Signal Transduction Chapter 3 PDF

CHAPTER 3 SIGNAL TRANSDUCTION Lloyd Cantley The evolution of multicellular organisms necessitated the development of mechanisms to tightly coordinate the activities among cells. Such communication is fundamental to all biological processes, ranging from the induction of embryonic development to th...

CHAPTER 3 SIGNAL TRANSDUCTION Lloyd Cantley The evolution of multicellular organisms necessitated the development of mechanisms to tightly coordinate the activities among cells. Such communication is fundamental to all biological processes, ranging from the induction of embryonic development to the integration of physiological responses in the face of environmental challenges. As our understanding of cellular and molecular physiology has increased, it has become evident that all cells can receive and process information. External signals such as odorants, chemicals that reflect metabolic status, ions, hormones, growth factors, and neurotransmitters can all serve as chemical messengers linking neighboring or distant cells. Even external signals that are not considered chemical in nature (e.g., light and mechanical or thermal stimuli) may ultimately be transduced into a chemical messenger. Most chemical messengers interact with specific cell surface receptors and trigger a cascade of secondary events, including the mobilization of diffusible intracellular second-messenger systems that mediate the cell’s response to that stimulus. However, hydrophobic messengers, such as steroid hormones and some vitamins, can diffuse across the plasma membrane and interact with cytosolic or nuclear receptors. It is now clear that cells use a number of different, often intersecting intracellular signaling pathways to ensure that the cell’s response to a stimulus is tightly controlled. MECHANISMS OF CELLULAR COMMUNICATION 48 However, many other cells and tissues not classically thought of as endocrine in nature also produce hormones. For example, the kidney produces 1,25-dihydroxyvitamin D3, and the salivary gland synthesizes nerve growth factor. It is now recognized that intercellular communication can involve the production of a “hormone” or chemical signal by one cell type that acts in any (or all) of three ways, as illustrated in Figure 3-1: on distant tissues (endocrine), on a neighboring cell in the same tissue (paracrine), or on the same cell that released the signaling molecule (autocrine). For paracrine and autocrine signals to be delivered to their proper targets, their diffusion must be limited. This restriction can be accomplished by rapid endocytosis of the chemical signal by neighboring cells, its destruction by extracellular enzymes, or its immobilization by the extracellular matrix. The events that take place at the neuromuscular junction are excellent examples of paracrine signaling. When an electrical impulse travels down an axon and reaches the nerve terminal (Fig. 3-2), it stimulates release of the neurotransmitter acetylcholine (ACh). In turn, ACh transiently activates a ligand-gated cation channel on the muscle cell membrane. The resultant transient influx of Na+ causes a localized positive shift of Vm (i.e., depolarization), initiating events that result in propagation of an action potential along the muscle cell. The ACh signal is rapidly terminated by the action of acetylcholinesterase, which is present in the synaptic cleft. This enzyme degrades the ACh that is released by the neuron. Cells can communicate with one another by chemical signals Soluble chemical signals interact with target cells by binding to surface or intracellular receptors Early insight into signal transduction pathways was obtained from studies of the endocrine system. The classic definition of a hormone is a substance that is produced in one tissue or organ and released into the blood and carried to other organs (targets), where it acts to produce a specific response. The idea of endocrine or ductless glands developed from the recognition that certain organs—such as the pituitary, adrenal, and thyroid gland—can synthesize and release specific chemical messengers in response to particular physiological states. Four types of chemicals can serve as extracellular signaling molecules: amines, such as epinephrine; peptides and proteins, such as angiotensin II and insulin; steroids, including aldosterone, estrogens, and retinoic acid; and other small molecules, such as amino acids, nucleotides, ions (e.g., Ca2+), and gases (e.g., nitric oxide). For a molecule to act as a signal, it must bind to a receptor. A receptor is a protein (or in some cases a lipoprotein) on the cell surface or within the cell that specifically binds a Chapter 3 • Signal Transduction A B ENDOCRINE Cell of endocrine tissue PARACRINE C AUTOCRINE Hormones Nucleus Blood vessel Hormones Signaling molecules Hormone receptor Nucleus Target receptors Nucleus Non-target cells Figure 3-1 Target receptor Target cells Modes of cell communication. are initiated by the binding of any one ligand to its receptor. Receptors can be divided into four categories on the basis of their associated mechanisms of signal transduction (Table 3-1). Axon Electrical stimulus Nerve terminal ACh + Na Muscle cell Acetylcholine receptor Arrival of an electrical stimulus triggers release of acetylcholine, which binds to the acetylcholine receptor on the muscle cell… …activating the entry of sodium, which causes a local membrane depolarization. Acetylcholinesterase degrades the transmitter, terminating the signal. Figure 3-2 Example of paracrine signaling. The release of ACh at the neuromuscular junction is a form of paracrine signaling because the nerve terminal releases a chemical (i.e., ACh) that acts on a neighboring cell (i.e., the muscle). signaling molecule (the ligand). In some cases, the receptor is itself an ion channel, and ligand binding produces a change in Vm. Thus, the cell can transduce a signal with no machinery other than the receptor. In most cases, however, interaction of the ligand with one or more specific receptors results in an association of the receptor with an effector molecule that initiates a cellular response. Effectors include enzymes, channels, transport proteins, contractile elements, and transcription factors. The ability of a cell or tissue to respond to a specific signal is dictated by the complement of receptors it possesses and by the chain of intracellular reactions that 1. Ligand-gated ion channels. Integral membrane proteins, these hybrid receptor/channels are involved in signaling between electrically excitable cells. The binding of a neurotransmitter such as ACh to its receptor—which in fact is merely part of the channel—results in transient opening of the channel, thus altering the ion permeability of the cell. 2. G protein–coupled receptors. These integral plasma membrane proteins work indirectly—through an intermediary—to activate or to inactivate a separate membrane-associated enzyme or channel. The intermediary is a heterotrimeric guanosine triphosphate (GTP)–binding complex called a G protein. 3. Catalytic receptors. When activated by a ligand, these integral plasma membrane proteins are either enzymes themselves or part of an enzymatic complex. 