Introduction

G-protein-linked receptors are the largest family of cell-surface receptors. More than 100 members have already been defined in mammals. Many of these have been identified by homology cloning, in which low stringency hybridization with existing cDNA probes is used to detect related DNA sequences (see Figure7-17). Other family members have been found by expression cloning, using their ligand-binding or cell-activation properties to identify them. In one form of this approach, a library of cDNA molecules prepared from cells or tissues that express the receptor are copied into RNA molecules, which are then injected into Xenopus oocytes. The oocytes translate the RNA molecules into proteins. These proteins are inserted into the plasma membrane, where their ligand-binding or cell-activation properties allow them to be detected.

G-protein-linked receptors mediate the cellular responses to an enormous diversity of signaling molecules, including hormones, neurotransmitters, and local mediators, which are as varied in structure as they are in function: the list includes proteins and small peptides, as well as amino acid and fatty acid derivatives. The same ligand can activate many different 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 serotonin.

Despite the chemical and functional diversity of the signaling molecules that bind to them, all of the G-protein-linked receptors whose amino acid sequences are known from DNA sequencing studies have a similar structure and are almost certainly evolutionarily related. They consist of a single polypeptide chain that threads back and forth across the lipid bilayer seven times ( Figure 15-17). As we discuss later, this superfamily of seven-pass transmembrane receptor proteins includes rhodopsin, the light-activated protein in the vertebrate eye, as well as olfactory receptors in the vertebrate nose. Other family members are found in unicellular organisms: the receptors in yeasts that recognize the yeast mating factors are an example. This ancient structural motif is also shared by bacteriorhodopsin, a bacterial light-activated H⁺ pump discussed in Chapter 10, although, unlike the other family members, bacteriorhodopsin is not a receptor and does not act via a G protein. Taken together, these findings suggest that the G-protein-linked receptors that mediate cell-cell signaling in multicellular organisms may have evolved from sensory receptors possessed by their unicellular ancestors. The members of this receptor family have conserved not only their amino acid sequence but also their functional relationship to G proteins by means of which they broadcast into the interior of the cell the message that an extracellular ligand is present. It is the intracellular sequence of events beginning with the activation of G proteins that mainly concern us in this section.

Figure 15-17

A schematic drawing of a G-protein-linked receptor. Receptors that bind protein ligands have a large extracellular ligand-binding domain formed by the part of the polypeptide chain shown in light green. Receptors for small ligands such as adrenaline have small (more...)

Trimeric G Proteins Relay the Intracellular Signal from G-Protein-linked Receptors ^{11, 12}

The trimeric GTP-binding proteins (G proteins) that functionally couple these receptors to their target enzymes or ion channels in the plasma membrane are structurally distinct from the single-chain GTP-binding proteins (called monomeric GTP-binding proteins or monomeric GTPases) that help relay intracellular signals and regulate vesicular traffic and many other processes in eucaryotic cells. The monomeric GTPases are discussed later in this chapter as well as in other chapters. Both classes of GTP-binding proteins, however, are GTPases and function as molecular switches that can flip between two states: active, when GTP is bound, and inactive, when GDP is bound. "Active" in this context usually means that the molecule acts as a signal to trigger other events in the cell. When an extracellular ligand binds to a G-protein-linked receptor, the receptor changes its conformation and switches on the trimeric G proteins that associate with it by causing them to eject their GDP and replace it with GTP. The switch is turned off when the G protein hydrolyzes its own bound GTP, converting it back to GDP. But before that occurs, the active protein has an opportunity to diffuse away from the receptor and deliver its message for a prolonged period to its downstream target.

Most G-protein-linked receptors activate a chain of events that alters the concentration of one or more small intracellular signaling molecules. These small molecules, often referred to as intracellular mediators (also called intracellular messengers or second messengers), in turn pass the signal on by altering the behavior of selected cellular proteins. Two of the most widely used intracellular mediators are cyclic AMP (cAMP) and Ca²⁺: changes in their concentrations are stimulated by distinct pathways in most animal cells, and most G-protein-linked receptors regulate one or the other of them, as outlined in Figure 15-18.

Figure 15-18

Two major pathways by which G-protein-linked cell-surface receptors generate small intracellular mediators. In both cases the binding of an extracellular ligand alters the conformation of the cytoplasmic domain of the receptor, causing it to bind to a (more...)

Some Receptors Increase Intracellular Cyclic AMP by Activating Adenylyl Cyclase via a Stimulatory G Protein (G_s) ¹³

Cyclic AMP ( Figure 15-19) was first identified as an intracellular mediator of hormone action in 1959 and has since been found to act as an intracellular signaling molecule in all procaryotic and animal cells that have been studied. For cyclic AMP to function as an intracellular mediator, its intracellular concentration (normally ≤ 10^-7 M) must be able to change up or down in response to extracellular signals: upon hormonal stimulation, cyclic AMP levels can change fivefold in seconds. As explained earlier (see Figure 15-10), such responsiveness requires that rapid synthesis of the molecule be balanced by rapid breakdown or removal. Cyclic AMP is synthesized from ATP by a plasma-membrane-bound enzyme adenylyl cyclase, and it is rapidly and continuously destroyed by one or more cyclic AMP phosphodiesterases, which hydrolyze cyclic AMP to adenosine 5'-monophosphate (5'-AMP) ( Figure 15-20).

Figure 15-19

Cyclic AMP. It is shown as a formula, a ball-and-stick model, and as a space-filling model. (C, H, N, O, and P indicate carbon, hydrogen, nitrogen, oxygen, and phosphorus atoms, respectively.)

Figure 15-20

The synthesis and degradation of cyclic AMP (cAMP). A pyrophosphatase makes the synthesis of cyclic AMP an irreversible reaction by hydrolyzing the released pyrophosphate - (not shown).

Many extracellular signaling molecules work by controlling cyclic AMP levels, and they do so by altering the activity of adenylyl cyclase ( Figure 15-21) rather than the activity of phosphodiesterase. Just as the same steroid hormone produces different effects in different target cells, so different target cells respond very differently to external signals that change intracellular cyclic AMP levels ( Table 15-1). All ligands that activate adenylyl cyclase in a given type of target cell, however, usually produce the same effect: 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 (see Table 15-1). The different receptors for these hormones activate a common pool of adenylyl cyclase molecules, to which they are coupled by a trimeric G protein. Because this G protein is involved in enzyme activation, it is called stimulatory G protein (G_s). Individuals who are genetically deficient in G_s show decreased responses to certain hormones and, consequently, have metabolic abnormalities, abnormal bone development, and are mentally retarded.

Figure 15-21

Adenylyl cyclase. In vertebrates the enzyme usually contains about 1100 amino acid residues and is thought to have two clusters of six transmembrane segments separating two similar cytoplasmic catalytic domains. There are at least six types of this form (more...)

