Signal-Transduction Pathways

Transcription

1 CHATER 12 Signal-Transduction athways 12.1 Signal Transduction Depends on Molecular Circuits 12.2 Receptor roteins Transmit Information Into the Cell 12.3 Metabolism in Context: Insulin Signaling Regulates Metabolism 12.4 Calcium Ion Is a Ubiquitous Cytoplasmic Messenger 12.5 Defects in Signaling athways Can Lead to Diseases Signal transduction is an important facility in all life forms. It allows an organism to sense the environment and formulate the proper biochemical response. Just as the computer chip has on off switches that allow the transmission of information, cells have molecular on off switches that allow the transmission of information in the cell and between cells. [Courtesy of Intel.] T his chapter provides an overview of how cells receive, process, and respond to information from the environment. Signal-transduction cascades mediate the sensing and processing of these stimuli. These molecular circuits detect, amplify, and integrate diverse external signals to generate responses such as changes in enzyme activity, gene expression, or ion-channel activity. This chapter introduces some of the basic principles of signal transduction and important classes of molecules that participate in common signal-transduction pathways. 173

2 Signal-Transduction athways Amplification Signal Reception Transduction Response(s) Figure 12.1 rinciples of signal transduction. An environmental signal is first received by interaction with a cellular component, most often a cell-surface receptor. The information that the signal has arrived is then converted into other chemical forms, or transduced. The transduction process often comprises many steps. The signal is often amplified before evoking a response. Feedback pathways regulate the entire signaling process Signal Transduction Depends on Molecular Circuits Signal-transduction pathways follow a broadly similar course that can be viewed as a molecular circuit (Figure 12.1). All such circuits contain certain key steps: 1. Release of the rimary Messenger. A stimulus such as a wound or digested meal triggers the release of the signal molecule, also called the primary messenger. 2. Reception of the rimary Messenger. Most signal molecules are too large and too polar to pass through the cell membrane or through transporters. Thus, the information presented by signal molecules must be transmitted across the cell membrane without the molecules themselves entering the cell. Membrane receptors transfer information from the environment to a cell s interior. Such receptors are intrinsic membrane proteins that have both extracellular and intracellular domains. A binding site on the extracellular domain specifically recognizes the signal molecule (often referred to as the ligand). The formation of the receptor ligand complex alters the tertiary or quaternary structure of the receptor, including the intracellular domain. However, structural changes in the few receptors that are bound to ligands are not sufficient to yield a response from the cell. The information conveyed by the receptor must be transduced into other forms of information that can alter the biochemistry of the cell. 3. Relay of Information by the Second Messenger. Structural changes in receptors lead to changes in the concentration of small molecules, called second messengers, which are used to relay information from the receptor ligand complex. articularly important second messengers include cyclic AM (cam) and cyclic GM (cgm, or cyclic guanosine monophosphate), calcium ion, inositol 1,4,5-trisphosphate, (I 3 ), and diacylglycerol (DAG; Figure 12.2). The use of second messengers has several consequences. First, second messengers are often free to diffuse to other compartments of the cell, such as the nucleus, where they can influence gene expression and other processes. Second, the signal may be amplified significantly in the generation of second messengers. Each activated receptor ligand complex can lead to the generation of many second messengers within the cell. Thus, a low concentration of signal in the environment, even as little as a single molecule, can yield a large intracellular signal and response. 4. Activation of Effectors That Directly Alter the hysiological Response. The ultimate effect of the signal pathway is to activate (or inhibit) the pumps, enzymes, and gene-transcription factors that directly control metabolic pathways, gene activation, and processes such as nerve transmission. A or G H Cyclic AM (cam), Cyclic GM (cgm) 2+ H 2 H 2 H 2 H 2 Ca H 2 H 2 H 2 Calcium ion H H H Inositol 1,4,5-trisphosphate (I 3 ) Figure 12.2 Common second messengers. Second messengers are intracellular molecules that change in concentration in response to environmental signals. That change in concentration conveys information inside the cell. Diacylglycerol (DAG) H H

3 5. Termination of the Signal. After a signaling process has been initiated and the information has been transduced to affect other cellular processes, the signaling processes must be terminated. Without such termination, cells lose their responsiveness to new signals. Moreover, signaling processes that fail to be terminated properly may lead to uncontrolled cell growth and the possibility of cancer Receptor roteins Essentially every biochemical process presented in the remainder of this book either is a component of a signal-transduction pathway or can be affected by one Receptor roteins Transmit Information Into the Cell Most receptor proteins that mediate information transfer into the cell interior fall into three classes: seven-transmembrane-helix receptors, dimeric receptors that recruit protein kinases, and dimeric receptors that are protein kinases. We begin by considering the largest and one of the most important classes of receptor, the seven-transmembrane-helix receptors. Seven-Transmembrane-Helix Receptors Change Conformation in Response to Ligand Binding and Activate G roteins The seven-transmembrane-helix (7TM) receptors are responsible for transmitting information initiated by signals as diverse as photons, odorants, tastants, hormones, and neurotransmitters (Table 12.1). Several thousand such receptors are known, and the list continues to grow. Indeed, approximately 50% of the drugs that we use alter receptors of this class. As the name indicates, these receptors contain seven helices that span the membrane bilayer (Figure 12.3). An example of a 7TM receptor that responds to chemical signals is the b-adrenergic receptor. This protein binds epinephrine (also called adrenaline), a hormone responsible for the fight or flight response. We will address the biochemical roles of this hormone in more detail later (p. 375). A variety of evidence reveals that the 7TM receptors, particularly their cytoplasmic domains, change conformation in response to ligand binding. Thus, the binding of a ligand from outside the cell induces a conformational change in the 7TM receptor that can be detected inside the cell. As we shall see, 7TM receptors also have in common the next step in their signal-transduction cascades. Table 12.1 Biological functions mediated by 7TM receptors Hormone action Hormone secretion Neurotransmission Chemotaxis Exocytosis Control of blood pressure Embryogenesis Cell growth and differentiation Development Smell Taste Vision Viral infection Source: After J. S. Gutkind, J. Biol. Chem. 273: , H H H H H N Epinephrine CH 3 (A) N (B) N Ligand-binding site C C Cytoplasmic loops Figure 12.3 The structure of 7TM receptors. (A) Schematic representation of a 7TM receptor showing how it passes through the membrane seven times. (B) Three-dimensional structure of rhodopsin, a 7TM receptor taking part in visual signal transduction. Notice the ligand-binding site near the extracellular surface. As the first 7TM receptor for which the structure was determined, rhodopsin provides a framework for understanding other 7TM receptors. [Drawn from 1F88.pdb.]

