lodish molecular cell biology 7th_15 signal transduction and g protein-coupled receptors

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Lodish Molecular Cell Biology 7th_15 Signal Transduction and G Protein-Coupled Receptors

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  • .

    The mouse retina contains photoreceptors (purple) that sense light

    using G protein-coupled receptors and four other types of neurons

    stained yellow, green, pink, and blue, which connect the photoreceptor cells to the brain. [Rachel Wong, University of Washington]

    N o cell lives in isolation . Cellular communication is a fundamental property of all cells and shapes the devel-opment and function of every living organism. Even

    single-celled eukaryotic microorganisms, such as yeasts, slime molds, and protozoans, communicate through extracellular signals: secreted molecules called pheromones coordinate the aggregation of free-living cells for sexual mating or differentia-tion under certain environmental conditions. More important in plants and animals are hormones and other extracellular signaling molecules that function within an organism to con-trol a variety of processes, including the metabolism of sugars, fats, and amino acids; the growth and differentiation of tissues; the synthesis and secretion of proteins; and the composition of intracellular and extracellular fluids. Many types of cells also respond to signals from the external environment, including light, oxygen, odorants, anJi tastants in food.

    In any system, for a signal to have an effect on a target, it has to be received. In cells, a signal produce~ a specific re-sponse only in target cells with receptors for that signal. For

    OUTLINE

    15.1 Signal Transduction: From Extracellular Signal to Cellular Response

    1 5.2 Studying Cell-Surface Receptors and Signal Transduction Proteins

    15.3 G Protein-Coupled Receptors: Structure and Mechanism

    675

    681

    687

    CHAPTER

    SIGNAL TRANSDUCTION AND G PROTEIN-COUPLED RECEPTORS

    some receptors, this signal is a physical stimulus such as light, touch, or heat. For others, it is a chemical molecule. Many types of chemicals are used as signals: small molecules (e.g., amino acid or lipid derivatives, acetylcholine), gases (nitric oxide), peptides (e.g., ACTH and vasopressin), solu-ble proteins (e.g., insulin and growth hormone), and pro-teins that are tethered to the surface of a cell or bound to the extracellular matrix. Many of these extracellular signaling molecules are synthesized and released by specialized signal-ing cells within multicellular organisms. Most receptors bind a single molecule or a group of closely related molecules.

    Some signaling molecules, especially hydrophobic mole-cules such as steroids, retinoids, and thyroxine, spontane-ously diffuse through the plasma membrane and bind to intracellular receptors; signaling from such intracellular re-ceptors is discussed in detail in Chapter 7.

    Most extracellular signaling molecules, however, are too large and too hydrophilic to penetrate through the plasma membrane. How, then, can they affect intracellular processes?

    15.4 G Protein- Coupled Receptors That Regulate lon Channels 693

    15.5 G Protein- Coupled Receptors That Activate or Inhibit Adenylyl Cyclase 699

    15.6 G Protein-Coupled Receptors That Trigger Elevations in Cytosolic Ca2+ 707

  • 0 OVERVIEW ANIMATION: Extracellular Signaling FIGURE 1 5-1 Overview of signaling by cell-surface receptors. Communication by extracellular signals usually involves the following steps: synthesis of the signaling molecule by the signaling cell and its

    incorporation into small intracellular vesicles (step Ol. its release into the extracellular space by exocytosis (step 8), and transport of the signal to the target cell (step iJ). Binding of the signaling molecule to a specific cell-surface receptor protein triggers a conformational change in the receptor, thus activating it (step B). The activated receptor then activates one or more downstream signal transduction proteins or small-molecule second messengers (step B), eventually leading to activation of one or more effector proteins (step mJ. The end result of a signaling cascade can be either a short-term change in cellular function, metabolism, or movement (step m or a long-term change in gene expression or development (step fm). Termination or down-modulation of the cellular response is caused by negative feedback from intracellular signaling molecules (step [i)) and by removal of the extracellular signal (step m).

    These signaling molecules bind to cell-surface receptors that are integral membrane proteins embedded in the plasma mem-brane. Cell-surface receptors generally consist of three discrete domains, or segments: an extracellular domain facing the extracellular fluid, a membrane-spanning (transmembrane) domain that spans the plasma membrane, and the intracellular domain segment facing the cytosol. The signaling molecule acts as a ligand, which binds to a structurally complementary site on the extracellular or the membrane-spanning domains of the receptor. Binding of the ligand to its site on the receptor induces a conformational change in the receptor that is trans-mitted through the membrane-spanning domain to the cyto-solic domain, resulting in binding to and subsequent activation or inhibition of other proteins in the cytosol or attached to the plasma membr~ne. In many cases, these activated proteins catalyze the synthesis of certain small molecules or change the concentration of an intracellular ion such as Ca2 These intra-cellular proteins or small molecule second messengers then carry the signal to one or more effector proteins. The overall process of converting extracellular signals into intracellular re-sponses, as well as the individual steps in this process, is termed signal transduction (Figure 15-1).

    In eukaryotes, there are about a dozen classes of cell-surface receptors, which activate several types of intracellu-lar signal transduction pathways. Our knowledge of signal transduction has advanced greatly in recent years, in large measure because these receptors and pathways are highly conserved and function in essentially the same way in organ-tsms as diverse as worms, flies, mice, and humans. Genetic studies combined with biochemical analyses have enabled researchers to trace many entire signaling pathways from binding of ligand to final cellular responsr.

    Perhaps the most numerous class of receptors-found in organisms from yeasts to humans-are G protein-coupled receptors (GPCRs). As their name implies, G protein-coupled receptors consist of an integral membrane receptor protein coupled to an intracellular G protein that transmits signals to the interior of the cell. The human genome encodes about

    0

    ~!r'';" :o~ o o o o /mo . ~ 0 /_ Active cell-surface

    lnact1ve cell-surface .,. receptor receptor

    ~~~~ cell 1 Responding {(J

    Signal transduction t proteins and second Q

    messengers

    m~

    Modification of fl! 0 Effector protein ce llu lar metabolism, ___ ,. ',

    . -

  • (

    15.1 Signal Transduction: From Extracellular Signal to Cellular Response

    As shown in Figure 15-1, signal transduction begins when extracellular signaling molecules bind to cell-surface recep-tors. Binding of signaling molecules to their receptors induces two major types of cellular responses: ( l) changes in the ac-tivity or function of specific enzymes and other proteins that preexist in rhe cell and (2) changes in the amounts of specific proteins produced by a cell, most commonly by modification of transcription factors that stimulate or repress gene expres-sion (see Figure 15-1, steps fl1 and fl!l). In general, the first type of response occurs more rapidly than the second. Tran-scription factors activated in the cytosol by these pathways move into the nucleus, where they stimulate (or occasionally repress) transcription of specific target genes.

    The connection between an activated receptor and a cel-lular response is not direct and generally involves several in-termediate proteins or small molecules. Collectively, this chain of intermediates is called a signal transduction path-way because it transduces, or converts, information from one form into another as a signal is relayed from a receptor to its targets. Some signal transduction pathways contain just two or three intermediates; others can involve over a dozen. Regardless, most pathways contain members of cer-tain classes of signal transduction proteins that have been highly conserved throughout evolution.

    In this section, we provide an overview of the major steps in signal transduction, starting with the signaling molecules themselves. We explore the molecular basis for ligand-receptor binding and the chain of events initiated in the target cell by binding of the signal to its receptor, focusing on a few compo-nents that are central to many signal transduction pathways.

    Signaling Molecules Can Act Locally or at a Distance

    Cells respond to many different types of signals-some orig-inating from outside the organism, some internally gener-ated. Those that are generated internally can be described by how they reach their target. Some signaling molecules are transported long distancflS by the blood; others have more local effects. In animals, signaling by extracellular molecules can be classified into three types-endocrine, paracrine, or autocrine-based on the distance over which the signal acts (Figure 15-2a-c). In addition, certain membrane-bound pro-teins on one cell can directly signal an adjacent cell.

    In endocrine signaling, the signaling molecules are synthe-sized and secreted by signaling cells (for example, those found in endocrine glands), transported through the circulatory sys-tem of the organism, and tinally act on target cells Jistant from their site of synthesis. The term hormone generally re-fers to signaling molecules that mediate endocrine signaling. Insulin secreted by the pancreas and epinephrine secreted by the adrenal glands are examples of hormones that travel through the blood and thus mediate endocrine signaling.

    ln paracrine signaling, the signaling molecules released by a cell affect only those target cells in close proximity. A nerve cell releasing a neurotransmitter (e.g., acetylcholine) that acts on an atljacent nerve cell or on a muscle cell (inducmg or in-hibiting muscle contraction) is an example of paracrine sig-naling. In addition to neurotransmitters, many protein growth factors regulating development in multicellular or-ganisms act at short range. Some of these growth-factor pro-teins bind tightly to components of the extracellular matrix

    (a) Endocrine signaling

    Hormone secretion into blood by endocrine gland

    (b) Paracrine signaling

    0

    0 0 0

    0 () () 0

    / oJo\ 0 0 0

    Distant target cells

    o oQ 0 0

    Secretory cell

    (c) Autocrine signaling

    Target sites on same cell

    Adjacent target cell

    Key:

    o Extracellular signal

    Receptor

    T Membrane-attached signal

    (d) Signaling by plasma-membrane-attached proteins

    Signaling cell

    \ )

    Adjacent target cell

    FIGURE 1 S-2 Types of extracellular signaling. (a-c) Cell-to-cell signaling by extracellular chemicals occurs over distances from a few micrometers in autocrine and paracrine signaling to several meters in endocrine signaling. (d) Proteins attached to the plasma membrane of one cell can interact directly with cell-surface receptors on adjacent cells.