Table 15-1

Some Hormone-induced Cellular Responses Mediated by Cyclic AMP.

The best-studied examples of receptors coupled to the activation of adenylyl cyclase are the β-adrenergic receptors, which mediate some of the actions of adrenaline and noradrenaline ( Figure 15-22, and see Table 15-1). An adrenaline-activated adenylyl cyclase system can be reconstituted in synthetic phospholipid vesicles using purified β-adrenergic receptors, G_s, and adenylyl cyclase molecules, indicating that no other proteins are required for the activation process. But precisely how does G_s mediate the coupling? The answer depends on the trimeric structure of the G protein, as we now discuss.

Figure 15-22

Adrenaline. This hormone (also called epinephrine) is made from tyrosine and is secreted by the adrenal gland when a mammal is stressed.

Trimeric G Proteins Are Thought to Disassemble When Activated ^{11, 12, 14}

A trimeric G protein is composed of three different polypeptide chains, called α, β, and γ. The G_s α chain(α_s) binds and hydrolyzes GTP and activates adenylyl cyclase. The G_s β chain and γ chain form a tight complex (βγ), which anchors G_s to the cytoplasmic face of the plasma membrane, at least partly by a lipid chain (a prenyl group) that is covalently attached to the γ subunit. In its inactive form G_s exists as a trimer with GDP bound to α_s. When stimulated by binding to a ligand-activated receptor, α_s exchanges its GDP for GTP. This is thought to cause α_s to dissociate from βγ, allowing α_sto bind instead to an adenylyl cyclase molecule, which it activates to produce cyclic AMP.

If cells are to be able to respond rapidly to changes in the concentration of an extracellular signaling molecule, the activation of adenylyl cyclase must be reversed quickly once the signaling ligand dissociates from its receptor. This ability to respond rapidly to change is assured because the lifetime of the active form of α_s is short: the GTPase activity of α_s is stimulated when α_s binds to adenylyl cyclase, so that the bound GTP is hydrolyzed to GDP, rendering both α_s and the adenylyl cyclase inactive. The α_s then reassociates with βγ to re-form an inactive G_s molecule ( Figure 15-23).

Figure 15-23

A current model of how G_s couples receptor activation to adenylyl cyclase activation. As long as the extracellular signaling ligand remains bound, the receptor protein can continue to activate molecules of G_s protein, thereby amplifying the response. More (more...)

The importance of the GTPase activity of α_s in shutting off the response can be readily 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, cyclic AMP production after hormone treatment is greatly prolonged. A similar phenomenon is seen in patients suffering from cholera, where the bacterial toxin responsible for the symptoms of the disease inhibits the self-inactivating mechanism of α_s. Cholera toxin is an enzyme that catalyzes the transfer of ADP ribose from intracellular NAD⁺ to α_s. The ADP ribosylation alters the α_s so that it can no longer hydrolyze its bound GTP. An adenylyl cyclase molecule activated by such an altered α_s subunit thus remains in the active state indefinitely. The resulting prolonged elevation in cyclic AMP levels within intestinal epithelial cells causes a large efflux of Na⁺ and water into the gut, which is responsible for the severe diarrhea that is characteristic of cholera.

Some Receptors Decrease Cyclic AMP by Inhibiting Adenylyl Cyclase via an Inhibitory Trimeric G Protein (G_i) ^{11, 12, 15}

The same signaling molecule can either increase or decrease the intracellular concentration of cyclic AMP depending on the type of receptor to which it binds. When adrenaline binds to β-adrenergic receptors, for example, it activates adenylyl cyclase, whereas when it binds to α₂-adrenergic receptors, it inhibits the enzyme. The difference reflects the type of G proteins that couple these receptors to the cyclase. While the β-adrenergic receptors are functionally coupled to adenylyl cyclase by G_s, the α₂-adrenergic receptors are coupled to this enzyme by an inhibitory G protein (G_i). G_i can contain the same β-γ complex as G_s, but it has a different α subunit (α_i). When activated, α₂-adrenergic receptors bind to G_i, causing α_i to bind GTP and dissociate from the βγ complex. Both the released α_i and βγ are thought to contribute to the inhibition of adenylyl cyclase. α_i inhibits the cyclase, probably indirectly, whereas βγ may inhibit cyclic AMP synthesis in two ways - directly, by binding to the cyclase itself, and indirectly, by binding to any free α_s subunits in the same cell, thereby preventing them from activating cyclase molecules. We see later that G_i also acts to open K⁺ channels in the plasma membrane, and it seems likely that this function is more important than the inhibition of adenylyl cyclase.

Whereas cholera toxin catalyzes the ADP ribosylation of α_s and thereby inactivates the GTPase activity of α_s, pertussis toxin, made by the bacterium that causes pertussis (whooping cough), catalyzes the ADP ribosylation of α_i. The ADP ribosylation of α_i prevents the G_i complex from interacting with receptors, and so the complex remains bound to GDP and is unable to inhibit adenylyl cyclase or open K⁺ channels.

The trimeric G proteins are remarkably versatile intracellular signaling molecules. In the examples considered so far, either the α subunit or both the α and the β-γ subunits are the active components. But in other cases receptors are coupled to their target proteins only by the released β-γ complex. Moreover, β-γ complexes can also act as conditional regulators of effector proteins: they can enhance the activation of some forms of adenylyl cyclase, for example, but only if the cyclase has already been activated by α_s.

Cyclic-AMP-dependent Protein Kinase (A-Kinase) Mediates the Effects of Cyclic AMP ¹⁶

Cyclic AMP exerts its effects in animal cells mainly by activating the enzyme cyclic-AMP-dependent protein kinase (A-kinase), which catalyzes the transfer of the terminal phosphate group from ATP to specific serines or threonines of selected proteins. The amino acids phosphorylated by A-kinase are marked by the presence of two or more basic amino acids on their amino-terminal side. Covalent phosphorylation of the appropriate amino acids in turn regulates the activity of the target protein.

A-kinase is found in all animal cells and is thought to account for all of the effects of cyclic AMP in most of these cells. (The only other known function of cyclic AMP in animals is to regulate a special class of ion channels in smell-responsive olfactory neurons, as we discuss later.) The substrates for A-kinase differ in different cell types, explaining why the effects of cyclic AMP vary depending on the target cell.

In the inactive state A-kinase consists of a complex of two catalytic subunits and two regulatory subunits that bind cyclic AMP. The binding of cyclic AMP alters the conformation of the regulatory subunits, causing them to dissociate from the complex. The released catalytic subunits are thereby activated to phosphorylate specific substrate protein molecules ( Figure 15-24).

