G-protein-linked receptors form the largest family of cell-surface receptors and are found in all eucaryotes. About 5% of the genes in the nematode C. elegans, for example, encode such receptors, and thousands have already been defined in mammals; in mice, there are about 1000 concerned with the sense of smell alone. G-protein-linked receptors mediate the responses to an enormous diversity of signal molecules, including hormones, neurotransmitters, and local mediators. These signal molecules that activate them are as varied in structure as they are in function: the list includes proteins and small peptides, as well as derivatives of amino acids and fatty acids. The same ligand can activate many different receptor family members; at least 9 distinct G-protein-linked receptors are activated by adrenaline, for example, another 5 or more by acetylcholine, and at least 15 by the neurotransmitter serotonin.
Despite the chemical and functional diversity of the signal molecules that bind to them, all G-protein-linked receptors have a similar structure. They consist of a single polypeptide chain that threads back and forth across the lipid bilayerseven times and are therefore sometimes called serpentine receptors (Figure 15-26). In addition to their characteristic orientation in the plasma membrane, they have the same functional relationship to the G proteins they use to signal the cell interior that an extracellular ligand is present.
A G-protein-linked receptor. Receptors that bind protein ligands have a large extracellular domain formed by the part of the polypeptide chain shown inlight green. This domain, together with some of the transmembrane segments, binds the protein ligand. (more...)
As we discuss later, this superfamily of seven-pass transmembrane proteins includes rhodopsin, the light-activatedprotein 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. In fact, it is thought that the G-protein-linked receptors that mediate cell-cell signaling in multicellular organisms evolved from sensory receptors that were possessed by their unicellular eucaryotic ancestors.
It is remarkable that about half of all known drugs work through G-protein-linked receptors. Genome sequencing projects are revealing vast numbers of new family members, many of which are likely targets for new drugs that remain to be discovered.
When extracellular signaling molecules bind to serpentine receptors, the receptors undergo a conformational change that enables them to activate trimeric GTP-binding proteins (G proteins). These G proteins are attached to the cytoplasmic face of the plasma membrane, where they serve as relay molecules, functionally coupling the receptors to enzymes or ion channels in this membrane. There are various types of G proteins, each specific for a particular set of serpentine receptors and for a particular set of downstream target proteins in the plasma membrane. All have a similar structure, however, and they operate in a similar way.
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 15-27). When stimulated by an activated receptor, the α subunit releases its bound GDP, allowing GTP to bind in its place. This exchange causes the trimer to dissociate into two activated components--an α subunit and a βγ complex (Figure 15-28).
The structure of an inactive G protein. (A) Note that both the α and the γ subunits have covalently attached lipid molecules (red) that help to bind them to the plasma membrane, and the α subunit has GDP bound. (B) The three-dimensional (more...)
The disassembly of an activated G-protein into two signaling components. (A) In the unstimulated state, the receptor and the G protein are both inactive. Although they are shown here as separate entities in the plasma membrane, in some cases, at least, (more...)
The dissociation of the trimeric G protein activates its two components in different ways. GTP binding causes a conformational change that affects the surface of the α subunit that associates with the βγ complex in the trimer. This change causes the release of the βγ complex, but it also causes and the α subunit to adopt a new shape that allows it to interact with its target proteins. The βγ complex does not change its conformation, but the surface previously masked by the α subunit is now available to interact with a second set of target proteins. The targets of the dissociated components of the G protein are either enzymes or ion channels in the plasma membrane, and they relay the signal onward.
The α subunit is a GTPase, and once it hydrolyzes its bound GTP to GDP, it reassociates with a βγ complex to re-form an inactive G protein, reversing the activation process (Figure 15-29). The time during which the α subunit and βγ complex remain apart and active is usually short, and it depends on how quickly the α subunit hydrolyzes its bound GTP. An isolated α subunit is an inefficient GTPase, and, left to its own devices, the subunit would inactivate only after several minutes. Its activation is usually reversed much faster than this, however, because the GTPase activity of the α subunit is greatly enhanced by the binding of a second protein, which can be either its target protein or a specific modulator known as a regulator of G protein signaling (RGS). RGS proteins act as α-subunit-specific GTPase activating proteins (GAPs), and they are thought to have a crucial role in shutting off G-protein-mediated responses in all eucaryotes. There are about 25 RGS proteins encoded in the human genome, each of which is thought to interact with a particular set of G proteins.
