each of which leads to the phosphorylation of protein Y but at different sites on the protein. Protein Y is activated only when both of these sites are phosphorylated, and therefore it becomes active only when signals A and B are simultaneously present. Such proteins are often called coincidence detectors.


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Cell_Biology_Alberts. because the signal exerts its effects by altering the concentrations of intracellular molecules that are short-lived (unstable), undergoing continual turnover. Thus, once the extracellular signal is gone, the degradation of the old molecules quickly wipes out all traces of the signal’s action. It follows that the speed with which a cell responds to signal removal depends on the rate of destruction, or turnover, of the intracellular molecules that the signal affects.


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Cell_Biology_Alberts. It is also true, although much less obvious, that this turnover rate can determine the promptness of the response when an extracellular signal arrives. Consider, for example, two intracellular signaling molecules, X and Y, both of which are normally maintained at a steady-state concentration of 1000 molecules per cell. The cell synthesizes and degrades molecule Y at a rate of 100 molecules per second, with each molecule having an average lifetime of 10 seconds. Molecule X has a turnover rate that is 10 times slower than that of Y: it is both synthesized and degraded at a rate of 10 molecules per second, so that each molecule has an average lifetime in the cell of 100 seconds. If a signal acting on the cell causes a tenfold increase in the synthesis rates of both X and Y with no change in the molecular lifetimes, at the end of 1 second the concentration of Y will have increased by nearly 900 molecules per cell (10 × 100 – 100), while the concentration of X will have increased by only


Cell_Biology_Alberts

Cell_Biology_Alberts. the molecular lifetimes, at the end of 1 second the concentration of Y will have increased by nearly 900 molecules per cell (10 × 100 – 100), while the concentration of X will have increased by only 90 molecules per cell. In fact, after a molecule’s synthesis rate has been either increased or decreased abruptly, the time required for the molecule to shift halfway from its old to its new equilibrium concentration is equal to its half-life—that is, equal to the time that would be required for its concentration to fall by half if all synthesis were stopped (Figure 15–14).


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Cell_Biology_Alberts. The same principles apply to proteins and small molecules, whether the molecules are in the extracellular space or inside cells. Many intracellular proteins have short half-lives, some surviving for less than 10 minutes. In most cases, these are key regulatory proteins whose concentrations are rapidly controlled in the cell by changes in their rates of synthesis. As we have seen, many cell responses to extracellular signals depend on the conversion of intracellular signaling proteins from an inactive to an active form, rather than on their synthesis or degradation. Phosphorylation or the binding of GTP, for example, commonly activates signaling proteins. Even in these cases, however, the activation must be rapidly and continuously reversed (by dephosphorylation or GTP hydrolysis to GDP, respectively, in these examples) to make rapid signaling possible. These inactivation processes play a crucial part in determining the magnitude, rapidity, and duration of the response.


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Cell_Biology_Alberts. Figure 15–13 Slow and rapid responses to an extracellular signal. Certain types of signal-induced cellular responses, such as increased cell growth and division, involve changes in gene expression and the synthesis of new proteins; they therefore occur slowly, often starting an hour or more after the signal is received. Other responses—such as changes in cell movement, secretion, or metabolism— need not involve changes in gene transcription and therefore occur much more quickly, often starting in seconds or minutes; they may involve the rapid phosphorylation of effector proteins in the cytoplasm, for example. Synaptic responses mediated by changes in membrane potential are even quicker and can occur in milliseconds (not shown). Some signaling systems generate both rapid and slow responses as shown here, allowing the cell to respond quickly to a signal while simultaneously initiating a more long-term, persistent response.


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Cell_Biology_Alberts. minutes after the synthesis rate has minutes after the synthesis rate has been decreased by a factor of 10 been increased by a factor of 10 relative concentration of intracellular molecule Cells Can Respond Abruptly to a Gradually Increasing Signal


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Cell_Biology_Alberts. Some signaling systems are capable of generating a smoothly graded response over a wide range of extracellular signal concentrations (Figure 15–15, blue line); such systems are useful, for example, in the fine tuning of metabolic processes by some hormones. Other signaling systems generate significant responses only when the signal concentration rises beyond some threshold value. These abrupt responses are of two types. One is a sigmoidal response, in which low concentrations of stimulus do not have much effect, but then the response rises steeply and continuously at intermediate stimulus levels (Figure 15–15, red line). Such systems provide a filter to reduce inappropriate responses to low-level background signals but respond with high sensitivity when the stimulus falls within a small range of physiological signal concentrations. A second type of abrupt response is the discontinuous or all-or-none response, in which the response switches on completely (and often irreversibly) when


Cell_Biology_Alberts

Cell_Biology_Alberts. range of physiological signal concentrations. A second type of abrupt response is the discontinuous or all-or-none response, in which the response switches on completely (and often irreversibly) when the signal reaches some threshold concentration (Figure 15–15, green line). Such responses are particularly useful for controlling the choice between two alternative cell states, and they generally involve positive feedback, as we describe in more detail shortly.


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Cell_Biology_Alberts. Cells use a variety of molecular mechanisms to produce a sigmoidal response to increasing signal concentrations. In one mechanism, more than one intracellular signaling molecule must bind to its downstream target protein to induce a response. As we discuss later, for example, four molecules of the second messenger cyclic AMP must bind simultaneously to each molecule of cyclic-AMP-dependent protein kinase (PKA) to activate the kinase. A similar sharpening of response is seen when the activation of an intracellular signaling protein requires phosphorylation at more than one site. Such responses become sharper as the number of required molecules or phosphate groups increases, and if the number is large enough, responses become almost all-or-none (Figure 15–16).


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Cell_Biology_Alberts. Responses are also sharpened when an intracellular signaling molecule activates one enzyme and also inhibits another enzyme that catalyzes the opposite reaction. A well-studied example of this common type of regulation is the stimulation of glycogen breakdown in skeletal muscle cells induced by the hormone adrenaline (epinephrine). Adrenaline’s binding to a G-protein-coupled


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Cell_Biology_Alberts. Figure 15–14 The importance of rapid turnover. The graphs show the predicted relative rates of change in the intracellular concentrations of molecules with differing turnover times when their synthesis rates are either (A) decreased or (B) increased suddenly by a factor of 10. In both cases, the concentrations of those molecules that are normally degraded rapidly in the cell (red lines) change quickly, whereas the concentrations of those that are normally degraded slowly (green lines) change proportionally more slowly. The numbers (in blue) on the right are the half-lives assumed for each of the different molecules.


Cell_Biology_Alberts

Cell_Biology_Alberts. switchlike responses. Some cell responses increase gradually as the concentration of extracellular signal molecule increases, eventually reaching a plateau as the signaling pathway is saturated, resulting in a hyperbolic response curve (blue line). In other cases, the signaling system reduces the response at low signal concentrations and then produces a steeper response at some intermediate signal concentration—resulting in a sigmoidal response curve (red line). In still other cases, the response is more abrupt and switchlike; the cell switches completely between a low and high response, without any stable intermediate response (green line). cell-surface receptor increases the intracellular concentration of cyclic AMP, which both activates an enzyme that promotes glycogen breakdown and inhibits an enzyme that promotes glycogen synthesis.


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Cell_Biology_Alberts. Like intracellular metabolic pathways (discussed in Chapter 2) and the systems controlling gene activity (Chapter 7), most intracellular signaling systems incorporate feedback loops, in which the output of a process acts back to regulate that same process. We discussed the mathematical analysis of feedback loops in Chapter 8. In positive feedback, the output stimulates its own production; in negative feedback, the output inhibits its own production (Figure 15–17). Feedback loops are of great general importance in biology, and they regulate many chemical and physical processes in cells. Those that regulate cell signaling can either operate exclusively within the target cell or involve the secretion of extracellular signals. Here, we focus on those feedback loops that operate entirely within the target cell; even the simplest of these loops can produce complex and interesting effects.


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Cell_Biology_Alberts. Positive feedback in a signaling pathway can transform the behavior of the responding cell. If the positive feedback is of only moderate strength, its effect will be simply to steepen the response to the signal, generating a sigmoidal response like those described earlier; but if the feedback is strong enough, it can produce an all-or-none response (see Figure 15–15). This response goes hand in hand with a further property: once the responding system has switched to the high level of activation, this condition is often self-sustaining and can persist even after the 0.5 1.0 1.5 2.0 1 2 8 16 % of maximum activation of target protein an allosteric protein as a function of effector molecule concentration.


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Cell_Biology_Alberts. The curves show how the sharpness of the activation response increases with an increase in the number of allosteric effector molecules that must be bound simultaneously to activate the target protein. The curves shown are those expected, under certain conditions, if the 0 activation requires the simultaneous binding relative concentration of effector molecule of 1, 2, 8, or 16 effector molecules. signal strength drops back below its critical value. In such a case, the system is said to be bistable: it can exist in either a “switched-off” or a “switched-on” state, and a transient stimulus can flip it from the one state to the other (Figure 15–18A and B).


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Cell_Biology_Alberts. Through positive feedback, a transient extracellular signal can induce longterm changes in cells and their progeny that can persist for the lifetime of the organism. The signals that trigger muscle-cell specification, for example, turn on the transcription of a series of genes that encode muscle-specific transcription regulatory proteins, which stimulate the transcription of their own genes, as well as genes encoding various other muscle-cell proteins; in this way, the decision to become a muscle cell is made permanent. This type of cell memory, which depends on positive feedback, is one of the basic ways in which a cell can undergo a lasting change of character without any alteration in its DNA sequence.