4 Signal-Transduction athways Epinephrine β-adrenergic receptor Adenylate cyclase GT α GD Figure 12.4 The activation of protein kinase A by a G-protein pathway. Hormone binding to a 7TM receptor initiates a signal-transduction pathway that acts through a G protein and cam to activate protein kinase A. β γ AT rotein kinase A Cyclic AM rotein kinase A Ligand Binding to 7TM Receptors Leads to the Activation of G roteins Let us focus on the -adrenergic receptor as a model of the 7TM receptor class. What is the next step in the pathway after the binding of epinephrine by the -adrenergic receptor? The conformational change in the cytoplasmic domain of the receptor activates a GT-binding protein. This signal-coupling protein is termed a G protein (G for guanyl nucleotide). The activated G protein stimulates the activity of adenylate cyclase, an enzyme that increases the concentration of the second messenger cam by forming it from AT (Figure 12.4). How do these G proteins operate? In the unactivated state, the guanyl nucleotide bound to the G protein is GD. In this form, the G protein exists as a heterotrimer consisting of,, and subunits; the subunit (referred to as G a ) binds the nucleotide (Figure 12.5). The and subunits are usually anchored to the membrane by covalently attached fatty acids. The exchange of the bound GD for GT is catalyzed by the hormone-bound receptor. The hormone receptor complex interacts with the heterotrimeric G protein and opens the nucleotide-binding site so that GD can depart and GT from solution can bind. The subunit simultaneously dissociates from the dimer (G bg ), see Figure 12.4). The dissociation of the G-protein heterotrimer into G a and G bg units transmits the signal that the receptor has bound its ligand. (A) (B) γ α GD β Figure 12.5 A heterotrimeric G protein. (A) A ribbon diagram shows the relation between the three subunits. In this complex, the subunit (gray and purple) is bound to GD. Notice that GD is bound in a pocket close to the surface at which the subunit interacts with the dimer. (B) A schematic representation of the heterotrimeric G protein. [Drawn from 1GT.pdb.] GD

5 A single hormone receptor complex can stimulate nucleotide exchange in many G-protein heterotrimers. Thus, hundreds of G molecules are converted from their GD into their GT forms for each bound molecule of hormone, giving an amplified response. All 7TM receptors appear to be coupled to G proteins, and so the 7TM receptors are sometimes referred to as G-protein-coupled receptors or GCRs Receptor roteins Activated G roteins Transmit Signals by Binding to ther roteins As described in the preceding subsection, the formation of the hormone receptor complex activates a G protein. How does the G protein propagate the message that the hormone is present? It does so by a variety of means, depending on the specific type of G protein. We will begin by examining one target of a G protein, the enzyme adenylate cyclase (Figure 12.6). The adenylate cyclase enzyme that is activated by the -adrenergic signaling pathway is a membrane protein that contains 12 presumed membrane-spanning helices. The G protein binds to adenylate cyclase on the G surface that had bound the dimer when the G protein was in its GD form. G s (where s stands for stimulatory ) stimulates adenylate cyclase activity, thus increasing cam production. The net result is that the binding of epinephrine to the receptor on the cell surface increases the rate of cam production inside the cell. (A) (B) G αs (GT form) N C Adenylate cyclase Adenylate cyclase fragment Figure 12.6 Adenylate cyclase activation. (A) Adenylate cyclase is a membrane protein with two large intracellular domains (red and orange) that contain the catalytic apparatus. (B) The structure of the complex between G in its GT form bound to a catalytic fragment of adenylate cyclase. Notice that the surface of G that had been bound to the dimer (see Figure 12.5) now binds adenylate cyclase. [Drawn from 1AZS.pdb.] Cyclic AM Stimulates the hosphorylation of Many Target roteins by Activating rotein Kinase A The increased concentration of cam can affect a wide range of cellular processes, depending on the cell type. For example, it enhances the degradation of storage fuels, increases the secretion of acid by the gastric mucosa in the cells of the stomach and intestines, leads to the dispersion of melanin pigment granules in skin cells, diminishes the aggregation of blood platelets, and induces the opening of chloride channels in the pancreas. How does cam influence so many cellular processes? Is there a common denominator for its diverse effects? Indeed there is. Most effects of cyclic AM in eukaryotic cells are mediated by the activation of a single protein kinase. This key enzyme is called protein kinase A (KA). Kinases are enzymes that phosphorylate a substrate at the expense of a molecule of AT. KA