The switching off of the G-protein α subunit by the hydrolysis of its bound GTP. After a G-protein α subunit activates its target protein, it shuts itself off by hydrolyzing its bound GTP to GDP. This inactivates the α subunit,(more...)
The importance of the GTPase activity in shutting off the response can be easily demonstrated in a test tube. If cells are broken open and exposed to an analogue of GTP (GTPγS) in which the terminal phosphate cannot be hydrolyzed, the activated α subunits remain active for a very long time.
Cyclic AMP (cAMP) was first identified as a small intracellular mediator in the 1950s. It has since been found to act in this role in all procaryotic and animal cells that have been studied. The normal concentration of cyclic AMP inside the cell is about 10-7 M, but an extracellular signal can cause cyclic AMP levels to change by more than twentyfold in seconds (Figure 15-30). As explained earlier (see Figure 15-10), such a rapid response requires that a rapid synthesis of the molecule be balanced by its rapid breakdown or removal. In fact, cyclic AMP is synthesized from ATP by a plasma-membrane-bound enzyme adenylyl cyclase, and it is rapidly and continuously destroyed by one or morecyclic AMP phosphodiesterases that hydrolyze cyclic AMP to adenosine 5′-monophosphate (5′-AMP) (Figure 15-31).
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 G-protein-linked receptor to cause a rapid rise in the intracellular concentration of cyclic (more...)
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 (
--); a pyrophosphatase (more...)
Many extracellular signal molecules work by increasing cyclic AMP content, and they do so by increasing the activity of adenylyl cyclase rather than decreasing the activity of phosphodiesterase. Adenylyl cyclase is a large multipass transmembrane 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+. All receptors that act via cyclic AMP are coupled to a stimulatory G protein (Gs), which activates adenylyl cyclase and thereby increases cyclic AMP concentration. Another G protein, called inhibitory G protein (Gi), inhibits adenylyl cyclase, but it mainly acts by directly regulating ion channels (as we discuss later) rather than by decreasing cyclic AMP content. Although it is usually the α subunit that regulates the cyclase, the βγ complex sometimes does so as well, either increasing or decreasing the enzyme's activity, depending on the particular βγ complex and the isoform of the cyclase.
Both Gs and Gi are targets for some 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 cyclic AMP levels 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 subunit from interacting with receptors; as a result, this α subunit retains its bound GDP and is unable to regulate its target proteins. These two toxins are widely used as tools to determine whether a cell's response to a signal is mediated by Gs or by Gi.
Some of the responses mediated by a Gs-stimulated increase in cyclic AMP concentration are listed in Table 15-1. It is clear that different cell types respond differently to an increase in cyclic AMP concentration, and that any one cell type usually responds in the same way, even if different extracellular signals induce the increase. At least four hormones activate adenylyl cyclase in fat cells, for example, and all of them stimulate the breakdown of triglyceride (the storage form of fat) to fatty acids.
Some Hormone-induced Cell Responses Mediated by Cyclic AMP.
Individuals who are genetically deficient in a particular Gs α subunit show decreased responses to certain hormones. As a consequence, they display metabolic abnormalities, have abnormal bone development, and are mentally retarded.
Although cyclic AMP can directly activate certain types of ion channels in the plasma membrane of some highly specialized cells, in most animal cells it exerts its effects mainly by activating cyclic-AMP-dependent protein kinase (PKA). This enzyme catalyzes the transfer of the terminal phosphate group from ATP to specific serines or threonines of selected target proteins, thereby regulating their activity.
PKA is found in all animal cells and is thought to account for the effects of cyclic AMP in most of these cells. The substrates for PKA differ in different cell types, which explains why the effects of cyclic AMP vary so markedly depending on the cell type.
In the inactive state, PKA consists of a complex of two catalytic subunits and two regulatory subunits. The binding of cyclic AMP to the regulatory subunits alters their conformation, causing them to dissociate from the complex. The released catalytic subunits are thereby activated to phosphorylate specific substrate protein molecules (Figure 15-32). The regulatory subunits of PKA also are important for localizing the kinase inside the cell: special PKA anchoring proteins bind both to the regulatory subunits and to a membrane or a component of the cytoskeleton, thereby tethering the enzyme complex to a particular subcellular compartment. Some of these anchoring proteins also bind other kinases and some phosphatases, creating a signaling complex.
The activation of cyclic-AMP-dependent protein kinase (PKA). The binding of cyclic AMP to the regulatory subunits induces a conformational change, causing these subunits to dissociate from the catalytic subunits, thereby activating the kinase activity (more...)