4. Nuclear receptors. These proteins, located in the cytosol or nucleus, are ligand-activated transcription factors. These receptors link extracellular signals to gene transcription. In addition to these four classes of membrane signaling molecules, some other transmembrane proteins act as messengers even though they do not fit the classic definition of a receptor. In response to certain physiological changes, they undergo regulated intramembrane proteolysis within the plane of the membrane, liberating cytosolic fragments that enter the nucleus to modulate gene expression. We discuss this process later in the chapter. Signaling events initiated by plasma membrane receptors can generally be divided into six steps: Step 1: Recognition of the signal by its receptor. The same signaling molecule can sometimes bind to more than one 49 50 Section II • Physiology of Cells and Molecules Table 3-1 Classification of Receptors and Associated Signal Transduction Pathways Class of Receptor Subunit Composition of Receptor Ligand Signal Transduction Pathway Downstream from Receptor Ligand-gated ion channels (ionotropic receptors) Heteromeric or homomeric oligomers Extracellular GABA Glycine ACh: muscle ACh: nerve 5-HT Glutamate: non-NMDA Glutamate: NMDA ATP (opening) Intracellular cGMP (vision) cAMP (olfaction) ATP (closes channel) IP3 Ca2+ or ryanodine Ion Current Cl− > HCO3− Cl− > HCO3− Na+, K+, Ca2+ Na+, K+, Ca2+ Na+, K+ Na+, K+, Ca2+ Na+, K+, Ca2+ Ca2+, Na+, Mg2+ Na+, K+ Na+, K+ K+ Ca2+ Ca2+ Receptors coupled to heterotrimeric (αβγ) G proteins Single polypeptide that crosses the membrane seven times Small transmitter molecules ACh Norepinephrine Peptides Oxytocin Parathyroid hormone Neuropeptide Y Gastrin Cholecystokinin Odorants Certain cytokines, lipids, and related molecules bg Directly activates downstream effector: Muscarinic ACh receptor activates atrial K+ channel α Activates an enzyme: Cyclases that make cyclic nucleotides (cAMP, cGMP) Phospholipases that generate IP3 and diacylglycerols Phospholipases that generate arachidonic acid and its metabolites Catalytic receptors Single polypeptide that crosses the membrane once May be dimeric or may dimerize after activation ANP TGF-β Receptor guanylyl cyclase Receptor serine/threonine kinases Receptor tyrosine kinase Tyrosine kinase–associated receptor Receptor tyrosine phosphatase Intracellular (or nuclear) receptors Homodimers of polypeptides, each with multiple functional domains Heterodimers of polypeptides, each with multiple functional domains NGF, EGF, PDGF, FGF, insulin, IGF-1 IL-3, IL-5, IL-6, EPO, LIF, CNTF, GH, IFN-α, IFN-β, IFN-γ, GM-CSF CD45 Steroid hormones Mineralocorticoids Glucocorticoids Androgens Estrogens Progestins Others Thyroid hormones Retinoic acid Vitamin D Prostaglandin kind of receptor. For example, ACh can bind to both ligand-gated channels and G protein–coupled receptors. Binding of a ligand to its receptor involves the same three types of weak, noncovalent interactions that characterize substrate-enzyme interactions. Ionic bonds are formed between groups of opposite charge. In van der Waals interactions, a transient dipole in one atom generates the opposite dipole in an adjacent atom, thereby creating an Bind to regulatory DNA sequences and directly or indirectly increase or decrease the transcription of specific genes electrostatic interaction. Hydrophobic interactions occur between nonpolar groups. Step 2: Transduction of the extracellular message into an intracellular signal or second messenger. Ligand binding causes a conformational change in the receptor that triggers the catalytic activities intrinsic to the receptor or causes the receptor to interact with membrane or cytoplasmic enzymes. The final consequence is the generation Chapter 3 • Signal Transduction of a second messenger or the activation of a catalytic cascade. Step 3: Transmission of the second messenger’s signal to the appropriate effector. These effectors represent a diverse array of molecules, such as enzymes, ion channels, and transcription factors. Step 4: Modulation of the effector. These events often result in the activation of protein kinases (which put phosphate groups on proteins) and phosphatases (which take them off), thereby altering the activity of other enzymes and proteins. Step 5: Response of the cell to the initial stimulus. This collection of actions represents the summation and integration of input from multiple signaling pathways. Step 6: Termination of the response by feedback mechanisms at any or all levels of the signaling pathway. Cells can also communicate by direct interactions gap Junctions Neighboring cells can be electrically and metabolically coupled by means of gap junctions formed between apposing cell membranes. These water-filled channels facilitate the passage of inorganic ions and small molecules, such as Ca2+ and 3′,5′-cyclic adenosine monophosphate (cAMP), from the cytoplasm of one cell into the cytoplasm of an adjacent cell. Mammalian gap junctions permit the passage of molecules that are less than ∼1200 Da but restrict the movement of molecules that are greater than ∼2000 Da. Gap junctions are also excellent pathways for the flow of electrical current between adjacent cells, playing a critical role in cardiac and smooth muscle. The permeability of gap junctions can be rapidly regulated by changes in cytosolic concentrations of Ca2+, cAMP, and H+ as well as by the voltage across the cell membrane or membrane potential (Vm) (see Chapter 5). This type of modulation is physiologically important for cell-to-cell communication. For example, if a cell’s plasma membrane is damaged, Ca2+ passively moves into the cell and raises [Ca2+]i to toxic levels. Elevated intracellular [Ca2+] in the damaged cell triggers closure of the gap junctions, thus preventing the flow of excessive amounts of Ca2+ into the adjacent cell. Adhering and Tight Junctions Adhering junctions form as the result of the Ca2+-dependent interactions of the extracellular domains of transmembrane proteins called cadherins (see Chapter 2). The clustering of cadherins at the site of interaction with an adjacent cell causes secondary clustering of intracellular proteins known as catenins, which in turn serve as sites of attachment for the intracellular actin cytoskeleton. Thus, adhering junctions provide important clues for the maintenance of normal cell architecture as well as the organization of groups of cells into tissues. In addition to a homeostatic role, adhering junctions can serve a signaling role during organ development and remodeling. In a cell that is stably associated with its neighbors, a catenin known as β-catenin is mainly sequestered at the adhering junctions, minimizing concentration of free βcatenin. However, disruption of adhering junctions by certain growth factors, for example, causes β-catenin to disassociate from cadherin. The resulting rise in free β-catenin levels promotes the translocation of β-catenin to the nucleus. There, β-catenin regulates the transcription of multiple genes, including ones that promote cell proliferation and migration. Similar to adhering junctions, tight junctions (see Chapter 2) comprise transmembrane proteins that link with their counterparts on adjacent cells as well as intracellular proteins that stabilize the complex and also have a signaling role. The transmembrane proteins—including claudins, occludin, and junctional adhesion molecule—and their extracellular domains create the diffusion barrier of the tight junction. One of the integral cytoplasmic proteins