6 cam C R R C + 4 cam C + R R + C Active Active cam-binding domains Figure 12.7 The regulation of protein kinase A. The binding of four molecules of cam activates protein kinase A by dissociating the inhibited holoenzyme (R 2 C 2 ) into a regulatory subunit (R 2 ) and two catalytically active subunits (C). consists of two regulatory (R) chains and two catalytic (C) chains. In the absence of cam, the R 2 C 2 complex is catalytically inactive (Figure 12.7). The binding of cam to the regulatory chains releases the catalytic chains, which are enzymatically active on their own. Activated KA then phosphorylates specific serine and threonine residues in many targets to alter their activity. The cam cascade is turned off by cam phosphodiesterase, an enzyme that converts cam into AM, which does not activate KA. The C and R subunits subsequently rejoin to form the inactive enzyme. G roteins Spontaneously Reset Themselves Through GT Hydrolysis The ability to shut down signal-transduction pathways is as critical as the ability to turn them on. How is the signal initiated by activated 7TM receptors switched off? G a subunits have intrinsic GTase activity, hydrolyzing bound GT to GD and i (inorganic orthophosphate) and thereby deactivating itself. This hydrolysis reaction is slow, however, requiring from seconds to minutes and thus allowing the GT form of G to activate downstream components of the signal-transduction pathway before GT hydrolysis deactivates the subunit. In essence, the bound GT acts as a built-in clock that spontaneously resets the G a subunit after a short time period. After GT hydrolysis and the release of i, the GDbound form of G then reassociates with G to reform the heterotrimeric protein (Figure 12.8). Adenylate cyclase GT H 2 i GD GD 178 Figure 12.8 Resetting G. n hydrolysis of the bound GT by the intrinsic GTase activity of G, G reassociates with the dimer to form the heterotrimeric G protein, thereby terminating the activation of adenylate cyclase.

7 Dissociation Receptor roteins Figure 12.9 Signal termination. Signal transduction by the 7TM receptor is halted, in part, by dissociation of the signal molecule (yellow) from the receptor. The hormone-bound activated receptor must be reset as well to prevent the continuous activation of G proteins. A key step in the inactivation of the receptor rests on the fact that the receptor ligand interaction is reversible (Figure 12.9). When the hormone dissociates, the receptor returns to its initial, unactivated state. The likelihood that the receptor remains in its unbound state depends on the concentration of hormone in the environment. QUICK QUIZ 1 List the means by which the -adrenergic pathway is terminated. Clinical Insight Cholera and Whooping Cough Are Due to Altered G-rotein Activity The alteration of G-protein-dependent signal pathways can result in pathological conditions. Let us first consider the mechanism of action of the cholera toxin, secreted by the intestinal bacterium Vibrio cholera. Cholera is an acute diarrheal disease that can be life threatening. It causes a voluminous secretion of electrolytes and fluids from the intestines of infected persons (Figure 12.10). The cholera toxin, choleragen, is a protein composed of two functional units a B subunit that binds to cells of the intestinal epithelium and a catalytic A subunit that enters the cell. The A subunit catalyzes the covalent modification of a G s protein. This modification stabilizes the active GTbound form of G s, trapping the molecule in the active conformation. The active G protein, in turn, continuously activates protein kinase A. KA opens a chloride channel (a CFTR channel) and inhibits the Na H exchanger by phosphorylation. The net result of the phosphorylation of these channels is an excessive loss of NaCl and the loss of large amounts of water into the intestine. atients suffering from cholera for 4 to 6 days may pass as much as twice their body weight in fluid. Treatment consists of rehydration with a glucose electrolyte solution. Whereas cholera is a result of a G protein trapped in the active conformation, causing the signal-transduction pathway to be perpetually stimulated, pertussis, or whooping cough, is a result of the opposite situation. The toxin also modifies a G protein called G i, which normally inhibits adenylate cyclase, closes Ca 2 channels, and opens K channels. The effect of this modification is to lower the G protein s affinity for GT, effectively trapping it in the off conformation. The symptoms of whooping cough, such as prolonged coughing that ends with a whoop as the patient gasps for air, have not yet been traced to the inhibition of any single target of the G i protein. ertussis toxin is secreted by Bordetella pertussis, the bacterium responsible for whooping cough. Figure Death s dispensary. An 1866 cartoon illustrating that contaminated water is a frequent source of cholera infection. [The Granger Collection.]

8 Signal-Transduction athways The Hydrolysis of hosphatidyl Inositol Bisphosphate by hospholipase C Generates Two Second Messengers Cyclic AM is not the only second messenger employed by 7TM receptors and the G proteins. We turn now to another ubiquitous second-messenger cascade used by many hormones to evoke a variety of responses. The phosphoinositide cascade, like the adenylate cyclase cascade, converts extracellular signals into intracellular ones. The intracellular messengers formed by activation of this pathway arise from the cleavage of phosphatidylinositol 4,5-bisphosphate (I 2 ), a phospholipid present in cell membranes. The binding of a hormone such as vasopressin to a 7TM receptor leads to the activation of phospholipase C. The G protein that activates phospholipase C is called G q. The activated enzyme then hydrolyzes the phosphodiester linkage joining the phosphorylated inositol unit to the acylated glycerol moiety. The cleavage of I 2 produces two messengers: inositol 1,4,5-trisphosphate (I 3 ), a soluble molecule that can diffuse from the membrane, and diacylglycerol (DAG), which stays in the membrane (Figure 12.11). What are the biochemical effects of the second messenger I 3? Unlike cam, I 3 does not cause a cascade of phosphorylation to elicit a response from the cell. I 3 directly causes the rapid release of Ca 2 from intracellular stores the endoplasmic reticulum and, in smooth muscle cells, the sarcoplasmic reticulum. I 3 associates with a membrane protein called the I 3 -gated channel or I 3 receptor to allow the flow of Ca 2 from the endoplasmic reticulum into the cell cytoplasm. The elevated level of Ca 2 in the cytoplasm then triggers a variety of biochemical processes such as smooth-muscle contraction, glycogen breakdown, and vesicle release. The lifetime of I 3 in the cell is very short less than a few seconds. It is rapidly converted into derivatives that have no effect on the I 3 -gated channel. H H H H hospholipase C hosphatidylinositol 4,5-bisphosphate (I 2 ) H H + H 2 3 H H Diacylglycerol (DAG) Inositol 1,4,5-trisphosphate (I 3 ) Figure The phospholipase C reaction. hospholipase C cleaves the membrane lipid phosphatidylinositol 4,5-bisphosphate into two second messengers: diacylglycerol, which remains in the membrane, and inositol 1,4,5-trisphosphate, which diffuses away from the membrane.