Some responses mediated by cyclic AMP are rapid while others are slow. In skeletal muscle cells, for example, activated PKA phosphorylates enzymes involved in glycogen metabolism, which simultaneously triggers the breakdown of glycogen to glucose and inhibits glycogen synthesis, thereby increasing the amount of glucose available to the muscle cell within seconds (see also Figure 15-30). At the other extreme are responses that take hours to develop fully and involve changes in the transcription of specific genes. In cells that secrete the peptide hormonesomatostatin, for example, cyclic AMP activates the gene that encodes this hormone. The regulatory region of the somatostatin gene contains a short DNA sequence, called the cyclic AMP response element (CRE), that is also found in the regulatory region of many other genes activated by cyclic AMP. A specific gene regulatory protein calledCRE-binding (CREB) protein recognizes this sequence. When CREB is phosphorylated by PKA on a single serine, it recruits a transcriptional coactivator called CREB-binding protein (CBP), which stimulates the transcription of these genes (Figure 15-33). If this serine is mutated, CREB cannot recruit CBP, and it no longer stimulates gene transcription in response to a rise in cyclic AMP levels.
How gene transcription is activated by a rise in cyclic AMP concentration. The binding of an extracellular signal molecule to its G-protein-linked receptor leads to the activation of adenylyl cyclase and a rise in cyclic AMP concentration. The increase (more...)
Since the effects of cyclic AMP are usually transient, cells must be able to dephosphorylate the proteins that have been phosphorylated by PKA. Indeed, the activity of any protein regulated by phosphorylation depends on the balance at any instant between the activities of the kinases that phosphorylate it and the phosphatases that are constantly dephosphorylating it. In general, the dephosphorylation of phosphorylated serines and threonines is catalyzed by four types of serine/threonine phosphoprotein phosphatases--protein phosphatases I, IIA, IIB, and IIC. Except forprotein phosphatase-IIC (which is a minor phosphatase, unrelated to the others), all of these phosphatases are composed of a homologous catalytic subunit complexed with one or more of a large set of regulatory subunits; the regulatory subunits help to control the phosphatase activity and enable the enzyme to select specific targets. Protein phosphatase I is responsible for dephosphorylating many of the proteins phosphorylated by PKA. It inactivates CREB, for example, by removing its activating phosphate, thereby turning off the transcriptional response caused by a rise in cyclic AMP concentration. Protein phosphatase IIA has a broad specificity and seems to be the main phosphatase responsible for reversing many of the phosphorylations catalyzed by serine/threonine kinases. Protein phosphatase IIB, also called calcineurin, is activated by Ca2+ and is especially abundant in the brain.
Having discussed how trimeric G proteins link activated receptors to adenylyl cyclase, we now consider how they couple activated receptors to another crucial enzyme, phospholipase C. The activation of this enzyme leads to an increase in the concentration of Ca2+ in the cytosol, which helps to relay the signal onward. Ca2+ is even more widely used as an intracellular mediator than is cyclic AMP.
Many G-protein-linked receptors exert their effects mainly via G proteins that activate the plasma-membrane-boundenzyme phospholipase C-β. Several examples of responses activated in this way are listed in Table 15-2. The phospholipase acts on an 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 15-34). Receptors that operate through this inositol phospholipid signaling pathway mainly activate a G protein called Gq, which in turn activates phospholipase C-β, in much the same way that Gs activates adenylyl cyclase. The activated phospholipase cleaves PI(4,5)P2 to generate two products: inositol 1,4,5-trisphosphate and diacylglycerol (Figure 15-35). At this step, the signaling pathway splits into two branches.
Some Cell Responses in Which G-Protein-linked Receptors Activate the Inositol-Phospholipid Signaling Pathway.
Three types of inositol phospholipids (phosphoinositides). The polyphosphoinositides--PI(4)P and PI(4,5)P2--are produced by the phosphorylation of phosphatidylinositol (PI) and PI(4)P, respectively. Although all three inositol phospholipids (more...)
The hydrolysis of PI(4,5)P2 by phospholipase C-β. Two intracellular mediators are produced when PI(4,5)P2 is hydrolyzed: inositol 1,4,5-trisphosphate (IP3), which diffuses through the cytosol and releases Ca2+ from the ER, and diacylglycerol, (more...)