    15.1 Signal Transduction: From Extracellular Signal to Cellular Response 675

  • and are unable to signal to adjacent cells; subsequent degra-dation of these matrix components, triggered by injury or infection, will release the active growth factor and enable it to signal. Many developmentally important signaling proteins diffuse away from the signaling cell, forming a concentration gradient and inducing different cellular responses depending on the concentration of the signaling protein.

    In autocrine signaling, cells respond to substances that they themselves release. Some growth factors act in this fash-ion, and cultured cells often secrete growth factors that stim-ulate their own growth and proliferation. This type of signaling is particularly characteristic of tumor cells, many of which overproduce and release growth factors that stimu-late inappropriate, unregulated self-proliferation, a process that may lead to formation of a tumor.

    Integral membrane proteins located on the cell surface also play an important role in signaling (Figure 15-2d). In some cases, such membrane-bound signals on one cell bind receptors on the surface of an adjacent target cell, triggering its differentiation. In other cases, proteolytic cleavage of a membrane-bound signaling protein releases the extracellular segment, which functions as a soluble signaling molecule.

    Some signaling molecules can act at both short and long ranges. For example, epinephrine (also known as adrenaline) functions as a systemic hormone (endocrine signaling) and as a neurotransmitter (paracrine signaling). Another example is epidermal growth factor (EGF), which is synthesized as an integral plasma membrane protein. Membrane-bound EGF

    (a)

    Growth hormone

    (b)

    EXPERIMENTAL FIGURE 15-3 Growth hormone binds to its receptor through molecular complementary. (a) As determined from the three-dimensional structure of the growth hormone-growth hormone receptor complex, 28 amino acids in the hormont' dre at the binding interface with one receptor. To determine which amino acids are

    important in ligand-receptor binding, researchers mutated each of these amino acids one at a time, to alanine, and measured the effect on receptor binding. From this study, it was found that only eight amino

    acids on growth hormone (pink) contribute 85 percent of the energy

    that is responsible for tight receptor binding; these amino acids are distant from each other in the primary sequence but adjacent in the

    can bind to receptors on an adjacent cell. In addition, cleavage by an extracellular protease releases a soluble form of EGF, which can signal in either an autocrine or a paracrine manner.

    Binding of Signaling Molecules Activates Receptors on Target Cells Receptor proteins for all hydrophilic extracellular small mole-cules and protein ~ignaling molecules a re located on the sur-face of the target cell. The signaling molecule, or ligand, binds to a site on the extracellular domain of the receptor with high specificity and affinity. Each receptor generally binds only a single signaling molecule or a group of structurally very closely related molecules. The binding specificity of a receptor refers to its abi lity to bind or not bind closely related substances.

    Ligand binding depends on weak, multiple noncovalent forces (i.e., ionic, van der Waals, and hydrophobic interac-tions) and molecular complementarity between the interacting surfaces of a receptor and ligand (see Figure 2-12). For exam-ple, the growth hormone receptor (Figure 15-3) binds to growth hormone but not to other hormones with very similar, though not identical, structures. Similarly, acetylcholine recep-tors bind only this small molecule and not others that differ only slightly in chemical structure, while the insulin receptor binds insulin and related hormones called insulin-like growth factors 1 and 2 (TGF-1 and IGF-2), but no other hormones.

    Binding of ligand to receptor causes a conformational change in the receptor that initiates a sequence of reactions

    (c)

    folded protein. Similar studies showed that two tryptophan residues (blue) in the receptor contribute most of the energy responsible for tight

    binding of growth hormone, although other amino acids at the interface with the hormone (yellow) are also important. (b) Binding of growth hormone to one receptor molecule is followed by (c) binding of a second

    receptor (purple) to the opposing side of the hormone; this involves the same set of yellow and blue amino acids on the receptor but different

    residues on the hormone. As we see in the next chapter, such hormone-

    induced receptor dimerization is a common mechanism for activation of receptors for protein hormones. [After B. Cunningham and J. Wells, 1993, J. Mol. Bioi. 234:554, and T. C1ackson and J. Wells, 1995, Science 267:383.]

    676 CHAPTER 15 Signal Transduction and G Protein- Coupled Receptors

    .

  • leading to a specific response inside the cell. Organisms have evolved to be able to use a single ligand to stimulate different cells to respond in distinct ways. For example, different cell types may have different sets of receptors for the same ligand, each of which induces a different intracellular signal response pathway. Alternatively, the same receptor can be found on various cell types in an organism, but binding of a particular ligand to the receptor triggers a different response in each type of cell, given the unique complement of proteins expressed by the cell. In these ways, the same ligand can induce different cells to respond in a variety of ways. This is what's known as the effector specificity of the receptor-ligand complex.

    For instance, the surfaces of skeletal muscle cells, heart muscle cells, and the pancreatic acinar cells that produce hy-drolytic digestive enzymes each have different types of recep-tors for acetylcholine. In a skeletal muscle cell, release of acetylcholine from a motor neuron innervating the cell triggers muscle contraction by activating an acetylcholine-gated ion channel. In heart muscle, the release of acetylcholine by certain neurons activates a G protein-coupled receptor and slows the rate of contraction and thus the heart rate. Acetylcholine stim-ulation of pancreatic acinar cells triggers a rise in cytosolic l Ca2 J that induces exocytosis of the digestive enzymes stored in secretory granules to facilitate digestion of a meal. Thus for-mation of different acetylcholine-receptor complexes in differ-ent cell types leads to different cellular responses.

    Protein Kinases and Phosphatases Are Employed in Virtually All Signaling Pathways

    Activation of virtually all cell-surface receptors leads directly or indirectly to changes in protein phosphorylation through the activation of protein kinases, which add phosphate groups to specific residues of specific target proteins. Some receptors activate protein phosphatases, which remove phos-phate groups frqm specific residues on target proteins. Phos-phatases act in concert with kinases to switch the function of various proteins on or off (Figure 15-4).

    At last count, the human genome encodes about 600 pro-tein kinases and 100 different phosphatases. In general, each protein kinase phosphorylates specific amino acid residues in a set of target, or substrate, proteins whose patterns of expres-sion generally differ in different cell types. Animal cells con-tain two types of protein kinases: those that add phosphate to the hydroxyl group on tyrosine residues and those that add phosphate to the hydroxyl group on serine or threonine (or both) residues. All kinases also bind to specific amino acid sequences surrounding the phosphorylated residue, and thus one can look at amino acid sequences surrounding tyrosine, serine, and threonine residues in a protein and make a good guess as to which kinases might phosphorylate this re~.;idue.

    In some signaling pathways, the receptor itself possesses intrinsic kinase activity or the receptor is tightly bound to a cytosolic kinase. Figure 15-5 illustrates a simple signal trans-duction pathway involving one kinase tightly bound to a receptor and one predominant target protein. In the absence of a bound ligand the kinase is held in the inactive state. Ligand binding triggers a conformational change in the receptor,

    Protein~ kinase V

    0 I

    0 -P=O I

    0

    Inactive

    (\Protein v phosphatast

    FIGURE 15-4 Regulation of protein activity by a kinase/ phosphatase switch. The cyclic phosphorylation and dephosphorylation of a protein is a common cellular mechanism for regulating protein activity. In this example, the target, or substrate, protein is inactive (light green) when not phosphorylated and active (dark green) when phosphorylated; some proteins have the opposite pattern. Both the protein kinase and the phosphatase act only on .specific target proteins, and their activities are usually highly regulated.

    leading to activation of the appended kinase. The kinase then phosphorylates the monomeric, inactive form of a spe-cific transcription factor, leading to its dimerization and movement from the cytosol into the nucleus, where it acti -vates transcription of target genes. A phosphatase in the nucleus subsequently removes the phosphate group from the transcription factor, causing it to form two inactive mono-mers and then move back into the cytosol, where it can be reactivated by a receptor-associated kinase.

    As this example illustrates, the activity of all protein ki-nases is opposed by the activity of protein phosphatases, some of which are themselves regulated by extracellular stg-nals. Thus the activity of a protein in a cell can be a complex function of the activities of the usually multiple kinases and phosphatases that act on it, either directly or indirectly through phosphorylation of another protein. Several exam-ples of this phenomenon occur in regulation of the cell cycle and are described in Chapter 19.

    Many proteins are substrates for multiple kinases, each of which phosphorylates different amino acids. Each phosphory-lation event can modify the activity of a particular target pro-tein in different ways, some activating its function, others inhibiting it. An example we encounter later is glycogen pho~phorylase kinase, a key regulatory enzyme in glucose metabo-lism. ln many cases, addition of a phosphate group to an amino acid creates a binding surface that allows a second protein to bind; in the following chapter we will encounter many exam-ples of such kinase-driven assembly of multiprotein complexes.

    Commonly the catalytic activity of a protein kinase itself is modulated by phosphorylation by other kinases, by the

    15.1 Signal Transduction: From Extracellular Signal to Cellular Response 677

  • FIGURE 15-5 A simple signal transduction pathway involving one kinase and one target protein. The receptor is tightly bound to a protein kinase that, in the absence of a bound ligand, is held in the inactive state. Ligand binding triggers a conformational change in the receptor, leading to activation of the appended kinase (0 ). The kinase then phosphorylates the monomeric, inactive form of a specific transcrip-tion factor (f)), leading to its dimerization (IJ) and movement from the cytosol into the nucleus (m ). where it activates transcription oftarget genes. A phosphatase in the nucleus will remove the phosphate group from the transcription factor (if). causing it to form the inactive monomer and then move back into the cytosol (1";1).

    Ligand-Ligand binding sites

    Cytosol

    binding of other proteins to it, and by changes in the levels of various small intracellular signaling molecules and me-tabolites. The resulting cascades of kinase activity are a com-mon feature of many signaling pathways.