9 Cell membrane Diacylglycerol (DAG) Receptor roteins DAG I 2 hospholipase C cleavage I 3 Calcium ion rotein kinase C I 3 receptor Cytoplasm ER membrane Calcium ion Figure The phosphoinositide cascade. The cleavage of phosphatidylinositol 4,5- bisphosphate (I 2 ) into diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (I 3 ) results in the release of calcium ions (owing to the opening of the I 3 receptor ion channels) and the activation of protein kinase C (owing to the binding of protein kinase C to free DAG in the membrane). Calcium ions bind to protein kinase C and help facilitate its activation. Lithium ion, widely used to treat bipolar affective disorder, may act by inhibiting the recycling of I 3, although the details of lithium action remain to be determined. Diacylglycerol, the other molecule formed by the receptor-triggered hydrolysis of I 2, also is a second messenger that, in conjunction with Ca 2,activates protein kinase C (KC), a protein kinase that phosphorylates serine and threonine residues in many target proteins (Figure 12.12). Some Receptors Dimerize in Response to Ligand Binding and Recruit Tyrosine Kinases The 7TM receptors initiate signal-transduction pathways through changes in tertiary structure that are induced by ligand binding. A fundamentally different mechanism is utilized by a number of other classes of receptors. For these receptors, ligand binding leads to changes in quaternary structures specifically, the formation of receptor dimers. We consider human growth hormone and its receptor as an example. Growth hormone is a monomeric protein of 217 amino acids. The growth-hormone receptor has an extracellular domain, a single membrane-spanning helix, and an intracellular domain. In the absence of bound hormone, the receptor is present as a monomer. Each monomeric hormone binds to the extracellular domains of two receptor molecules, thus promoting the formation of a dimer of the receptor (Figure 12.13). Dimerization of the extracellular domains of the receptor brings together the intracellular domains as well. Associated with each intracellular domain is

10 (A) Growth hormone (B) Extracellular domain Growth hormone Growthhormone receptor (extracellular domains) Intracellular domain Dimerized receptor (activated) Figure The binding of growth hormone leads to receptor dimerization. (A) A single growth-hormone molecule (yellow) interacts with the extracellular domains of two receptors (red and orange). (B) The binding of one hormone molecule to two receptors leads to the formation of a receptor dimer. Dimerization is a key step in this signal-transduction pathway. verstimulation of the growth-hormone signal-transduction pathway can lead to pathological conditions. Acromegaly is a rare hormonal disorder resulting from the overproduction of growth hormone in middle age. A common characteristic of acromegaly is enlargement of the face, hands, and feet. Excessive production of growth hormone in children results in gigantism. André the Giant, who played the beloved giant Fezzik in Rob Reiner s classic film The rincess Bride suffered from gigantism. a molecule of a protein kinase termed Janus kinase 2 (JAK2) in an unactivated form (Figure 12.14). Dimerization of the growth-hormone receptors brings together the JAK2 proteins associated with each intracellular domain. Each of the kinases phosphorylates its partner, resulting in the activation of the kinases. When activated by cross-phosphorylation, JAK2 can phosphorylate other substrates, such as a regulator of gene expression called STAT5 (STAT for signal transducers and activators of transcription). hosphorylated STAT5 moves to the nucleus, where it binds to the DNA binding sites to regulate gene expression. A signal received on the outside of the cell membrane is forwarded to the nucleus for action. Some Receptors Contain Tyrosine Kinase Domains Within Their Covalent Structures Some growth factors and hormones such as epidermal growth factor (EGF), platelet-derived growth factor, and insulin bind to the extracellular domains of transmembrane receptors that have kinase domains present within their Hormone-induced dimerization Crossphosphorylation Activated JAK Figure The cross-phosphorylation of two molecules of JAK2 induced by receptor dimerization. The binding of growth hormone (blue) leads to growth-hormone receptor dimerization, which brings two molecules of Janus kinase 2 (JAK2, yellow) together in such a way that each phosphorylates key residues on the other. The activated JAK2 molecules remain bound to the receptor.

11 intracellular domains. Such receptors have a specific kind of kinase called tyrosine kinase, which phosphorylates proteins on the hydroxyl group of tyrosine residues. These receptor tyrosine kinases (RTKs) signal by mechanisms quite similar to those described for the pathway initiated by the growth-hormone receptor discussed in the preceding subsection. f the more than 500 kinase genes in the human genome, fewer than 100 encode tyrosine kinases, and all of these tyrosine kinases appear to regulate the control of growth. Consider, for example, epidermal growth factor, a 6-kd polypeptide that stimulates the growth of epidermal and epithelial cells by binding to the epidermal growth factor receptor, a single polypeptide chain consisting of 1186 residues (Figure 12.15). The receptor tyrosine kinase is monomeric and enzymatically inactive in the absence of the growth factor. The binding of EGF to its extracellular domain causes the receptor to dimerize and undergo cross-phosphorylation and activation Receptor roteins EGF-binding domain Transmembrane helix Kinase domain C-terminal tail (tyrosine-rich) Figure The modular structure of the EGF receptor. This schematic view of the amino acid sequence of the EGF receptor shows the EGF-binding domain that lies outside the cell, a single transmembrane helix-forming region, the intracellular tyrosine kinase domain, and the tyrosine-rich domain at the carboxyl terminus. How is the signal transferred beyond the receptor tyrosine kinase? A key adaptor protein, called Grb-2, links the phosphorylation of the EGF receptor to the stimulation of cell growth through a chain of protein phosphorylations (Figure 12.16). n phosphorylation of the receptor, Grb-2 binds to the phosphotyrosine residues of the receptor tyrosine kinase. Grb-2 then recruits a protein called Sos. Sos, in turn, binds to Ras and activates it. Ras is a very prominent signal-transduction component that we will consider shortly. Finally, Ras, in its activated form, binds to other components of the molecular circuitry, leading to the activation of the specific protein kinases that phosphorylate specific targets that promote cell growth. We see here another example of how a signal-transduction pathway is constructed. Specific protein protein interactions link the original ligand-binding event to the final result the stimulation of cell growth. EGF EGF receptor Extracellular domain Intracellular domain Grb-2 Sos GD Ras GT GD GT Activated Ras Figure The EGF signaling pathway. The binding of epidermal growth factor (EGF) to its receptor leads to cross-phosphorylation of the receptor. The phosphorylated receptor binds Grb-2, which, in turn, binds Sos. Sos stimulates the exchange of GT for GD in Ras. Activated Ras binds to protein kinases and stimulates them (not shown).