Cell_Biology_Alberts

Cell_Biology_Alberts. Studies of signaling responses in large populations of cells can give the false impression that a response is smoothly graded, even when strong positive feedback is causing an abrupt, discontinuous switch in the response in individual cells. Only by studying the response in single cells is it possible to see its all-or-none character (Figure 15–19). The misleading smooth response in a cell population is due to the random, intrinsic variability in signaling systems that we described earlier: all cells in a population do not respond identically to the same concentration of extracellular signal, especially at intermediate signal concentrations where the receptor is only partially occupied. Negative Feedback is a Common Motif in Signaling Systems


Cell_Biology_Alberts

Cell_Biology_Alberts. Negative Feedback is a Common Motif in Signaling Systems By contrast with positive feedback, negative feedback counteracts the effect of a stimulus and thereby abbreviates and limits the level of the response, making the system less sensitive to perturbations (see Chapter 8). As with positive feedback, however, qualitatively different responses can be obtained when the feedback Figure 15–17 Positive and negative feedback. In these simple examples, a stimulus activates protein A, which, in turn, activates protein B. Protein B then acts back to either increase or decrease the activity of A.


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Cell_Biology_Alberts. Figure 15–18 Some effects of simple feedback. The graphs show the computed effects of simple positive and negative feedback loops (see Chapter 8). In each case, the input signal is an activated protein kinase (S) that phosphorylates and thereby activates another protein kinase (E); a protein phosphatase (I) dephosphorylates and inactivates the activated E kinase. In the graphs, the red line indicates the activity of the E kinase over time; the underlying blue bar indicates the time for which the input signal (activated S kinase) is present. (A) Diagram of the positive feedback loop, in which the activated E kinase acts back to promote its own phosphorylation and activation; the basal activity of the I phosphatase dephosphorylates activated E at a steady, low rate. (B) The top graph shows that, without feedback, the activity of the E kinase is simply proportional (with a


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Cell_Biology_Alberts. E kinase E kinase I phosphatase short lag) to the level of stimulation by the S kinase. The bottom graph shows that, with the positive feedback loop, the transient stimulation by S kinase switches the system from an “off” state to an “on” state, which then persists after the stimulus has been removed. (C) Diagram of the negative feedback loop, in which the activated E kinase phosphorylates and activates the I phosphatase, thereby increasing the rate at which the phosphatase dephosphorylates and inactivates the phosphorylated E kinase. (D) The top graph shows, again, the response in E kinase activity without feedback. The other graphs show the effects on E kinase activity of negative feedback operating after a short or long delay. With a short delay, the system shows a strong, brief response when the signal is abruptly changed, and the feedback then drives the response back down to a lower level. With a long delay, the feedback SIGNAL produces sustained oscillations for as long (D) as


Cell_Biology_Alberts

Cell_Biology_Alberts. when the signal is abruptly changed, and the feedback then drives the response back down to a lower level. With a long delay, the feedback SIGNAL produces sustained oscillations for as long (D) as the stimulus is present.


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Cell_Biology_Alberts. 0 0.001 0.01 0.1 1 10 progesterone (µM) increasing concentration of progesterone (A) operates more powerfully. A delayed negative feedback with a long enough delay can produce responses that oscillate. The oscillations may persist for as long as the stimulus is present (Figure 15–18C and D) or they may even be generated spontaneously, without need of an external signal to drive them. Many such oscillators also contain positive feedback loops that generate sharper oscillations. Later in this chapter, we will encounter specific examples of oscillatory behavior in the intracellular responses to extracellular signals; all of them depend on negative feedback, generally accompanied by positive feedback.


Cell_Biology_Alberts

Cell_Biology_Alberts. If negative feedback operates with a short delay, the system behaves like a change detector. It gives a strong response to a stimulus, but the response rapidly decays even while the stimulus persists; if the stimulus is suddenly increased, however, the system responds strongly again, but, again, the response rapidly decays. This is the phenomenon of adaptation, which we now discuss. Cells Can Adjust Their Sensitivity to a Signal


Cell_Biology_Alberts

Cell_Biology_Alberts. In responding to many types of stimuli, cells and organisms are able to detect the same percentage of change in a signal over a wide range of stimulus strengths. The target cells accomplish this through a reversible process of adaptation, or desensitization, whereby a prolonged exposure to a stimulus decreases the cells’ response to that level of stimulus. In chemical signaling, adaptation enables cells to respond to changes in the concentration of an extracellular signal molecule (rather than to the absolute concentration of the signal) over a very wide range of signal concentrations. The underlying mechanism is negative feedback that operates with a short delay: a strong response modifies the signaling machinery involved, such that the machinery resets itself to become less responsive to the same level of signal (see Figure 15–18D, middle graph). Owing to the delay, however, a sudden increase in the signal is able to stimulate the cell again for a short period before the negative


Cell_Biology_Alberts

Cell_Biology_Alberts. to the same level of signal (see Figure 15–18D, middle graph). Owing to the delay, however, a sudden increase in the signal is able to stimulate the cell again for a short period before the negative feedback has time to kick in.


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Cell_Biology_Alberts. Adaptation to a signal molecule can occur in various ways. It can result from inactivation of the receptors themselves. The binding of signal molecules to cell-surface receptors, for example, may induce the endocytosis and temporary sequestration of the receptors in endosomes. In some cases, such signal-induced receptor endocytosis leads to the destruction of the receptors in lysosomes, a process referred to as receptor down-regulation (in other cases, however, activated receptors continue to signal after they have been endocytosed). Receptors can also become inactivated on the cell surface—for example, by becoming phosphorylated—with a short delay following their activation. Adaptation can also occur at sites downstream of the receptors, either by a change in intracellular signaling proteins involved in transducing the extracellular signal or by the production of an inhibitor protein that blocks the signal transduction process. These various adaptation mechanisms are compared in


Cell_Biology_Alberts

Cell_Biology_Alberts. proteins involved in transducing the extracellular signal or by the production of an inhibitor protein that blocks the signal transduction process. These various adaptation mechanisms are compared in Figure 15–20.


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Cell_Biology_Alberts. Though bewildering in their complexity, the multiple cross-regulatory signaling pathways and feedback loops that we describe in this chapter are not just a haphazard tangle, but a highly evolved system for processing and interpreting


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Cell_Biology_Alberts. Figure 15–19 The importance of examining individual cells to detect all-or-none responses to increasing concentrations of an extracellular signal. In these experiments, immature frog eggs (oocytes) were stimulated with increasing concentrations of the hormone progesterone. The response was assessed by analyzing the activation of MAP kinase (discussed later), which is one of the protein kinases activated by phosphorylation in the response. The amount of phosphorylated (activated) MAP kinase in extracts of the oocytes was assessed biochemically. In (A), extracts of populations of stimulated oocytes were analyzed, and the activation of MAP kinase appeared to increase progressively with increasing progesterone concentration. There are two possible ways of explaining this result: (B) MAP kinase could have increased gradually in each individual cell with increasing progesterone concentration; or (C) individual cells could have responded in an all-or-none way, with the gradual increase in


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Cell_Biology_Alberts. could have increased gradually in each individual cell with increasing progesterone concentration; or (C) individual cells could have responded in an all-or-none way, with the gradual increase in total MAP kinase activation reflecting the increasing number of cells responding with increasing progesterone concentration. When extracts of individual oocytes were analyzed, it was found that cells had either very low amounts or very high amounts, but not intermediate amounts, of the activated kinase, indicating that the response was essentially all-or-none at the level of individual cells, as diagrammed in (C). Subsequent studies revealed that this allor-none response is due in part to strong positive feedback in the progesterone signaling system. (Adapted from J.E. Ferrell and E.M. Machleder, Science 280:895– 898, 1998. With permission from AAAS.)


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Cell_Biology_Alberts. Figure 15–20 Some ways in which target cells can become adapted (desensitized) to an extracellular signal molecule. The mechanisms shown here that operate at the level of the receptor often involve phosphorylation or ubiquitylation of the receptor proteins.


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Cell_Biology_Alberts. the vast number of signals that impinge upon animal cells. The whole molecular control network, leading from the receptors at the cell surface to the genes in the nucleus, can be viewed as a computing device; and, like that other biological computing device, the brain, it presents one of the hardest problems in biology. We can identify the components and discover how they work individually. We can understand how small subsets of components work together as regulatory modules, noise filters, or adaptation mechanisms, as we have seen. However, it is a much more difficult task to understand how the system works as a whole. This is not only because the system is complex; it is also because the way in which it behaves is strongly dependent on the quantitative details of the molecular interactions, and, for most animal cells, we have only rough qualitative information. A major challenge for the future of signaling research is to develop more sophisticated quantitative and computational


Cell_Biology_Alberts

Cell_Biology_Alberts. and, for most animal cells, we have only rough qualitative information. A major challenge for the future of signaling research is to develop more sophisticated quantitative and computational methods for the analysis of signaling systems, as described in Chapter 8.


Cell_Biology_Alberts

Cell_Biology_Alberts. Each cell in a multicellular animal is programmed to respond to a specific set of extracellular signal molecules produced by other cells. The signal molecules act by binding to a complementary set of receptor proteins expressed by the target cells. Most extracellular signal molecules activate cell-surface receptor proteins, which act as signal transducers, converting the extracellular signal into intracellular ones that alter the behavior of the target cell. Activated receptors relay the signal into the cell interior by activating intracellular signaling proteins. Some of these signaling proteins transduce, amplify, or spread the signal as they relay it, while others integrate signals from different signaling pathways. Some function as switches that are transiently activated by phosphorylation or GTP binding. Large signaling complexes form by means of modular interaction domains in the signaling proteins, which allow the proteins to form functional signaling networks.