Figure 15-24

The activation of cyclic-AMP-dependent protein kinase (A-kinase). The binding of cyclic AMP to the regulatory subunits induces a conformational change, causing these subunits to dissociate from the complex, thereby activating the catalytic subunits. Each (more...)

Cyclic-AMP-mediated protein phosphorylation was first demonstrated in studies of glycogen metabolism in skeletal muscle cells. Glycogen is the major storage form of glucose, and both its synthesis and degradation in skeletal muscle cells are regulated by adrenaline. When an animal is frightened or otherwise stressed, for example, the adrenal gland secretes adrenaline into the blood, "alerting" various tissues in the body. Among other effects, the circulating adrenaline induces muscle cells to break down glycogen to glucose 1-phosphate and at the same time to stop synthesizing glycogen. The glucose is then oxidized by glycolysis to provide ATP for sustained muscle contraction. In this way adrenaline prepares the muscle cells for anticipated strenuous activity. Adrenaline acts by binding to β-adrenergic receptors on the muscle cell surface, thereby causing an increase in the level of cyclic AMP in the cytosol. The cyclic AMP activates A-kinase, which phosphorylates two other enzymes. The first, phosphorylase kinase, which was the first protein kinase to be discovered (in 1956), phos-phorylates in turn the enzyme glycogen phosphorylase, thereby activating the phosphorylase to release glucose residues from the glycogen molecule ( Figure 15-25). The second enzyme phosphorylated by activated A-kinase is glycogen synthase, which performs the final step in glycogen synthesis from glucose. This phosphorylation inhibits the enzyme's activity, thereby shutting off glycogen synthesis. By means of this cascade of interactions, an increase in cyclic AMP levels both stimulates glycogen breakdown and inhibits glycogen synthesis, thus maximizing the amount of glucose available to the cell.

Figure 15-25

The stimulation of glycogen breakdown by cyclic AMP in skeletal muscle cells. The binding of cyclic AMP to A-kinase activates this enzyme to phosphorylate and thereby activate phosphorylase kinase, which in turn phosphorylates and activates glycogen phosphorylase, (more...)

In some animal cells an increase in cyclic AMP activates the transcription of specific genes. In cells that secrete the peptide hormone somatostatin, for example, cyclic AMP turns on 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 other genes that are activated by cyclic AMP. This sequence is recognized by a specific gene regulatory protein called CRE-binding (CREB) protein. When CREB is phosphorylated by A-kinase on a single serine residue, it is activated to turn on the transcription of these genes; the phosphorylation stimulates the transcriptional activity of CREB without affecting its DNA-binding properties. If this serine residue is mutated, CREB is inactivated and no longer stimulates gene transcription in response to a rise in cyclic AMP levels.

Serine/Threonine Protein Phosphatases Rapidly Reverse the Effects of A-Kinase ¹⁷

Since it is usually important that the effects of cyclic AMP are transient, cells must be able to dephosphorylate the proteins that have been phosphorylated by A-kinase. In general, the dephosphorylation of phosphorylated serines and threonines is catalyzed by four groups of serine/threonine phosphoprotein phosphatases - protein phosphatases I, IIA, IIB, and IIC. Except for protein 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 regulatory subunits. Protein phosphatase-I plays an important role in the response to cyclic AMP, as we discuss below. 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; it plays an important part in regulating the cell cycle. Protein phosphatase-IIB, also called calcineurin, is activated by Ca²⁺ and is especially abundant in the brain.

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. Protein phosphatase-I is responsible for dephosphorylating many of the proteins phosphorylated by A-kinase. It inactivates CREB, for example, by removing its activating phosphate, thereby turning off the transcriptional response caused by a rise in cyclic AMP. In skeletal muscle cells it dephosphorylates each of the three key enzymes in the glycogen pathway that, as mentioned earlier, are phosphorylated in response to adrenaline by A-kinase and switch the cells from synthesizing glycogen to degrading it. Protein phosphatase-I tends to counteract these phosphorylations, but its activity is suppressed in adrenaline-stimulated muscle cells by yet another target of A-kinase, which is a specific phosphatase inhibitor protein. When this inhibitor protein is phosphorylated by A-kinase, it binds to protein phosphatase-I and inactivates it ( Figure 15-26). By simultaneously activating phosphorylase kinase and inhibiting the opposing action of protein phosphatase-I, the A-kinase causes a much larger change in glycogen metabolism than could be obtained by its action on any one of these enzymes alone.

Figure 15-26

The role of protein phosphatase-I in the regulation of glycogen metabolism by cyclic AMP. Cyclic AMP inhibits protein phosphatase-I, which would otherwise oppose the phosphorylation reactions stimulated by cyclic AMP. It does so by activating A-kinase (more...)

Having discussed how trimeric G proteins couple receptors to adenylyl cyclase to alter the levels of cyclic AMP in cells, we now consider how G proteins couple receptors to another crucial enzyme - phospholipase C. The activation of this enzyme leads to an increase in the concentration of Ca²⁺ in the cytosol, and Ca²⁺ is even more widely used as an intracellular mediator than cyclic AMP.

To Use Ca²⁺ as an Intracellular Signal, Cells Must Keep Resting Cytosolic Ca²⁺ Levels Low ¹⁸

The concentration of free Ca²⁺ in the cytosol of any cell is extremely low (≤ 10^-7 M), whereas its concentration in the extracellular fluid (~10^-3 M) and in the endoplasmic reticulum (ER) 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, dramatically increasing the local Ca²⁺ concentration and triggering Ca²⁺-responsive proteins in the cell.

For this signaling mechanism to work, the resting concentration of Ca²⁺ in the cytosol must be kept low, and this is achieved in several ways. All eucaryotic cells have a Ca²⁺-ATPase 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⁺ pump in their plasma membrane that couples the efflux of Ca²⁺ to the influx of Na⁺. This Na⁺-Ca²⁺ exchanger has a relatively low affinity for Ca²⁺ and therefore begins to operate efficiently only when cytosolic Ca²⁺ levels rise to about 10 times their normal level, as occurs after repeated muscle or nerve cell stimulation. A Ca²⁺ pump in the ER membrane also plays an important part in keeping the cytosolic Ca²⁺ concentration low: this Ca²⁺-ATPase 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.

Normally, the concentration of free Ca²⁺ in the cytosol varies from about 10^-7 M, when the cell is at rest, to about 5 x 10^-6 M, when the cell is activated by an extracellular signal. But when a cell is damaged and cannot pump Ca²⁺ out of the cytosol efficiently, the Ca²⁺ concentration can rise beyond that to dangerously high levels (> 10^-5 M). In these circumstances a low-affinity, high-capacity Ca²⁺ pump in the inner mitochondrial membrane comes into action and uses the electrochemical gradient generated across this membrane during the electron-transfer steps of oxidative phosphorylation to take up Ca²⁺ from the cytosol. These mechanisms are summarized in Figure 15-27.