Inositol 1,4,5-trisphosphate (IP3) is a small, 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 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 15-36). We discuss later how Ca2+ acts to propagate the signal. Several mechanisms operate to terminate the initial Ca2+ response: (1) IP3 is rapidly dephosphorylated by specific phosphatases to form IP2; (2) IP3 is phosphorylated to IP4 (which may function as another intracellular mediator); and (3) Ca2+ that enters the cytosol is rapidly pumped out, mainly to the exterior of the cell.
The two branches of the inositol phospholipid pathway. The activated receptor stimulates the plasma-membrane-bound enzyme phospholipase C-β via a G protein. Depending on the isoform of the enzyme, it may be activated by the α subunit of (more...)
At the same time that the IP3 produced by the hydrolysis of PI(4,5)P2 is increasing the concentration of Ca2+ in thecytosol, the other cleavage product of PI(4,5)P2--diacylglycerol--is exerting different effects. Diacylglycerol remains embedded in the membrane, where it has two potential signaling roles. First, it can be further cleaved to release arachidonic acid, which can either act as a messenger in its own right or be used in the synthesis of other smalllipid messengers called eicosanoids. Eicosanoids, such as the prostaglandins, are made by most vertebrate cell types and have a wide variety of biological activities. They participate in pain and inflammatory responses, for example, and most anti-inflammatory drugs (such as aspirin, ibuprofen, and cortisone) act--in part, at least--by inhibiting their synthesis.
The second, and more important, function of diacylglycerol is to activate a crucial serine/threonine protein kinasecalled 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 phospholipidphosphatidylserine (see Figure 15-36). 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.
Each of the two branches of the inositol phospholipid signaling pathway can be mimicked by the addition of specific pharmacological agents to intact cells. The effects of IP3 can be mimicked by using a Ca 2+ ionophore, such as A23187 or ionomycin, which allows Ca2+ to move into the cytosol from the extracellular fluid (discussed in Chapter 11). The effects of diacylglycerol can be mimicked by phorbol esters, plant products that bind to PKC and activate it directly. Using these reagents, it has been shown that the two branches of the pathway often collaborate in producing a full cellular response. Some cell types, such as lymphocytes, for example, can be stimulated to proliferate in culture when treated with both a Ca2+ ionophore and a PKC activator, but not when they are treated with either reagent alone.
Many extracellular signals induce an increase in cytosolic Ca2+ level, not just those that work via G proteins. In eggcells, for example, a sudden rise in cytosolic Ca2+ concentration upon fertilization by a sperm triggers a Ca2+ wave that is responsible for the onset of embryonic development (Figure 15-37). In muscle cells, Ca2+ triggers contraction, and in many secretory cells, including nerve cells, it triggers secretion. Ca2+ can be used as a signal in this way because its concentration in the cytosol is normally kept very low (~10-7 M), whereas its concentration in the extracellular fluid (~10-3 M) and in the ER lumen is high. Thus, there is a large gradient tending to drive Ca2+ into the cytosol across both the plasma membrane and the ER membrane. When a signal transiently opens Ca2+ channels in either of these membranes, Ca2+ rushes into the cytosol, increasing the local Ca2+ concentration by 10-20-fold and triggering Ca2+-responsive proteins in the cell.
Fertilization of an egg by a sperm triggering an increase in 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 endoplasmic reticulum, is seen (more...)
Three main types of Ca2+ channels can mediate this Ca2+ signaling:
Voltage-dependent Ca2+ channels in the plasma membrane open in response to membrane depolarization and allow, for example, Ca2+ to enter activated nerve terminals and trigger neurotransmitter secretion.
IP3-gated Ca2+-release channels allow Ca2+ to escape from the ER when the inositol phospholipid signaling pathway is activated, as just discussed (see Figure 15-36).
Ryanodine receptors (so called because they are sensitive to the plant alkaloid ryanodine) react to a change inplasma membrane potential to release Ca2+ from the sarcoplasmic reticulum and thereby stimulate the contraction of muscle cells; they are also present in the ER of many nonmuscle cells, including neurons, where they can contribute to Ca2+ signaling.
The concentration of Ca2+ in the cytosol is kept low in resting cells by several mechanisms (Figure 15-38). Most notably, all eucaryotic cells have a Ca2+-pump in their plasma membrane that uses 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+ transport protein (exchanger) in their plasma membrane that couples the efflux of Ca2+ to the influx of Na+. A Ca2+ pump in the ER membrane also has an important role in keeping the cytosolic Ca2+concentration low: this Ca2+-pump enables the ER to take up large amounts of Ca2+ from the cytosol against a steep concentration gradient, even when Ca2+ levels in the cytosol are low. In addition, a low-affinity, high-capacity Ca2+pump in the inner mitochondrial membrane has an important role in returning the Ca2+ concentration to normal after a Ca2+ signal; it uses the electrochemical gradient generated across this membrane during the electron-transfer steps ofoxidative phosphorylation to take up Ca2+ from the cytosol.
