32.3 How Do Signal-Transducing Receptors Respond to the Hormonal Message? 1013 32.3 How Do Signal-Transducing Receptors Respond to the Hormonal Message? All receptors that mediate transmembrane signaling processes fit into one of three receptor superfamilies (Figure 32.7): 1. The G-protein–coupled receptors (see Section 32.4) are integral membrane pro- teins with an extracellular recognition site for ligands and an intracellular recog- nition site for a GTP-binding protein (see following discussion). 2. The single-transmembrane segment (1-TMS) catalytic receptors are proteins with only a single transmembrane segment and substantial globular domains on both the extracellular and the intracellular faces of the membrane. The extracellular domain in the ligand recognition site and the intracellular catalytic domain is ei- ther a tyrosine kinase or a guanylyl cyclase. 3. Oligomeric ion channels consist of associations of protein subunits, each of which contains several transmembrane segments. These oligomeric structures SH3 SH2 PH PDZ (dimer) Pumilio, complexed with RNA (green) FIGURE 32.5 Five of the protein modules found in cell signaling proteins.The binding specificity of these mod- ules is shown in Figure 32.6a.SH3 domains bind to proline-rich peptides; SH2 domains bind to phosphorylated tyrosine residues; PH domains bind to phosphoinositides (such as IP 3 ); PDZ domains bind to the terminal four or five residues of a target protein; pumilio domains bind to segments of RNA. A given protein may have a number of these protein modules, giving it the ability to interact with multiple partners. 1014 Chapter 32 The Reception and Transmission of Extracellular Information P SH3 (a) (b) PH PDZ SH2 WW IP 3 B D C PXXP COO ؊ COO ؊ N XXXXXSXXXXYXX PH IP 3 PDZ SH2 WW SH3 P X X P COO ؊ XXX P P P P A FIGURE 32.6 (a) Many signaling proteins consist of com- binations of protein modules, each with a specific bind- ing or enzymatic function. (b) Such multifunctional pro- teins can act as scaffolds that direct the assembly of large signaling complexes, termed signalsomes. Binding site for GTP-binding proteins Tyrosine kinase or guanylyl cyclase activity Channel opens and closes in response to extracellular ligand binding G-protein–coupled receptor N C Receptor tyrosine kinase or receptor guanylyl cyclase Oligomeric ion channels FIGURE 32.7 The membrane receptor superfamilies.The G-protein–coupled receptors are named for the GTP- binding proteins that mediate some of their effects.The receptor tyrosine kinases and receptor guanylyl cyclases contain intracellular enzymatic domains that respond to extracellular hormone binding.The oligomeric ion chan- nels (some of which were discussed in Chapter 9) open and close in response to ligand binding and/or changes of the transmembrane electrochemical potential. 32.3 How Do Signal-Transducing Receptors Respond to the Hormonal Message? 1015 are ligand-gated ion channels. Binding of the specific ligand typically opens the ion channel. The ligands for these ion channels are neurotransmitters. The G-Protein–Coupled Receptors Are 7-TMS Integral Membrane Proteins The G-protein–coupled receptors (GPCRs) have primary and secondary structure similar to that of bacteriorhodopsin (see Chapter 9), with seven transmembrane ␣-helical segments; they are thus known as 7-transmembrane segment (7-TMS) pro- teins. Rhodopsin and the -adrenergic receptors, for which epinephrine is a ligand, are good examples (Figure 32.8). The site for binding of cationic catecholamines to the adrenergic receptors is located within the hydrophobic core of the receptor. Binding of hormone to a GPCR induces a conformation change that activates a GTP-binding protein, also known as a G protein (discussed in Section 32.4). Acti- vated G proteins trigger a variety of cellular effects, including activation of adenylyl and guanylyl cyclases (which produce cAMP and cGMP from ATP and GTP), activation of phospholipases (which produce second messengers from phospho- lipids) and activation of Ca 2ϩ and K ϩ channels (which leads to elevation of intra- cellular [Ca 2ϩ ] and [K ϩ ]). (All of these effects are described in Section 32.4.) The Single TMS Receptors Are Guanylyl Cyclases or Tyrosine Kinases Receptor proteins that span the plasma membrane with a single helical trans- membrane segment possess an external ligand recognition site and an internal do- main with enzyme activity—either receptor tyrosine kinase (RTK) or receptor Membrane Outside  2 -Adrenergic receptor Inside NH 3 + COO – Asp 113 (a) (b) FIGURE 32.8 (a) The arrangement of the  2 -adrenergic receptor in the membrane. Substitution of Asp 113 in the third hydrophobic domain of the -adrenergic receptor with an Asn or Gln by site-directed mutagenesis results in a dramatic decrease in affinity of the receptor for both agonists and antagonists. Significantly, this Asp residue is conserved in all other GPCRs that bind biogenic amines but is absent in receptors whose ligands are not amines. Asp 113 appears to be the counterion of the amine moiety of adrenergic ligands. (b) The structure of a  2 - adrenergic receptor (pdb id ϭ 2RH1).The flexible third intracellular loop and C-terminal segment are not shown. (From Figure 2 from Palczewski, K.,et al., 2000. Crystal structure of rhodopsin: A G-protein-coupled receptor. Science 289:739–745.) 