Cell_Biology_Alberts

Cell_Biology_Alberts. Target cells use various mechanisms, including feedback loops, to adjust the ways in which they respond to extracellular signals. Positive feedback loops can help cells to respond in an all-or-none fashion to a gradually increasing concentration of an extracellular signal and to convert a short-lasting signal into a long-lasting, or even irreversible, response. Negative feedback allows cells to adapt to a signal molecule, which enables them to respond to small changes in the concentration of the signal molecule over a large concentration range.


Cell_Biology_Alberts

Cell_Biology_Alberts. G-protein-coupled receptors (GPCRs) form the largest family of cell-surface receptors, and they mediate most responses to signals from the external world, as well as signals from other cells, including hormones, neurotransmitters, and local mediators. Our senses of sight, smell, and taste depend on them. There are more than 800 GPCRs in humans, and in mice there are about 1000 concerned with the sense of smell alone. The signal molecules that act on GPCRs are as varied in structure as they are in function and include proteins and small peptides, as well as derivatives of amino acids and fatty acids, not to mention photons of light and all the molecules that we can smell or taste. The same signal molecule can activate many different GPCR family members; for example, adrenaline activates at least 9 distinct GPCRs, acetylcholine another 5, and the neurotransmitter serotonin at least 14. The different receptors for the same signal are usually expressed in different cell types and elicit


Cell_Biology_Alberts

Cell_Biology_Alberts. least 9 distinct GPCRs, acetylcholine another 5, and the neurotransmitter serotonin at least 14. The different receptors for the same signal are usually expressed in different cell types and elicit different responses.


Cell_Biology_Alberts

Cell_Biology_Alberts. Despite the chemical and functional diversity of the signal molecules that activate them, all GPCRs have a similar structure. They consist of a single polypeptide chain that threads back and forth across the lipid bilayer seven times, forming a cylindrical structure, often with a deep ligand-binding site at its center (Figure 15–21). In addition to their characteristic orientation in the plasma membrane, they all use G proteins to relay the signal into the cell interior. The GPCR superfamily includes rhodopsin, the light-activated protein in the vertebrate eye, as well as the large number of olfactory receptors in the vertebrate nose. Other family members are found in unicellular organisms: the receptors in yeasts that recognize secreted mating factors are an example. It is likely that the GPCRs that mediate cell–cell signaling in multicellular organisms evolved from the sensory receptors in their unicellular eukaryotic ancestors.


Cell_Biology_Alberts

Cell_Biology_Alberts. It is remarkable that almost half of all known drugs work through GPCRs or the signaling pathways GPCRs activate. Of the many hundreds of genes in the human genome that encode GPCRs, about 150 encode orphan receptors, for which the ligand is unknown. Many of them are likely targets for new drugs that remain to be discovered. When an extracellular signal molecule binds to a GPCR, the receptor undergoes a conformational change that enables it to activate a trimeric GTP-binding protein (G protein), which couples the receptor to enzymes or ion channels in the membrane. In some cases, the G protein is physically associated with the receptor before the receptor is activated, whereas in others it binds only after receptor activation. There are various types of G proteins, each specific for a particular set of GPCRs and for a particular set of target proteins in the plasma membrane. They all have a similar structure, however, and operate similarly.


Cell_Biology_Alberts

Cell_Biology_Alberts. G proteins are composed of three protein subunits—α, β, and γ. In the unstimulated state, the α subunit has GDP bound and the G protein is inactive (Figure 15–22). When a GPCR is activated, it acts like a guanine nucleotide exchange factor (GEF) and induces the α subunit to release its bound GDP, allowing GTP to bind in its place. GTP binding then causes an activating conformational change in the Gα subunit, releasing the G protein from the receptor and triggering dissociation of the GTP-bound Gα subunit from the Gβγ pair—both of which then interact with various targets, such as enzymes and ion channels in the plasma membrane, which relay the signal onward (Figure 15–23).


Cell_Biology_Alberts

Cell_Biology_Alberts. The α subunit is a GTPase and becomes inactive when it hydrolyzes its bound GTP to GDP. The time required for GTP hydrolysis is usually short because the GTPase activity is greatly enhanced by the binding of the α subunit to a second protein, which can be either the target protein or a specific regulator of G protein signaling (RGS). RGS proteins act as α-subunit-specific GTPase-activating proteins (GAPs) (see Figure 15–8), and they help shut off G-protein-mediated responses in all eukaryotes. There are about 25 RGS proteins encoded in the human genome, each of which interacts with a particular set of G proteins.


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Cell_Biology_Alberts. Figure 15–21 A G-protein-coupled receptor (GPCR). (A) GPCRs that bind small ligands such as adrenaline have small extracellular domains, and the ligand usually binds deep within the plane of the membrane to a site that is formed by amino acids from several transmembrane segments. GPCRs that bind protein ligands have a large extracellular domain (not shown here) that contributes to ligand binding. (B) The structure of the β2-adrenergic receptor, a receptor for the neurotransmitter adrenaline, illustrates the typical cylindrical arrangement of the seven transmembrane helices in a GPCR. The ligand (orange) binds in a pocket between the helices, resulting in conformational changes on the cytoplasmic surface of the receptor that promote G-protein activation (not shown). (PDB code: 3P0G.) Some G Proteins Regulate the Production of Cyclic AMP


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Cell_Biology_Alberts. Some G Proteins Regulate the Production of Cyclic AMP Cyclic AMP (cAMP) acts as a second messenger in some signaling pathways. An extracellular signal can increase cAMP concentration more than twentyfold in seconds (Figure 15–24). As explained earlier (see Figure 15–14), such a rapid response requires balancing a rapid synthesis of the molecule with its rapid breakdown or removal. Cyclic AMP is synthesized from ATP by an enzyme called


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Cell_Biology_Alberts. Figure 15–22 The structure of an inactive G protein. (A) Note that both the α and the γ subunits have covalently attached lipid molecules (red tails) that help bind them to the plasma membrane, and the α subunit has GDP bound. (B) The three-dimensional structure of the inactive, GDP-bound form of a G protein called Gs, which interacts with numerous GPCRs, including the β2-adrenergic receptor shown in Figures 15–21 and 15–23. The α subunit contains the GTPase domain and binds to one side of the β subunit. The γ subunit binds to the opposite side of the β subunit, and the β and γ subunits together form a single functional unit. The GTPase domain of the α subunit contains two major subdomains: the “Ras” domain, which is related to other GTPases and provides one face of the nucleotide-binding pocket; and the alpha-helical or “AH” domain, which clamps the nucleotide in place. (B, based on D.G. Lombright et al., Nature 379:311– 319, 1996. With permission from Macmillan Publishers Ltd.)


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Cell_Biology_Alberts. D.G. Lombright et al., Nature 379:311– 319, 1996. With permission from Macmillan Publishers Ltd.) Figure 15–23 Activation of a G protein by an activated GPCR. Binding of an extracellular signal molecule to a GPCR changes the conformation of the receptor, which allows the receptor to bind and alter the conformation of a trimeric G protein. The AH domain of the G protein α subunit moves outward to open the nucleotide-binding site, thereby promoting dissociation of GDP. GTP binding then promotes closure of the nucleotide-binding site, triggering conformational changes that cause dissociation of the α subunit from the receptor and from the βγ complex. The GTP-bound α subunit and the βγ complex each regulate the activities of downstream signaling molecules (not shown). The receptor stays active while the extracellular signal molecule is bound to it, and it can therefore catalyze the activation of many G-protein molecules (Movie 15.1).


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Cell_Biology_Alberts. adenylyl cyclase, and it is rapidly and continuously destroyed by cyclic AMP phosphodiesterases (Figure 15–25). Adenylyl cyclase is a large, multipass trans-membrane protein with its catalytic domain on the cytosolic side of the plasma membrane. There are at least eight isoforms in mammals, most of which are regulated by both G proteins and Ca2+. Many extracellular signals work by increasing cAMP concentrations inside the cell. These signals activate GPCRs that are coupled to a stimulatory G protein (Gs). The activated α subunit of Gs binds and thereby activates adenylyl cyclase. Other extracellular signals, acting through different GPCRs, reduce cAMP levels by activating an inhibitory G protein (Gi), which then inhibits adenylyl cyclase.


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Cell_Biology_Alberts. Both Gs and Gi are targets for medically important bacterial toxins. Cholera toxin, which is produced by the bacterium that causes cholera, is an enzyme that catalyzes the transfer of ADP ribose from intracellular NAD+ to the α subunit of Gs. This ADP ribosylation alters the α subunit so that it can no longer hydrolyze its bound GTP, causing it to remain in an active state that stimulates adenylyl cyclase indefinitely. The resulting prolonged elevation in cAMP concentration within intestinal epithelial cells causes a large efflux of Cl– and water into the gut, thereby causing the severe diarrhea that characterizes cholera. Pertussis toxin, which is made by the bacterium that causes pertussis (whooping cough), catalyzes the ADP ribosylation of the α subunit of Gi, preventing the protein from interacting with receptors; as a result, the G protein remains in the inactive GDP-bound state and is unable to regulate its target proteins. These two toxins are widely used in experiments to


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Cell_Biology_Alberts. from interacting with receptors; as a result, the G protein remains in the inactive GDP-bound state and is unable to regulate its target proteins. These two toxins are widely used in experiments to determine whether a cell’s GPCR-dependent response to a signal is mediated by Gs or by Gi.