Figure 15-27

Controls on cytosolic Ca²⁺. The schematic drawing shows the main ways in which cells maintain a very low concentration of free Ca²⁺ in the cytosol in the face of high concentrations of Ca²⁺ in the extracellular fluid. Ca²⁺ is actively pumped out of the (more...)

Ca²⁺ Functions as a Ubiquitous Intracellular Messenger ¹⁹

The first direct evidence that Ca²⁺functions as an intracellular mediator came from an experiment done in 1947 showing that the intracellular injection of a small amount of Ca²⁺ causes a skeletal muscle cell to contract. In recent years it has become clear that Ca²⁺ also acts as an intracellular messenger in a wide variety of other cellular responses, including secretion and cell proliferation. Two pathways of Ca²⁺ signaling have been well defined, one used mainly by electrically active (excitable) cells and the other used by almost all eucaryotic cells. The first of these pathways has been particularly well studied in nerve cells, in which depolarization of the plasma membrane causes an influx of Ca²⁺ into the nerve terminal, initiating the secretion of neurotransmitter; the Ca²⁺ enters through voltage-gated Ca²⁺ channels that open when the plasma membrane of the nerve terminal is depolarized by an invading action potential (see Figure 11-34). In the second, ubiquitous pathway the binding of extracellular signaling molecules to cell-surface receptors causes the release of Ca²⁺ from the ER. The events at the cell surface are coupled to the opening of Ca²⁺ channels in the ER through yet another intracellular messenger molecule, inositol trisphosphate ( Figure 15-28) , as we discuss next.

Figure 15-28

Two common pathways by which Ca²⁺ can enter the cytosol in response to extracellular signals. In (A) Ca²⁺ enters a nerve terminal from the extracellular fluid through voltage-gated Ca²⁺ channels when the nerve terminal membrane is depolarized by an action (more...)

Some G-Protein-linked Receptors Activate the Inositol Phospholipid Signaling Pathway by Activating Phospholipase C-β ²⁰

A role for inositol phospholipids ( phosphoinositides) in signal transduction was first suggested in 1953, when it was found that some extracellular signaling molecules stimulate the incorporation of radioactive phosphate into phosphatidylinositol (PI), a minor phospholipid in cell membranes. It was later shown that this incorporation results from the breakdown and subsequent resynthesis of inositol phospholipids. The inositol phospholipids found to be most important in signal transduction were two phosphorylated derivatives of PI, PI phosphate (PIP) and PI bisphosphate (PIP₂), which are thought to be located mainly in the inner half of the plasma membrane lipid bilayer ( Figure 15-29). Although PIP₂ is less plentiful in animal cell membranes than PI, it is the hydrolysis of PIP₂ that matters most.

Figure 15-29

Inositol phospholipids (phosphoinositides). The polyphosphoinositides (PIP and PIP₂) are produced by the phosphorylation of phosphatidylinositol (PI). Although all three inositol phospholipids may be broken down in the signaling response, it is the breakdown (more...)

The chain of events leading to PIP₂ breakdown begins with the binding of a signaling molecule to a G-protein-linked receptor in the plasma membrane. More than 25 different cell-surface receptors have been shown to utilize this transduction pathway; several examples of responses mediated in this way are given in Table 15-2. Although the details of the activation process are not as well understood as they are in the cyclic AMP pathway, the same type of multistep mechanism is thought to operate in the plasma membrane. An activated receptor stimulates a trimeric G protein called G_q, which in turn activates a phospho-inositide-specific phospholipase C called phospholipase C- β. In less than a second, this enzyme cleaves PIP₂ to generate two products: inositol trisphosphate and diacylglycerol ( Figure 15-30). At this step the signaling pathway splits into two branches. Since both molecules play crucial parts in signaling the cell, we consider them in turn.

Table 15-2

Some Cellular Responses Mediated by G-Protein-linked Receptors Coupled to the Inositol-Phospholipid Signaling Pathway.

Figure 15-30

The hydrolysis of PIP₂. Two intracellular mediators are produced when PIP₂ is hydrolyzed: inositol trisphosphate (IP₃), which diffuses through the cytosol and releases Ca²⁺ from the ER, and diacylglycerol, which remains in the membrane and helps activate the (more...)

Inositol Trisphosphate (IP₃) Couples Receptor Activation to Ca²⁺ Release from the ER ²¹

The inositol trisphosphate (IP₃) produced by PIP₂ hydrolysis is a small water-soluble molecule that leaves the plasma membrane and diffuses rapidly through the cytosol. There it releases Ca²⁺ from the ER by binding to IP₃-gated Ca²⁺-release channels in the ER membrane. The channels are structurally similar to the Ca²⁺-release channels ( ryanodine receptors) in the sarcoplasmic reticulum of muscle cells, which release the Ca²⁺ that triggers muscle contraction (see Figure 16-92). Both types of channels are regulated by positive feedback, in which the released Ca²⁺ can bind back to the channels to increase the Ca²⁺ release, which tends to make the release occur in a sudden, all-or-none fashion. In many cells, including muscle cells, both types of Ca²⁺-release channels are present.

Two mechanisms operate to terminate the initial Ca²⁺ response: (1) IP₃ is rapidly dephosphorylated (and thereby inactivated) by specific phosphatases, and (2) Ca²⁺ that enters the cytosol is rapidly pumped out, mainly out of the cell.

Not all of the IP₃ is dephosphorylated, however: some is instead phosphorylated to form inositol 1,3,4,5-tetrakisphosphate (IP₄), which may mediate slower and more prolonged responses in the cell or promote the refilling of the intracellular Ca²⁺ stores from the extracellular fluid, or both. The enzyme that catalyzes the production of IP₄ is activated by the increase in cytosolic Ca²⁺ induced by IP₃, providing a form of negative feedback regulation on IP₃ levels.

Ca²⁺ Oscillations Often Prolong the Initial IP₃-induced Ca²⁺ Response ²²

When Ca²⁺-sensitive fluorescent indicators, such as aequorin or fura-2 (discussed in Chapter 4), are used to monitor cytosolic Ca²⁺ in individual cells in which the inositol phospholipid signaling pathway has been activated, the initial Ca²⁺ signal is often seen to propagate as a wave through the cytosol from a localized region of the cell. Moreover, the initial transient increase in Ca²⁺ is often followed by a series of Ca²⁺ "spikes," each lasting seconds or minutes; these Ca²⁺ oscillations can persist for as long as receptors are activated on the cell surface ( Figure 15-31).

Figure 15-31

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 increasing concentration (more...)