The main ways eucaryotic cells maintain a very low concentration of free Ca2+in their cytosol. (A) Ca2+ is actively pumped out of the cytosol to the cell exterior. (B) Ca2+ is pumped into the ER and mitochondria, and various molecules in the cell bind (more...)
Ca2+-sensitive fluorescent indicators, such as aequorin or fura-2 (discussed in Chapter 9), are often used to monitor cytosolic Ca2+ in individual cells after the inositol phospholipid signaling pathway has been activated. When viewed in this way, the initial Ca2+ signal is often seen to be small and localized to one or more discrete regions of the cell. These signals have been called Ca2+ blips, quarks, puffs, or sparks, and they are thought to reflect the local opening of individual (or small groups of) Ca2+-release channels in the ER and to represent elementary Ca2+ signaling units. If the extracellular signal is sufficiently strong and persistent, this localized signal can propagate as a regenerative Ca2+wave through the cytosol, much like an action potential in an axon (see Figure 15-37). Such a Ca2+ "spike” is often followed by a series of further spikes, each usually lasting seconds (Figure 15-39). These Ca2+ oscillations can persist for as long as receptors are activated at the cell surface. Both the waves and the oscillations are thought to depend, in part at least, on a combination of positive and negative feedback by Ca2+ on both the IP3-gated Ca2+-release channels and the ryanodine receptors: the released Ca2+ initially stimulates more Ca2+ release, a process known as Ca2+-induced Ca2+ release. But then, as its concentration gets high enough, the Ca2+ inhibits further release.
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 vasopressin. Note that the frequency of the Ca2+ spikes increases with an increasing concentration (more...)
The frequency of the Ca2+ oscillations reflects the strength of the extracellular stimulus (see Figure 15-39), and it 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. The frequency-dependent response can also be nonoscillatory. 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.
Ca 2+ -binding proteins serve as transducers of the cytosolic Ca2+ signal. The first such protein to be discovered wastroponin C in skeletal muscle cells; its role in muscle contraction is discussed in Chapter 16. A closely related Ca2+-binding protein, known as calmodulin, is found in all eucaryotic cells, where it can constitute as much as 1% of the total protein mass. Calmodulin functions as a multipurpose intracellular Ca2+ receptor, mediating many Ca2+-regulated processes. It consists of a highly conserved, single polypeptide chain with four high-affinity Ca2+-binding sites (Figure 15-40A). When activated by binding Ca2+, it undergoes a conformational change. Because two or more Ca2+ ions must bind before calmodulin adopts its active conformation, the protein responds in a switchlike manner to increasing concentrations of Ca2+ (see Figure 15-22): a tenfold increase in Ca2+ concentration, for example, typically causes a fiftyfold increase in calmodulin activation.
The structure of Ca2+/calmodulin based on x-ray diffraction and NMR studies. (A) The molecule has a "dumbbell” shape, with two globular ends connected by a long, exposed α helix. Each end has two Ca2+-binding domains, each with (more...)
The allosteric activation of calmodulin by Ca2+ is analogous to the allosteric activation of PKA by cyclic AMP, except that Ca2+/calmodulin has no enzymic activity itself but instead acts by binding to other proteins. In some cases, calmodulin serves as a permanent regulatory subunit of an enzyme complex, but mostly the binding of Ca2+enables calmodulin to bind to various target proteins in the cell to alter their activity.
When an activated molecule of Ca2+/calmodulin binds to its target protein, it undergoes a marked change inconformation (Figure 15-40B). Among the targets regulated by calmodulin binding are many enzymes and membrane transport proteins. As one example, Ca2+/calmodulin binds to and activates the plasma membrane Ca2+-pump that pumps 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 normal.