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Cell_Biology_Alberts. Some of the responses mediated by a Gs-stimulated increase in cAMP concentration are listed in Table 15–1. As the table shows, different cell types respond differently to an increase in cAMP concentration. Some cell types, such as fat cells, activate adenylyl cyclase in response to multiple hormones, all of which thereby stimulate the breakdown of triglyceride (the storage form of fat) to fatty acids. Individuals with genetic defects in the Gs α subunit show decreased responses to certain hormones, resulting in metabolic abnormalities, abnormal bone development, and mental retardation. Cyclic-AMP-Dependent Protein Kinase (PKA) Mediates Most of the Effects of Cyclic AMP In most animal cells, cAMP exerts its effects mainly by activating cyclic-AMPdependent protein kinase (PKA). This kinase phosphorylates specific serines or


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Cell_Biology_Alberts. In most animal cells, cAMP exerts its effects mainly by activating cyclic-AMPdependent protein kinase (PKA). This kinase phosphorylates specific serines or Figure 15–25 The synthesis and degradation of cyclic AMP. In a reaction catalyzed by the enzyme adenylyl cyclase, cyclic AMP (cAMP) is synthesized from ATP through a cyclization reaction that removes two phosphate groups as pyrophosphate (PPi); a pyrophosphatase drives this synthesis by hydrolyzing the released pyrophosphate to phosphate (not shown). Cyclic AMP is short-lived (unstable) in the cell because it is hydrolyzed by specific phosphodiesterases to form 5ʹ-AMP, as indicated. Figure 15–24 An increase in cyclic AMP in response to an extracellular signal.


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Cell_Biology_Alberts. Figure 15–24 An increase in cyclic AMP in response to an extracellular signal. This nerve cell in culture is responding to the neurotransmitter serotonin, which acts through a GPCR to cause a rapid rise in the intracellular concentration of cyclic AMP. To monitor the cyclic AMP level, the cell has been loaded with a fluorescent protein that changes its fluorescence when it binds cyclic AMP. Blue indicates a low level of cyclic AMP, yellow an intermediate level, and red a high level. (A) In the resting cell, the cyclic AMP level is about 5 × 10–8


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Cell_Biology_Alberts. M. (B) Twenty seconds after the addition of serotonin to the culture medium, the intracellular level of cyclic AMP has increased to more than 10–6 M in the relevant parts of the cell, an increase of more than twentyfold. (From B.J. Bacskai et al., Science 260:222–226, 1993. With permission from AAAS.) threonines on selected target proteins, including intracellular signaling proteins and effector proteins, thereby regulating their activity. The target proteins differ from one cell type to another, which explains why the effects of cAMP vary so markedly depending on the cell type (see Table 15–1).


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Cell_Biology_Alberts. In the inactive state, PKA consists of a complex of two catalytic subunits and two regulatory subunits. The binding of cAMP to the regulatory subunits alters their conformation, causing them to dissociate from the complex. The released catalytic subunits are thereby activated to phosphorylate specific target proteins (Figure 15–26). The regulatory subunits of PKA (also called A-kinase) are important for localizing the kinase inside the cell: special A-kinase anchoring proteins (AKAPs) bind both to the regulatory subunits and to a component of the cytoskeleton or a membrane of an organelle, thereby tethering the enzyme complex to a particular subcellular compartment. Some AKAPs also bind other signaling proteins, forming a signaling complex. An AKAP located around the nucleus of heart muscle cells, for example, binds both PKA and a phosphodiesterase that hydrolyzes cAMP. In unstimulated cells, the phosphodiesterase keeps the local cAMP concentration low, so that the bound PKA is


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Cell_Biology_Alberts. muscle cells, for example, binds both PKA and a phosphodiesterase that hydrolyzes cAMP. In unstimulated cells, the phosphodiesterase keeps the local cAMP concentration low, so that the bound PKA is inactive; in stimulated cells, cAMP concentration rapidly rises, overwhelming the phosphodiesterase and activating the PKA. Among the target proteins that PKA phosphorylates and activates in these cells is the adjacent phosphodiesterase, which rapidly lowers the cAMP concentration again. This negative feedback arrangement converts what might otherwise be a prolonged PKA response into a brief, local pulse of PKA activity.


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Cell_Biology_Alberts. Whereas some responses mediated by cAMP occur within seconds (see Figure 15–24), others depend on changes in the transcription of specific genes and take hours to develop fully. In cells that secrete the peptide hormone somatostatin, regulatory inactive complex of Figure 15–26 The activation of cyclicAMP-dependent protein kinase (PKA).


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Cell_Biology_Alberts. The binding of cAMP to the regulatory subunits of the PKA tetramer induces a conformational change, causing these subunits to dissociate from the catalytic subunits, thereby activating the kinase activity of the catalytic subunits. The release of the catalytic subunits requires the binding of more than two cAMP molecules to the regulatory subunits in the tetramer. This requirement greatly sharpens the response of the kinase to changes in cAMP concentration, as discussed earlier (see Figure 15–16). Mammalian cells have at least two types of PKAs: type I is mainly in the cytosol, whereas type II is bound via its regulatory subunits and special anchoring proteins to the plasma membrane, nuclear membrane, mitochondrial outer membrane, and microtubules. In both types, once the catalytic subunits are freed and active, they can migrate into the nucleus (where they can phosphorylate transcription regulatory proteins), while the regulatory subunits remain in the cytoplasm.


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Cell_Biology_Alberts. signal molecule activated ˜ subunit of stimulatory G plasma protein (G ) membranes activated, phosphorylated CREB Figure 15–27 How a rise in intracellular cyclic AMP concentration can alter gene transcription. The binding of an extracellular signal molecule to its GPCR activates adenylyl cyclase via Gs and thereby increases cAMP concentration in the cytosol. This rise activates PKA, and the released catalytic subunits of PKA can then enter the nucleus, where they phosphorylate the transcription regulatory protein CREB. Once phosphorylated, CREB recruits the coactivator CBP, which stimulates gene transcription. In some cases, at least, the inactive CREB protein is bound to the cyclic AMP response element (CRE) in DNA before it is phosphorylated (not shown). See Movie 15.2.


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Cell_Biology_Alberts. for example, cAMP activates the gene that encodes this hormone. The regulatory region of the somatostatin gene contains a short cis-regulatory sequence, called the cyclic AMP response element (CRE), which is also found in the regulatory region of many other genes activated by cAMP. A specific transcription regulator called CRE-binding (CREB) protein recognizes this sequence. When PKA is activated by cAMP, it phosphorylates CREB on a single serine; phosphorylated CREB then recruits a transcriptional coactivator called CREB-binding protein (CBP), which stimulates the transcription of the target genes (Figure 15–27). Thus, CREB can transform a short cAMP signal into a long-term change in a cell, a process that, in the brain, is thought to play an important part in some forms of learning and memory.


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Cell_Biology_Alberts. Many GPCRs exert their effects through G proteins that activate the plasma-membrane-bound enzyme phospholipase C-β (PLCβ). Table 15–2 lists some examples of responses activated in this way. The phospholipase acts on a phosphorylated inositol phospholipid (a phosphoinositide) called phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], which is present in small amounts in the inner half of the plasma membrane lipid bilayer (Figure 15–28). Receptors that activate this inositol phospholipid signaling pathway mainly do so via a G protein called Gq, which activates phospholipase C-β in much the same way that Gs activates adenylyl cyclase. The activated phospholipase then cleaves the PI(4,5)P2 to generate two products: inositol 1,4,5-trisphosphate (IP3) and diacylglycerol. At this step, the signaling pathway splits into two branches.


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Cell_Biology_Alberts. IP3 is a water-soluble molecule that leaves the plasma membrane and diffuses rapidly through the cytosol. When it reaches the endoplasmic reticulum (ER), it binds to and opens IP3-gated Ca2+-release channels (also called IP3 receptors) in the ER membrane. Ca2+ stored in the ER is released through the open channels, quickly raising the concentration of Ca2+ in the cytosol (Figure 15–29). The increase in cytosolic Ca2+ propagates the signal by influencing the activity of Ca2+sensitive intracellular proteins, as we describe shortly.


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Cell_Biology_Alberts. At the same time that the IP3 produced by the hydrolysis of PI(4,5)P2 is increasing the concentration of Ca2+ in the cytosol, the other cleavage product of the PI(4,5)P2, diacylglycerol, is exerting different effects. It also acts as a second messenger, but it remains embedded in the plasma membrane, where it has several potential signaling roles. One of its major functions is to activate a protein kinase called protein kinase C (PKC), so named because it is Ca2+-dependent. The initial rise in cytosolic Ca2+ induced by IP3 alters the PKC so that it translocates from the cytosol to the cytoplasmic face of the plasma membrane. There it is activated by the combination of Ca2+, diacylglycerol, and the negatively charged membrane phospholipid phosphatidylserine (see Figure 15–29). Once activated, PKC phosphorylates target proteins that vary depending on the cell type. The principles are the same as discussed earlier for PKA, although most of the target proteins are different.