in tight junctions, zonula occludin 1 (ZO-1), colocalizes with a serine/threonine kinase known as WNK1, which is found in certain renal tubule epithelial cells that reabsorb Na+ and Cl− from the tubule lumen. Because WNK1 is important for determining the permeability of the tight junctions to Cl−, mutations in WNK1 can increase the movement of Cl− through the tight junctions (see Chapter 35) and thereby lead to hypertension. Membrane-Associated Ligands Another mechanism by which cells can directly communicate is by the interaction of a receptor in the plasma membrane with a ligand that is itself a membrane protein on an adjacent cell. Such membrane-associated ligands can provide spatial clues in migrating cells. For example, an ephrin ligand expressed on the surface of one cell can interact with an Eph receptor on a nearby cell. The resulting activation of the Eph receptor can in turn provide signals for regulating such developmental events as axonal guidance in the nervous system and endothelial cell guidance in the vasculature. Second-messenger systems amplify signals and integrate responses among cell types Once a signal has been received at the cell surface, it is typically amplified and transmitted to specific sites within the cells through second messengers. For a molecule to function as a second messenger, its concentration, or window of activity, must be finely regulated. The cell achieves this control by rapidly producing or activating the second messenger and then inactivating or degrading it. To ensure that the system returns to a resting state when the stimulus is removed, counterbalancing activities function at each step of the cascade. The involvement of second messengers in catalytic cascades provides numerous opportunities to amplify a signal. For example, the binding of a ligand to its receptor can generate hundreds of second-messenger molecules, which can in turn alter the activity of thousands of downstream effectors. This modulation usually involves the conversion of an inactive species into an active molecule or vice versa. An example of such a cascade is the increased intracellular concentration of the second messenger cAMP. Receptor occupancy activates a G protein, which in turn stimulates a membrane-bound enzyme, adenylyl cyclase. This enzyme catalyzes the synthesis of cAMP from adenosine triphosphate (ATP), and a 5-fold increase in the intracellular concentration of cAMP is achieved in ∼5 seconds. This sudden rise in cAMP levels is rapidly counteracted by its 51 52 Section II • Physiology of Cells and Molecules breakdown to adenosine 5′-monophosphate by cAMP phosphodiesterase. Second-messenger systems also allow specificity and diversity. Ligands that activate the same signaling pathways in cells usually produce the same effect. For example, epinephrine, adrenocorticotropic hormone (ACTH), glucagon, and thyroid-stimulating hormone induce triglyceride breakdown through the cAMP messenger system. However, the same signaling molecule can produce distinct responses in different cells, depending on the complement of receptors and signal transduction pathways that are available in the cell as well as the specialized function that the cell carries out in the organism. For example, ACh induces contraction of skeletal muscle cells but inhibits contraction of heart muscle. It also facilitates the exocytosis of secretory granules in pancreatic acinar cells. This signaling molecule achieves these different endpoints by interacting with distinct receptors. The diversity and specialization of second-messenger systems are important to a multicellular organism, as can be seen in the coordinated response of an organism to a stressful situation. Under these conditions, the adrenal gland releases epinephrine. Different organ systems respond to epinephrine in a distinct manner, such as activation of glycogen breakdown in the liver, constriction of the blood vessels of the skin, dilation of the blood vessels in skeletal muscle, and increased rate and force of heart contraction. The overall effect is an integrated response that readies the organism for attack, defense, or escape. In contrast, complex cell behaviors, such as proliferation and differentiation, are generally stimulated by combinations of signals rather than by a single signal. Integration of these stimuli requires crosstalk among the various signaling cascades. As discussed later, most signal transduction pathways use elaborate cascades of signaling proteins to relay information from the cell surface to effectors in the cell membrane, the cytoplasm, or the nucleus. In Chapter 4, we discuss how signal transduction pathways that lead to the nucleus can affect the cell by modulating gene transcription. These are genomic effects. Signal transduction systems that project to the cell membrane or to the cytoplasm produce nongenomic effects, the focus of this chapter. stoichiometry of 2 : 1 : 1 : 1. This receptor is called nicotinic because the nicotine contained in tobacco can activate or open the channel and thereby alter Vm. Note that the nicotinic AChR is very different from the muscarinic AChR discussed later, which is not a ligand-gated channel. Additional examples of ligand-gated channels are the IP3 receptor and the Ca2+ release channel (also known as the ryanodine receptor). Both receptors are tetrameric Ca2+ channels located in the membranes of intracellular organelles. RECEPTORS COUPLED TO G PROTEINS G protein–coupled receptors (GPCRs) constitute the largest family of receptors on the cell surface, with more than 1000 members. GPCRs mediate cellular responses to a diverse array of signaling molecules, such as hormones, neurotransmitters, vasoactive peptides, odorants, tastants, and other local mediators. Despite the chemical diversity of their ligands, most receptors of this class have a similar structure (Fig. 3-3). They consist of a single polypeptide chain with seven membrane-spanning α-helical segments, an extracellular N terminus that is glycosylated, a large cytoplasmic loop that is composed mainly of hydrophilic amino acids between helices 5 and 6, and a hydrophilic domain at the cytoplasmic C terminus. Most small ligands (e.g., epinephrine) bind in the plane of the membrane at a site that involves several membrane-spanning segments. In the case of larger protein ligands, a portion of the extracellular N terminus also participates in ligand binding. The 5,6-cytoplasmic loop appears to be the major site of interaction with the intracellular G protein, although the 3,4-cytoplasmic loop and the cytoplasmic C terminus also contribute to binding in some cases. Binding of the GPCR to its extracellular ligand regulates this interaction between the receptor and the G proteins, thus transmitting a signal to downstream effectors. In the next four sections of this subchapter, we discuss the general principles of how G proteins function; three major Extracellular space RECEPTORS THAT ARE ION CHANNELS N Ligand-gated ion channels transduce a chemical signal into an electrical signal The property that defines this class of multisubunit membrane-spanning receptors is that the signaling molecule itself controls the opening and closing of an ion channel by binding to a site on the receptor. Thus, these receptors are also called ionotropic receptors to distinguish them from the metabotropic receptors, which act through “metabolic” pathways. One superfamily of ligand-gated channels includes the ionotropic receptors for ACh, serotonin, γ-aminobutyric acid (GABA), and glycine. Most structural and functional information for ionotropic receptors comes from the nicotinic ACh receptor (AChR) present in skeletal muscle (Fig. 3-2). The nicotinic AChR is a cation channel that consists of four membrane-spanning subunits, α, β, γ, and δ, in a C G protein binding Cytosol Figure 3-3 Receptor coupled to a G protein. Chapter 3 • Signal Transduction second-messenger systems that are triggered by G proteins are then considered. GENERAL PROPERTIES OF G PROTEINS G proteins are heterotrimers that exist in many combinations of different α, b, and g subunits G proteins are members of a superfamily of GTP-binding proteins. This superfamily includes the classic heterotrimeric G proteins that bind to GPCRs as well as the so-called small GTP-binding proteins, such as Ras. Both the heterotrimeric and small G proteins can hydrolyze GTP and switch between an active GTP-bound state and an inactive guanosine diphosphate (GDP)–bound state. Heterotrimeric G proteins are composed of three subunits, α, β, and γ. At least 16 different α subunits (∼42 to Table 3-2 50 kDa), 5 β subunits (∼33 to 35 kDa), and 11 γ subunits (∼8 to 10 kDa) are present in mammalian tissue. The α subunit binds and hydrolyzes GTP and also interacts with “downstream” effector proteins such as adenylyl cyclase. Historically, the α subunits were thought to provide the principal specificity to each type of G protein, with the βγ complex functioning to anchor the trimeric complex to the membrane. However, it is now clear that the βγ complex also functions in signal transduction by interacting with certain effector molecules. Moreover, both the α and γ subunits are involved in anchoring the complex to the membrane. The α subunit is held to the membrane by either a myristyl or a palmitoyl group; the γ subunit is held by a prenyl group. The multiple α, β, and γ subunits demonstrate distinct tissue distributions and interact with different receptors and effectors (Table 3-2). Because of the potential for several hundred combinations of the known α, β, and γ subunits, G proteins are ideally suited to link a diversity of receptors to a diversity of effectors. The many classes of G proteins, in Families of G Proteins Family/Subunit % Identity Toxin Distribution Receptor Effector/Role αs αs(s) αs(l) 100 CTX Ubiquitous β-adrenergic, TSH, glucagon ↑ Adenylyl cyclase ↑ Ca2+ channel ↑ Na+ channel αolf 88 CTX Olfactory epithelium Odorant ↑ Adenylyl cyclase Open K+ channel Gi αi1 αi2 αi3 100 88 PTX PTX PTX ∼Ubiquitous Ubiquitous ∼Ubiquitous M2, α2-adrenergic, others ↑ IP3, DAG, Ca2+, and AA release ↓ Adenylyl cyclase αO1A αO1B 73 73 PTX PTX Brain, others Brain, others Met-enkephalin, α2adrenergic, others αt1 αt2 68 68 PTX, CTX PTX, CTX Retinal rods Retinal cones Rhodopsin Cone opsin ↑ cGMP-phosphodiesterase αg αz 67 60 PTX, CTX (?) Taste buds Brain, adrenal, platelet Taste (?) M2 (?), others (?) ? ↓ Adenylyl cyclase Gq αq α11 α14 α15 α16 100 88 79 57 58 ∼Ubiquitous ∼Ubiquitous Lung, kidney, liver B cell, myeloid T cell, myeloid M1, α1-adrenergic, others ↑ PLCβ1, β2, β3 Several receptors ↑ PLCβ1, β2, β3 G12 α12 α13 100 67 Ubiquitous Ubiquitous CTX, cholera toxin; M1 and M2, muscarinic cholinergic receptors; PTX, pertussis toxin; TSH, thyrotropin (thyroid-stimulating hormone). 53 54 Section II • Physiology of Cells and Molecules conjunction with the presence of several receptor types for a single ligand, provide a mechanism whereby a common signal can elicit the appropriate physiological changes in different tissues. For example, when epinephrine binds β1adrenergic receptors in the heart, it stimulates adenylyl cyclase, which increases heart rate and the force of contraction. However, in the periphery, epinephrine acts on α2adrenergic receptors coupled to a G protein that inhibits adenylyl cyclase, thereby increasing peripheral vascular resistance and consequently increasing venous return and blood pressure. Among the first effectors found to be sensitive to G proteins was the enzyme adenylyl cyclase. The heterotrimeric G protein known as Gs was so named because it stimulates adenylyl cyclase. A separate class of G proteins was given the name Gi because it is responsible for the hormone-dependent inhibition of adenylyl cyclase. Identification of these classes of G proteins was greatly facilitated by the observation that the α subunits of individual G proteins are substrates for adenosine diphosphate (ADP) ribosylation catalyzed by bacterial toxins. The toxin from Vibrio cholerae activates Gs, whereas the toxin from Bordetella pertussis inactivates the cyclase-inhibiting Gi (see the box titled Action of Toxins on Heterotrimeric G Proteins). For their work in identifying G proteins and elucidating the physiological role of these proteins, Alfred Gilman and Martin Rodbell received the 1994 Nobel Prize in Physiology or Medicine. G protein activation follows a cycle In their inactive state, heterotrimeric G proteins are a complex of α, β, and γ subunits in which GDP occupies the guanine nucleotide–binding site of the α subunit. After ligand binding to the GPCR (Fig. 3-4, step 1), the activated receptor interacts with the αβγ heterotrimer to promote a conformational change that facilitates the release of bound GDP and simultaneous binding of GTP (step 2). This GDP-GTP exchange stimulates dissociation of the complex from the receptor (step 3) and causes disassembly of the trimer into a free α subunit and βγ complex (step 4). The free, active GTP-bound α subunit can now interact in the plane of the membrane with downstream effectors such as adenylyl cyclase and phospholipases (step 5). Similarly, the βγ subunit can now activate ion channels or other effectors. The α subunit terminates the signaling events that are mediated by the α and βγ subunits by hydrolyzing GTP to GDP and inorganic phosphate (Pi). The result is an inactive α-GDP complex that dissociates from its downstream effector and reassociates with a βγ subunit (Fig. 3-4, step 6), thus completing the cycle (step 1). The βγ subunit stabilizes αGDP and thereby substantially slows the rate of GDP-GTP exchange (step 2) and dampens signal transmission in the resting state. The RGS (for “regulation of G protein signaling”) family of proteins appears to enhance the intrinsic guanosine triphosphatase (GTPase) activity of some but not all α subunits. Investigators have identified at least 15 mammalian RGS proteins and shown that they interact with specific α subunits. RGS proteins bind the complex Gα/GDP/AlF4−, which is the structural analogue of the GTPase transition state. By stabilizing the transition state, RGS proteins may promote GTP hydrolysis and thus the termination of signaling. As noted earlier, α subunits can be anchored to the cell membrane by myristyl or palmitoyl groups. Activation can result in the removal of these groups and the release of the α subunit into the cytosol. Loss of the α subunit from the membrane may decrease the interaction of G proteins with receptors and downstream effectors (e.g., adenylyl cyclase). Activated α subunits couple to a variety of downstream effectors, including enzymes, ion channels, and membrane trafficking machinery Activated α subunits can couple to a variety of enzymes. A major enzyme that acts