    GTP-Binding Proteins Are Frequently Used in Signal Transduction as On/Off Switches

    Many signal transduction pathways utilize intracellular "switch" proteins that turn downstream proteins on or off. The most important group of intracellular switch proteins is the GTPase super family. All the GTPase switch proteins exist in two forms (Figure 15-6): (1 ) an active ("on") form with bound GTP (guanosine triphosphate) that modulates the activity of specific target proteins and (2) an inactive ("off") form with bound GDP (guanosine diphosphate).

    Conversion of the inactive to active state is triggered by a signal (e.g., a hormone binding to a receptor) and is mediated by a guanine nucleotide exchange factor (GEF), which causes release of GDP from the switch protein. Subsequent binding of GTP, favored by its high intracellular concentration rela-tive to its binding affinity, induces a conformational change to the active form. The principal conformational changes in-volve two highly conserved segments of the protein, termed switch I and switch II, that allow the protein to bind to and activate other downstream signaling proteins (figure 15-7). Conversion of the active form back to the inactive state is

    Inactive kinase

    Into nucleus; binds DNA and activates transcription

    Bound ligand

    Cytosotic inactive non phosphorylated transcription factor

    Act~

  • (a) GTP-bound "on" state l (b) GOP-bound "off" state FIGURE 15-7 Switching mechanism of G proteins. The ability of a G protein to interact with other proteins and thus transduce a signal differs in the GTP-bound "on" state and GOP-bound "off"

    state. (a) In the active "on" state, two domains, termed switch I (green) and switch II (blue), are

    bound to the terminal gamma phosphate of GTP through interactions with the backbone amide

    groups of a conserved threonine and glycine residue. When bound to GTP in this way, lh~ two

    switch domains are in a conformation such that

    they can bind to and thus activate specific downstream effector proteins. (b) Release of the

    gamma phosphate by GTPase-catalyzed hydrolysis

    causes switch I and switch II to relax into a different conformation, the inactive "off" state; in

    this state they are unable to bind to effector proteins. The ribbon models shown here represent

    both conformations of Ras, a monomeric G protein. A similar spring-loaded mechanism switches the alpha subunit in trimeric G proteins

    between the active and inactive conformations by movement of three switch segments. [Adapted from

    I. Vetter and A. Wittinghofer, 2001. SCience 294:1299.]

    Gly-60 Thr-35

    Switch II I I

    y GOP

    able to signal its downstream target proteins: the slower the rate of GTP hydrolysis, the longer the protein remains in the active state. The rate of GTP hydrolysis is often modulated by other proteins. for instance, both GTPase-activating pro-tems (GAP) and regulator of G protein signaling (RGS) pro-teins accelerate GTP hydrolysis. Many regulators of G protein activity are themselves controlled by extracellular signals.

    Two large classes of GTPase switch proteins are used in signaling. Trimeric (large) G proteins directly bind to and are activated by cettain cell-surface receptors. As we will see in Section 15.3, G protein-coupled receptors function as guanine nucleotide-exchange factors (GEFs)-triggering release of GDP and binding of GTP, thus activating the G protein. Monomeric (small) G proteins, such as Ras and various Ras-like proteins, are not bound to receptors but play crucial roles in many pathways that re.gulate cell division and cell motility, as is evidenced by the fact that mutations in genes encoding these G proteins frequently lead to cancer. Other members of both GTPase classes, by switching between GTP-bound "on" and GDP-bound "off" forms, function in protein synthesis, the transport of proteins between the nucleus and the cytoplasm, the formation of coated vesicles and their fusion with target membranes, and rearrangements of the actin cytoskeleton.

    Intracellular "Second Messengers" Transmit and Amplify Signals from Many Receptors

    The binding of ligands ("first messengers") to many cell-surface receptors leads to a short-lived increase (or decrease) in the concentration of certain low-molecular-weight intracellular

    signaling molecules termed second messengers. These, in turn, bind to other proteins, modifying their activity.

    One second messenger used in virtually all metazoan cells is Ca1+ ions. We noted in Chapter 11 that the concen-tration of free Ca2+ in the cytosol is kept very low (< 10 "M) by ATP-powered pumps that continually transport Ca2 out of the cell or into the endoplasmic reticulum (ER). The cyto-solic Ca2+ level can increase from 10- to 100-fold by a signal-induced release of Ca 1 from ER stores or by its import through calcium channels from the extracellular environment; this change can be detected by fluorescent dyes introduced into the cell (see Figure 9-11 ). In muscle, a signal-induced rise in cytosolic Cah triggers contraction (see Figure 17-35) . In endocrine cells, a similar increase in Ca2+ induces exocytosis of secretory vesicles containing hormones, which are thus released into the circulation. In nerve cells, an increase in cytosolic Ca2+ leads to the exocytosis of neurotransmitter-conta ining vesicles (see Chapter 22). In all cells, this rise in cytosolic Ca2+ is sensed by Ca2+ -binding proteins, particu-larly those of the EF hand family, such as calmodulin, all of which contain the helix-loop-helix motif (see Figure 3-9b). The binding of Ca1 to calmodulin and other EF hand pro-teins causes a conformationa l change that permits the pro-tein to bind various target proteins, thereby switchrng their activities on or off (see Figure 3-31 ).

    Another nearly universal second messenger is cyclic AMP (cAMP). In many eukaryotic cells, a rise in cAMP triggers activation of a particular protein kinase, protein kinase A, that in turn phosphorylates specific target proteins to induce specific changes in cell metabolism. In some cells, cAMP

    1S.1 Signal Transduction: From Extracellular Signal to Cellular Response 679

  • 0 FOCUS ANIMATION: Second Messengers in Signaling Pathways NH2 0

    N~N 5 N__. ' N-:7

    0 1;>'), t ~NH

    N---lN ~NH O- CH2 2 1

    CH3 -(CH2 )n - C 0 CH2 PO 2

    ~I

    3

    6 o;03 2

    r-i O=P--0 OH

    0

    3',5'-CyclicAMP (cAMP)

    Activates protein kinase A {PKA)

    ~0~1 H'

    O=P--0 OH

    0

    3',5 '-Cyclic GMP (cGMP)

    Activates protein kinase G {PKG) and opens cation channels in

    rod cells

    FIGURE 15-8 Four common intracellular second messengers. The major direct effect or effects of each compound are indicated below its

    regulates the activity of certain ion channels. The structures of cAMP and three other common second messengers are shown in Figure 15-8. Later in this chapter, we examine the specific roles of second messengers in signa ling pathways ac-tivated by various G protein-coupled receptors.

    Because second messengers such as Ca2 and cAMP dif-fuse through the cytosol much faster than do proteins, they arc employed in pathways where the downstream ta rget is located in an intracellular organelle (such as a secreto ry ves-icle or the nucleus) distant from the plasma membra ne re-ceptor where the messenger is generated.

    Another advantage of second messengers is that they fa-cilitate amplification of an extracellular signal. Activation of a single cell-surface receptor molecule can result in an increase in perhaps thousand!> of cAMP molecules o r Ca2 ions in the cytosol. Each of these, in turn, by activating its target protein affects the activity of multiple downstream proteins. In many signal transduction pathways, amplification is necessary be-cause cell surface receptors are typica lly low-abundance pro-teins, present in only a thousand or so copies per cell. Yet the cellular responses induced by the binding of a relatively small

    FIGURE 15-9 Amplification of an extracellular signal. In this example, binding of a sing le epinephrine molecule to one G protein-coupled receptor molecule induces activation of several molecules of adenylyl cyclase, the enzyme that catalyzes the synthesis of cyclic AMP, and each of these enzyme synthesizes a large number of cAMP molecules, the first level of amplification. Two molecules of cAMP activate one molecule of protein kinase A (PKA), but each activated PKA phosphorylates and activates multip le target proteins. This second level of amplification may involve severa l sequential reactions in which the product of one reaction activates the enzyme catalyzing the next reaction. The more steps in such a cascade, the greater the signal amplification possible.

    0 2

    CH3 -{CH2 )n c - CH

    0 Fatty acyl groups 3

    CH20H Glyc: rol

    1,2-Diacylglycerol (DAG)

    Activates protein kinase C {PKC)

    OH 1 . '

    OH HO / opo 2 'h---3-r 3

    Inositol 1.4,5-trisphosphate

    (IP3 )

    Opens Ca2+ channels in the endoplasmic reticu lu m

    structural formula. Calcium ions (Ca2+) a~d several membrane-bound phosphatidylinositol derivatives also act as second messengers.

    number of hormones to the available receptors often require production of tens o r hundreds of thousand's of activated ef-fector molecules per cell. In the case of G protein-coupled hormone receptors, signal amplification is possible in part be-cause a single receptor can activa te multiple G proteins, each of which in turn activates an effector protein. For example, a single epinephrine-GPCR complex causes activation of up to 100 adenylyl cyclase molecu les, each of which in turn cata-lyzes synthesis of many cAMP molecules during the time it remains in the active state. Two cAMP molecules activate one molecule of protein kinase A that in turn phosphorylate~ and act ivates multiple target product molecules (Figure 15-9). Later in th is chapter, we see how this amplification cascade allows blood levels of epinephrine as low as I o-to M to stim-ulate glycogenolysis (conversion of glycogen to glucose) by the liver and release of glucose into the blood.

    Epinephrine {1o -1o M) Amplification ~t --.... ~

    Adenylyl .6 A /.:::,.

    ~1~ cyclase

    Amplification

    cAMP {10- 6 M) l l l l l Protein 0 0 0 0 0 kinase A

    ~1~ Amplification Activated 0 enzyme ~1~ Amplification Product

    680 CHAPTER 15 Signal Transduction and G Protein- Coupled Receptors

  • .

    .

    KEY CONCEPTS of Section 15.1

    Signal Transduction: From Extracellular Signal to Cellular Response

    All cells communicate through extracellular signals. In unicellular organisms, extracellular signaling molecules reg-ulate interactions between individuals, while in multicellular organisms, they regulate physiology and development.