The mechanisms responsible for the propagation of Ca²⁺ waves and for generating the oscillations are uncertain, although a number of models have been proposed. In most models both the propagation and oscillations depend on positive feedback, whereby Ca²⁺ activates its own release, thereby producing an all-or-none Ca²⁺ spike. The models differ mainly in whether Ca²⁺ acts directly on the Ca²⁺-release channels in the ER to stimulate its own release or whether it acts indirectly, by increasing the activity of phospholipase C, thereby generating surges of IP₃, which in turn induce surges of Ca²⁺ release.

The biological significance of the Ca²⁺ oscillations is also uncertain. Their frequency often depends on the concentration of the extracellular signaling ligand (see Figure 15-31) and might, in principle, be translated into a frequency-dependent cellular response. In hormone-secreting pituitary cells, for example, stimulation by an extracellular signaling molecule induces repeated Ca²⁺ spikes, each of which is associated with a burst of hormone secretion. It has been suggested that this arrangement might maximize secretory output while avoiding the toxic effects of a sustained rise in cytosolic Ca²⁺.

Diacylglycerol Activates Protein Kinase C (C-Kinase) ²³

At the same time that the IP₃ produced by hydrolysis of PIP₂ is increasing the concentration of Ca²⁺ in the cytosol, the other cleavage product of PIP₂ - diacylglycerol - is exerting different effects. Diacylglycerol has two potential signaling roles. First, it can be further cleaved to release arachidonic acid, which either can act as a messenger in its own right or be used in the synthesis of eicosanoids (see Figure 15-6). Second, and more important, it activates a crucial serine/threonine protein kinase that phosphorylates selected proteins in the target cell.

The enzyme activated by diacylglycerol is called protein kinase C (C-kinase, or PKC) because it is Ca²⁺-dependent. The initial rise in cytosolic Ca²⁺ induced by IP₃ is thought to alter the C-kinase 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. Of the eight or more distinct isoforms of C-kinase in mammals, at least four are activated by diacylglycerol.

Because the diacylglycerol produced initially by the cleavage of PIP₂ is rapidly metabolized, it cannot sustain the activity of C-kinase, as would be required for long-term responses such as cell proliferation or differentiation. Prolonged activation of C-kinase depends on a second wave of diacylglycerol production, catalyzed by phospholipases that cleave the major membrane phospholipid phosphatidylcholine. It is uncertain how these later-acting phospholipases become activated.

When activated, C-kinase phosphorylates specific serine or threonine residues on target proteins that vary depending on the cell type. The highest concentrations of C-kinase are found in the brain, where (among other things) it phosphorylates ion channels in nerve cells, thereby changing their properties and altering the excitability of the nerve cell plasma membrane.

In many cells the activation of C-kinase increases the transcription of specific genes. At least two pathways are known. In one, C-kinase activates a protein kinase cascade that leads to the phosphorylation and activation of a DNA-bound gene regulatory protein; in another, C-kinase activation leads to the phosphorylation of an inhibitor protein, thereby releasing a cytoplasmic gene regulatory protein so that it can migrate into the nucleus and stimulate the transcription of specific genes ( Figure 15-32).

Figure 15-32

Two intracellular pathways by which activated C-kinase can activate the transcription of specific genes. In one ( red arrows) C-kinase activates a phosphorylation cascade that leads to the phosphorylation of a pivotal protein kinase called MAP-kinase (more...)

The two branches of the inositol phospholipid signaling pathway are summarized in Figure 15-33. As indicated in the figure, each branch of the pathway can be mimicked by the addition of specific pharmacological agents to intact cells. The effects of IP₃ can be mimicked by using a Ca²⁺ 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 C-kinase 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. A number of cell types, for example, can be stimulated to proliferate in culture when treated with both a Ca²⁺ ionophore and a C-kinase activator but not when they are treated with either reagent alone.

Figure 15-33

The two branches of the inositol phospholipid pathway. The activated receptor binds to a specific trimeric G protein (G_q), causing the α subunit to dissociate and activate phospholipase C-β, which cleaves PIP₂ to generate IP₃ and diacylglycerol. (more...)

Calmodulin Is a Ubiquitous Intracellular Ca²⁺ Receptor ²⁴

Since the free Ca²⁺ concentration in the cytosol is usually ≤ 10^-7 M and generally does not rise above 6 x 10^-6 M even when the cell is activated by an influx of Ca²⁺, any structure in the cell that is to serve as a direct target for Ca²⁺-dependent regulation must have an affinity constant ( K_a) for Ca²⁺ of around 10⁶ liters/mole. Moreover, since the concentration of free Mg²⁺ in the cytosol is relatively constant at about 10^-3 M, these Ca²⁺-binding sites must have a selectivity for Ca²⁺ over Mg²⁺ of at least 1000-fold. Several specific Ca²⁺-binding proteins fulfill these criteria.

The first such protein to be discovered was troponin 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 that have been examined. A typical animal cell contains more than 10⁷ molecules of calmodulin, which can constitute as much as 1% of the total protein mass of the cell. Calmodulin functions as a multipurpose intracellular Ca²⁺ receptor, mediating many Ca²⁺-regulated processes. It is a highly conserved, single polypeptide chain of about 150 amino acids, with four high-affinity Ca²⁺-binding sites ( Figure 15-34A), and it undergoes a conformational change when it binds Ca²⁺.

Figure 15-34

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 a loop of 12 amino (more...)

The allosteric activation of calmodulin by Ca²⁺ is analogous to the allosteric activation of A-kinase by cyclic AMP, except that Ca²⁺/calmodulin has no enzyme activity itself but acts by binding to other proteins. In some cases calmodulin serves as a permanent regulatory subunit of an enzyme complex, but in most cases the binding of Ca²⁺ enables calmodulin to bind to various target proteins in the cell and thereby alter their activity. When Ca²⁺/calmodulin binds to its target protein, it can undergo a further and more dramatic change in conformation ( Figure 15-34B).

Among the targets regulated by Ca²⁺/calmodulin are various enzymes and membrane transport proteins. In many cells, for example, Ca²⁺/calmodulin binds to and activates the plasma membrane Ca²⁺-ATPase that pumps Ca²⁺ out of the cell. Thus, if the concentration of Ca²⁺ in the cytosol rises, the pump is activated, which helps return the cytosolic Ca²⁺level to normal. Most effects of Ca²⁺/calmodulin, however, are more indirect and are mediated by Ca²⁺/calmodulin-dependent protein kinases.

Ca²⁺/Calmodulin-dependent Protein Kinases (CaM-Kinases) Mediate Most of the Actions of Ca²⁺ in Animal Cells ²⁵

Most of the effects of Ca²⁺ in cells are mediated by protein phosphorylations catalyzed by a family of Ca²⁺/calmodulin-dependent protein kinases (CaM-kinases). These kinases phosphorylate serines or threonines in proteins, and, as in the case of cyclic AMP, the response of a target cell to an increase in free Ca²⁺ concentration in the cytosol 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 glycogen breakdown - have narrow substrate specificities. More recently, however, a number of CaM-kinases have been identified that have much broader specificities, and these seem to be responsible for mediating many of the actions of Ca²⁺ in animal cells.