as an effector downstream of activated α subunits is adenylyl cyclase (Fig. 3-5A). This enzyme can be either activated or inhibited by G protein signaling, depending on whether it associates with the GTP-bound form of Gαs (stimulatory) or Gαi (inhibitory). Thus, different hormones—acting through different G protein complexes—can have opposing effects on the same intracellular messenger. G proteins can also activate enzymes that break down cyclic nucleotides. For example, the G protein called transducin, which plays a key role in phototransduction (see Chapter 15), activates the cyclic guanosine monophosphate (cGMP) phosphodiesterase, which catalyzes the breakdown of cGMP to GMP (Fig. 3-5B). Thus, in retinal cells expressing transducin, light leads to a decrease in [cGMP]i. G proteins can also couple to phospholipases. These enzymes catabolize phospholipids, as discussed in detail later in the section on G protein second messengers. This superfamily of phospholipases can be grouped into phospholipases A2, C, or D on the basis of the site at which the enzyme cleaves the phospholipid. The G protein αq subunit activates phospholipase C, which breaks phosphatidylinositol bisphosphate (PIP2) into two intracellular messengers, membraneassociated diacylglycerol and cytosolic IP3 (Fig. 3-5C). Diacylglycerol stimulates protein kinase C, whereas IP3 binds to a receptor on the endoplasmic reticulum membrane and triggers the release of Ca2+ from intracellular stores. Some G proteins interact with ion channels. Agonists that bind to the β-adrenergic receptor activate the L-type Ca2+ channel in the heart and skeletal muscle (see Chapter 7). The G protein Gs directly stimulates this channel as the α subunit of Gs binds to the channel, and Gs also indirectly stimulates this channel through a signal transduction cascade that involves cAMP-dependent protein kinase. A clue that G proteins serve additional functions in membrane trafficking (see Chapter 2) in the cell comes from the observation that many cells contain intracellular pools of heterotrimeric G proteins, some bound to internal membranes and some free in the cytosol. Experiments involving toxins, inhibitors, and cell lines harboring mutations in G protein subunits have demonstrated that these intracellular G proteins are involved in vesicular transport. G proteins have been implicated in the budding of secretory vesicles from the trans-Golgi network, fusion of endosomes, recruitment of non–clathrin coat proteins, and transcytosis and apical secretion in polarized epithelial cells. The receptors Chapter 3 • Signal Transduction N Receptor (R) consists of seven membranespanning segments. C 2 Receptor interacts with the G protein to promote a conformational change and the exchange of GDP for GTP. Extracellular space 1 Ligand binds, receptor activates. E1 E1 E1 γ R R β α E2 γ β R α E2 E3 Cytosol G protein 3 G protein dissociates from the receptor. 4 α-GTP and βγ subunits dissociate. E1 R R γ β α E2 γ R R E1 α β E2 6 5 Both α-GTP and βγ can now interact with their appropriate effectors (E1, E2). α-catalyzed hydrolysis of GTP to GDP inactivates α and promotes reassembly of the trimer. E1 R R α E1 γ β E2 R γ α β Pi Figure 3-4 Enzymatic cycle of heterotrimeric G proteins. RGS E2 Members of the RGS family of G-protein regulators stimulate GTP hydrolysis with some but not all α subunits. 55 56 Section II • Physiology of Cells and Molecules A G PROTEINS ACTING VIA ADENYLYL CYCLASE Extracellular space Adenylyl cyclase γ αs β αs αi AC G protein complex (stimulatory) cAMP β γ G protein complex (inhibitory) NH2 Cyclic AMP activates protein kinase A. Cytosol B PKA Adenine N N N N CH2 O G PROTEIN ACTING VIA A PHOSPHODIESTERASE Light H O H H H OH O Extracellular space P O – O Phosphodiesterase Cyclic AMP γ Cytosol αt αt β G protein complex (transducin) PDE cGMP GMP O The breakdown of cGMP leads to the closure of cGMP-dependent channels. O O– H2N P O N H H H H GMP Phospholipase C β N CH2 O OH OH G PROTEIN ACTING VIA A PHOSPHOLIPASE γ O – Extracellular space C N N cGMP Guanine C αq αq DAG activates the enzyme protein kinase C. PIP2 PKC PLC PKC Ca G protein complex DAG 2+ IP3 IP3 signals the release of Ca2+ from the ER. ER Figure 3-5 Downstream effects of activated G protein α subunits. A, When a ligand binds to a receptor coupled to αs, adenylyl cyclase (AC) is activated, whereas when a ligand binds to a receptor coupled to αi, the enzyme is inhibited. The activated enzyme converts ATP to cAMP, which then can activate protein kinase A (PKA). B, In phototransduction, a photon interacts with the receptor and activates the G protein transducin. The αt activates phosphodiesterase (PDE), which in turn hydrolyzes cGMP and lowers the intracellular concentrations of cGMP and therefore closes the cGMP-activated channels. C, In this example, the ligand binds to a receptor that is coupled to αq, which activates phospholipase C (PLC). This enzyme converts PIP2 to IP3 and diacylglycerol (DAG). The IP3 leads to the release of Ca2+ from intracellular stores, whereas the diacylglycerol activates protein kinase C (PKC). ER, endoplasmic reticulum. Chapter 3 • Signal Transduction Action of Toxins on Heterotrimeric G Proteins I nfectious diarrheal disease has a multitude of causes. Cholera toxin, a secretory product of the bacterium Vibrio cholerae, is responsible in part for the devastating characteristics of cholera. The toxin is an oligomeric protein composed of one A subunit and five B subunits (AB5). After cholera toxin enters intestinal epithelial cells, the A subunit separates from the B subunits and becomes activated by proteolytic cleavage. The resulting active A1 fragment catalyzes the ADP ribosylation of Gαs. This ribosylation, which involves transfer of the ADP-ribose moiety from the oxidized form of nicotinamide adenine dinucleotide (NAD+) to the α subunit, inhibits the GTPase activity of Gαs. As a result of this modification, Gαs remains in its activated, GTP-bound form and can activate adenylyl cyclase. In intestinal epithelial cells, the constitutively activated Gαs elevates levels of cAMP, which causes an increase in Cl− conductance and water flow and thereby contributes to the large fluid loss characteristic of this disease. A related bacterial product is pertussis toxin, which is also an AB5 protein. It is produced by Bordetella pertussis, the causative agent of whooping cough. Pertussis toxin ADPribosylates Gαi. This ADP-ribosylated Gαi cannot exchange its bound GDP (inactive state) for GTP. Thus, αi remains in its GDP-bound inactive state. As a result, receptor occupancy can no longer release the active αi-GTP, so adenylyl cyclase cannot be inhibited. Thus, both cholera toxin and pertussis toxin increase the generation of cAMP. and effectors that interact with these intracellular G proteins have not been determined. The bg subunits of G proteins can also activate downstream effectors Considerable evidence now indicates that the βγ subunits can also interact with downstream effectors. The neurotransmitter ACh released from the vagus nerve reduces the rate and strength of heart contraction. This action in the atria of the heart is mediated by muscarinic M2 AChRs (see Chapter 14). These receptors can be activated by muscarine, an alkaloid found in certain poisonous mushrooms. Muscarinic AChRs are very different from the nicotinic AChRs discussed earlier, which are ligand-gated