12 Signal-Transduction athways Table 12.2 Ras superfamily of GTases Subfamily Function Ras Rho Arf Rab Ran Regulates cell growth through serine or threonine protein kinases Reorganizes cytoskeleton through serine or threonine protein kinases Activates the AD-ribosyltransferase of the cholera toxin A subunit; regulates vesicular trafficking pathways; activates phospholipase D lays a key role in secretory and endocytotic pathways Functions in the transport of RNA and protein into and out of the nucleus Ras Belongs to Another Class of Signaling G rotein The signal-transduction protein Ras is member of an important family of signal proteins the small G proteins, or small GTases. This large superfamily of proteins grouped into subfamilies called Ras, Rho, Arf, Rab, and Ran plays a major role in a host of cell functions including growth, differentiation, cell motility, cytokinesis, and the transport of materials throughout the cell (Table 12.2). Like their relatives the heterotrimeric G proteins (p. 176), the small G proteins cycle between an active GT-bound form and an inactive GD-bound form. They differ from the heterotrimeric G proteins in being smaller (20 25 kd compared with kd) and monomeric. In their activated GT-bound form, small G proteins such as Ras stimulate cell growth and differentiation. Recall that Sos is the immediate upstream link to Ras in the circuit conveying the EGF signal. How does Sos activate Ras? Sos binds to Ras, reaches into the nucleotide-binding pocket, and opens it up, allowing GD to escape and GT to enter in its place. Sos is referred to as a guanine-nucleotide exchange factor (GEF). Like the G protein, Ras possesses an intrinsic GTase activity, which serves to terminate the signal and return the system to the inactive state. This activity is slow but is augmented by helper proteins termed GTase-activating proteins (GAs). The guanine-nucleotide exchange factors and the GTase-activating proteins allow the G-protein cycle to proceed with rates appropriate for a balanced level of downstream signaling Metabolism in Context: Insulin Signaling Regulates Metabolism Insulin is among the principal hormones that regulate metabolism, and we will examine the effects of this hormone on many metabolic pathways later. This section presents an overview of its signal-transduction pathway. Insulin is the hormone released after eating a full meal and is the biochemical signal designating the fed state. In all of its detail, this multibranched pathway is quite complex; so we will focus solely on the major branch. This branch leads to the mobilization of glucose transporters to the cell surface. These transporters allow the cell to take up the glucose that is plentiful in the bloodstream after a meal. Figure Insulin structure. Notice that insulin consists of two chains (shown in blue and yellow) linked by two interchain disulfide bonds. The chain (blue) also has an intrachain disulfide bond. [Drawn from IB2F.pdb.] The Insulin Receptor Is a Dimer That Closes Around a Bound Insulin Molecule Insulin is a peptide hormone that consists of two chains, linked by two disulfide bonds (Figure 12.17). The insulin receptor is a member of the receptor tyrosine kinase class of membrane proteins. The identical subunits form dimers on

13 insulin binding, and this change in quaternary structure results in cross-phosphorylation by the two kinase domains, activating the kinase activity (Figure 12.18). Each subunit consists of one chain and one chain linked to one another by a single disulfide bond. Each subunit lies completely outside the cell, whereas each subunit lies primarily inside the cell, spanning the membrane with a single transmembrane segment. The two subunits move together to form a binding site for a single insulin molecule. The Activated Insulin Receptor Kinase Initiates a Kinase Cascade n phosphorylation, the insulin receptor tyrosine kinase is activated. Because the two units of the receptor are held in close proximity to each other, additional sites within the receptor also are phosphorylated. These phosphorylated sites act as docking sites for other substrates, including a class of molecules referred to as insulin-receptor substrates (IRSs). The IRS proteins are subsequently phosphorylated by the tyrosine kinase activity of the insulin receptor. The signal is conveyed to the cell interior by the IRS protein through a series of membrane-anchored molecules to a protein kinase that finally leaves the membrane (Figure 12.19). Insulin Insulin receptor Insulin-binding site β subunit α subunit Figure The insulin receptor. The receptor consists of two units, each of which consists of an subunit and a subunit linked by a disulfide bond. The subunit lies outside the cell and two subunits come together to form a binding site for insulin. Each subunit lies primarily inside the cell and includes a protein kinase domain. I 2 IRS-1 hosphoinositide 3-kinase Akt I 3 AT AD DK1 (I 3 -dependent protein kinase) Activated Akt Figure Insulin signaling. The binding of insulin results in the crossphosphorylation and activation of the insulin receptor. hosphorylated sites on the receptor act as binding sites for insulin-receptor substrates such as IRS-1. The lipid kinase phosphoinositide 3-kinase binds to phosphorylated sites on IRS-1 through its regulatory domain and then converts I 2 into I 3. Binding to I 3 activates I 3 -dependent protein kinase (DK1), which phosphorylates and activates kinases such as Akt. Activated Akt can then diffuse throughout the cell to continue the signal-transduction pathway. In their phosphorylated form, the IRS molecules act as adaptor proteins.the phosphotyrosine residues in the IRS proteins are recognized by other proteins, the most important of which is a lipid kinase that phosphorylates phosphatidylinositol 4,5-bisphosphate to form phosphatidylinositol 3,4,5-trisphosphate (I 3 ; Figure 12.20, see also Figure 12.19). This lipid product, in turn, activates a protein R H H H H AT hosphatidylinositide R' 3-kinase R' hosphatidylinositol 4,5-bisphosphate (I 2 ) hosphatidylinositol 3,4,5-trisphosphate (I 3 ) AD R H H H Figure The action of a lipid kinase in insulin signaling. hosphorylated IRS-1 and IRS-2 activate the enzyme phosphatidylinositide 3-kinase, an enzyme that converts I 2 into I