1016 Chapter 32 The Reception and Transmission of Extracellular Information guanylyl cyclase (RGC). Each of these enzyme activities is manifested in two dif- ferent cellular forms. Thus, guanylyl cyclase activity is found in both membrane- bound receptors and in soluble, cytoplasmic proteins. Tyrosine kinase activity, on the other hand, is exhibited by two different types of membrane proteins: The RTKs are integral transmembrane proteins, whereas the non-RTKs are peripheral, lipid-anchored proteins. RTKs and RGCs Are Membrane-Associated Allosteric Enzymes The binding of polypeptide hormones and growth factors to RTKs and RGCs acti- vates the intracellular enzyme activity of these proteins. These catalytic receptors are composed of three domains (Figure 32.9): an extracellular receptor-binding domain (which may itself include several subdomains), a transmembrane domain consisting of a single transmembrane ␣-helix, and an intracellular domain. This intracellular portion includes a tyrosine kinase or guanylyl cyclase domain that mediates the bio- logical response to the hormone or growth factor via its catalytic activity and a regu- latory domain that contains multiple phosphorylation sites. The human genome contains at least 58 different RTKs, which can be grouped into about 20 families on the basis of their kinase domain sequences and the various extracellular subdomains. The epidermal growth factor (EGF) receptor and the insulin receptor are represen- tative of this class of receptor proteins. Given that the extracellular and intracellular domains of RTKs and RGCs are joined by only a single transmembrane helical segment, how does extracellular hormone binding activate intracellular enzyme activity? How is the signal transduced? As shown in Figure 32.10, signal transduction occurs by hormone-induced oligomeric associa- tion of receptors. Hormone binding triggers a conformational change in the extracel- lular domain, which induces oligomeric association. Oligomeric association allows ad- jacent cytoplasmic domains to interact, leading to phosphorylation of the cytoplasmic domains and stimulation of cytoplasmic enzyme activity. By virtue of these ligand- Outside (a) (b) (c) Inside N I II III IV C Fn3 Fn2b Fn2a Fn1 Tyrosine kinase Tyrosine kinase L2 CR L1 Fn3 Fn2b CC Hormone- binding domain Juxta- membrane domain Protein kinase–like domain Protein kinase domains Guanylyl cyclase ANP-R (d) FIGURE 32.9 (a) The EGF receptor and (b) the insulin re- ceptor are receptor tyrosine kinases. EGF receptors are activated by ligand-induced dimerization, whereas the insulin receptor is a glycoprotein composed of two kinds of subunits in an ␣ 2  2 tetramer stabilized by disul- fide bonds.The extracellular portions of the EGF and in- sulin receptors consist of multiple modules or domains. Fn refers to a series of FnIII-type domains numbered 1, 2a, 2b, and 3.(c) The atrial natriuretic peptide receptor is a receptor guanylyl cyclase with a large extracellular hormone-binding domain and two intracellular do- mains, and activation typically involves ligand-induced dimerization. (d) The growth factor receptor Ret is a re- ceptor tyrosine kinase that mediates the effects of neu- ronal growth factors known as neurotrophins.The Ret receptor domains (yellow) requires a GPI-anchored coreceptor (blue) for binding of ligands (pink). 32.3 How Do Signal-Transducing Receptors Respond to the Hormonal Message? 1017 induced conformation changes and oligomeric interactions, RTKs and RGCs are membrane-associated allosteric enzymes. EGF Receptor Is Activated by Ligand-Induced Dimerization Human EGF is a 53-amino acid peptide that stimulates proliferation of epithelial cells (cells that cover a surface or line a cavity in biological tissues). The human EGF receptor is a 1186–amino acid RTK. The extracellular domain, where EGF binds, contains 622 amino acids and is divided into four domains (Figure 32.9). Domains I and III are similar -helical barrels, whereas domains II and IV are long, slender, cysteine-rich domains stabilized by disulfide bonds. Domain II is characterized by a -hairpin structure that protrudes from the middle of the domain (Figure 