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Cell_Biology_Alberts. Diacylglycerol can be further cleaved to release arachidonic acid, which can either act as a signal in its own right or be used in the synthesis of other small lipid signal molecules called eicosanoids. Most vertebrate cell types make eicosanoids, including prostaglandins, which have many biological activities. They participate RETICULUMPI 4,5-bisphosphate [PI(4,5)P2] inositol 1,4,5-trisphosphate (IP3) Figure 15–28 The hydrolysis of PI(4,5) P2 by phospholipase C-β. Two second messengers are produced directly from the hydrolysis of PI(4,5)P2: inositol 1,4,5-trisphosphate (IP3), which diffuses through the cytosol and releases Ca2+ from the endoplasmic reticulum, and diacylglycerol, which remains in the membrane and helps to activate protein kinase C (PKC; see Figure 15–29). There are several classes of phospholipase


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Cell_Biology_Alberts. C: these include the β class, which is activated by GPCRs; as we see later, the γ class is activated by a class of enzyme-coupled receptors called receptor tyrosine kinases (RTKs). signal molecule PI 4,5-bisphosphate activated GPCR activated Gqprotein activated phospholipase C-˜diacylglycerol plasma membrane [PI(4,5)P2] activated protein kinase C Ca inositol 1,4,5-trisphosphate (IP3) lumen of endoplasmic reticulum open IP3-gated Ca2+-release channel 2+ GTPP P P P P P ˜°˛ in pain and inflammatory responses, for example, and many anti-inflammatory drugs (such as aspirin, ibuprofen, and cortisone) act in part by inhibiting their synthesis. Ca2+ Functions as a Ubiquitous Intracellular Mediator


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Cell_Biology_Alberts. Ca2+ Functions as a Ubiquitous Intracellular Mediator Many extracellular signals, and not just those that work via G proteins, trigger an increase in cytosolic Ca2+ concentration. In muscle cells, Ca2+ triggers contraction, and in many secretory cells, including nerve cells, it triggers secretion. Ca2+ has numerous other functions in a variety of cell types. Ca2+ is such an effective signaling mediator because its concentration in the cytosol is normally very low (~10–7 M), whereas its concentration in the extracellular fluid (~10–3 M) and in the lumen of the ER [and sarcoplasmic reticulum (SR) in muscle] is high. Thus, there is a large gradient tending to drive Ca2+ into the cytosol across both the plasma membrane and the ER or SR membrane. When a signal transiently opens Ca2+ channels in these membranes, Ca2+ rushes into the cytosol, and the resulting 10–20-fold increase in the local Ca2+ concentration activates Ca2+-responsive proteins in the cell.


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Cell_Biology_Alberts. Some stimuli, including membrane depolarization, membrane stretch, and certain extracellular signals, activate Ca2+ channels in the plasma membrane, resulting in Ca2+ influx from outside the cell. Other signals, including the GPCR-mediated signals described earlier, act primarily through IP3 receptors to stimulate Ca2+ release from intracellular stores in the ER (see Figure 15–29). The ER membrane also contains a second type of regulated Ca2+ channel called the ryanodine receptor (so called because it is sensitive to the plant alkaloid ryanodine), which opens in response to rising Ca2+ levels and thereby amplifies the Ca2+ signal, as we describe shortly.


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Cell_Biology_Alberts. Several mechanisms rapidly terminate the Ca2+ signal and are also responsible for keeping the concentration of Ca2+ in the cytosol low in resting cells. Most importantly, there are Ca2+-pumps in the plasma membrane and the ER membrane that use the energy of ATP hydrolysis to pump Ca2+ out of the cytosol. Cells such as muscle and nerve cells, which make extensive use of Ca2+ signaling, have an additional Ca2+ transporter (a Na+-driven Ca2+ exchanger) in their plasma membrane that couples the efflux of Ca2+ to the influx of Na+. The IP3 receptors and ryanodine receptors of the ER membrane have an important feature: they are both stimulated by low to moderate cytoplasmic Ca2+ concentrations. This Ca2+-induced calcium release (CICR) results in positive feedback,


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Cell_Biology_Alberts. Figure 15–29 How GPCRs increase cytosolic Ca2+ and activate protein kinase C. The activated GPCR stimulates the plasma-membrane-bound phospholipase C-β (PLCβ) via a G protein called Gq. The α subunit and βγ complex of Gq are both involved in this activation. Two second messengers are produced when PI(4,5)P2 is hydrolyzed by activated PLCβ. Inositol 1,4,5-trisphosphate (IP3) diffuses through the cytosol and releases Ca2+ from the ER by binding to and opening IP3-gated Ca2+-release channels (IP3 receptors) in the ER membrane. The large electrochemical gradient for Ca2+ across this membrane causes Ca2+ to escape into the cytosol when the release channels are opened. Diacylglycerol remains in the plasma membrane and, together with phosphatidylserine (not shown) and Ca2+, helps to activate protein kinase C (PKC), which is recruited from the cytosol to the cytosolic face of the plasma membrane. Of the 10 or more distinct isoforms of PKC in humans, at least 4 are activated by diacylglycerol


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Cell_Biology_Alberts. kinase C (PKC), which is recruited from the cytosol to the cytosolic face of the plasma membrane. Of the 10 or more distinct isoforms of PKC in humans, at least 4 are activated by diacylglycerol (Movie 15.3).


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Cell_Biology_Alberts. which has a major impact on the properties of the Ca2+ signal. The importance of this feedback is seen clearly in studies with Ca2+-sensitive fluorescent indicators, such as aequorin or fura-2 (discussed in Chapter 9), which allow researchers to monitor cytosolic Ca2+ in individual cells under a microscope (Figure 15–30 and Movie 15.4).


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Cell_Biology_Alberts. When cells carrying a Ca2+ indicator are treated with a small amount of an extracellular signal molecule that stimulates IP3 production, tiny bursts of Ca2+ are seen in one or more discrete regions of the cell. These Ca2+ puffs or sparks reflect the local opening of small groups of IP3-gated Ca2+-release channels in the ER. Because various Ca2+-binding proteins act as Ca2+ buffers and restrict the diffusion of Ca2+, the signal often remains localized to the site where the Ca2+ enters the cytosol. If the extracellular signal is sufficiently strong and persistent, however, the local Ca2+ concentration can reach a sufficient level to activate nearby IP3 receptors and ryanodine receptors, resulting in a regenerative wave of Ca2+ release that moves through the cytosol (Figure 15–31), much like an action potential in an axon.


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Cell_Biology_Alberts. Figure 15–30 The fertilization of an egg by a sperm triggers a wave of cytosolic Ca2+. This starfish egg was injected with a Ca2+-sensitive fluorescent dye before it was fertilized. A wave of cytosolic Ca2+ (red), released from the ER, sweeps across the egg from the site of sperm entry (arrow). This Ca2+ wave changes the egg cell surface, preventing the entry of other sperm, and it also initiates embryonic development (Movie 15.5). The initial increase in Ca2+ is thought to be caused by a sperm-specific form of PLC (PLCζ) that the sperm brings into the egg cytoplasm when it fuses with the egg; the PLCζ cleaves PI(4,5)P2 to produce IP3, which releases Ca2+ from the egg ER. The released Ca2+ stimulates further Ca2+ release from the ER, producing the spreading wave, as we explain in Figure 15–31. (Courtesy of Stephen A. Stricker.)


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Cell_Biology_Alberts. Figure 15–31 Positive and negative feedback produce Ca2+ waves and oscillations. This diagram shows IP3 receptors and ryanodine receptors on a portion of the ER membrane: active receptors are in green; inactive receptors are in red. When a small amount of cytosolic IP3 activates a cluster of IP3 receptors at one site on the ER membrane (top), the local release of Ca2+ promotes the opening of nearby IP3 and ryanodine receptors, resulting in more Ca2+ release. This positive feedback (indicated by positive signs) produces a regenerative wave of Ca2+ release that spreads across the cell (see Figure 15–30). These waves of Ca2+ release move more quickly across the cell than would be possible by simple diffusion. Also, unlike a diffusing burst of Ca2+ ions, which will become more dilute as it spreads, the regenerative wave produces a high Ca2+ concentration across the entire cell. Eventually, the local Ca2+ concentration inactivates IP3 receptors and ryanodine receptors (middle; indicated by


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Cell_Biology_Alberts. the regenerative wave produces a high Ca2+ concentration across the entire cell. Eventually, the local Ca2+ concentration inactivates IP3 receptors and ryanodine receptors (middle; indicated by red negative signs), shutting down the Ca2+ release. Ca2+-pumps reduce the local cytosolic Ca2+ concentration to its normal low levels. The result is a Ca2+ spike: positive feedback drives a rapid rise in cytosolic Ca2+, and negative feedback sends it back down again. The Ca2+ channels remain refractory to further stimulation for some period of time, delaying the generation of another Ca2+ spike (bottom). Eventually, however, the negative feedback wears off, allowing IP3 to trigger another Ca2+ wave. The end result is repeated Ca2+ oscillations (see Figure 15–32). Under some conditions, these oscillations can be seen as repeating narrow waves of Ca2+ moving across the cell.


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Cell_Biology_Alberts. Another important property of IP3 receptors and ryanodine receptors is that they are inhibited, after some delay, by high Ca2+ concentrations (a form of negative feedback). Thus, the rise in Ca2+ in a stimulated cell leads to inhibition of Ca2+ release; because Ca2+ pumps remove the cytosolic Ca2+, the Ca2+ concentration falls (see Figure 15–31). The decline in Ca2+ eventually relieves the negative feedback, allowing cytosolic Ca2+ to rise again. As in other cases of delayed negative feedback (see Figure 15–18), the result is an oscillation in the Ca2+ concentration. These oscillations persist for as long as receptors are activated at the cell surface, and their frequency reflects the strength of the extracellular stimulus (Figure 15–32). The frequency, amplitude, and breadth of oscillations can also be modulated by other signaling mechanisms, such as phosphorylation, which influence the Ca2+ sensitivity of Ca2+ channels or affect other components in the signaling system.


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Cell_Biology_Alberts. The frequency of Ca2+ oscillations can be translated into a frequency-dependent cell response. In some cases, the frequency-dependent response itself is also oscillatory: in hormone-secreting pituitary cells, for example, stimulation by an extracellular signal induces repeated Ca2+ spikes, each of which is associated with a burst of hormone secretion. In other cases, the frequency-dependent response is non-oscillatory: in some types of cells, for instance, one frequency of Ca2+ spikes activates the transcription of one set of genes, while a higher frequency activates the transcription of a different set. How do cells sense the frequency of Ca2+ spikes and change their response accordingly? The mechanism presumably depends on Ca2+-sensitive proteins that change their activity as a function of Ca2+-spike frequency. A protein kinase that acts as a molecular memory device seems to have this remarkable property, as we discuss next.