Many effects of Ca2+, however, are more indirect and are mediated by protein phosphorylations catalyzed by a family of Ca 2+ /calmodulin-dependent protein kinases (CaM-kinases). These kinases, just like PKA and PKC, phosphorylate serines or threonines in proteins, and, as with PKA and PKC, the response of a target cell depends on which CaM-kinase-regulated target proteins are present in the cell. The first CaM-kinases to be discovered--myosin light-chain kinase, which activates smooth muscle contraction, and phosphorylase kinase, which activates glycogenbreakdown--have narrow substrate specificities. A number of CaM-kinases, however, have much broader specificities, and these seem to be responsible for mediating many of the actions of Ca2+ in animal cells. Some phosphorylate gene regulatory proteins, such as the CREB protein discussed earlier, and in this way activate or inhibit the transcription of specific genes.
The best-studied example of such a multifunctional CaM-kinase is CaM-kinase II, which is found in all 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 at least two remarkable properties that are related. First, it can 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 the kinase phosphorylates itself (a process called autophosphorylation) as well as other cell proteins when it is activated by Ca2+/calmodulin. In its autophosphorylated state, the enzyme remains active even in the absence of Ca2+, thereby prolonging the duration of the kinase activity beyond that of the initial activating Ca2+ signal. This activity is maintained until phosphatases overwhelm the autophosphorylating activity of the enzyme and shut it off (Figure 15-41). CaM-kinase II activation can thereby serve as a memory trace of a prior Ca2+ pulse, and it seems to have an important role in some types of memory and learning in the vertebrate nervous system. Mutant mice that lack the brain-specific subunit illustrated in Figure 15-41 have specific defects in their ability to remember where things are in space. A point mutation in CaM-kinase II that removes its autophosphorylation site, but otherwise leaves the kinase activity intact, produces the same learning defect, revealing that the autophosphorylation is critical in these animals.
The activation of CaM-kinase II. The enzyme is a large protein complex of about 12 subunits, although, for simplicity, only one subunit is shown. The subunits are of four homologous kinds (α, β, γ, and σ), which are expressed(more...)
The second remarkable property of CaM-kinase II is that it can use its memory mechanism to act as a frequency decoder of Ca2+ oscillations. This property is thought to be especially important at a nerve cell synapse, where changes in intracellular Ca2+ levels in an activated postsynaptic cell can lead to long-term changes in the subsequent effectiveness of that synapse (discussed in Chapter 11). When CaM-kinase II is immobilized on a solid surface and 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 15-42). Moreover, the frequency response of this multisubunit enzyme depends on its exact subunit composition, so that a cell can tailor its response to Ca2+ oscillations to particular needs by adjusting the composition of the CaM-kinase II enzyme that it makes.
CaM-kinase II as a frequency decoder of Ca2+oscillations. (A) At low frequencies of Ca2+ spikes (gray bars), the enzyme becomes inactive after each spike, as the autophosphorylation induced by Ca2+/calmodulin binding does not maintain the enzyme's activity (more...)
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 protein that converts a monomeric GTPase of the Rho family (discussed in Chapter 16) into its active form, which then alters the actin cytoskeleton. In some other cases, G proteins directly activate or inactivate ion channels in theplasma membrane of the target cell, thereby altering the ion permeability--and hence the excitability of the membrane. Acetylcholine released by the vagus nerve, for example, reduces both the rate and strength of heart muscle cell contraction (see Figure 15-9A). A special class of acetylcholine receptors that activate the Gi protein discussed earlier mediates this effect. Once activated, the α subunit of Gi inhibits adenylyl cyclase (as described previously), while the βγ complex binds to K+ channels in the heart muscle cell plasma membrane to open them. The opening of these K+ channels makes it harder to depolarize the cell, which contributes to the inhibitory effect of acetylcholine on the heart. (These acetylcholine receptors, which can be activated by the fungal alkaloid muscarine, are calledmuscarinic acetylcholine receptors to distinguish them from the very different nicotinic acetylcholine receptors,which are ion-channel-linked receptors on skeletal muscle and nerve cells that can be activated by the binding of nicotine, as well as by acetylcholine.)
Other trimeric G proteins regulate the activity of ion channels less directly, either by stimulating channelphosphorylation (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. The 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 are detected by specialized olfactory receptorneurons in the lining of the nose. These cells recognize odors by means of specific G-protein-linked olfactory receptors, which are displayed on the surface of the modified cilia that extend from each cell (Figure 15-43). The receptors act through cyclic AMP. When stimulated by odorant binding, they activate an olfactory-specific G protein (known as Golf), which in turn activates adenylyl cyclase. The resulting increase in cyclic AMP 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.
Olfactory receptor neurons. (A) This drawing shows 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 (more...)