channels. Binding of ACh to the muscarinic M2 receptor in the atria activates a heterotrimeric G protein, resulting in the generation of both activated Gαi as well as a free βγ subunit complex. The βγ complex then interacts with a particular class of K+ channels, increasing their permeability. This increase in K+ permeability keeps the membrane potential relatively negative and thus renders the cell more resistant to excitation. The βγ subunit complex also modulates the activity of adenylyl cyclase and phospholipase C and stimulates phospholipase A2. Such effects of βγ can be independent of, synergize with, or antagonize the action of the α subunit. For example, studies using various isoforms of adenylyl cyclase have demonstrated that purified βγ stimulates some isoforms, inhibits others, and has no effect on still others. Different combinations of βγ isoforms may have different activities. For example, β1γ1 is one tenth as efficient at stimulating type II adenylyl cyclase as is β1γ2. An interesting action of some βγ complexes is that they bind to a special protein kinase called the β-adrenergic receptor kinase (βARK). As a result of this interaction, βARK translocates to the plasma membrane, where it phosphorylates the ligand-receptor complex (but not the unbound receptor). This phosphorylation results in the recruitment of β-arrestin to the GPCR, which in turn mediates disassociation of the receptor-ligand complex and thus attenuates the activity of the same β-adrenergic receptors that gave rise to the βγ complex in the first place. This action is an example of receptor desensitization. These phosphorylated receptors eventually undergo endocytosis, which transiently reduces the number of receptors that are available on the cell surface. This endocytosis is an important step in resensitization of the receptor system. Small GTP-binding proteins are involved in a vast number of cellular processes A distinct group of proteins that are structurally related to the α subunit of the heterotrimeric G proteins are the small GTP-binding proteins. More than 100 of these have been identified to date, and they have been divided into five groups including the Ras, Rho, Rab, Arf, and Ran families. These 21-kDa proteins can be membrane associated (e.g., Ras) or may translocate between the membrane and the cytosol (e.g., Rho). The three isoforms of Ras (N, Ha, and Ki) relay signals from the plasma membrane to the nucleus through an elaborate kinase cascade (see Chapter 4), thereby regulating gene transcription. In some tumors, mutation of the genes encoding Ras proteins results in constitutively active Ras. These mutated genes are called oncogenes because the altered Ras gene product promotes the malignant transformation of a cell and can contribute to the development of cancer (oncogenesis). In contrast, Rho family members are primarily involved in rearrangement of the actin cytoskeleton; Rab and Arf proteins regulate vesicle trafficking. Similar to the α subunit of heterotrimeric G proteins, the small GTP-binding proteins switch between an inactive GDP-bound form and an active GTP-bound form. Two classes of regulatory proteins modulate the activity of these small GTP-binding proteins. The first of these includes the GTPase-activating proteins (GAPs) and neurofibromin (a product of the neurofibromatosis type 1 gene). GAPs increase the rate at which small GTP-binding proteins hydrolyze bound GTP and thus result in more rapid inactivation. Counteracting the activity of GAPs are guanine nucleotide exchange proteins (GEFs) such as “son of sevenless” or SOS, which promote the conversion of inactive RasGDP to active Ras-GTP. Interestingly, cAMP directly activates several GEFs, such as Epac (exchange protein activated by cAMP), demonstrating crosstalk between a classical heterotrimeric G protein signaling pathway and the small Ras-like G proteins. 57 58 Section II • Physiology of Cells and Molecules G PROTEIN SECOND MESSENGERS: CYCLIC NUCLEOTIDES cAMP-dependent kinase (PKA) is composed of two regulatory (R) and 2 catalytic (C) subunits. Binding of cAMP to the regulatory subunits induces a conformational change that reduces their affinity for the catalytic subunits. cAMP usually exerts its effect by increasing the activity of protein kinase A Activation of Gs-coupled receptors results in the stimulation of adenylyl cyclase and a rise in intracellular concentrations of cAMP (Fig. 3-5A). The downstream effects of this increase in [cAMP]i depend on the specialized functions that the responding cell carries out in the organism. For example, in the adrenal cortex, ACTH stimulation of cAMP production results in the secretion of aldosterone and cortisol; in the kidney, vasopressin-induced changes in cAMP levels facilitate water reabsorption (see Chapters 38 and 50). Excess cAMP is also responsible for certain pathologic conditions. One is cholera (see the box on page 57, titled Action of Toxins on Heterotrimeric G Proteins). Another pathologic process associated with excess cAMP is McCune-Albright syndrome, characterized by a triad of (1) variable hyperfunction of multiple endocrine glands, including precocious puberty in girls, (2) bone lesions, and (3) pigmented skin lesions (café au lait spots). This disorder is caused by a somatic mutation that constitutively activates the G protein αs subunit in a mosaic pattern. cAMP exerts many of its effects through cAMP-dependent protein kinase A (PKA). This enzyme catalyzes transfer of the terminal phosphate of ATP to certain serine or threonine residues within selected proteins. PKA phosphorylation sites are present in a multitude of intracellular proteins, including ion channels, receptors, and signaling pathway proteins. Phosphorylation of these sites can influence either the localization or the activity of the substrate. For example, phosphorylation of the β2-adrenergic receptor causes receptor desensitization in neurons, whereas phosphorylation of the cystic fibrosis transmembrane conductance regulator (CFTR) increases its Cl− channel activity. To enhance regulation of phosphorylation events, the cell tightly controls the activity of PKA so that the enzyme can respond to subtle—and local—variations in cAMP levels. One important control mechanism is the use of regulatory subunits that constitutively inhibit PKA. In the absence of cAMP, two catalytic subunits of PKA associate with two of these regulatory subunits, resulting in a heterotetrameric protein complex that has a low level of catalytic activity (Fig. 3-6). Binding of cAMP to the regulatory subunits induces a conformational change that diminishes their affinity for the catalytic subunits, and the subsequent dissociation of the complex results in activation of kinase activity. In addition to the short-term effects of PKA activation noted before, the free catalytic subunit of PKA can also enter the nucleus, where substrate phosphorylation can activate the transcription of specific PKA-dependent genes (see Chapter 4). Although most cells use the same catalytic subunit, different regulatory subunits are found in different cell types. Another mechanism that contributes to regulation of PKA is the targeting of the enzyme to specific subcellular locations. Such targeting promotes the preferential phosphorylation of substrates that are confined to precise locations within the cell. PKA targeting is achieved by the