14 Crossphosphorylation Insulin + Insulin receptor Enzymatic reaction Activated receptor hosphorylated IRS proteins rotein protein interaction Localized phosphoinositide 3-kinase Enzymatic reaction hosphatidyl inositol-3,4,5-trisphosphate (I 3 ) rotein lipid interaction Amplification Activated I 3 -dependent protein kinase Enzymatic reaction Amplification Amplification Activated Akt protein kinase Increased glucose transporter on cell surface Figure Insulin signaling pathway. Key steps in the signal-transduction pathway initiated by the binding of insulin to the insulin receptor. QUICK QUIZ 2 Why does it make good physiological sense for insulin to increase the number of glucose transporters in the cell membrane? Figure An EF hand. Formed by a helix-loop-helix unit, an EF hand is a binding site for Ca 2+ in many calcium-sensing proteins. Here, the E helix is yellow, the F helix is blue, and calcium is represented by the green sphere. Notice that the calcium ion is bound in a loop connecting two nearly perpendicular helices. [Drawn from 1CLL.pdb.] 186 kinase, DK1. This activated protein kinase phosphorylates and activates Akt, another protein kinase. Akt is not membrane anchored and moves through the cell to phosphorylate enzymes that stimulate glycogen synthesis as well as components that control the trafficking of the glucose transporter GLUT4 to the cell surface. At the cell surface, GLUT4, one of a family of five glucose transporters, allows the entry of glucose down its concentration gradient into the cell. The cascade initiated by the binding of insulin to the insulin receptor is summarized in Figure The signal is amplified at several stages along this pathway. Because the activated insulin receptor itself is a protein kinase, each activated receptor can phosphorylate multiple IRS molecules Activated enzymes further amplify the signal in at least two of the subsequent steps. Thus, a small increase in the concentration of circulating insulin can produce a robust intracellular response. Note that, as complicated as the pathway described here is, it is substantially less elaborate than the full network of pathways initiated by insulin. Insulin Signaling Is Terminated by the Action of hosphatases We have seen that the activated G protein promotes its own inactivation by the release of a phosphoryl group from GT. In contrast, proteins phosphorylated on serine, threonine, or tyrosine residues, such as insulin-receptor substrates, do not hydrolyze spontaneously; they are extremely stable kinetically. Specific enzymes, called protein phosphatases, are required to hydrolyze these phosphorylated proteins and convert them back into the states that they were in before the initiation of signaling. Similarly, lipid phosphatases are required to remove phosphoryl groups from inositol lipids that had been phosphorylated as part of a signaling cascade. In insulin signaling, three classes of enzymes are of particular importance: protein tyrosine phosphatases that remove phosphoryl groups from tyrosine residues on the insulin receptor, lipid phosphatases that hydrolyze phosphatidylinositol 3,4,5-trisphosphate to phosphatidylinositol 3,4-bisphosphate, and protein serine phosphatases that remove phosphoryl groups from activated protein kinases such as Akt. Many of these phosphatases are activated or recruited as part of the response to insulin. Thus, the binding of the initial signal sets the stage for the eventual termination of the response Calcium Ion Is a Ubiquitous Cytoplasmic Messenger We have already seen that Ca 2 is an important component of one signaltransduction circuit, the phosphoinositide cascade. Indeed, Ca 2 is itself an intracellular messenger in many eukaryotic signal-transducing pathways. Calmodulin (CaM), a 17-kd protein with four calcium-binding sites, serves as a calcium sensor in nearly all eukaryotic cells. Calmodulin is activated by the binding of Ca 2 when the cytoplasmic calcium level is raised. Calmodulin is a member of the EFhand protein family. The EF hand is a Ca 2 -binding motif that consists of a helix, a loop, and a second helix. This Ca 2+ motif, originally discovered in the protein parvalbumin, was named the EF hand because the helices designated E and F in parvalbumin that form the calcium-binding motif are positioned like the forefinger and thumb of the EF hand right hand (Figure 12.22).