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, where it is highly concentrated in synapses. When neurons that use catecholamines (dopamine, noradrenaline, or adrenaline) as their neurotransmitter are activated, for example, the influx of Ca²⁺ through voltage-gated Ca²⁺ channels in the plasma membrane stimulates the cells to secrete their neurotransmitter. The Ca²⁺influx also activates CaM-kinase II to phosphorylate, and thereby activate, tyrosine hydroxylase, which is the rate-limiting enzyme in catecholamine synthesis. In this way, both the secretion and resynthesis of the neurotransmitter are stimulated when the cell is activated.

CaM-kinase II has a remarkable property: 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⁺ is withdrawn. 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 in the absence of Ca²⁺, thereby prolonging the duration of the kinase activity beyond the duration of the initial activating Ca²⁺ signal; the activity is maintained until phosphatases overwhelm the autophosphorylating activity of the enzyme and shut it off ( Figure 15-35).

Figure 15-35

The activation of CaM-kinase II. The enzyme is a protein complex of about 12 subunits. The subunits are of four homologous kinds (α, β, γ, and delta), which are expressed in different proportions in different cell types. Only the (more...)

Because of these properties, CaM-kinase II activation can serve as a memory trace of a prior Ca²⁺ pulse, and it seems to play an important part in some types of memory and learning in the vertebrate nervous system. Mutant mice that are missing the brain-specific subunit illustrated in Figure 15-35 have specific defects in their ability to remember the location of an object - that is, in spatial learning.

The Cyclic AMP and Ca²⁺ Pathways Interact ²⁶

The cyclic AMP and Ca²⁺ intracellular signaling pathways interact at several levels in the hierarchy of control. First, cytosolic Ca²⁺ and cyclic AMP levels can influence each other. For example, some forms of the enzymes that break down and make cyclic AMPcyclic AMP phosphodiesterase and adenylyl cyclase, respectively - are regulated by Ca²⁺-calmodulin complexes. Conversely, A-kinase can phosphorylate some Ca²⁺ channels and pumps and alter their activity; A-kinase phosphorylates the IP₃ receptor in the ER, for example, which can either inhibit or promote IP₃-induced Ca²⁺ release, depending on the cell type. Second, the enzymes directly regulated by Ca²⁺ and cyclic AMP can influence each other. Some CaM-kinases are phosphorylated by A-kinase, for example. Third, these enzymes can have interacting effects on shared downstream target molecules. Thus A-kinase and CaM-kinases frequently phosphorylate different sites on the same proteins, which are thereby regulated by both cyclic AMP and Ca²⁺; the CREB gene regulatory protein, which we discussed earlier (see p. 741), is one example.

As an example of how Ca²⁺and cyclic AMP pathways can interact, consider the phosphorylase kinase of skeletal muscle, whose role in glycogen degradation we have already discussed. This kinase phosphorylates glycogen phosphorylase, causing it to break down glycogen (see Figure 15-25). The kinase is a multisubunit enzyme, but only one of its four subunits actually catalyzes the phosphorylation reaction: the other three subunits are regulatory and enable the enzyme complex to be activated both by cyclic AMP and by Ca²⁺. The four subunits are designated α, β, γ, and delta. The γ subunit carries the catalytic activity; the delta subunit is calmodulin and is largely responsible for the Ca²⁺ dependence of the enzyme; the α and β subunits are targets for cyclic AMP-mediated regulation, both being phosphorylated by the A-kinase ( Figure 15-36).

Figure 15-36

Phosphorylase kinase. This highly schematized drawing shows the four subunits of the enzyme from mammalian muscle. The γ subunit has the catalytic activity of the active enzyme; the α and β subunits and the delta subunit (calmodulin) (more...)

The same Ca²⁺ signal that initiates muscle contraction also ensures that there is adequate glucose to power the contraction. The large influx of Ca²⁺ into the cytosol discussed in Chapter 16 alters the conformation of the calmodulin subunit of phosphorylase kinase, increasing kinase activity and thereby increasing the rate of the glycogen breakdown catalyzed by glycogen phosphorylase several hundredfold within seconds. In addition, the Ca²⁺ influx activates CaM-kinases that phosphorylate and inhibit glycogen synthase, thereby shutting off glycogen synthesis. By contrast, the adrenaline-induced A-kinase phosphorylations previously discussed adjust muscle cell metabolism in anticipation of an increased energy demand, allowing the enzyme to be activated when fewer calcium ions are bound to calmodulin, thereby making it more sensitive to Ca²⁺.

Some Trimeric G Proteins Directly Regulate Ion Channels ²⁷

Trimeric G proteins do not act exclusively by regulating the activity of enzymes and altering the concentration of cyclic nucleotides or Ca²⁺ in the cytosol. In some cases they directly activate or inactivate ion channels in the plasma 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. The effect is mediated by a special class of acetylcholine receptors that activate the inhibitory G protein, G_i, discussed previously. (These 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 ion-channel-linked receptors on skeletal muscle cells and nerve cells that can be activated by nicotine.) Once activated, the α subunit of G_i not only inhibits adenylyl cyclase (as described previously), it also directly opens K⁺ channels in the muscle cell plasma membrane. The opening of these K⁺ channels makes it harder to depolarize the cell, which contributes to the inhibitory effect of acetylcholine on the heart.

Other trimeric G proteins regulate the activity of ion channels less directly, either by regulating channel phosphorylation (by A-kinase, C-kinase, or CaM-kinase, for example) or by causing the production or destruction of cyclic nucleotides that directly activate or inactivate ion channels. Such cyclic-nucleotide-gated ion channels play a crucial role in both smell (olfaction) and vision.

Smell and Vision Depend on G-Protein-linked Receptors and Cyclic-Nucleotide-gated Ion Channels ²⁸

Humans can distinguish more than 10,000 different smells ( odorants), which are detected by specialized olfactory receptor neurons in the lining of the nose. These cells recognize odorants 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 15-37). Many of these receptors act through cyclic AMP: when stimulated by odorant binding, they activate an olfactory-specific trimeric G protein ( G_olf), which in turn activates adenylyl cyclase; the resulting increase in cyclic AMP opens cyclic-AMP-gated cation channels, which allows an influx of Na⁺ that depolarizes the cell and initiates a nerve impulse that travels along the axon to the brain. Other olfactory receptors act via the inositol phospholipid pathway and IP₃-gated Ca²⁺ channels in the plasma membrane, but less is known about this transduction mechanism.

Figure 15-37

Olfactory receptor neurons. (A) Schematic drawing of olfactory epithelium in the nose. The 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 (more...)