cAMP cAMP R C cAMP cAMP C R R C R cAMP cAMP cAMP PKA C cAMP The complex dissociates and the catalytic subunits are free to catalyze the phosphorylation of protein substrates. Figure 3-6 Activation of protein kinase A by cAMP. association of a PKA regulatory subunit with an A kinase anchoring protein (AKAP), which in turn binds to cytoskeletal elements or to components of cellular subcompartments. More than 35 AKAPs are known. The specificity of PKA targeting is highlighted by the observation that in neurons, PKA is localized to postsynaptic densities through its association with AKAP79. This anchoring protein also targets calcineurin—a protein phosphatase—to the same site. This targeting of both PKA and calcineurin to the same postsynaptic site makes it possible for the cell to tightly regulate the phosphorylation state of important neuronal substrates. The cAMP generated by adenylyl cyclase does not interact only with PKA. For example, olfactory receptors (see Chapter 15) interact with a member of the Gs family called Golf. The rise in [cAMP]i that results from activation of the olfactory receptor activates a cation channel, a member of the family of cyclic nucleotide–gated (CNG) ion channels. Na+ influx through this channel leads to membrane depolarization and the initiation of a nerve impulse. For his work in elucidating the role played by cAMP as a second messenger in regulating glycogen metabolism, Earl Sutherland received the 1971 Nobel Prize in Physiology or Medicine. In 1992, Edmond Fischer and Edwin Krebs shared the prize for their part in demonstrating the role of protein phosphorylation in the signal transduction process. This coordinated set of phosphorylation and dephosphorylation reactions has several physiological advantages. First, it allows a single molecule (e.g., cAMP) to regulate a range of enzymatic reactions. Second, it affords a large amplification to a small signal. The concentration of epinephrine needed to stimulate glycogenolysis in muscle is ∼10−10 M. This subnanomolar level of hormone can raise [cAMP]i to ∼10−6 M. Thus, the catalytic cascades amplify the hormone signal 10,000-fold, resulting in the liberation of enough glucose to raise blood glucose levels from ∼5 to ∼8 mM. Although the effects of cAMP on the synthesis and degradation of glycogen are confined to muscle and liver, a Chapter 3 • Signal Transduction wide variety of cells use cAMP-mediated activation cascades in the response to a wide variety of hormones. Protein phosphatases reverse the action of kinases As discussed, one way that the cell can terminate a cAMP signal is to use a phosphodiesterase to degrade cAMP. In this way, the subsequent steps along the signaling pathway can also be terminated. However, because the downstream effects of cAMP often involve phosphorylation of effector proteins at serine and threonine residues by kinases such as PKA, another powerful way to terminate the action of cAMP is to dephosphorylate these effector proteins. Such dephosphorylation events are mediated by enzymes called serine/threonine phosphoprotein phosphatases. Four groups of serine/threonine phosphoprotein phosphatases (PP) are known, 1, 2a, 2b, and 2c. These enzymes themselves are regulated by phosphorylation at their serine, threonine, and tyrosine residues. The balance between kinase and phosphatase activity plays a major role in the control of signaling events. PP1 dephosphorylates many proteins phosphorylated by PKA, including those phosphorylated in response to epinephrine (see Chapter 58). Another protein, phosphoprotein phosphatase inhibitor 1 (I-1), can bind to and inhibit PP1. Interestingly, PKA phosphorylates and thus activates I-1 (Fig. 3-7), thereby inhibiting PP1 and preserving the phosphate groups added by PKA in the first place. PP2a, which is less specific than PP1, appears to be the main phosphatase responsible for reversing the action of other protein serine/threonine kinases. The Ca2+-dependent PP2b, also known as calcineurin, is prevalent in the brain, skeletal muscle, and cardiac muscle and is also the target of the immunosuppressive reagents FK-506 and cyclosporine. The importance of PP2c is presently unclear. cAMP R cGMP is another cyclic nucleotide that is involved in G protein signaling events. In the outer segments of rods and cones in the visual system, the G protein does not couple to an enzyme that generates cGMP but, as noted earlier, couples to an enzyme that breaks it down. As discussed further in Chapter 15, light activates a GPCR called rhodopsin, which activates the G protein transducin, which in turn activates the cGMP phosphodiesterase that lowers [cGMP]i. The fall in [cGMP]i closes cGMP-gated nonselective cation channels that are members of the same family of CNG ion channels that cAMP activates in olfactory signaling (see Chapter 15). Many messengers bind to receptors that activate phosphoinositide breakdown C R cAMP cAMP cGMP exerts its effect by stimulating a nonselective cation channel in the retina G PROTEIN SECOND MESSENGERS: PRODUCTS OF PHOSPHOINOSITIDE BREAKDOWN PKA (active) cAMP In addition to serine/threonine kinases such as PKA, a second group of kinases involved in regulating signaling pathways (discussed later in this chapter) are known as tyrosine kinases because they phosphorylate their substrate proteins on tyrosine residues. The enzymes that remove phosphates from these tyrosine residues are much more variable than the serine and threonine phosphatases. The first phosphotyrosine phosphatase (PTP) to be characterized was the cytosolic enzyme PTP1B from human placenta. PTP1B has a high degree of homology with CD45, a membrane protein that is both a receptor and a tyrosine phosphatase. cDNA sequence analysis has identified a large number of PTPs that can be divided into two classes: membranespanning receptor-like proteins such as CD45 and cytosolic forms such as PTP1B. A number of intracellular PTPs contain so-called Src homology 2 (SH2) domains, a peptide sequence or motif that interacts with phosphorylated tyrosine groups. Several of the PTPs are themselves regulated by phosphorylation. C I-1 I-1 I-1 P P PP1 Inactive PP1 Phosphoprotein phosphatase (active) Figure 3-7 Activation of phosphoprotein phosphatase 1 (PP1) by PKA. I-1, inhibitor of PP1. Although the phosphatidylinositols (PIs) are minor constituents of cell membranes, they are largely distributed in the internal leaflet of the membrane and play an important role in signal transduction. The inositol sugar moiety of PI molecules (see Fig. 2-2A) can be phosphorylated to yield the two major phosphoinositides that are involved in signal transduction: phosphatidylinositol 4,5-bisphosphate (PI4,5P2 or PIP2) and phosphatidylinositol 3,4,5-trisphosphate (PI3,4,5P3). Certain membrane-associated receptors act though G proteins (e.g., Gq) that stimulate phospholipase C (PLC) to cleave PIP2 into inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG), as shown in Figure 3-8A. PLCs are classified into three families (β, γ, δ) that differ in their catalytic properties, cell type–specific expression, and modes of activation. PLCβ is typically activated downstream of certain G proteins (e.g., Gq), whereas PLCγ contains an SH2 domain 59 60 Section II • Physiology of Cells and Molecules PIP2 DAG 13p6 A Binding of a hormone to a cell surface G protein–coupled receptor activates phospholipase Cβ. O C PRODUCTION