15 The Ca 2 calmodulin complex stimulates a wide array of enzymes, pumps, and other target proteins. Two targets are especially noteworthy: one that propagates the Ca 2 signal and another that abrogates it. The binding of Ca 2 calmodulin to a calmodulin-dependent protein kinase (CaM kinase) activates the kinase and enables it to phosphorylate a wide variety of target proteins. CaM kinases regulate the metabolism of fuel, ionic permeability, neurotransmitter synthesis, and neurotransmitter release through the action of the Ca 2 calmodulin complex. The plasma membrane Ca 2 ATase pump is another important target of Ca 2 calmodulin. Stimulation of the pump by Ca 2 calmodulin drives the calcium level down to restore a low-calcium basal state to the cell, thus helping to terminate the signal Signaling athway Defects 12.5 Defects in Signal-Transduction athways Can Lead to Disease In light of their complexity, it comes as no surprise that signal-transduction pathways occasionally fail, leading to pathological or disease states. Cancer, a set of diseases characterized by uncontrolled or inappropriate cell growth, is strongly associated with defects in signal-transduction proteins. Indeed, the study of cancer has contributed greatly to our understanding of signal-transduction proteins and pathways. Genes that, when mutated, cause cancer often normally regulate cell growth. The unmutated, normally expressed versions of these genes are termed protooncogenes, and the proteins that they encode are signal-transduction proteins that regulate cell growth. If a proto-oncogene suffers a mutation that leads to unrestrained growth by the cell, the gene is then referred to as an oncogene. The gene encoding Ras, a component of the EGF-initiated pathway, is one of the genes most commonly mutated in human tumors. Mammalian cells contain three Ras proteins (H-, K-, and N-Ras), each of which cycles between inactive GD and active GT forms. The most-common mutations in tumors lead to a loss of the ability to hydrolyze GT. Thus, the Ras protein is trapped in the on position and continues to stimulate cell growth, even in the absence of a continuing signal. Mutated, or overexpressed, receptor tyrosine kinases also are frequently observed in tumors. For instance, the epidermal-growth-factor receptor (EGFR) is overexpressed in some human epithelial cancers, including breast, ovarian, and colorectal cancer. Because some small amount of the receptor can dimerize and activate the signaling pathway even without binding to EGF, the overexpression of the receptor increases the likelihood that a grow and divide signal will be inappropriately sent to the cell. ther genes can contribute to cancer development only when both copies of the gene normally present in a cell are deleted or otherwise damaged. Such genes are called tumor-suppressor genes. These genes encode proteins that either inhibit cell growth by turning off growth-promoting pathways or trigger the death of tumor cells. For example, genes for some of the phosphatases that participate in the termination of EGF signaling are tumor suppressors. Without any functional phosphatase present, EGF signaling persists after its initiation, stimulating inappropriate cell growth. c-abl gene Chromosome 9 Chromosome 22 Translocation bcr gene bcr-abl gene Clinical Insight rotein Kinase Inhibitors May Be Effective Anticancer Drugs The widespread occurrence of overactive protein kinases in cancer cells suggests that molecules that inhibit these enzymes might act as antitumor agents. Recent results have dramatically supported this concept. More that 90% of patients with chronic myelogenous leukemia (CML) have a specific chromosomal defect in affected cells (Figure 12.23). The translocation of genetic material between Figure The formation of the bcr-abl gene by translocation. In chronic myelogenous leukemia, parts of chromosomes 9 and 22 are reciprocally exchanged, causing the bcr and abl genes to fuse. The protein kinase encoded by the bcr-abl gene is expressed at higher levels in cells having this translocation than is the c-abl gene in normal cells.

16 Signal-Transduction athways chromosomes 9 and 22 causes the c-abl ( c for cellular) gene, which encodes a tyrosine kinase, to be inserted into the bcr gene on chromosome 22. The result is the production of a fusion protein called Bcr-Abl that consists primarily of sequences for the c-abl kinase. However, the bcr-abl gene is expressed at higher levels than the gene encoding the normal c-abl kinase, leading to an excess of signals for cell growth. In addition, the Bcr-Abl protein may have regulatory properties that are subtly different from those of the c-abl kinase itself. Thus, leukemia cells express a unique target for drugs. Recent clinical trials of a specific inhibitor of the Bcr-Abl kinase, imatinib mesylate (called Gleevec commercially), have shown dramatic results; more than 90% of patients responded well to the treatment. Thus, our understanding of signal-transduction pathways is leading to conceptually new disease treatments. SUMMARY 12.1 Signal Transduction Depends on Molecular Circuits Most signal-transduction pathways are constructed with a similar set of components. A primary messenger, such as a hormone, binds to the extracellular part of a membrane-bound receptor. The messenger receptor complex generates a second messenger inside the cell, which activates proteins that alter the biochemical environment inside the cell. Finally, means exist to terminate the signal-transduction pathway Receptor roteins Transmit Information Into the Cell Seven-transmembrane-helix receptors operate in conjunction with heterotrimeric G proteins. The binding of a hormone to a 7TM receptor triggers the exchange of GT for GD bound to the subunit of the G protein. G proteins can transmit information in a number of ways. G s -GT activates adenylate cyclase, an integral membrane protein that catalyzes the synthesis of cam. Cyclic AM then activates protein kinase A by binding to its regulatory subunit, thus unleashing its catalytic chains. 7TM receptors activate G q proteins and the phosphoinositide pathway. The receptortriggered activation of phospholipase C generates two intracellular messengers by hydrolysis of phosphatidylinositol 4,5-bisphosphate. Inositol trisphosphate opens calcium channels in the endoplasmic and sarcoplasmic reticulum membranes. Diacylglycerol activates protein kinase C. Some ligands induce dimerization of the receptors to which they bind. Such a receptor contains an extracellular domain that binds the ligand, a transmembrane region, and a cytoplasmic domain that either binds a protein kinase or is a protein kinase. The growth-hormone receptor participates in an example of this type of signal-transduction pathway. Dimerization of the receptor activates Janus kinase 2, a protein kinase associated with the intracellular part of the receptor. Intrinsic tyrosine kinases are covalently incorporated in the intracellular domains of some receptors, such as the epidermal-growth-factor receptor and the insulin receptor. When such receptor tyrosine kinases dimerize and are activated, cross-phosphorylation takes place. The phosphorylated tyrosines in activated receptor tyrosine kinases serve as docking sites for signaling proteins and permit further propagation of the signal. A prominent component of such pathways is the small GTase Ras. The Ras protein, like the G subunit, cycles between an inactive form bound to GD and an active form bound to GT Metabolism in Context: Insulin Signaling Regulates Metabolism The hormone insulin is secreted when blood levels of glucose are high. Insulin binds to the insulin receptor, which is a receptor tyrosine kinase.