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Cell_Biology_Alberts. Ca2+/Calmodulin-Dependent Protein Kinases Mediate Many Responses to Ca2+ Signals Various Ca2+-binding proteins help to relay the cytosolic Ca2+ signal. The most important is calmodulin, which is found in all eukaryotic cells and can constitute as much as 1% of a cell's total protein mass. Calmodulin functions as a multipurpose intracellular Ca2+ receptor, governing many Ca2+-regulated processes. It consists of a highly conserved, single polypeptide chain with four high-affinity Ca2+-binding sites (Figure 15–33A). When activated by Ca2+ binding, it undergoes a conformational change. Because two or more Ca2+ ions must bind before 0.4 nM 0.6 nM 0.9 nM


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Cell_Biology_Alberts. Figure 15–32 Vasopressin-induced Ca2+ oscillations in a liver cell. The cell was loaded with the Ca2+-sensitive protein aequorin and then exposed to increasing concentrations of the peptide signal molecule vasopressin, which activates a GPCR and thereby PLCβ (see Table 15–2). Note that the frequency of the Ca2+ spikes increases with an increasing concentration of vasopressin but that the amplitude of the spikes is not affected. Each spike lasts about 7 seconds. (Adapted from N.M. Woods, K.S.R. Cuthbertson and P.H. Cobbold, Nature 319:600–602, 1986. With permission from Macmillan Publishers Ltd.) peptide portion of target protein calmodulin adopts its active conformation, the protein displays a sigmoidal response to increasing concentrations of Ca2+ (see Figure 15–16).


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Cell_Biology_Alberts. The allosteric activation of calmodulin by Ca2+ is analogous to the activation of PKA by cyclic AMP, except that Ca2+/calmodulin has no enzymatic activity itself but instead acts by binding to and activating other proteins. In some cases, calmodulin serves as a permanent regulatory subunit of an enzyme complex, but usually the binding of Ca2+ instead enables calmodulin to bind to various target proteins in the cell to alter their activity.


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Cell_Biology_Alberts. When an activated molecule of Ca2+/calmodulin binds to its target protein, the calmodulin further changes its conformation, the nature of which depends on the specific target protein (Figure 15–33B). Among the many targets calmodulin regulates are enzymes and membrane transport proteins. As one example, Ca2+/ calmodulin binds to and activates the plasma membrane Ca2+-pump that uses ATP hydrolysis to pump Ca2+ out of cells. Thus, whenever the concentration of Ca2+ in the cytosol rises, the pump is activated, which helps to return the cytosolic Ca2+ level to resting levels. Many effects of Ca2+, however, are more indirect and are mediated by protein phosphorylations catalyzed by a family of protein kinases called Ca2+/calmodulin-dependent kinases (CaM-kinases). Some CaM-kinases phosphorylate transcription regulators, such as the CREB protein (see Figure 15–27), and in this way activate or inhibit the transcription of specific genes.


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Cell_Biology_Alberts. One of the best-studied CaM-kinases is CaM-kinase II, which is found in most animal cells but is especially enriched in the nervous system. It constitutes up to 2% of the total protein mass in some regions of the brain, and it is highly concentrated in synapses. CaM-kinase II has several remarkable properties. To begin with, it has a spectacular quaternary structure: twelve copies of the enzyme are assembled into a stacked pair of rings, with kinase domains on the outside linked to a central hub (Figure 15–34). This structure helps the enzyme function as a molecular memory device, switching to an active state when exposed to Ca2+/ calmodulin and then remaining active even after the Ca2+ signal has decayed. This is because adjacent kinase subunits can phosphorylate each other (a process called autophosphorylation) when Ca2+/calmodulin activates them (Figure 15–34). Once a kinase subunit is autophosphorylated, it remains active even in the absence of Ca2+, thereby prolonging the


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Cell_Biology_Alberts. called autophosphorylation) when Ca2+/calmodulin activates them (Figure 15–34). Once a kinase subunit is autophosphorylated, it remains active even in the absence of Ca2+, thereby prolonging the duration of the kinase activity beyond that of the initial activating Ca2+ signal. The enzyme maintains this activity until a protein phosphatase removes the autophosphorylation and shuts the kinase off. CaM-kinase II activation can thereby serve as a memory trace of a prior Ca2+ pulse, and it seems to have a role in some types of memory and learning in the vertebrate nervous system. Mutant mice that lack a brain-specific form of the enzyme have specific defects in their ability to remember where things are.


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Cell_Biology_Alberts. Figure 15–33 The structure of Ca2+/calmodulin. (A) The molecule has a dumbbell shape, with two globular ends, which can bind to many target proteins. The globular ends are connected by a long, exposed α helix, which allows the protein to adopt a number of different conformations, depending on the target protein it interacts with. Each globular head has two Ca2+binding sites (Movie 15.6). (B) Shown is the major structural change that occurs in Ca2+/calmodulin when it binds to a target protein (in this example, a peptide that consists of the Ca2+/calmodulin-binding domain of a Ca2+/calmodulin-dependent protein kinase). Note that the Ca2+/calmodulin has “jack-knifed” to surround the peptide. When it binds to other targets, it can adopt different conformations. (A, based on x-ray crystallographic data from Y.S. Babu et al., Nature 315:37–40, 1985. With permission from Macmillan Publishers Ltd; B, based on x-ray crystallographic data from W.E. Meador, A.R. Means, and


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Cell_Biology_Alberts. W.E. Meador, A.R. Means, and F.A. Quiocho, Science 257:1251–1255, 1992, and on nuclear magnetic resonance (NMR) spectroscopy data from M. Ikura et al., Science 256:632–638, 1992.)


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Cell_Biology_Alberts. Figure 15–34 The stepwise activation of CaM-kinase II. (A) Each CaM-kinase II protein has two major domains: an amino-terminal kinase domain (green) and a carboxyl-terminal hub domain (blue), linked by a regulatory segment. Six CaM-kinase II proteins are assembled into a giant ring in which the hub domains interact tightly to produce a central structure that is surrounded by kinase domains. The complete enzyme contains two stacked rings, for a total of 12 kinase proteins, but only one ring is shown here for clarity. When the enzyme is inactive, the ring exists in a dynamic equilibrium between two states. The first (upper left) is a compact state, in which the kinase domains interact with the hub, so that the regulatory segment is buried in the kinase active site and thereby blocks catalytic activity. In the second inactive state (upper middle), a kinase domain has popped out and is linked to the central hub by its regulatory segment, which continues to inhibit the kinase but is now


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Cell_Biology_Alberts. activity. In the second inactive state (upper middle), a kinase domain has popped out and is linked to the central hub by its regulatory segment, which continues to inhibit the kinase but is now accessible to Ca2+/calmodulin. If present, Ca2+/calmodulin will bind the regulatory segment and prevent it from inhibiting the kinase, thereby locking the kinase in an active state (upper right). If the adjacent kinase subunit also pops out from the hub, it will also be activated by Ca2+/calmodulin, and the two kinases will then phosphorylate each other on their regulatory segments (lower right). This autophosphorylation further activates the enzyme. It also prolongs the activity of the enzyme in two ways. First, it traps the bound Ca2+/calmodulin so that it does not dissociate from the enzyme until cytosolic Ca2+ levels return to basal values for at least 10 seconds (not shown). Second, it converts the enzyme to a Ca2+-independent form, so that the kinase remains active even after the


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Cell_Biology_Alberts. until cytosolic Ca2+ levels return to basal values for at least 10 seconds (not shown). Second, it converts the enzyme to a Ca2+-independent form, so that the kinase remains active even after the Ca2+/calmodulin dissociates from it (lower left). This activity continues until the action of a protein phosphatase overrides the autophosphorylation activity of CaM-kinase II. (B) This structural model of the enzyme is based on x-ray crystallographic analysis.


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Cell_Biology_Alberts. The remarkable dodecameric structure of the enzyme allows it to achieve a broad range of intermediate activity states in response to different Ca2+ oscillation frequencies: higher frequencies tend to cause more subunits in the enzyme to reach the phosphorylated active state (see Figure 15–35). The behavior of CaM-kinase II is also controlled by the length of the linker segment between the kinase and hub domains. The linker is longer in some isoforms of the enzyme; in these isoforms, the kinase domains tend to pop out of the ring more frequently, making it more sensitive to Ca2+. These and other mechanisms allow the cell to tailor the responsiveness of the enzyme to the needs of different types of neurons. (Adapted from L.H. Chao et al., Cell 146:732–745, 2011. PDB code: 3SOA.)


Cell_Biology_Alberts

Cell_Biology_Alberts. Another remarkable property of CaM-kinase II is that the enzyme can use its intrinsic memory mechanism to decode the frequency of Ca2+ oscillations. This property is thought to be especially important at a nerve cell synapse, where changes in intracellular Ca2+ levels in a postsynaptic cell as a result of neural activity can lead to long-term changes in the subsequent effectiveness of that synapse (discussed in Chapter 11). When CaM-kinase II is exposed to both a protein phosphatase and repetitive pulses of Ca2+/calmodulin at different frequencies that mimic those observed in stimulated cells, the enzyme’s activity increases steeply as a function of pulse frequency (Figure 15–35).