There are about 1000 different olfactory receptors in a mouse, each encoded by a different gene and each recognizing a different set of odorants. All of these receptors belong to the G-protein-linked receptor superfamily. Each olfactory receptor neuron produces only one of these 1000 receptors, and the neuron responds to a specific set of odorants by means of the specific receptor it displays. The same receptor also has a crucial role in directing 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 more than 100 G-protein-linked receptors acts in a similar way to mediate a mouse's responses to pheromones,chemical signals detected in a different part of the nose that are used in communication between members of the same species.
Vertebrate vision involves a similarly elaborate, highly sensitive, signal-detection process. Cyclic-nucleotide-gatedion channels are also involved, but the crucial cyclic nucleotide is cyclic GMP (Figure 15-44) rather than cyclic AMP. As with cyclic AMP, a continuous rapid synthesis (by guanylyl cyclase) and rapid degradation (by cyclic GMPphosphodiesterase) controls the concentration of cyclic GMP in cells.
Cyclic GMP.
In visual transduction responses, which are the fastest G-protein-mediated responses known in vertebrates, thereceptor activation caused by light leads to 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; this nerve cell in turn relays the signal along the visual pathway (Figure 15-45). The phototransduction apparatus is in the outer segment, which contains a stack of discs, each formed by a closed sac of membrane in which many photosensitive rhodopsin molecules are embedded. Theplasma membrane surrounding the outer segment contains cyclic-GMP-gated Na + channels. These channels are kept open in the dark by cyclic GMP that has bound to them. Paradoxically, light causes a hyperpolarization (which inhibits synaptic signaling) rather than a depolarization of the plasma membrane (which could stimulate synaptic signaling). Hyperpolarization (an increase in the membrane potential--discussed in Chapter 11) results because the activation by light of rhodopsin molecules in the disc membrane leads to a fall in cyclic GMP concentration and theclosure of the special Na+ channels in the surrounding plasma membrane (Figure 15-46).
A rod photoreceptor cell. There are about 1000 discs in the outer segment. The disc membranes are not connected to the plasma membrane.
The response of a rod photoreceptor cell to light. Rhodopsin molecules in the outer-segment discs absorb photons. Photon absorption leads to the closure of Na+ channels in the plasma membrane, which hyperpolarizes the membrane and reduces the rate of (more...)
Rhodopsin is a seven-pass transmembrane molecule homologous to other members of the G-protein-linked receptorfamily, and, like its cousins, it acts through a trimeric G protein. The activating extracellular signal, however, 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 G-protein transducin (Gt), causing its α subunit to dissociate and 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 leads to a decrease in the amount of cyclic GMP bound to the plasma membrane Na+ channels, allowing more of these highly 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.
A number of mechanisms operate in rods 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 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 then binds to the phosphorylated rhodopsin, further inhibiting rhodopsin's activity. If the gene encoding RK is inactivated by mutation in mice or humans, the light response of rods is greatly prolonged, and the rods eventually die.
At the same time as rhodopsin is being shut off, an RGS protein (see p. 854) binds to activated transducin, stimulating the transducin to hydrolyze its bound GTP to GDP, which returns transducin to its inactive state. In addition, the Na+channels that close in response to light are also permeable to Ca2+, 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 a 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.
These shut-off mechanisms do more than just return the rod to its resting state after a light flash; they also help to enable the photoreceptor to adapt, stepping down the response when it 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.
The various trimeric G proteins we have discussed in this chapter are summarized in Table 15-3.
Three Major Families of Trimeric G Proteins*.
Despite the differences in molecular details, the signaling systems that are triggered by G-protein-linked receptors share certain features and are governed by similar general principles. They depend on relay chains of intracellular signaling proteins and small intracellular mediators. In contrast to the more direct signaling pathways used by nuclear receptors discussed earlier, and by ion-channel-linked receptors discussed in Chapter 11, these relay chains provide numerous opportunities for amplifying the responses to extracellular signals. In the visual transduction cascade just described, 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 Na2+ channels in the plasma membrane (Figure 15-47). As a result, a rod cell can respond to a single photon of light, in a way that is highly reproducible in its timing and magnitude.
Amplification in the light-induced catalytic cascade in vertebrate rods. The divergent arrows indicate the steps where amplification occurs.
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 cyclic AMP molecules. A similar amplification operates in the inositol-phospholipid pathway. A nanomolar (10-9 M) change in the concentration of an extracellular signal can thereby induce micromolar (10-6 M) changes in the concentration of asmall intracellular mediator such as cyclic AMP or Ca2+. Because these mediators function as allosteric effectors to activate specific enzymes or ion channels, a single extracellular signal molecule can cause many thousands of protein molecules to be altered within the target cell.