17 The activated tyrosine kinase then phosphorylates insulin-receptor substrate. The signaling pathway continues, with the key result being an increase in glucose transporters in the cell membrane. 189 roblems 12.4 Calcium Ion Is a Ubiquitous Cytoplasmic Messenger Calcium ion acts by binding to calmodulin and other calcium sensors. Calmodulin contains four calcium-binding modules called EF hands that recur in other proteins. Ca 2 calmodulin activates target proteins by binding to positively charged amphipathic helices Defects in Signaling athways Can Lead to Disease If the genes encoding components of the signal-transduction pathways are altered by mutation, pathological conditions, most notably cancer, may result. In their mutated form, these genes are called oncogenes. The normal counterparts are called proto-oncogenes and function in pathways that control cell growth and replication. Mutated versions of ras are frequently found in human cancers. Key Terms primary messenger (p. 174) ligand (p. 174) second messenger (p. 174) seven-transmembrane-helix (7TM) receptor) (p. 175) G protein (p. 176) G (p. 176) G (p. 176) G-protein-coupled receptor (GCR) (p. 177) adenylate cyclase (p. 177) protein kinase A (KA) (p. 177) phosphoinositide cascade (p. 180) phospholipase C (p. 180) protein kinase C (KC) (p. 181) receptor tyrosine kinase (RTK) (p. 183) Ras (p. 183) calmodulin (CaM) (p. 186) EF hand (p. 186) calmodulin-dependent protein (CaM) kinase (p. 187) oncogene (p. 187) proto-oncogene (p. 187) Answers to QUICK QUIZZES 1. Dissociation of epinephrine from the receptor. Conversion of cam into AM by phosphodiesterase and the subsequent inhibition of KA. Conversion GT into GD by G and the subsequent reformation of the inactive heterotrimeric G protein. 2. Insulin signifies the fed state. Its presence leads to the removal of glucose from the blood for storage or metabolism. Increasing the number of glucose transporters available makes these biochemical processes more efficient. roblems 1. A reappearance. Ligand-gated channels can be thought of as receptors. Explain. 2. Magnification. Explain how a small number of hormones binding to the extracellular surface of a cell can have a large biochemical effect inside the cell. 3. n off. Why is the GTase activity of G proteins crucial to the proper functioning of a cell? 4. Specificity. Hormones affect the biochemistry of a distinct set of tissues. What accounts for the specificity of hormone action? 5. Making connections. Suppose that you were investigating a newly discovered growth-factor signal-transduction pathway. You found that, if you added a GT analog in which the terminal phosphate was replaced by sulfate, the duration of the hormonal response was increased. What can you conclude? 6. Viva la différence. Why is the fact that a monomeric hormone binds to two identical receptor molecules, thus promoting the formation of a dimer of the receptor, considered remarkable?

18 Signal-Transduction athways 7. Chimeras. In an elegant experiment on the nature of receptor tyrosine kinase signaling, a gene was synthesized that encoded a chimeric receptor the extracellular part came from the insulin receptor, and the membrane-spanning and cytoplasmic parts came from the EGF receptor. The striking result was that the binding of insulin induced tyrosine kinase activity, as evidenced by rapid autophosphorylation. What does this result tell you about the signaling mechanisms of the EGF and insulin receptors? 8. Active mutants. Some protein kinases are inactive unless they are phosphorylated on key serine or threonine residues. In some cases, active enzymes can be generated by mutating these serine or threonine residues to glutamate. ropose an explanation. 9. Antibodies mimicking hormones. An antibody has two identical antigen-binding sites. Remarkably, antibodies to the extracellular parts of growth-factor receptors often lead to the same cellular effects as does exposure to growth factors. Explain this observation. 10. Facile exchange. A mutated form of the subunit of the heterotrimeric G protein has been identified; this form readily exchanges nucleotides even in the absence of an activated receptor. What would be the effect on a signaling pathway containing the mutated subunit? 11. Diffusion rates. Normally, rates of diffusion vary inversely with molecular weights; so smaller molecules diffuse faster than do larger ones. In cells, however, calcium ion diffuses more slowly than does cam. ropose a possible explanation. 12. Awash with glucose. Glucose is mobilized for AT generation in muscle in response to epinephrine, which activates G s. Cyclic AM phosphodiesterase is an enzyme that converts cam into AM. How would inhibitors of cam phosphodiesterase affect glucose mobilization in muscle? 13. Many defects. Considerable effort has been directed toward determining the genes in which sequence variation contributes to the development of type 2 diabetes, a disease that results from a loss of sensitivity of cells to insulin. Approximately 800 genes have been implicated. ropose an explanation for this observation. 14. Growth-factor signaling. Human growth hormone binds to a cell-surface membrane protein that is not a receptor tyrosine kinase. The intracellular domain of the receptor can bind other proteins inside the cell. Furthermore, studies indicate that the receptor is monomeric in the absence of hormone but dimerizes on hormone binding. ropose a possible mechanism for growth-hormone signaling. 15. Total amplification. Suppose that each -adrenergic receptor bound to epinephrine converts 100 molecules of G s into their GT forms and that each molecule of activated adenylate cyclase produces 1000 molecules of cam per second. With the assumption of a full response, how many molecules of cam will be produced in 1 s after the formation of a single complex between epinephrine and the -adrenergic receptor? Data Interpretation roblems 16. Establishing specificity. You wish to determine the hormone-binding specificity of a newly identified membrane receptor. Three different hormones, X, Y, and Z, were mixed with the receptor in separate experiments, and the percentage of binding capacity of the receptor was determined as a function of hormone concentration, as shown in graph A. (A) Binding to receptor as a percentage of the maximum X Hormone concentration (M) (a) What concentrations of each hormone yield 50% maximal binding? (b) Which hormone shows the highest binding affinity for the receptor? You next wish to determine whether the hormone receptor complex stimulates the adenylate cyclase cascade. To do so, you measure adenylate cyclase activity as a function of hormone concentration, as shown in graph B. (B) Stimulation of adenylate cyclase as a percentage of maximum X Hormone concentration (M) Y Y Z Z 10 2 (c) What is the relation between the binding affinity of the hormone receptor complex and the ability of the hormone to enhance adenylate cyclase activity? What can you conclude about the mechanism of action of the hormone receptor complex?