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Cell_Biology_Alberts. G proteins do not act exclusively by regulating the activity of membrane-bound enzymes that alter the concentration of cyclic AMP or Ca2+ in the cytosol. The α subunit of one type of G protein (called G12), for example, activates a guanine nucleotide exchange factor (GEF) that activates a monomeric GTPase of the Rho family (discussed later and in Chapter 16), which regulates the actin cytoskeleton.


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Cell_Biology_Alberts. In some other cases, G proteins directly activate or inactivate ion channels in the plasma membrane of the target cell, thereby altering the ion permeability— and hence the electrical excitability—of the membrane. As an example, acetylcholine released by the vagus nerve reduces the heart rate (see Figure 15–5B). This effect is mediated by a special class of acetylcholine receptors that activate the Gi protein discussed earlier. Once activated, the αsubunit of Gi inhibits adenylyl cyclase (as described previously), while the βγ subunits bind to K+ channels in the heart muscle cell plasma membrane and open them. The opening of these K+ channels makes it harder to depolarize the cell and thereby contributes to the inhibitory effect of acetylcholine on the heart. (These acetylcholine receptors, which can be activated by the fungal alkaloid muscarine, are called muscarinic acetylcholine receptors to distinguish them from the very different nicotinic acetylcholine receptors, which are


Cell_Biology_Alberts

Cell_Biology_Alberts. which can be activated by the fungal alkaloid muscarine, are called muscarinic acetylcholine receptors to distinguish them from the very different nicotinic acetylcholine receptors, which are ion-channel-coupled receptors on skeletal muscle and nerve cells that can be activated by the binding of nicotine, as well as by acetylcholine.)


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Cell_Biology_Alberts. Other G proteins regulate the activity of ion channels less directly, either by stimulating channel phosphorylation (by PKA, PKC, or CaM-kinase, for example) or by causing the production or destruction of cyclic nucleotides that directly activate or inactivate ion channels. These cyclic-nucleotide-gated ion channels have a crucial role in both smell (olfaction) and vision, as we now discuss. Humans can distinguish more than 10,000 distinct smells, which they detect using specialized olfactory receptor neurons in the lining of the nose. These cells use specific GPCRs called olfactory receptors to recognize odors; the receptors are displayed on the surface of the modified cilia that extend from each cell (Figure 15–36). The receptors act through cAMP. When stimulated by odorant binding, Figure 15–35 CaM-kinase II as a frequency decoder of Ca2+ oscillations.


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Cell_Biology_Alberts. (A) At low frequencies of Ca2+ spikes, the enzyme becomes inactive after each spike, as the autophosphorylation induced by Ca2+/calmodulin binding does not maintain the enzyme’s activity long enough for the enzyme to remain active until the next Ca2+ spike arrives. (B) At higher spike frequencies, however, the enzyme fails to inactivate completely between Ca2+ spikes, so its activity ratchets up with each spike. If the spike frequency is high enough, this progressive increase in enzyme activity will continue until the enzyme is autophosphorylated on all subunits and is therefore maximally activated. Although not shown, once enough of its subunits are autophosphorylated, the enzyme can be maintained in a highly active state even with a relatively low frequency of Ca2+ spikes (a form of cell memory). The binding of Ca2+/ calmodulin to the enzyme is enhanced by the CaM-kinase II autophosphorylation (an additional form of positive feedback), helping to generate a more switchlike response


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Cell_Biology_Alberts. memory). The binding of Ca2+/ calmodulin to the enzyme is enhanced by the CaM-kinase II autophosphorylation (an additional form of positive feedback), helping to generate a more switchlike response to repeated Ca2+ spikes. (From


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Cell_Biology_Alberts. P.I. Hanson, T. Meyer, L. Stryer, and H. Schulman, Neuron 12:943–956, 1994. With permission from Elsevier.) they activate an olfactory-specific G protein (known as Golf), which in turn activates adenylyl cyclase. The resulting increase in cAMP opens cyclic-AMP-gated cation channels, thereby allowing an influx of Na+, which depolarizes the olfactory receptor neuron and initiates a nerve impulse that travels along its axon to the brain.


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Cell_Biology_Alberts. There are about 1000 different olfactory receptors in a mouse and about 350 in a human, each encoded by a different gene and each recognizing a different set of odorants. Each olfactory receptor neuron produces only one of these receptors; the neuron responds to a specific set of odorants by means of the specific receptor it displays, and each odorant activates its own characteristic set of olfactory receptor neurons. The same receptor also helps direct the elongating axon of each developing olfactory neuron to the specific target neurons that it will connect to in the brain. A different set of GPCRs acts in a similar way in some vertebrates to mediate responses to pheromones, chemical signals detected in a different part of the nose that are used in communication between members of the same species. Humans, however, are thought to lack functional pheromone receptors.


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Cell_Biology_Alberts. Vertebrate vision employs a similarly elaborate, highly sensitive, signal-detection process. Cyclic-nucleotide-gated ion channels are also involved, but the crucial cyclic nucleotide is cyclic GMP (Figure 15–37) rather than cAMP. As with cAMP, a continuous rapid synthesis (by guanylyl cyclase) and rapid degradation (by cyclic GMP phosphodiesterase) controls the concentration of cyclic GMP in the cytosol.


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Cell_Biology_Alberts. In visual transduction responses, which are the fastest G-protein-mediated responses known in vertebrates, the receptor activation stimulated by light causes a fall rather than a rise in the level of the cyclic nucleotide. The pathway has been especially well studied in rod photoreceptors (rods) in the vertebrate retina. Rods are responsible for noncolor vision in dim light, whereas cone photoreceptors (cones) are responsible for color vision in bright light. A rod photoreceptor is a highly specialized cell with outer and inner segments, a cell body, and a synaptic region where the rod passes a chemical signal to a retinal nerve cell (Figure 15–38). This nerve cell relays the signal to another nerve cell in the retina, which in turn relays it to the brain.


Cell_Biology_Alberts

Cell_Biology_Alberts. The phototransduction apparatus is in the outer segment of the rod, which contains a stack of discs, each formed by a closed sac of membrane that is densely packed with photosensitive rhodopsin molecules. The plasma membrane surrounding the outer segment contains cyclic-GMP-gated cation channels. Cyclic GMP bound to these channels keeps them open in the dark. Paradoxically, light causes a hyperpolarization (which inhibits synaptic signaling) rather than a depolarization of the plasma membrane (which would stimulate synaptic signaling). Hyperpolarization (that is, the membrane potential moves to a more negative value—discussed in Chapter 11) results because the light-induced activation of rhodopsin molecules in the disc membrane decreases the cyclic GMP concentration and closes the cation channels in the surrounding plasma membrane (Figure 15–39).


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Cell_Biology_Alberts. Figure 15–36 Olfactory receptor neurons. (A) A section of olfactory epithelium in the nose. Olfactory receptor neurons possess modified cilia, which project from the surface of the epithelium and contain the olfactory receptors, as well as the signal transduction machinery. The axon, which extends from the opposite end of the receptor neuron, conveys electrical signals to the brain when an odorant activates the cell to produce an action potential. In rodents, at least, the basal cells act as stem cells, producing new receptor neurons throughout life, to replace the neurons that die. (B) A scanning electron micrograph of the cilia on the surface of an olfactory neuron. (B, from E.E. Morrison and R.M. Costanzo, J. Comp. Neurol. 297:1–13, 1990. With permission from Wiley-Liss.) H2NNNNHNCH2OOOHOO_OPGUANINEPHOSPHATESUGAROFigure 15–37 Cyclic GMP.


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Cell_Biology_Alberts. E.E. Morrison and R.M. Costanzo, J. Comp. Neurol. 297:1–13, 1990. With permission from Wiley-Liss.) H2NNNNHNCH2OOOHOO_OPGUANINEPHOSPHATESUGAROFigure 15–37 Cyclic GMP. Figure 15–38 A rod photoreceptor cell. There are about 1000 discs in the outer segment. The disc membranes are not connected to the plasma membrane. The inner and outer segments are specialized parts of a primary cilium (discussed in Chapter 16). A primary cilium extends from the surface of most vertebrate cells, where it serves as a signaling organelle.


Cell_Biology_Alberts

Cell_Biology_Alberts. Rhodopsin is a member of the GPCR family, but the activating extracellular signal is not a molecule but a photon of light. Each rhodopsin molecule contains a covalently attached chromophore, 11-cis retinal, which isomerizes almost instantaneously to all-trans retinal when it absorbs a single photon. The isomerization alters the shape of the retinal, forcing a conformational change in the protein (opsin). The activated rhodopsin molecule then alters the conformation of the G protein transducin (Gt), causing the transducin α subunit to activate cyclic GMP phosphodiesterase. The phosphodiesterase then hydrolyzes cyclic GMP, so that cyclic GMP levels in the cytosol fall. This drop in cyclic GMP concentration decreases the amount of cyclic GMP bound to the plasma membrane cation channels, allowing more of these cyclic-GMP-sensitive channels to close. In this way, the signal quickly passes from the disc membrane to the plasma membrane, and a light signal is converted into an electrical one,


Cell_Biology_Alberts

Cell_Biology_Alberts. more of these cyclic-GMP-sensitive channels to close. In this way, the signal quickly passes from the disc membrane to the plasma membrane, and a light signal is converted into an electrical one, through a hyperpolarization of the rod cell plasma membrane.


Cell_Biology_Alberts

Cell_Biology_Alberts. Rods use several negative feedback loops to allow the cells to revert quickly to a resting, dark state in the aftermath of a flash of light—a requirement for perceiving the shortness of the flash. A rhodopsin-specific protein kinase called rhodopsin kinase (RK) phosphorylates the cytosolic tail of activated rhodopsin on multiple serines, partially inhibiting the ability of the rhodopsin to activate transducin. An inhibitory protein called arrestin (discussed later) then binds to the phosphorylated rhodopsin, further inhibiting rhodopsin’s activity. Mice or humans with a mutation that inactivates the gene encoding RK have a prolonged light response.