It is thought that there are hundreds of different olfactory receptors, each encoded by a different gene and each recognizing different odorants but all belonging to the G-protein-linked receptor superfamily. Although it is known that each olfactory cell responds to a specific set of odorants, it is not yet clear if each cell contains only one type of receptor that recognizes a set of odorants or whether each cell contains a set of receptors, each specific for a single odorant. G-protein-linked receptors also seem to mediate some forms of taste, but less is known about them.

Cyclic-nucleotide-gated ion channels are also involved in signal transduction in vertebrate vision, but here the crucial cyclic nucleotide is cyclic GMP ( Figure 15-38) rather than cyclic AMP. Like cyclic AMP, the concentration of cyclic GMP in cells is controlled by rapid synthesis (by guanylyl cyclase) and rapid degradation (by cyclic GMP phosphodiesterase).

Figure

Cyclic GMP.

In visual transduction, receptor activation is caused by light, and it 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 monochromatic 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 an outer and an inner segment, a cell body, and a synaptic region where the rod passes a chemical signal to a retinal nerve cell, which relays the signal along the visual pathway ( Figure 15-39). The phototransduction apparatus is in the outer segment, which contains a stack of discs, each formed by a closed sac of membrane in which photosensitive rhodopsin molecules are embedded. The plasma membrane surrounding the outer segment contains cyclic-GMP-gated Na⁺ channels. These Na⁺ channels are kept open in the dark by cyclic GMP molecules bound to the channels. Paradoxically, light causes a hyperpolarization (which inhibits synaptic signaling) rather than a depolarization of the plasma membrane (which could stimulate synaptic signaling), because the activation by light of rhodopsin molecules in the disc membrane leads to the closure of the Na⁺ channels in the surrounding plasma membrane ( Figure 15-40).

Figure 15-39

Drawing of a rod photoreceptor cell. There are about 1000 discs in the outer segment, and the disc membranes are not connected to the plasma membrane.

Figure 15-40

The response of a rod photoreceptor cell to light. Photons are absorbed by rhodopsin molecules in the outer-segment discs. This leads to the closure of Na⁺ channels in the plasma membrane, which hyperpolarizes the membrane and reduces the rate of neurotransmitter release (more...)

Rhodopsin, as we noted earlier, is a seven-pass transmembrane molecule homologous to other members of the G-protein-linked receptor family, 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 slower conformational change in the protein (opsin). The activated protein then binds to the trimeric G-protein transducin (G_t), causing the α subunit (α_t) to dissociate and activate cyclic GMP phosphodiesterase, which hydrolyzes cyclic GMP, so that cyclic GMP levels in the cytosol drop. As a consequence, cyclic GMP dissociates from the plasma membrane Na⁺ channels, allowing them to close. In this way the signal passes from the disc membrane to the plasma membrane, and a light signal is converted into an electrical one.

The Na⁺ channels 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 fall in Ca²⁺ stimulates guanylyl cyclase to replenish the cyclic GMP, rapidly returning the cell toward the state it was in before the light was switched on. The activation of guanylyl cyclase by the fall in Ca²⁺ is mediated by a Ca²⁺-sensitive protein called recoverin, which, in contrast to calmodulin, is inactive when Ca²⁺ is bound to it and active when it is Ca²⁺-free; it stimulates the cyclase when Ca²⁺ levels are low following a light response. This Ca²⁺-dependent mechanism is of crucial importance in two ways. First, it allows the photoreceptor to revert quickly to its resting, dark state in the aftermath of a flash of light, making it possible to perceive the shortness of the flash. Second, it helps to enable the photoreceptor to adapt, stepping down the response when it is exposed to light continuously. Adaptation, as we explain later, means that the receptor cell can 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 that we have discussed in this chapter are summarized in Table 15-3.

Table 15-3

The Major Families of Trimeric G proteins*.

Extracellular Signals Are Greatly Amplified by the Use of Intracellular Mediators and Enzymatic Cascades ²⁹

Despite the differences in molecular details, all of the signaling systems that are triggered by G-protein-linked receptors share certain features and are governed by similar general principles. Most of them depend on complex cascades, or relay chains, of intracellular mediators. By contrast with the more direct signaling pathways used by intracellular receptors discussed earlier and by ion-channel-linked receptors discussed in Chapter 11, catalytic cascades of intracellular mediators 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, which transiently closes hundreds of Na²⁺ channels in the plasma membrane ( Figure 15-41).

Figure 15-41

Amplification in the light-induced catalytic cascade in vertebrate rods. The divergent arrows indicate the steps where amplification occurs.

Similarly, when an extracellular signaling molecule binds to a receptor that indirectly activates adenylyl cyclase via G_s, each receptor protein may activate many molecules of G_s protein, each of which can activate a cyclase molecule. As each G_s molecule activated persists in its active form for seconds before it hydrolyzes its bound GTP to shut itself off, it can keep its bound cyclase molecule active for seconds, so that the cyclase can catalyze the conversion of a large number of ATP molecules to cyclic AMP molecules ( Figure 15-42). The same type of amplification operates in the inositol-phospholipid pathway. As a result, a nanomolar (10^-9 M) concentration of an extracellular signal often induces micromolar (10^-6 M) concentrations of an intracellular second messenger such as cyclic AMP or Ca²⁺. Since these messengers themselves function as allosteric effectors to activate specific enzymes or ion channels, a single extracellular signaling molecule can cause many thousands of molecules to be altered within the target cell. Moreover, each protein in the relay chain of signals can be a separate target for regulation, as, for example, in the glycogen breakdown cascade in skeletal muscle cells.

Figure 15-42

Amplification in a ligand-induced catalytic cascade. The first amplification step requires that the signaling ligand remain bound to the receptor long enough for the complex to activate many G_s molecules; in many cases the ligand will dissociate too quickly (more...)

Any such amplifying cascade of stimulatory signals requires that there should 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 transport proteins 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 takes off. As we saw earlier (see p. 727), in general the response to stimulation can be rapid only if the inactivating mechanisms also are rapid.

Cells Can Respond Suddenly to a Gradually Increasing Concentration of an Extracellular Signal ³⁰

Some cellular responses to signaling ligands are smoothly graded in simple proportion to the concentration of the ligand. The primary responses to steroid hormones (see Figure 15-13) often follow this pattern, presumably because each intracellular hormone receptor protein binds a single molecule of hormone and each specific DNA recognition sequence in a steroid-hormone-responsive gene acts independently. As the concentration of hormone increases, the concentration of hormone-receptor complexes increases proportionally, as does the number of complexes bound to specific recognition sequences in the responsive genes; the cellular response is therefore a gradual and linear one.