32.11a). Prior to EGF binding, EGF receptors exist as inactive monomers in the plasma membrane. In this state, the four domains are folded so that domains II and IV lie parallel, with the -hairpin of domain II in contact with domain IV (Figure 32.11a). Binding of EGF to domain I induces a conformation change that rotates domains I and II so that the bound EGF is brought into contact with domain III and the -hairpin is extended away from the rest of the structure. Pairs of such receptor monomers then dimerize by mutual association of the -hairpin structures (Figure 32.11b). The next event is the critical step in transmembrane signal transduction. Dimer- ization of the extracellular domains brings the intracellular domains together, acti- vating the tyrosine kinase activity. Thus, EGF-induced dimerization allows an extra- cellular signal (EGF) to exert an intracellular response. EGF Receptor Activation Forms an Asymmetric Tyrosine Kinase Dimer The tyrosine kinase domain of the EGF receptor consists of an N-terminal domain built around a twisted -sheet and a C-terminal domain that is primarily ␣-helical (Figure 32.12a). In the inactive, monomeric EGF receptor, the active site of the tyrosine kinase domain is blocked by a 30-residue loop of the protein (residues 831–860) that is termed the activation loop. Extracellular dimer formation by the Ligand(a) (a) (d) SS SS Ligand(b) S S ANIMATED FIGURE 32.10 Ligand-induced oligomerization of membrane receptors can occur in several ways. (a) EGF receptor dimerization and activation involves binding of hormone to the extracellular domains of two receptor molecules. (b) The insulin receptor is a preexisting tetramer. Two insulin- binding sites are created by association of the extracellular domains. (c) RGCs bind a single hormone ligand at the dimer interface. (Ligand dimers could presumably act in a similar manner). (d) Neurotrophin activation of Ret involves association of two molecules of the Ret kinase and two molecules of a GPI-anchored coreceptor protein. See this figure animated at www.cengage.com/login. Ligand monomer or dimer (c) (b) 1018 Chapter 32 The Reception and Transmission of Extracellular Information EGF receptor appears to promote formation of an asymmetric dimer of the intra- cellular tyrosine kinase domains (Figure 32.12b), with the C-terminal lobe of one kinase domain juxtaposed with the N-terminal lobe of the other kinase domain. In this asymmetric dimer, one monomer is inactive but acts as an activator of kinase ac- tivity in the other monomer. Conversion of the kinase domain from its inactive state to the active conformation involves rotation of the activation loop out of the active site, making room for a peptide substrate to enter the site (Figure 32.12c). The activated tyrosine kinase domain of the EGF receptor can phosphorylate several tyrosine residues at its own C-terminus (Figure 32.13), a process termed autophosphorylation. These phosphorylated tyrosines are binding sites for a variety of other signaling proteins that contain phosphotyrosine-binding SH2 domains (see Figures 32.5 and 32.6). Each of these SH2-domain–containing proteins can initiate several signal transduction cascades. Binding site for EGF on domain III (a) (b) Domain I/II rigid body rotation Dimerization 1 1 EGF 2 2 E 3 3 4 4 E 11 22 33 44 DimerAuto-inhibited EE II III IV I I III II IV FIGURE 32.11 (a) In the absence of hormone, the extracellular domain of the EGF recep- tor is folded with the hairpin loop and dimerization interface on domain II (green) asso- ciated with domain IV (orange). Binding of EGF (red) induces a conformation change that exposes the dimerization interface and promotes formation of the activated dimer complex (pdb id ϭ 1NQL). (b) Dimerization involves dove-tailing of the domain II hair- pin loops (pdb id ϭ 1IVO). 1019 (a) (c) (b) Phosphorylated tyrosines Downstream signaling molecules Ligand (e.g., EGF) Ligand-induced dimerization Y992 Y1173 Y1148 Y1068 Y1045 1. Activation of kinase domain Extracellular region (1–621) Kinase domain (686–960) Sites of tyrosine phosphorylation Juxtamembrane segment (645–685) Transmembrane segment (622–644) 2. FIGURE 32.13 Hormone-induced dimerization of the EGF receptor promotes autophosphorylation of five tyrosine residues near the C-terminus of each EGF receptor subunit. Signaling proteins that contain phosphotyrosine-binding SH2 domains can be activated by binding to these phosphotyrosines. FIGURE 32.12 (a) The kinase domain of the EGF receptor consists of an N-terminal lobe built around a twisted -sheet and an ␣-helical C-terminal lobe. Bound ATP is pink, and the activation loop, shown blocking the active site, is blue. (b) Hormone-induced receptor dimerization promotes formation of an asymmetric dimer of the intracellular kinase domains (yellow and green). (c) Before hormonal activation (left), the kinase active site is blocked by a 30-residue activation loop (green). In the activated complex (right), the activation loop (green) has rotated away from the active site in one of the subunits, making room for binding of a peptide substrate (blue) (a, b, c [right]: pdb id ϭ 2GS6; c [left]: pdb id ϭ 2GS7). 