    External signals include membrane-anchored and secreted proteins or peptides (e.g., vasopressin and insulin), small hy-drophobic molecules (e.g., steroid hormones and thyroxine), small hydrophilic molecules (e.g., epinephrine), gases (e.g., 0 2, nitric oxide), and physical stimuli (e.g., light).

    Binding of extracellular signaling molecules to cell-surface receptors triggers a conformational change in the receptor, which in turn leads to activation of intracellular signal trans-duction pathways that ultimately modulate cellular metabo-lism, function, or gene expression (see Figure 15-1 ).

    Signals from one cell act on distant cells in endocrine sig-naling, on nearby cells in paracrine signaling, or on the sig-naling cell itself in autocrine signaling (see Figure 15-2).

    Protein phosphorylation and de-phosphorylation, cata-lyzed by protein kinases and phosphatases, are employed in virtually all signaling pathways. The activities of kinases and phosphatases are highly regulated by many receptors and signal transduction proteins (see Figures 15-4 and 15-5).

    GTP-binding proteins of the GTPase superfamily act as switches regulating many signal transduction pathways (see Figures 15-6 and 15-7).

    Ca2+ , cAMP, and other nonprotein, low-molecular-weight intracellular molecules (see Figure 15-8) act as "second mes-sengers," relaying and often amplifying the signa l of the "first messenger," that is, the ligand. Binding of ligand to cell-surface receptors often results in a rapid increase (or, occasionally, decrease) in the intracellular concentration of these ions or molecules.

    ' 15.2 Studying Cell-Surface Receptors and Signal Transduction Proteins

    The response of a cell to an external signal depends on the cell's complement of receptors that recognize the signal and the signal transduction pathways activated by those recep-tors. In this section, we explore the biochemical basis for the specificity of receptor-ligand binding, as well as the ability of different concentrations of hgand to activate a pathway. We also examine experimental techniques used to characterize receptor proteins. Many of these methods are also applicable to receptors that mediate endocytosis (see Chapter 14) or cell adhesion (see Chapter 20). We conclude the section with a discussion of techniques commonly used to measure the

    activity of signal transduction components, such as kinases and GTP-binding "switch" proteins.

    The Dissociation Constant Is a Measure of the Affinity of a Receptor for Its Ligand

    Ligand binding to a receptor usually can be viewed as a sim-ple reversible reaction, where the receptor is represented as R, the ligand as L, and the receptor-ligand complex as RL:

    R+L RL (15-1)

    koff is the rate constant for dissociation of a ligand from its re-ceptor, and kon is the rate constant for formation of a receptor-ligand complex from free ligand and receptor.

    At equilibrium, the rate of formation of the receptor-ligand complex is equal to the rate of its dissociation and can be de-scribed by the simple equilibrium-binding equation

    [R LJ K - - -u- RLl (15-2)

    where [R] and [L] are the concentrations of free receptor (that is, receptor without bound ligand) and ligand, respec-tively, at equilibrium, and [RL] is the concentration of the receptor-ligand complex. Kd, the dissociation constant, is a measure of the affinity (or tightness of binding) of the recep-tor for its ligand (see also Chapter 2 ). For a simple binding reaction, Kd = korrlkon The lower k off is relative to ko"' the more stable the RL complex-the tighter the binding-and thus the lower the value of Kd. Another way of seeing this key point is that Ku equals the concentration of ligand at which half of the receptors have a ligand bound when the system is at equilibrium; at this ligand concentration [R] = [RL] and thus, from Equation 15-2, Kd = [L]. The lower the Kd, the lower the ligand concentration required to bind 50 percent of the cell-surface receptors. The Kd for a binding reaction here is essentially equivalent to the Michaelis con-stant Km, which reflects the affinity of an enzyme for its sub-strate (see Chapter 3). Like all equilibrium constants, however, the value of Ku does not depend on the absolute values of koff and k0 n, only on their ratio. In the next section, we learn how Kd values are experimentally determined.

    Hormone receptors are characterized by their high af-finity and specificity for their ligands. Because of their

    high affinity and great specificity for their target hormone, the extracellular, ligand-binding domains of cell c;urface re-ceptors can be converted into powerful drugs. Consider the hormone tumor necrosis factor alpha (TNFa ), which is se-creted by a number of immune system cells. TNfa induces inflammation by recruiting various immune cells to a site of injury or infection; abnormal levels of TN Fa cause the exces-sive inflammation seen in patients with autoimmune diseases

    15.2 Studying Cell-Surface Receptors and Signal Transduction Proteins 681

  • such as the hlistering skin disease psoriasis or the joint disease rheumatoid arthritis. These diseases are being treated with a chimeric "fusion" protein, generated by recombinant DNA, that contains the extracellular domain of a TNFa. receptor fused to the constant (Fe) region of a human immunoglohin (see Figures 3-19 and 23-8). The drug binds tightly to free TNFa. and prevents it from binding to its cell-surface recep-tors and causing inflammation; the fused Fe domain causes the protein to be stable when injected into the body.

    Binding Assays Are Used to Detect Receptors and Determine Their Affinity and Specificity for ligands Usually receptors are detected and measured hy their ahil ity to bind radioactive or fluorescent ligands to intact cells or to cell fragments. Figure 15-10 illustrates such a binding assay for interaction of the red-cell-forming hormone erythropoie-tin (Epo) with Epo receptors that are expressed by recombi-nant DNA in a line of cultured cells. The amounts of radioactive Epo bound to its receptor on growing cells (verti-cal axis) were measured as a function of increasing concen-tration of 12 'I-Iabeled Epo added to the extracellular fluid (horizontal axis). Both the number of ligand-binding sites per cell and the Kd value are easily determined from the specific hinding curve (curve C). Assuming each receptor hinds just one ligand molecule, the total number of ligand-binding sites on a cell equals the number of active receptors per cell. In the example shown in Figure 15-10, the value of Kd is about 1.1 X 1 0 10 M, or 0.1 nM. In other words, an Epo concen-tration of 1.1 X 10 10 M in the extracellular fluid is required for 50 percent of a cell's Epo receptors to have a bound Epo.

    Direct binding assays like the one in rigure 15-10 are feasible with receptors that have a high affinity for their li-gands, such as t.he erythropoietin receptor and the insulin receptor on liver cells (Kd = 1.4 X 10 10 M). However, many ligands, such as epinephrine and other catecholamines, hind to their receptors with much lower affinity. If the Kd for binding is greater than -1 X 10 ., M, a case when the rate constant koff is relatively large compared to k""' then it is likely that during the seconds to minutes required to mea-sure the amount of bound ligand, some of the receptor-hound ligand will dissociate and thus the observed binding values will be systematically too low.

    One way to measure relatively weak binding of a ligand to its receptor is in a comfJetition assay with another ligand that binds to the same receptor with high affinity (low Kd value). In this type of assay, increasing amounts of an unla-beled, low-affinity ligand (the competitor) arc added to a cell sample with a constant amount of the radiolabeled, high-affinity ligand (Figure 15-11 ). Binding of unlabeled competi-tor to the receptor blocks binding of the radioactive ligand to the receptor. The concentration dependence of this com-petition can be used together with the Kd value of the radio-active ligand to calculate the inhibitory constant, K., which is very close to the Kd value for binding of the competitor to the receptor. It is possible to accurately measure the amount

    5.0

    4.5

    Iii 4.0 Qi --------------------c u 3.5

  • 100

    ~ Ol 80 c :0 c

    :0 0 60 0 c Q) .... 0.

    "' 0 40 c 0 .= :0 .r= 20 E

    Competitor concentration (M)

    EXPERIMENTAL FIGURE 15 11 For low-affinity ligands, binding can be detected in competition assays. In this example, the synthetic ligand alprenolol, which binds with high affinity to the epinephrine receptor on liver cells (Kd- 3 x 10 9 M), is used to detect the binding of two low-affinity ligands, the natural hormone epineph-rine (EP) and a synthetic ligand called isoproterenol (IP). Assays are performed as described in Figure 15-10 but in reactions containing a constant amount of [3H) alprenolol and increasing amounts of unlabeled epinephrine or isoproterenol. At each competitor concentra-tion, the amount of bound labeled alprenolol is determined. In a plot of

    Consider for instance the drug isoproterenol, used to treat asthma. Isoproterenol is made by the chemical addition of two methyl groups to epinephrine (see Figure 15-ll, right). Isoproterenol, an agonist of the epinephrine-responsive G protein-coupled receptors on bronchial smooth muscle cells, binds about tenfold more strongly (tenfold lower Kd) than does epinephrine"(see Figure 15-11, left). Because activation of these receptors promotes relaxation of bronchial smooth mus-cle and thus opening of the air passages in the lungs, isopro-terenol is used in treating bronchial asthma, chronic bronchitis, and emphysema. ln contrast, activation of a different type of epinephrine-responsive G protein-coupled receptors on car-diac muscle cells (called [3-,adrenergic receptors) increases the heart contraction rate. Antagonists of this receptor, such as alprenolol and related compounds, are referred to as beta-blockers; such antagonists are used to slow heart contractions in the treatment of cardiac arrhythmias and angina.