Other responses to signaling ligands, however, begin more abruptly as the concentration of ligand increases. Some may even occur in a nearly all-or-none manner, being undetectable below a threshold concentration of ligand and then reaching a maximum as soon as this concentration is exceeded. What might be the molecular basis for such steep or even switchlike responses to graded signals?

One mechanism for steepening the response is to require that more than one intracellular effector molecule or complex bind to some target macromolecule in order to induce a response. In some steroid-hormone-induced responses, for example, it appears that more than one hormone-receptor complex must bind simultaneously to specific regulatory sequences in the DNA in order to activate a particular gene. As a result, as the hormone concentration rises, gene activation begins more abruptly than it would if only one bound complex were sufficient for activation ( Figure 15-43). A similar mechanism operates in the activation of A-kinase and calmodulin, as discussed earlier. Two or more Ca²⁺ ions, for example, must bind before calmodulin adopts its activating conformation; as a result, a fiftyfold increase in activation occurs when the free intracellular Ca²⁺ concentration increases only tenfold. Such responses become sharper as the number of cooperating molecules increases, and if the number is large enough, responses approaching the all-or-none type can be achieved ( Figures15-44and 15-45).

Figure 15-43

Response of chick oviduct cells to the steroid sex hormone estradiol. When activated, estradiol receptors turn on the transcription of several genes. Dose-response curves for two of these genes, one coding for the egg protein conalbumin and the other (more...)

Figure 15-44

Activation curves as a function of signal-molecule concentration. The curves show how the sharpness of the response increases as the number of effector molecules that must bind simultaneously to activate a target macromolecule increases. The curves shown (more...)

Figure 15-45

One type of signaling mechanism expected to show a steep thresholdlike response. Here the simultaneous binding of eight molecules of a signaling ligand to a set of eight subunits is required to form an active protein complex: the ability of the subunits (more...)

Responses are also sharpened when a ligand activates one enzyme and at the same time inhibits another that catalyzes the opposite reaction. We have already discussed one example of this common type of regulation in the stimulation of glycogen breakdown in skeletal muscle cells, where a rise in the intracellular cyclic AMP level both activates phosphorylase kinase and inhibits the opposing action of phosphoprotein phosphatase.

The above mechanisms can produce responses that are very steep but, nevertheless, always smoothly graded according to the concentration of the signaling ligand. Another mechanism, however, can produce true all-or-none responses, such that raising the signal above a critical threshold level trips a sudden switch in the responding system. All-or-none threshold responses of this type generally depend on positive feedback. Thus, by positive feedback nerve and muscle cells generate all-or-none action potentials in response to neurotransmitters (discussed in Chapter 11). The activation of acetylcholine receptors at a neuromuscular junction, for example, opens cation channels in the muscle cell plasma membrane. The result is a net influx of Na⁺ that locally depolarizes the membrane. This causes voltage-gated Na⁺ channels to open in the same membrane region, producing a further influx of Na⁺, which further depolarizes the membrane and thereby opens more Na⁺ channels. If the initial depolarization exceeds a certain threshold, this positive feedback has an explosive "runaway" effect, producing an action potential that propagates to involve the entire muscle membrane. As discussed earlier, a similar phenomenon occurs when Ca²⁺ is released from the ER or sarcoplasmic reticulum by Ca²⁺-release channels: the released Ca²⁺ can bind back to the channels, increasing Ca²⁺ release, thereby producing an all-or-none Ca²⁺ spike.

The Effect of Some Signals Can Be Remembered by the Cell ³¹

An accelerating positive feedback mechanism can operate through signaling proteins that are enzymes rather than ion channels. Suppose, for example, that a particular signaling ligand activates an enzyme located downstream in the signal relay pathway and that two or more molecules of the product of the enzymatic reaction bind back to the same enzyme to activate it further ( Figure 15-46). The consequence will be a very low rate of synthesis of the enzyme product in the absence of the ligand, increasing slowly with the concentration of ligand until, at some threshold level of ligand, enough of the product is being synthesized to activate the enzyme in a self-accelerating, runaway fashion; the concentration of the enzyme product then suddenly increases to a much higher level. In this way the cell can translate a gradual change in the concentration of a signaling ligand into a switchlike change in the level of a particular enzyme product, creating an all-or-none response by the cell.

Figure 15-46

An accelerating positive feedback mechanism. The initial binding of the signaling ligand activates the enzyme to generate a product that binds back to the enzyme, further increasing the enzyme's activity.

This type of mechanism has an important property that makes it unsuitable for some purposes and uniquely valuable for others. If such a system has been switched on by raising the concentration of signaling ligand above threshold, it will generally remain switched on even when the signal disappears: instead of faithfully reflecting the current level of signal, the response system displays a memory. We have already discussed one example - CaM-kinase II, which is activated by Ca²⁺/calmodulin to phosphorylate itself and other proteins. The autophosphorylation keeps the kinase active long after Ca²⁺ levels return to normal and Ca²⁺/calmodulin has dissociated from the enzyme (see Figure 15-35). A self-activating memory mechanism can also operate further downstream in a signaling pathway, at the level of gene transcription. The signals that trigger muscle cell determination, for example, turn on a series of muscle-specific gene regulatory proteins that stimulate the transcription of their own genes as well as genes producing many other muscle cell proteins (see p. 445).

Summary

G-protein-linked receptors indirectly activate or inactivate plasma-membrane-bound enzymes or ion channels via trimeric GTP-binding regulatory proteins (G proteins) that shut themselves off by hydrolyzing their bound GTP. Some G-protein-linked receptors 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-b), which hydrolyzes phosphatidylinositol bisphosphate (PIP₂) to generate two intracellular mediators - inositol trisphosphate (IP₃), which releases Ca²⁺ from the ER and thereby increases the concentration of Ca²⁺ in the cytosol, and diacylglycerol, which remains in the plasma membrane and activates C-kinase. A rise in cyclic AMP or Ca²⁺ levels affects cells by stimulating A-kinase and CaM-kinases, respectively. C-kinase, A-kinase, and CaM-kinases phosphorylate specific target proteins on serine or threonine residues 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 these intracellular mediators. Through the intracellular signaling cascades activated by G-protein-linked receptors, the responses to extracellular signals can be greatly amplified.

The various responses mediated by these receptors are rapidly turned off when the extracellular signaling ligand is removed. This is because the G proteins self-inactivate by hydrolyzing their bound GTP, IP₃ is rapidly dephosphorylated by a phosphatase (or phosphorylated by a kinase), diacylglycerol is rapidly broken down, cyclic nucleotides are hydrolyzed by phosphodiesterases, Ca²⁺ is rapidly pumped out of the cytosol, and proteins are dephosphorylated by protein phosphatases. The continuous rapid turnover of these intracellular mediators makes possible rapid increases in their concentrations when cells respond to extracellular signals. In addition, cells make use of both cooperativity and positive feedback to sharpen their responses.