OF IP3 AND DAG CH2 PLC cleaves the polar head group here. O O CH CH2 – P C O CH2 O O O C O O P O C O O CH CH2 Plasma membrane O Cytosol O OH O – O – O O O P O O O P O OH HO OH HO O O O – OH OH Phospholipase Cβ hydrolyzes PIP2 into IP3 and DAG. O O P – O IP3 P – O O O Extracellular space DAG Plasma membrane Cytosol γ α β α PKC PLCβ PKC PIP2 Active Receptor–G protein complex IP3 C IP3 interacts with a receptor in the membrane of the ER, which allows + the release of Ca2 into the cytosol. ER + H BREAKDOWN OF PHOSPHATIDYLCHOLINE BY PLC AND PLD R1 R2 The SERCA Ca2+ pump transports the + Ca2 back into the SR. 2+ Ca CH2 C B TIME COURSE OF IP3 AND DAG LEVELS IP3 The early DAG peak is caused by DAG released from PIP2 by PLCβ. DAG H2C PLC – O O CH CH2 P O CH2 Choline CH2 H3C N + CH3 CH3 Seconds Minutes O O PLD The slow DAG wave is caused by DAG released by PLCβ and PLD from phosphatidylcholine (PC). C O O Response CH2 O Hours Figure 3-8 Second messengers in the DAG/IP3 pathway. ER, endoplasmic reticulum; SERCA, sarcoplasmic and endoplasmic reticulum Ca2+-ATPase. Chapter 3 • Signal Transduction and is activated downstream of certain tyrosine kinases. Stimulation of PLCβ results in a rapid increase in cytosolic IP3 levels as well as an early peak in DAG levels (Fig. 3-8B). Both products are second messengers. DAG remains in the plane of the membrane to activate protein kinase C, which migrates from the cytosol and binds to DAG in the membrane. The water-soluble IP3 travels through the cytosol to stimulate Ca2+ release from intracellular stores. It is within this system that Ca2+ was first identified as a messenger that mediates the stimulus-response coupling of endocrine cells. Phosphatidylcholines (PCs), which—unlike PI—are an abundant phospholipid in the cell membrane, are also a source of DAG. The cell can produce DAG from PC by either of two mechanisms (Fig. 3-8C). First, PLC can directly convert PC to phosphocholine and DAG. Second, phospholipase D (PLD), by cleaving the phosphoester bond on the other side of the phosphate, converts PC to choline and phosphatidic acid (PA; also phospho-DAG). This PA can then be converted to DAG by PA-phosphohydrolase. Production of DAG from PC, either directly (by PLC) or indirectly (by PLD), produces the slow wave of increasing cytosolic DAG shown in Figure 3-8B. Thus, in some systems, the formation of DAG is biphasic and consists of an early peak that is transient and parallels the formation of IP3, followed by a late phase that is slow in onset but sustained for several minutes. Factors such as tumor necrosis factor α (TNF-α), interleukin 1 (IL-1), interleukin 3 (IL-3), interferon α (IFN-α), and colony-stimulating factor stimulate the production of DAG from PC. Once generated, some DAGs can be further cleaved by DAG lipase to arachidonic acid, which can have signaling activity itself or can be metabolized to other signaling molecules, the eicosanoids. We cover arachidonic acid metabolism later in this chapter. Inositol triphosphate liberates Ca2+ from intracellular stores As discussed earlier, IP3 is generated by the metabolism of membrane phospholipids and then travels through the cytosol to release Ca2+ from intracellular stores. The IP3 receptor (ITPR) is a ligand-gated Ca2+ channel located in the membrane of the endoplasmic reticulum (Fig. 3-8A). This Ca2+ channel is structurally related to the Ca2+ release channel (or ryanodine receptor), which is responsible for releasing Ca2+ from the sarcoplasmic reticulum of muscle and thereby switching on muscle contraction (see Chapter 9). The IP3 receptor is a tetramer composed of subunits of ∼260 kDa. At least three genes encode the subunits of the receptor. These genes are subject to alternative splicing, which further increases the potential for receptor diversity. The receptor is a substrate for phosphorylation by protein kinases A and C and calcium-calmodulin (Ca2+-CaM)–dependent protein kinases. Interaction of IP3 with its receptor results in passive efflux of Ca2+ from the endoplasmic reticulum and thus a rapid rise in the free cytosolic Ca2+ concentration. The IP3-induced changes in [Ca2+]i exhibit complex temporal and spatial patterns. The rise in [Ca2+]i can be brief or persistent and can oscillate repetitively, spread in spirals or waves within a cell, or spread across groups of cells that are coupled by gap junctions. In at least some systems, the frequency of [Ca2+]i oscillations seems to be physiologically important. For example, in isolated pancreatic acinar cells, graded increases in the concentration of ACh produce graded increases in the frequency—but not the magnitude—of repetitive [Ca2+]i spikes. The mechanisms responsible for [Ca2+]i oscillations and waves are complex. It appears that both propagation and oscillation depend on positive feedback mechanisms, in which low [Ca2+]i facilitates Ca2+ release, as well as on negative feedback mechanisms, in which high [Ca2+]i inhibits further Ca2+ release. The dephosphorylation of IP3 terminates the release of Ca2+ from intracellular stores; an ATP-fueled Ca2+ pump (SERCA; see Chapter 5) then moves the Ca2+ back into the endoplasmic reticulum. Some of the IP3 is further phosphorylated to IP4, which may mediate a slower and more prolonged response of the cell or may promote the refilling of intracellular stores. In addition to IP3, cyclic ADP ribose (cADPR) can mobilize Ca2+ from intracellular stores and augment a process known as calcium-induced Ca2+ release. Although the details of these interactions have not been fully elucidated, cADPR appears to bind to the Ca2+ release channel (ryanodine receptor) in a Ca2+-CaM–dependent manner. In addition to the increase in [Ca2+]i produced by the release of Ca2+ from intracellular stores, [Ca2+]i can also rise as a result of enhanced influx of this ion through Ca2+ channels in the plasma membrane. For Ca2+ to function as a second messenger, it is critical that [Ca2+]i be normally maintained at relatively low levels (at or below ∼100 nM). Leakage of Ca2+ into the cell through Ca2+ channels is opposed by the extrusion of Ca2+ across the plasma membrane by both an ATP-dependent Ca2+ pump and the Na-Ca exchanger (see Chapter 5). As discussed later, increased [Ca2+]i exerts its effect by binding to cellular proteins and changing their activity. Some Ca2+-dependent signaling events are so sensitive to Ca2+ that a [Ca2+]i increase of as little as 100 nM can trigger a vast array of cellular responses. These responses include secretion of digestive enzymes by pancreatic acinar cells, release of insulin by β cells, contraction of vascular smooth muscle, conversion of glycogen to glucose in the liver, release of histamine by mast cells, aggregation of platelets, and DNA synthesis and cell division in fibroblasts. Calcium activates calmodulin-dependent protein kinases How does an increase in [Ca2+]i lead to downstream responses in the signal transduction cascade? The effects of changes in [Ca2+]i are mediated by Ca2+-binding proteins, the most important of which is calmodulin (CaM). CaM is a highaffinity cytoplasmic Ca2+-binding protein of 148 amino acids. Each molecule of CaM cooperatively binds four calcium ions. Ca2+ binding induces a major conformational change in CaM that allows it to bind to other proteins (Fig. 3-9). Although CaM does not have intrinsic enzymatic activity, it forms a complex with a number of enz