    Maximal Cellular Response to a Signaling Molecule Usually Does Not Require Activation of All Receptors

    All signaling systems evolved such that a rise in the level of extracellular signaling molecules induces a proportional re-sponse in the responding cell. For this to happen, the binding affinity (Kd value) of a cell-surface receptor for a signaling molecule must be greater than the norma l (unstimulated)

    CH2 OH CH II I I 3 CH + I O-CH2-CH-CH2-NH2-CH

    CD2~ ~H I

    3

    Alprenolol (API ~

    the inhibition of [3H) alprenolol binding versus epinephrine or isoproterenol concentration, such as shown here, the concentration of the competitor that inhibits alprenolol binding by SO percent approxi-mates the Kd value for competitor binding. Note that the concentra-tions of competitors are plotted on a logarithmic scale. The Kd for binding of epinephrine to its receptor on liver cells is only- 5 X 10 5 M and would not be measurable by a direct binding assay with eHJ epinephrine. The Kd for binding of isoproterenol, which induces the normal cellular response, is more than tenfold lower.

    level of that molecule in the extracellular fluids or blood. We can see this principle in practice by comparing the levels of insulin present in the body and the Kd for binding of insulin to its receptor on liver cells, 1 .4 X l 0 10 M. Suppose, for instance, that the norma l concentration of insulin in the blood is 5 X 10 12 M. By substituting this value and the in-sulin K..1 into Equation 15-2, we can calculate the fraction of insulin receptors with bound insulin

    RL/( RL + Rj)

    at equilibrium as 0.0344; that is, about 3 percent of the total insulin receptors will be bound with insulin. If the insulin concentration rises fivefold to 2.5 X 10 11 M, the number of receptor-hormone complexes will rise proportionately, al-most fivefold, so that about 15 percent of the total receptors will have bound insulin. If the extent of the induced cellular response parallels the number of insulin-receptor complexes, [RLj, as is often the case, then the cellular responses also will increase by about fivefold.

    On the other hand, suppose that the normal concentra-tion of insulin in the blood were the same as the Kd value of 1.4 X 10 10 M; in this case, 50 percent of the total receptors would have a bound insulin. A fivefold increase in the insu-lin concentration to 7 X 10- 10 M would result in 83 percent of all insulin receptors having insulin bound (a 66 percent increase). Thus, in order for a rise in hormone concentration

    15.2 Studying Cell -Su rface Receptors and Signal Transduction Proteins 683

  • Cl c ~Q) - (/)

    1.0

    ~ 0.8 roo. -~ ~ ~ ~ 0.6 E~

    - :J 0~ c u 0.4 0 ~ ; 0 u ~

    LL 0.2

    0

    Physiological response

    :yt:1 Ligand concentration : I for 50% physiological response I I

    I 1 1/ Kd for ligand binding I I

    1 2 3 4

    Relative concentration of ligand

    EXPERIMENTAL FIGURE 15-12 The maximal physiological response to an external signal occurs when only a fraction of the receptors are occupied by ligand. For signaling pathways that exhibit this behavior, plots of the extent of ligand binding to the receptor and of physiological response at different ligand concentrations differ. In the example shown here, SO percent of the maximal physiological response is induced at a ligand concentration at which only 18 percent of the receptors are occupied. Likewise, 80 percent of the maximal response is induced when the ligand concentration equals the Kd value, at which SO percent of the receptors are occupied.

    to cause a proportional increase in the fraction of receptors with bound ligand, the normal concentration of the hor-mone must be well below the Kd value.

    In general, the maximal cellular response to a particular ligand is induced when much less than 100 percent of its re-ceptors are bound to the ligand. This phenomenon can be revealed by determining the extent of the response and of receptor-ligand binding at different concentrations of ligand (Figure 15-12). For example, a typical red blood (erythroid) progenitor cell has - 1000 surface receptors for erythropoi-etin, the protein hormone that induces these cells to prolifer-ate and differentiate into red blood cells. Because only 100 of these receptors need to bind erythropoietin to induce divi-sion of a progenitor cell, the ligand concentration needed to induce 50 percent of the maximal cellular response is pro-portionally lower than the Kd value for binding. In such cases, a plot of the percentage of maximal binding versus li-gand concentration differs from a plot of the percentage of maximal cellular response versus ligand concentration.

    Sensitivity of a Cell to External Signals Is Determined by the Number of Surface Receptors and Their Affinity for Ligand

    Because the cellular response to a particular signaling mole-cule depends on the number of receptor-ligand complexes, the fewer receptors present on the surface of a cell, the less sensi-tive the cell is to that ligand. As a consequence, a higher ligand concentration is necessary to induce the physiological re-sponse than would be the case if more receptors were present.

    To illustrate the important relationship between receptor number and ligand sensitivity, let's extend our example of a

    typical erythroid progenitor cell. The Kd for binding of erythropoietin (Epo) to its receptor is about 10- 10 M. As we noted above, only 10 percent of the -1000 erythropoietin receptors on the surface of a cell must be bound to ligand to induce the maximal cellular response. We can determine the ligand concentration, [L], needed to indllce the maximal re-sponse by rewriting Equation 15-2 as follows:

    (15-3)

    where RT = [R] + [RL], the total number of receptors per cell. If the total number of Epo receptors per cell, RT, is 1000, Kd is 10 10 M, and [RL] is 100 (the number of Epa-occupied receptors needed to indoce the maximal response), then an Epo concentration ([L]) of 1.1 X 10-ll M will elicit the maximal response. If the total number of Epo receptors (RT) is reduced to 200 per cell, then a ninefold-higher Epo concentration (10 10 M) is required to occupy 100 receptors and induce the maximal response. Clearly, therefore, a cell's sensitivity to a signaling molecule is heav,ily influenced by the number of receptors for that ligand that are present as well as the Kd.

    A Epithelial growth factor (EGF), as its name implies, 11..11 stimulates the proliferation of many types of epithelial cells, including those that line the ducts of the mammary gland. In about 25 percent of breast cancers, the tumor cells produce elevated levels of one particular EGF receptor called HER2. The overproduction of HER2 makes the cells hyper-sensitive to ambient levels of EGF that normally are too low to stimulate cell proliferation; as a consequence, growth of these tumor cells is inappropriately stimulated by EGF. We will see in Chapter 16 that an understanding of the role of HER2 in certain breast cancers led to development of mono-clonal antibodies that bind HER2 and thereby block signal-ing by EGF; these antibodies have proved useful in treatment of these breast cancer patients.

    The HER2-breast cancer connection vividly demon-strates that regulation of the number of receptors for a given signaling molecule expressed by a cell plays a key role in di-recting physiological and developmental events. Such regula-tion can occur at the levels of transcription, translation, and post-translational processing or by controlling the rate of re-ceptor degradation. Alternatively, endocytosis of receptors on the cell surface can sufficiently reduce the number present such that the cellular response is term ina ted. As we discuss in later sections, other mechanisms can reduce a receptor's affinity for ligand and so reduce the cell's response to a given concentration of ligand. Thus reduction in a cell's sensitivity to a particular ligand, called desensitization, can result from various mechanisms and is critica l to the ability of cells to respond appropriately to external signals.

    684 CHAPTER 15 Signal Transduction and G Protein-Coupled Receptors

  • .

    Receptors Can Be Purified by Affinity Techniques

    In order to fully understand how receptors function, it IS necessary to purify them and analyze their biochemical properties. Determining their molecular structures with and without a bound ligand, for instance, can elucidate the con-formational changes that occur on ligand binding that acti-vate downstream signal transduction proteins. But this can be challenging. A "typical" mammalian cell has 1000 to 50,000 copies of a single type of cell-surface receptor. This may seem like a large number, but when you consider that this same cell contains -10 10 total protein molecules and -106 proteins in the plasma membrane alone, you realize that these receptors constitute only 0.1 to 5 percent of plasma-membrane proteins. This low abundance compli-cates the isolation and purification of cell-surface receptors. Purification of receptors is also difficult because these inte-gral membrane proteins first must be solubilized from the membrane with a non-ionic detergent (see Figure 10-23) and then separated from other cellular proteins.

    As we saw with the Epo receptor discussed earlier, recom-binant DNA techniques can be used to generate cells that ex-press large amounts of these proteins. But even when recombinant DNA techniques are used to generate cells that express receptors in large amounts, special techniques arc nec-essary to isolate and purify them from other membrane pro-teins . One technique often used in purifying cell-surface receptors that retain their ligand-binding ability when solubi-lized by detergents is similar to affinity chromatography using antibodies (see Figure 3-38c). To purify a receptor by this tech-nique, a ligand for the receptor of interest, rather than an anti-body, is chemically linked to the beads used to form a column. A crude, detergent-solubilized preparation of membrane pro-teins is passed through the column; only the receptor binds, while other proteins are washed away. Passage of an excess of the soluble ligand through the column causes the bound re-ceptor to be displaced from the beads and eluted from the column. In some cases, a receptor can be purified as much as 100,000-fold in a single affinity-chromatographic step.

    lmmunoprecipitation Assays and Affinity Techniques Can Be Used to Study the Activity of Signal Transduction Proteins

    Following ligand binding, receptors activate one or more sig-nal transduction proteins that, in turn, can affect the activity of multiple effector proteins (see Figure 15-1 ); to understand a signaling cascade requires the researcher to be able to quantify the activity of these signal transduction proteins. Kinases and GTP-binding proteins are found in many signal-ing cascades, and in this section we describe several assays used for measuring their activities.

    lmmunoprecipitation of Kinases Kinases function in virtu-ally all signaling pathways, and typical mammalian cells contain a hundred or more different kinases, each of which

    is highly regulated and can phosphorylate many target pro-teins. Immunoprecipitation assays are frequently used to measure the activity of a particular kinase in a cell extract. In one version of the method, an antibody specific for the de-sired kinase is first reacted with small beads coated with Pro-tein A; this causes the antibody to bind to the beads via its Fe segment (see Figure 9-29). The beads are then mixed with a preparation of cell cytosol or nucleus, then recovered by cen-trifugation and washed extensively with a salt solution to remove weakly bound proteins that arc unlikely to be bind-ing specifically to the antibody. Thus only cell proteins that specifically bind to the antibody-the kinase itself and pro-teins tightly bound to the kinase-are present on the beads. The beads are then incubated in a buffered solution with a substrate protein and -y-e2Pl ATP, where only the-y phos-phate is labeled. The amount of [uP] transferred to the sub-strate protein is a measure of kinase activity and can be quantified either by polyacrylamide gel electrophoresis fol-lowed by autoradiography (see Figure 3-36) or by tmmuno-precipitation with an antibody specific for the substrate followed by counting the radioactivity in the immunopre-cipitate. By comparing extracts from cells before and after ligand addition, for example, one can readily determine whether or not a particular kinase is activated in the signal transduction pathway triggered by that ligand.