1020 Chapter 32 The Reception and Transmission of Extracellular Information The Insulin Receptor Mediates Several Signaling Pathways Insulin, a small heterodimeric peptide (see Figure 5.8), is the most potent anabolic hormone known. It regulates blood glucose levels, and it promotes the synthesis and storage of carbohydrates, proteins, and lipids. Abnormalities of insulin pro- duction, action, or both lead to diabetes, as well as other serious health issues. In- sulin binding to receptors in liver, muscle, and other tissues triggers multiple sig- naling pathways, and insulin action is responsible for a variety of cellular effects. The insulin receptor is an RTK that catalyzes the phosphorylation of tyrosine residues of several intracellular substrates, including the insulin receptor substrate (IRS) proteins, and several other proteins known as Gab-1, Shc, and APS (Figure 32.14). Each of these phosphorylated substrates binds a particular family of proteins containing SH2 domains (see Figures 32.5 and 32.6). These SH2 proteins interact specifically with phosphotyrosine-containing IRS sequences. Each of these signaling pathways can be confined to distinct cellular locations and can proceed with a dif- ferent time course, thus providing spatial and temporal dimensions to insulin ac- tion in cells. The Insulin Receptor Adopts a Folded Dimeric Structure in the Membrane Unlike the majority of RTKs, which are single-chain receptors, the insulin receptor is an ␣ 2  2 tetramer. The ␣-chain contains two leucine-rich domains (L1 and L2) with a cysteine-rich domain (CR) between them, as well as an intact fibronectin do- main (Fn1) and a partial fibronectin domain (Fn2). The -chain contains the other half of Fn2, followed by a third fibronectin domain (Fn3), a transmembrane ␣-helix, and (inside the cell) the tyrosine kinase domain (see Figure 32.9). The ex- tracellular domain (the ectodomain) forms a folded dimer, with the L1 and L2 domains of one ␣-chain juxtaposed with the Fn2 domain of the other ␣/ chain pair, to create the insulin-binding site (Figure 32.15). Thus, each of the two insulin- binding sites of the ectodomain consists of portions of both ␣-chains. Autophosphorylation of the Insulin Receptor Kinase Opens the Active Site Binding of insulin to its receptor activates the tyrosine kinase activity of the intra- cellular domains. Like the EGF receptor kinase, the insulin receptor kinase con- tains an activation loop that lies across the kinase active site, excluding substrate peptides and thus inhibiting the kinase. Phosphorylation of three tyrosine residues CAP Grb2 Insulin receptor Shc Y-P Y-P Shp-2 MAP kinase pathway Gab-1 Y-P P-Y P13-K IRS1-4 CrkII Lipid raft AKT pathway Cbl P-Y APS FIGURE 32.14 Substrates of the insulin receptor tyrosine kinase include the insulin receptor substrate (IRS) pro- teins, as well as Gab-1, Shc, and APS.The phosphorylated substrates in turn bind to several families of SH2 domain–containing proteins, activating several signal- ing pathways. Insulin-binding sites L1 L1 L1 L2 L2 L2 CR Fn1 Fn2 Fn3 FIGURE 32.15 The ectodomain formed by the insulin receptor is constructed from the two ␣/ units. These two units are folded so that the L1 domain of one ␣-chain is juxtaposed with the Fn1 domain of the other ␣-chain. Each insulin-binding site is created by the L1 and L2 domains of one ␣-chain and the Fn2 domain of the other ␣/ chain pair (see Figure 32.9) (pdb id ϭ 2DTG). 32.3 How Do Signal-Transducing Receptors Respond to the Hormonal Message? 1021 on the activation loop—another case of autophosphorylation—causes the activa- tion loop to move out of and away from the active site (Figure 32.16). This opens the active site so that target proteins are bound and phosphorylated by the kinase, triggering the appropriate signaling pathways (see Figure 32.14). Receptor Guanylyl Cyclases Mediate Effects of Natriuretic Hormones When you eat a salty meal, your body secretes hormones that protect you from the harmful effects of excess salt intake. When your heart senses that blood volume is too great, it sends signals to the kidneys to excrete NaCl and water (processes termed natriuresis and diuresis, respectively). These are just two examples of the action of natriuretic hormones, which allow tissues and organs to communicate with one an- other to regulate the volumes of blood and