Any such amplifying cascade of stimulatory signals requires that there be counterbalancing mechanisms at every step of the cascade to restore the system to its resting state when stimulation ceases. 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, it is also important for defining the resting state from which a response begins. As we saw earlier, in general, the response to stimulation can be rapid only if the inactivating mechanisms are also rapid. Each protein in the relay chain of signals can be a separate target for regulation, including the receptor, as we discuss next.
As discussed earlier, target cells use a variety of mechanisms to desensitize, or adapt, when they are exposed to a high concentration of stimulating ligand for a prolonged period (see Figure 15-25). We discuss here only those mechanisms that involve an alteration in G-protein-linked receptors themselves.
These receptors can desensitize in three general ways:
They can become altered so that they can no longer interact with G proteins (receptor inactivation).
They can be temporarily moved to the interior of the cell (internalized) so that they no longer have access to theirligand (receptor sequestration).
They can be destroyed in lysosomes after internalization (receptor down-regulation).
In each case, the desensitization process depends on phosphorylation of the receptor, by PKA, PKC, or a member of the family of G-protein-linked receptor kinases (GRKs). (The GRKs include the rhodopsin-specific kinase involved in rod photoreceptor desensitization discussed earlier.) The GRKs phosphorylate multiple serine and threonines on a receptor, but they do so only after the receptor has been activated by ligand binding. As with rhodopsin, once a receptor has been phosphorylated in this way, it binds with high affinity to a member of thearrestin family of proteins (Figure 15-48).
The roles of G-protein-linked receptor kinases (GRKs) and arrestins in receptor desensitization. The binding of an arrestin to the phosphorylated receptor prevents the receptor from binding to its G protein and can direct its endocytosis. Mice that are (more...)
The bound arrestin can contribute to the desensitization process in at least two ways. First, it inactivates the receptorby preventing it from interacting with G proteins, an example of receptor uncoupling. Second, it can serve as anadaptor protein to couple the receptor to clathrin-coated pits (discussed in Chapter 13), inducing receptor-mediated endocytosis. Endocytosis results in either the sequestration or degradation (down-regulation) of the receptor, depending on the specific receptor and cell type, the concentration of the stimulating ligand, and the duration of the ligand's presence.
G-protein-linked receptors can indirectly activate or inactivate either plasma-membrane-bound enzymes or ionchannels via G proteins. When stimulated by an activated receptor, a G protein disassembles into an α subunit and a βγ complex, both of which can directly regulate the activity of target proteins in the plasma membrane. Some G-protein-linked receptors either activate or inactivate adenylyl cyclase, thereby altering the intracellular concentration of the intracellular mediator cyclic AMP. Others activate a phosphoinositide-specific phospholipase C (phospholipase C-β), which hydrolyzes phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] to generate two small intracellular mediators. One is inositol 1,4,5-trisphosphate (IP3), which releases Ca2+from the ER and thereby increases the concentration of Ca2+in the cytosol. The other is diacylglycerol, which remains in the plasma membrane and activatesprotein kinase C (PKC). A rise in cyclic AMP or Ca2+levels affects cells mainly by stimulating protein kinase A (PKA) and Ca2+/calmodulin-dependent protein kinases (CaM-kinases), respectively.
PKC, PKA, and CaM-kinases phosphorylate specific target proteins on serines or threonines and thereby alter the activity of the proteins. Each type of cell has characteristic sets of target proteins that are regulated in these ways, enabling the cell to make its own distinctive response to the small intracellular mediators. The intracellular signaling cascades activated by G-protein-linked receptors allow the responses to be greatly amplified, so that many target proteins are changed for each molecule of extracellular signaling ligand bound to its receptor.
The responses mediated by G-protein-linked receptors are rapidly turned off when the extracellular signaling ligand is removed. Thus, the G-protein α subunit is induced to inactivate itself by hydrolyzing its bound GTP to GDP, IP3is rapidly dephosphorylated by a phosphatase (or phosphorylated by a kinase), cyclic nucleotides are hydrolyzed by phosphodiesterases, Ca2+is rapidly pumped out of the cytosol, and phosphorylated proteins are dephosphorylated by protein phosphatases. Activated G-protein-linked receptors themselves are phosphorylated by GRKs, thereby trigging arrestin binding, which uncouples the receptors from G proteins and promotes receptor endocytosis.
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