    We noted that many proteins can be phosphorylated by ~everal different kinases, usually on different serine, threo-nine, or tyrosine residues. Thus it is important to measure the extent of phosphorylation of a single amino acid side chain in a specific protein, say before and after hormone stimulation. Antibodies play a crucial role in detecting such phosphorylation events. To generate an antibody that can recognize a specific phosphorylated amino acid in a specific protein, one first chemically synthesizes an approximately 15 amino acid peptide that has the amino acid sequence sur-rounding the phosphorylated amino acid of the specific pro-tein but where a phosphate group has been chemically linked to the desired serine, threonine, or tyrosine. After coupling this peptide to an adjuvant to increase its immunogeniciry, it is used to generate a set of monoclonal antibodies (see Figure 9-6). One then selects a particular monoclonal antibody that reacts only with the phosphorylated, but nor the nonphos-phorylated peptide; such an antibody generally will bind to the parent protein only when this specific amino acid is phosphorylated. This specificity is possible because the anti-body binds simultaneously to the phosphorylated amino acid and to side chains of adjacent amino acids. As an example of the use of such antibodies, Figure 15-13 shows that three signal transduction proteins in red-cell progenitors become phosphorylated on specific amino acid residues within 10 minutes of stimulation by varying concentrations of the hor-mone erythropoietin; phosphorylation increases with Epo concentration and is the first step in triggering the differen-tiation of these cells into red blood cells.

    Pulldown Assays of GTP-Binding Proteins We've seen that the GTPase superfamily of intracellular-switch proteins cycle

    15.2 Studying Cell-Surface Receptors and Signal Transduction Proteins 685

  • I. Ill

    I ....., __ ......,, --- ,......, .

    I -~-- 1

    1---

    Epo (U/ml)

    anti- Stat5

    anti-Stat5

    anti-Akt

    anti-Akt

    anti- p42/p44

    anti-p42/p44

    :nil Activation of three signal transduction proteins by phosphorylation. Mouse erythrocyte progenitor cells were treated for 1 0 min with different concentrations of the hormone erythropoietin (Epo). Extracts of the cell were analyzed by Western blotting with three different antibodies specific for the phosphorylated forms of three signal transduction proteins and three that recognize a nonphosphorylated segment of amino acids in the same protein. The data show that with increasing concentration of Epo, the three proteins become phosphorylated. Treatment with 1 unit Epo per ml is sufficient to maximally phosphorylate and thus activate all three pathways. Stat 5 = transcription factor phosphorylated on tyrosine 694; Akt = kinase phosphorylated on serine 473; p42/p44 =

    p42/p44 MAP kinase phosphorylated on threonine 202 and tyrosine 204. [Courtesy Jing Zhang; Zhang et al., 2003, Blood 1 02:3938.]

    between an active ("on") form with bound GTP that modu-lates the activity of specific target proteins and an inactive ("off") form with bound GDP. The principal assay for mea-suring activation of this class of proteins takes advantage of the fact that each such protein has one or more targets to

    TA u A pull-down assay shows that the small GTP-binding protein Rac1 is activated by platelet-derived growth factor (PDGF). Like other small GTPases, Racl regulates molecular events by cycling between an inactive GOP-bound form and an active GTPbound form. In its active (GTP-bound) state, Racl binds specifically to the p21-binding domain (PBD) of p21-activated protein kinase (PAK) to control downstream signaling cascades. (a) Assay principle: the Rae-binding PBD domain is generated by recombinant DNA techniques and attached to agarose beads, then mixed with cell extracts (step 0 ). The beads are recovered by centrifugation (step f)) and the amount of GTP-bound Racl is quantified by Western blotting using an anti-Racl antibody (step Ill. (b) Western blot showing activation of Rac1 after treatment of hematopoietic stem cells for 1 min with the hormone platelet-derived growth factor (PDGF). A Western blot for actin serves as a control that the same amount of total protein is loaded on each lane of the gel. [(a) After Cell Biolabs Inc.; (b) from G. Ghiaur et al., 2006, Blood 1 08:2087-2094.]

    which it binds only when it has a bound GTP; the target pro-tein usua lly has a specific binding domain that binds to the switch segments of the GTP-binding protein. Pull-down as-says used to quantify the activation of a specific GTP-binding protein are similar to immunoprecipi tations except that the specific binding domain of the target protein is immobilized on small beads (Figure 15-14). The beads are mixed with a cell extract and then recovered by centrifugation; the amount of the GTP-binding protein on the beaJ~ i~ quantified by Western blotting. The example in Figure 15-14 shows tha t

    {a) Assay Priciple

    Lysate # 1 (Low GTP-bound

    Rae content)

    Lysate# 1 {High GTP-bound

    Rae content)

    ~ .I GOP-bound Rae GTP-bound Rae I C. PAK1 PBD agarose J I

    1 D PAKl PBD agarose ! is added -=::::. I

    '='' ; ~"{ ~ = J: ' I. l l ) " )

    1 f) Mixing and 1 centifugat ion --:- {pull-down of

    GTP-bound Rae)

    1:~

    ~ ,p

    {b) Western blot of hematopoietic stem cells before and after treatment w ith PDGF

    PDGF 0 1'

    Rae GTP (visualized w ith ant i-Rae antibody)

    ............ ~ actin {visualized with ~ anti-actin antibody)

    686 CHAPTER 15 Signal Transduction and G Protein- Coupled Receptors

  • the fraction of the small GTPase Racl that has a bound GTP increases markedly after stimulation by the hormone platelet-derived growth factor (PDGF), indicating that Racl is a sig-nal transduction protein activated by the PDGF receptor.

    KEY ("ni\JriEPTS nf c;.oction 1 5-2

    Studying Cell-Surface Receptors and Signal Transduction Proteins

    The concentration of ligand at which half the ligand's re-ceptors are occupied, the Kd, can be determined experimen-tally and is a measure of the affinity of the receptor for the ligand (see Figure 15-1 0) .

    Because of receptors' high affinity for their target ligand, the extracellular domain of receptors can be used as a drug to reduce the level of free hormone.

    The maximal response of a cell to a particular ligand gener-ally occurs at ligand concentrations at which less than 100 percent of its receptors are bound to ligand (see Figure 15-12).

    Affinity chromatography techniques can be used to purify receptors even when they are present in low abundance.

    Immunoprecipitation assays using antibodies specific for protein kinases can measure kinase activity. lmmunoprecipi-tation assays using antibodies specific for phosphorylated peptides can measure phosphorylation of a specific amino acid on any desired protein within a cell (see Figure 15-13).

    Pull-down assays using the protein-binding domain of a tar-get protein can be used to quantify activation of a GTP-binding protein within a cell (see Figure 15-14).

    15.3 G Protein-Coupled Receptors: Structure and Mechanism

    As noted above, perhaps the most numerous class of recep-tors are the G protein-coupled receptors (GPCRs). In hu-mans, GPCRs are used to detect and respond to many different types of signals,)ncluding neurotransmitters, hor-mones involved in glycogen and fat metabolism, and even photons of light. All GPCR signal transduction pathways share the following common elements: ( 1) a recepror that contains seven membrane-spanning a helixes; (2) a coupled trimeric G protein, which functions as a switch by cycling between active and inactive forms; (3) a membrane-bound effector protein; and (4) proteins that participate in feedback regulation and desensitization of the signaling pathway. A second messenger also occurs in many GPCR pathways. GPCR pathways usually have short-term effects in the cell by quickly modifying existing proteins, either enzymes or ion channels. Thus these pathways allow cells to respond rapidly to a variety of signals, whether they are environmental stim-uli such as light or hormonal stimuli such as epinephrine.

    In this section, we discuss the basic structure and mecha-nism of GPCRs and their associated trimeric G proteins. In Sections 15.4 through 15.6, we describe GPCR pathways that activate several different effector proteins.

    All G Protein-Coupled Receptors Share the Same Basic Structure

    All G protein-coupled receptors have the same orientation in the membrane and contain seven transmembrane a-helical re-gions (H1-H7), four extracellular segments, and four cyto-solic segments (Figure 15-15). Invariably theN-terminus is on the exoplasmic face and the C-terminus is on the cytosolic face of the plasma membrane. The carboxyl-terminal segment (C4 ), the C3 loop, and, in some receptors, also the C2 loop are involved in interactions with a coupled trimeric G protein. Many subfamilies of G protein-coupled receptors have been conserved through evolution; members of these subfamilies are especially similar in amino acid sequence and structure.

    G protein-coupled receptors are stably anchored in the hydrophobic core of the plasma membrane by many hy-drophobic amino acids on the outer surfaces of the seven membrane-spanning segments. One group of G protein-coupled receptors whose structure is known in molecular detail is the P-adrenergic receptors, which bind hormones such as epinephrine and norepinephrine (Figure 15-16). In these and many other receptors, segments of several membrane-embedded a helices and extracellular loops form the ligand binding site that is open to the exoplasmic surface. The an-tagonist cyanopindolol, shown in Figure 15-16, binds with a much higher affinity to the receptor than most agomsts, and the receptor-ligand complex has been crystallized and its structure determined. Side chains of 15 amino acids located in four transmembrane a helices and extracellular loop 2 make

    Exterior

    Cytosol

    FIGURE 15-15 General struct ure of G protein-coupled receptors. All receptors of this type have the same orientation in the membrane and contain seven transmembrane a-helical regions (Hl-H7), four extracellular segments (E l-E4), and four cytosolic segments (Cl-(4). The carboxyl-terminal segment (C4), the C3 loop, and, in some receptors, also the C2 loop are involved in interactions with a coupled trim eric G protein.