other body fluids and the osmotic effects of Na ϩ , K ϩ , Cl Ϫ , and other ions. Guanylin and uroguanylin are produced in the in- testines after ingestion of a salty meal and are secreted into the intestinal lumen. Binding of guanylin and uroguanylin to receptor guanylyl cyclases on cell mem- branes lining the lumen activates the intracellular guanylyl cyclase (Figure 32.17a). cGMP (see Figure 10.12) produced in this reaction is a second messenger that in- hibits Na ϩ uptake from the lumen and activates Cl Ϫ export into the lumen. The re- sult is a beneficial enhanced excretion of NaCl and water. (This effect can be carried too far, however. Heat-stable enterotoxin (ST) produced by E. coli—with a sequence similar to guanylin and uroguanylin—causes violent diarrhea.) Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP, so named because it was discov- ered first in brain) are both produced primarily in the heart. When the heart mus- cle is stressed and stretched by increased blood volume, the heart secretes ANP and BNP into the blood. In the kidneys, ANP and BNP bind to RGCs in kidney tubules, activating intracellular guanylyl cyclase and producing cGMP (Figure 32.17b). As in the intestines, cGMP inhibits Na ϩ uptake, once again stimulating excretion of salt and water and reducing blood volume. A Symmetric Dimer Binds an Asymmetric Peptide Ligand RGC monomers associate as dimers in the membrane, even in the absence of their hormone ligands (Figure 32.18a). A dimeric receptor complex is activated by the binding of a single polypeptide hormone. This raises two important questions about the mechanism of action of these receptors: (1) How does an asymmetric hormone ligand (for example, ANP) bind to a symmetric homodimeric receptor? And (2) how does hormone binding to its extracellular dimeric receptor activate the intracellular guanylyl cyclase domain? Answers to these provocative questions have been provided FIGURE 32.16 In its inactive state, the insulin receptor tyrosine kinase is inhibited by an activation loop (yellow and red), which prevents substrate access to the active site (left—pdb id ϭ 1IRK). Autophosphorylation of three tyrosine residues on the activation loop induces a con- formation change that rotates the loop out of the active site (right—pdb id ϭ 1IR3), permitting access by insulin receptor substrates (blue) and ATP (ATP analog in orange). 1022 Chapter 32 The Reception and Transmission of Extracellular Information by Kunio Misono and his colleagues, who determined the structure of the ANP receptor–ANP complex (Figure 32.18b). Remarkably, there is no significant in- tramolecular conformational change in either of the ANP receptor monomers. How- ever, upon ANP binding, the two ANP receptor molecules undergo a twist motion in the membrane in order to insert the ANP hormone between them (Figure 32.19). The hormone lies like a disc between the two receptor subunits. The hormone- induced twist of the extracellular domains suggests a mechanism for activation of guanylyl cyclase activity across the membrane. Rotation of the transmembrane + E. coli ST NSSN NCCCCPA TGCCELYY Human guanylin PYTCACAA TGCCE IG Human uroguanylin Intestinal cell (enterocyte) Kidney tubule cell or Kidney cells Intestinal cells NNDCVLCVA TGCCE 115105 LD G R I G D F R I G S S R L S N S F R Y A S E C Atrial natriuretic peptide C G L G Cl – Na + H + cGMP BloodLumen GTP FIGURE 32.17 (a) Ingestion of a salty meal triggers excretion of NaCl and water in the intestines. Binding of guanylin and uroguanylin to RGC on cell membranes lining the lumen activates the intracellular guanylyl cyclase. cGMP produced by the cyclase inhibits Na ϩ uptake from the lumen and activates Cl Ϫ export into the lumen. (b) Atrial natriuretic peptide (ANP) protects the heart and circulatory system from the deleterious effects of increased blood volume.When the heart muscle is stressed and stretched, the heart secretes ANP. ANP binding to RGCs in kidney tubules activates intracellular guanylyl cyclase, producing cGMP. Inhibition of Na ϩ uptake in the kidneys stimulates excretion of salt and water, reducing blood volume. N-terminus N-terminus N-terminus N-terminus C-terminus C-terminus C-terminus C-terminus ANP (a) (b) FIGURE 32.18 Activation of the ANP receptor involves binding of an asymmetric ligand at the interface of two identical receptor domains. Comparison of the structures of the receptor domain in the absence (a) and presence (b) of ANP reveals no significant intramolecular conformational change in either of the receptor monomers. ANP binding induces a twist of the two ANP receptor molecules, allowing the ANP hormone to insert between them. (a) PDB file provided by Kunio Misono, University of Nevada, Reno; (b) pdb id ϭ 1T34. (a) (b)