Cell_Biology_Alberts

Cell_Biology_Alberts. At the same time as arrestin shuts off rhodopsin, an RGS protein (discussed earlier) binds to activated transducin, stimulating the transducin to hydrolyze its bound GTP to GDP, which returns transducin to its inactive state. In addition, the cation channels that close in response to light are permeable to Ca2+, as well as high rate of low rate of (signals to discs of photoreceptive outer


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Cell_Biology_Alberts. Figure 15–39 The response of a rod photoreceptor cell to light. Rhodopsin molecules in the outer-segment discs absorb photons. Photon absorption closes cation channels in the plasma membrane, which hyperpolarizes the membrane and reduces the rate of neurotransmitter release from the synaptic region. Because the neurotransmitter inhibits many of the postsynaptic retinal neurons, illumination serves to free the neurons from inhibition and thus, in effect, excites them. The neural connections of the retina lie between the light source and the outer segment, and so the light must pass through the synapses and rod cell nucleus to reach the light sensors.


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Cell_Biology_Alberts. to Na+, so that when they close, the normal influx of Ca2+ is inhibited, causing the Ca2+ concentration in the cytosol to fall. The decrease in Ca2+ concentration stimulates guanylyl cyclase to replenish the cyclic GMP, rapidly returning its level to where it was before the light was switched on. A specific Ca2+-sensitive protein mediates the activation of guanylyl cyclase in response to the fall in Ca2+ levels. In contrast to calmodulin, this protein is inactive when Ca2+ is bound to it and active when it is Ca2+-free. It therefore stimulates the cyclase when Ca2+ levels fall following a light response.


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Cell_Biology_Alberts. Negative feedback mechanisms do more than just return the rod to its resting state after a transient light flash; they also help the rod to adapt, stepping down the response when the rod is exposed to light continuously. Adaptation, as we discussed earlier, allows the receptor cell to function as a sensitive detector of changes in stimulus intensity over an enormously wide range of baseline levels of stimulation. It is why we can see faint stars in a dark sky, or a camera flash in bright sunlight. The various trimeric G proteins we have discussed in this chapter fall into four major families, as summarized in Table 15–3.


Cell_Biology_Alberts

Cell_Biology_Alberts. The various trimeric G proteins we have discussed in this chapter fall into four major families, as summarized in Table 15–3. Signaling molecules like cyclic nucleotides and calcium are hydrophilic small molecules that generally act within the cell where they are produced. Some signaling molecules, however, are hydrophobic enough, small enough, or both, to pass readily across the plasma membrane and carry signals to nearby cells. An important and remarkable example is the gas nitric oxide (NO), which acts as a signal molecule in many tissues of both animals and plants.


Cell_Biology_Alberts

Cell_Biology_Alberts. In mammals, one of NO’s many functions is to relax smooth muscle in the walls of blood vessels. The neurotransmitter acetylcholine stimulates NO synthesis by activating a GPCR on the membranes of the endothelial cells that line the interior of the vessel. The activated receptor triggers IP3 synthesis and Ca2+ release (see Figure 15–29), leading to stimulation of an enzyme that synthesizes NO. Because dissolved NO passes readily across membranes, it diffuses out of the cell where it is produced and into neighboring smooth muscle cells, where it causes muscle relaxation and thereby vessel dilation (Figure 15–40). It acts only locally because it has a short half-life—about 5–10 seconds—in the extracellular space before oxygen and water convert it to nitrates and nitrites.


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Cell_Biology_Alberts. The effect of NO on blood vessels provides an explanation for the mechanism of action of nitroglycerine, which has been used for about 100 years to treat patients with angina (pain resulting from inadequate blood flow to the heart muscle). The nitroglycerine is converted to NO, which relaxes blood vessels. This reduces the workload on the heart and, as a consequence, reduces the oxygen requirement of the heart muscle. NO is made by the deamination of the amino acid arginine, catalyzed by enzymes called NO synthases (NOS) (see Figure 15–40). The NOS in endothelial cells is called eNOS, while that in nerve and muscle cells is called nNOS. Both eNOS and nNOS are stimulated by Ca2+. Macrophages, by contrast, make yet another NOS, called inducible NOS (iNOS), that is constitutively active but synthesized only when the cells are activated, usually in response to an infection.


Cell_Biology_Alberts

Cell_Biology_Alberts. In some target cells, including smooth muscle cells, NO binds reversibly to iron in the active site of guanylyl cyclase, stimulating synthesis of cyclic GMP. NO can increase cyclic GMP in the cytosol within seconds, because the normal rate of turnover of cyclic GMP is high: rapid degradation to GMP by a phosphodiesterase constantly balances the production of cyclic GMP by guanylyl cyclase. The drug Viagra® and its relatives inhibit the cyclic GMP phosphodiesterase in the penis, thereby increasing the amount of time that cyclic GMP levels remain elevated in the smooth muscle cells of penile blood vessels after NO production is induced by local nerve terminals. The cyclic GMP, in turn, keeps the blood vessels relaxed and thereby the penis erect. NO can also signal cells independently of cyclic GMP. It can, for example, alter the activity of an intracellular protein by covalently nitrosylating thiol (–SH) groups on specific cysteines in the protein.


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Cell_Biology_Alberts. Figure 15–40 The role of nitric oxide (NO) in smooth muscle relaxation in a blood vessel wall. (A) Simplified cross section of a blood vessel, showing the endothelial cells lining the lumen and the smooth muscle cells around them. (B) The neurotransmitter acetylcholine stimulates blood vessel dilation by activating a GPCR—the muscarinic acetylcholine receptor—on the surface of endothelial cells. This receptor activates a G protein, Gq, thereby stimulating IP3 synthesis and Ca2+ release by the mechanisms illustrated in Figure 15–29. Ca2+ activates nitric oxide synthase, causing the endothelial cells to produce NO from arginine. The NO diffuses out of the endothelial cells and into the neighboring smooth muscle cells, where it activates guanylyl cyclase to produce cyclic GMP. The cyclic GMP triggers a response that causes the smooth muscle cells to relax, increasing blood flow through the vessel.


Cell_Biology_Alberts

Cell_Biology_Alberts. Despite the differences in molecular details, the different intracellular signaling pathways that GPCRs trigger share certain features and obey similar general principles. They depend on relay chains of intracellular signaling proteins and second messengers. These relay chains provide numerous opportunities for amplifying the responses to extracellular signals. In the visual transduction cascade, for example, a single activated rhodopsin molecule catalyzes the activation of hundreds of molecules of transducin at a rate of about 1000 transducin molecules per second. Each activated transducin molecule activates a molecule of cyclic GMP phosphodiesterase, each of which hydrolyzes about 4000 molecules of cyclic GMP per second. This catalytic cascade lasts for about 1 second and results in the hydrolysis of more than 105 cyclic GMP molecules for a single quantum of light absorbed, and the resulting drop in the concentration of cyclic GMP in turn transiently closes hundreds of cation


Cell_Biology_Alberts

Cell_Biology_Alberts. in the hydrolysis of more than 105 cyclic GMP molecules for a single quantum of light absorbed, and the resulting drop in the concentration of cyclic GMP in turn transiently closes hundreds of cation channels in the plasma membrane (Figure 15–41). As a result, a rod cell can respond to even a single photon of light in a way that is highly reproducible in its timing and magnitude.


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Cell_Biology_Alberts. Likewise, when an extracellular signal molecule binds to a receptor that indirectly activates adenylyl cyclase via Gs, each receptor protein may activate many molecules of Gs protein, each of which can activate a cyclase molecule. Each cyclase molecule, in turn, can catalyze the conversion of a large number of ATP molecules to cAMP molecules. A similar amplification operates in the IP3 signaling pathway. In these ways, a nanomolar (10–9 M) change in the concentration of an extracellular signal can induce micromolar (10–6 M) changes in the concentration of a second messenger such as cAMP or Ca2+. Because these messengers function as allosteric effectors to activate specific enzymes or ion channels, a single extracellular signal molecule can alter many thousands of protein molecules within the target cell.


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Cell_Biology_Alberts. Any such amplifying cascade of stimulatory signals requires counterbalancing mechanisms at every step of the cascade to restore the system to its resting state when stimulation ceases. As emphasized earlier, the response to stimulation can be rapid only if the inactivating mechanisms are also rapid. Cells therefore have efficient mechanisms for rapidly degrading (and resynthesizing) cyclic nucleotides and for buffering and removing cytosolic Ca2+, as well as for inactivating the responding enzymes and ion channels once they have been activated. This is not only essential for turning a response off, but is also important for defining the resting state from which a response begins. Each protein in the signaling relay chain can be a separate target for regulation, including the receptor itself, as we discuss next.


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Cell_Biology_Alberts. Each protein in the signaling relay chain can be a separate target for regulation, including the receptor itself, as we discuss next. As discussed earlier, when target cells are exposed to a high concentration of a stimulating ligand for a prolonged period, they can become desensitized, or adapted, in several different ways. An important class of adaptation mechanisms depends on alteration of the quantity or condition of the receptor molecules themselves. For GPCRs, there are three general modes of adaptation (see Figure 15–20): (1) In receptor sequestration, they are temporarily moved to the interior of the cell (internalized) so that they no longer have access to their ligand. (2) In receptor down-regulation, they are destroyed in lysosomes after internalization. (3) In receptor inactivation, they become altered so that they can no longer interact with G proteins.