    15.3 G Protein-Coupled Receptors : Structure and Mechanism 687

  • FIGURE 15 16 St ructure of the turkey Jl1-adrenergic receptor complexed with the antagonist cyanopindolol. (a) Side view

    showing the approximate location of the membrane phospholipid bilayer. A ribbon representation of the receptor structure is in rainbow coloration (N-terminus, blue; (-terminus, red), with cyanopindolol as a gray space-filling model. The extracellu lar loop 2 (E2) and cytoplasmic loops 1 and 2 ((1, C2) are labeled. (b) View from external face showing a close-up of the ligand-binding pocket that is formed by amino acids in helices 3, 5, 6, and 7, as well as extracellular loop 2, located between helite~ 4 and 5. Cyanopindolol atoms are colored grey (carbon), blue (nitrogen), and red (oxygen). The ligand-binding pocket comprises 15 side chains from amino acid residues in four transmembrane a-helices and extracellular loop 2. As examples of specific binding interactions, the positively charged N atom in the amino group found both in cyanopindolol and in epinephrine forms an ionic bond with the carboxylate side chain of aspartate 121 (0121) in helix 3 and the carboxylate of asparagine 329 (N32~ in helix 7. [From T. Wayne et al., 2008, Nature 454:486.]

    noncovalent contacts with the ligand. The amino acids that form the interior of different G protein-coupled receptors are diverse, allowing different receptors to bind very different small molecu les, whether they are hydrophilic such as epi-nephrine or hydrophobtc such as many odorants.

    While all G protein-coupled receptors share the same basic structure, different subtypes of GCPRs can bind the same hormone, with different cellular effects. To illustrate the versatility of these receptors, we will consider the set of G protein-coupled receptors for epinephrine found in different types of mammalian cells. The hormone epinephrine is par-ticularly important in mediating the body's response to stress, also known as the fight-or-flight response. During moments of fear or heavy exercise, when tissues may have an increased need to catabofize glucose and fatty acids to produce ATP, epinephrine signals the rapid breakdown of glycogen to glu-cose in the liver and of triacylglycerols to fatty acids in adi-pose (fat) cells; within seconds these principal metabolic fuels are supplied to the blood. In mammals, the liberation of glu-cose and fatty acids is triggered by binding of epinephrine (or its derivative norepinephrine) to [3-adrenergic receptors on the surface of hepatic (liver) and adipose cells.

    Epinephrine has other bodily effects as well. Epinephrine bound to [3-adrenergic receptors on heart muscle cells, for example, increases the contraction rate, which increases the blood supply to the tissues. In contrast, epinephrine stimula-tion of [3-adrenergic receptors on smooth muscle cells of the intestine causes them to relax. Another type of epinephrine GPCR, the a-adrenergtc receptor, is found on smooth mus-cle cells lining the blood vessels in the intestinal tract, skin, and kidneys. Binding of epinephrine to these receptors causes the arteries to constrict, cutting off circulation to these or-gans. These diverse effects of epinephrine help orchestrate integrated responses throughout the body all directed to a common end: supplying energy to major locomotor muscles, while at the same time diverting it from other organs not as crucial in executing a response to bodily stress.

    (a) ~-adrenergic receptor

    Exoplasmic face

    -ooc Cytosolic face

    (b) View from exoplasmic face

    688 CHAPTER 15 Signal Transduction and G Protein-Coupled Receptors

    C2

  • Ligand-Activated G Protein- Coupled Receptors Catalyze Exchange of GTP for GOP on the a Subunit of a Trimeric G Protein Trimeric G proteins contain three subunits designated a , 13, and 'Y Both the G" and G'Y subunits are linked to the mem-brane by covalently attached lipids. The 13 and 'Y subunits are always bound together and are usually referred to as the G~h subunit. In the resting state, when no ligand is bound to the receptor, the

  • from its a subunit, will sometimes transduce a signal by in-teracting with an effector protein.

    The active G":GTP state is short-lived because the bound GTP is hydrolyzed to GDP in minutes, catalyzed by the intrinsic GTPase activity of the G., subunit (see Figure 15-17, step DJ). The conformation of the G" thus switches back to the inactive G,:GDP state, blocking any further activation of effector pro-teins. The rate of GTP hydrolysis is sometimes further en-hanced by binding of the G"GTP complex to the effector; the effector thus functions as a GTPase-activating protein (GAP). This mechanism significantly reduces the duration of effector activation and avoids a cellular overreaction. In many cases, a second type of GAP protein called a regulator of G protein signaling (RGS) also accelerates GTP hydrolysis by the G" sub-unit, further reducing the time during which the effector re-mains activated. The resulting G.,GDP quickly reassociates with G~"Y and the complex becomes ready to interact with an activated receptor and start the process all over again. Thus the GPCR signal transduction system contains a built-in feedback mechanism that ensures the effector protein becomes activated only for a few seconds or minutes following receptor activa-tion; continual activation of receptors via ligand binding to-gether with subsequent activation of the corresponding G protein is essential for prolonged activation of the effector.

    Early evidence supporting the model shown in figure 15-17 came from studies with compounds called GTP analogs that are structurally similar to GTP and so can bmd to Gu subunits

    as well as GTP does but cannot be hydrolyzed by the intrinsic GTPase. In some of these compounds, the P-0-P phosphodies-ter linkage connecting the [3 and 'Y phosphates of GTP is re-placed by a nonhydrolyzable P-CHrP or P-NH-P linkage. Addition of such a GTP analog to a plasma membrane prepara-tion in the presence of an agonist for a particular receptor re-sults in a much longer-lived activation of the G protein and its associated effector protein than occurs with GTP. In this ex-periment, once the nonhydrulyzable GTP analog is exchanged for GDP bound to G", it remains permanently bound to Ga. Because the GaGTP-analog complex is as functional as the normal G.,GTP complex in activating the effector protein, the effector remains permanently active.

    GPCR-mediated dissociation of trimeric G proteins can be detected in living cells. These studies have exploited the phe-nomenon of fluorescence energy transfer, which changes the wavelength of emitted fluorescence,when two fluorescent pro-teins interact (see Figure 9-22). Figure 15-18 shows how this experimental approach has demonstrated the dissociation of the G"G~"Y complex within a few seconds of ligand addition, providing further evidence for the model of G protein cycling. This general experimental approach can be used to follow the formation and dissociation of other protein-protein complexes in living cells.

    ror many years, it was impossible to determine the struc-ture of the same GPCR in the active and inactive states. This has now been accomplished with the [3-adrenergic receptor (as

    0 PODCAST: Activation of G Proteins Measured by Fluorescence Resonance Energy Transfer (FRET) (a)

    cAMP 0

    (lf1 . Inactive

    D /~U~( ~

    (b)

    /~ gL_)

    Fluorescence l - P 527 nm ( ellow) Fluorescence Y energy

    / F Fluorescence l transfer Excitation light

    440 nm 490 nm Excitation light (cyan) 440 nm

    EXPERIMENTAL FIGURE 15-18 Activation of G proteins occurs within seconds of ligand binding in amoeba cells. in the amoeba Dictyostelium discoideum cell, cAMP acts as an extracellular signaling molecule and binds to a G protein- coupled receptor; it is

    not a second messenger. Amoeba cells were transfected with genes

    encoding two fusion proteins: a Ga fused to cyan fluorescent protein (CFP), a mutant form of green fluorescent protein (GFP), and a G~ fused

    to another GFP variant, yellow fluorescent protein (YFP). CFP normally fluoresces 490-nm light; YFP, 527-nm light. (a) When CFP and YFP are

    nearby, as in the resting G .. G~~ complex, fluorescence energy transfer can occur between CFP and YFP (left). As a result, irradiation of resting

    cells with 440-nm light (which directly excites CFP but not YFP) causes

    0

    Time (s)

    emission of 527-nm (yellow) light. characteristic ofYFP. However, if

    ligand binding leads to dissociation of the G" and G~1 subunits, then fluorescence energy transfer cannot occur. In this case, irradiation

    of cells at 440 nm causes emission of 490-nm light (cyan) characteristic of CFP (right). (b) Plot of the emission of yellow light (527 nm) from a

    single transfected amoeba cell before and after addition of extracellu-lar cyclic AMP (arrow), the ligand for the G protem-coupled receptor

    in these cells. The drop in yellow fluorescence, which results from the dissociation of the G,.-CFP fusion protein from the G~-YFP fusion protein, occurs within seconds of cAMP addition. [Adapted from

    C. Janetopoulos et al., 2001, Science 291 :2408.]

    690 CHAPTER 15 Signal Transduction and G Protein-Coupled Receptors .'

  • . (a) Side view p-adrenergic receptor

    Exterior

    Membrane

    Cytosol

    TM5 ~-

    (b) View from cytosolic surface

    (c) P-adrenergic receptor

    Exterior

    Membrane

    Cytosol

    Gas

    J GTP~

    well as with rhodopsin, discussed in Section 15.4 ). The seven membrane-embedded a helices of the ~-adrenergic receptor completely surround a central segment to which an agonist or antagonist is noncovalently bound (Figure 15- 19). Binding of an agonist to the receptor induces a major conformational

    FIGURE 15-19 Structure of the j3-adrenergic receptor in the inactive and active states and with its associated trim eric G protein, G us (a) Comparison of the three-dimensional structures of the activated [3-adrenergic receptor (gold) bound to a strong agonist and the inactive receptor (purple) bound to an antagonist. (b) View from the cytosolic surface. Note the major changes seen in the conforma tions of the intracellular domains of transmembrane helices 5 (TMS) and 6 (TM6). In the active state, TMS is extended by two helical turns, whereas TM6 is moved outward by 1.4 nm. (c) The overall structure of the active receptor complex shows the adrenergic receptor (gold) bound to an agonist (black and red spheres) and engaged in extensive

    interactions with a segment of G0 , (purple). G.,, together with G~ (green) and Gy (red) constitute the heterotrimeric G protein G,. [After S. Rasmussen et al., 2011, Nature 476:387-390.]

    change (Figure 15-19a) in which there are substantial move-ments of transmembrane helices 5 and 6 and changes in the structure of the C3 loop; together these create a surface that can now bind to a segment of the Gm subunit (Figure 15-19b).

    X-ray crystallographic studies of the complex of activated receptor and G, have also revealed how the subunits of a G protein interact with each other and provided clues about how binding of GTP leads to dissociation of the G('l from the Gp-y subunit. As revealed in the structural model in Figure 15-19b, a large surface of Ga GDP interacts with the G13 sub-unit; part of this surface is located in the aN alpha helix in theN-terminal segment of G('IGDP. Note that G" directly contacts Gil but not G'Y. Binding of theN-terminal alpha-helical segments aN and aS of the Ga, protein to transmembrane helices 5 and 6 of the activated receptor (figure 15- J9b) will, as with other G proteins, be followed by opening of the G" subunit, eviction of the bound GDP, and its replacement with GTP; this is immediately followed by conformational changes within switches I and II that disrupt the molecular interac-tions between Ga and G13-y, leading to their dissociation.

    Different G Proteins Are Activated by Different GPCRs and In Turn Regulate Different Effector Proteins

    All effector proteins in GPCR pathways are either mem-brane-bound ion channels or membrane-bound enzymes that catalyze formation of the second messengers shown in Figure 15-8. The variations on the theme of GPCR signaling that we examine in Sections 15.4 through 15.6 arise because multiple G proteins are encoded in eukaryotic genomes. At last count, humans have 2 1 different G('l subunits encoded by 16 genes, several of which undergo alternative spl icing; six G13 subunits; and 12 G'Y subunits. So far as is known, the dif-ferent Gll-v subunits are essentia lly interchangeable in their functions, while the different G('l subunits afford the various G proteins their specificity. Thus we can refer to the entire three-subunit G protein by the name of its alpha subunit.

    Table 15-"1 summarizes the functions of the major classes o f G proteins with different Gu subunits. For example, the different types of epinephrine receptors mentioned previously

    15.3 G Protein-Coupled Receptors: Structure and Mechanism 691

  • TABLE 1 S-1 Major Classes of Mammalian Trimeric G Proteins and Their Effectors

    Ga Class Associated Effector

    Adenylyl cyclase

    GQI

    2nd Messenger

    cAMP (increased)

    cA.\1P (decreased)

    Receptor Examples

    13-Adrenergic (epinephrine) receptor; receptors for glucagon, serotonin, vasopressin

    Adenylyl cyclase K+ channel (G~1 activates effector)

    Change in membrane potential arAdrenergic receptor Muscarinic acetylcholine receptor

    Adenylyl cyclase cAMP (increased) Odorant receptors in nose

    Phospholipase C IP3, DAG (increased) a 1-Adrenergic receptor

    G"o Phospholipase C IP3, DAG (increased ) Acetylcholine receptor in endothelial cells

    cGMP phosphodiesterase cGMP (decreased) Rhodopsin (light rece'ptor) in rod cells

    ,. A g1ven c. subclass may be assoc1ared w1rh more rhan one cffecror protein. To dare, only one major C,,_ has been identified, bur multiple C.4

    and C,. proteins have been described. Effector proteins commonly are regula red by C. bur in some cases by C~) or rhe combined acrion of G. and G~) IP3 = inositol 1,4,5-rnsphosphare; DAG = 1,2-dmcylglycerol. SOURCE~: See L. B1rnbaumer, 1992, Cell 71 :1069; Z. Farfel er al., 1999, New F11g. ]. Med. 340: 1012; and K. Pierce cr al., 2002, Nature Reu. Mol. Cell Bioi. 3:639.

    are coupled to different G" subunits that influence effector proteins differently and so have distinct effects on cell behav-ior in a target cell. Both subtypes of 13-adrenergic receptors, termed 13 1 and 132, arc coupled to a stimulatory G protein (G.) whose alpha subunit (Gnsl activates a membrane-bound ef-fector enzyme called adenylyl cyclase. Once activated, this enzyme catalyzes synthesis of the second messenger cAMP. In contrast, the a 2 subtype of 13-adrenergic receptor is coupled to an inhibitory G protein (G,) whose alpha subunit G"' inhibits adenylyl cyclase, the same effector enzyme associated with 13-adrenergic receptors. The Guq subunit, which is coupled to the a 1-adrenergic receptor, activates a different effector en-zyme, phospholipase C, which generates two other second messengers, DAG and IP3 (see Figure 15-8). Examples of sig-naling pathways that use each of the Gn subunits listed in Table 15-1 are described in the following three sections.

    .. Some bacterial toxins contain a subunit that penetrates the plasma membrane of target mammalian cells and in

    the cytosol catalyzes a chemical modification on Gn proteins that prevents hydrolysis of bound GTP to GDP. For example, toxins produced by the bacterium Vibrio cholera, which causes cholera, or certain strains of E. coli, modify the G,,. protein in intestinal epithelial cells. As a result, Ga, remains in the active state, continuously activating the effector adenylyl cyclase m the absence of hormonal stimulation. The resulting excessive rise in intracellular cAMP leads to the loss of elec-trolytes and water into the intestinal lumen, producing the watery diarrhea characteristic of infection by these bacteria. The toxin produced by Bordetella pertussis, a bacterium that commonly infects the respiratory tract and causes whooping cough, catalyzes a modification of Gni that prevents release of bound GDP. As a result, G ... i is locked in the inactive state,

    reducing the inhibition of adenylyl cyclase. The resulting in-crease in cAMP in epithelial cells of the airways promotes loss of fluids and electrolytes and mucus secretion.

    KEY CONCEPTS of Section 1 5.3

    G Protein-Coupled Receptors: Structure and Mechanism

    G protein-coupled receptors (GPCRs) are a large and di-verse family with a common structure of seven membrane-spanning a helices and an internal ligand-binding pocket that is specific for ligands (see Figures 15-15 and 15-16).

    GPCRs can have a range of cellular effects depending on the subtype of receptor that binds ligand. The hormone epi-nephrine, for example, which mediates the fight-or-flight response, binds to multiple subtypes of GPCRs in multiple cell types, with varying physiological effects.

    GPCRs are coupled to trimeric G proteins, which contain three subunits designated a, 13, and -y. The Ga subunit is a GTPase switch protein that alternates between an active ("on") state with bound GTP and inactive ("off" ) state with GDP. The "on" form separates from the 13 and -y subunits and activates a membrane-bound effector. The 13 and-y sub-units remain bound together and only occasionally trans-duce signals (see Figure 15-17) .

    Ligand binding causes a conformational change in certain membrane-spann ing helices and intracellular loops of the GPCR, allowing it to bind to and function as a guanine nu-cleotide exchange factor (GEF) for its coupled Ga subunit,

    692 CHAPTER 15 Signal Transduction and G Protein- Coupled Receptors

  • catalyzing dissociation of GDP and allowing GTP to bind. The resulting change in conformation of switch regions in Ga causes it to dissociate from the G 13'Y subunit and the recep-tor and interact with an effector protein (see Figure 15-17).

    Fluorescence energy-transfer experiments demonstrate receptor-mediated dissociation of coupled Ga and G13'Y sub-units in living cells (see Figure 15-18).

    The effector proteins activated (or ina~tivated) by trimeric G proteins are either enzymes that form second messengers (e.g., adenylyl cyclase, phospholipase C) or ion channels (see Table 15-1 ). In each case, it is the Ga subunit that determines the function of the G protein and affords its specificity.

    15.4 G Protein-Coupled Receptors

    That Regulate lon Channels

    One of the simplest cellular responses to a signal is the open-ing of ion channels essential for transmission of nerve im-pulses. Nerve impulses are essential to the sensory perception of environmental stimuli such as light and odors, to transmis-sion of information to and from the brain, and to the stimula-tion of muscle movement. During transmission of nerve impulses, the opening and closing of ion channels cause!> changes in the membrane potential. Many neurotransmitter receptors are ligand-gated ion channels, which open in re-sponse to binding of a ligand. Such receptors include some types of glutamate, serotonin, and acetylcholine receptors, including the acetylcholine receptor found at nerve-muscle synapses. Ligand-gated ion channels that function as neu-rotransmitter receptors are covered in Chapter 22.

    Many neurotransmitter receptors, however, are G protein-coupled receptors whose effector proteins are Na.,. or K'" chan-nels. Neurotransmitter binding to these receptors causes the associated ion channel to open or close, leading to changes in the membrane potential. Still other neurotransmitter receptors, as well as odorant receptors in the nose and photoreceptors in the eye, are G protein-coupled receptors that indirectly modu-late the activity of ion chapnels via the action of second mes-sengers. In this section, we consider two G protein-coupled receptors that illustrate the direct and indirect mechanisms for regulating ion channels: the muscarinic acetylcholine receptor of the heart and the light-activated rhodopsin protein in the eye.

    Acetylcholine Receptors in the Heart Muscle Activate a G Protein That Opens K+ Channels

    Muscarinic acetylcholine receptors are a type of GPCR found in cardiac muscle. When activated, these receptors slow the rate of heart muscle contraction. Because musca-rine, an acetylcholine analog, also activates these receptors, they are termed "muscarinic." This type of acetylcholine

    Acetylcholine

    Exterior

    Cytosol

    Active muscarinic acetylcholine receptor

    K- channel

    t +

    K

    J]~ + ... + + +

    FIGURE 1 5 -20 Activation of the muscarinic acetylcholine receptor and its effector K