Seven-Helix Receptors

Members of the largest family of plasma membrane receptors are built from a serpentine arrangement of seven transmembrane α-helices. These diverse receptors use trimeric GTP-binding proteins (see Fig. 25-9) to relay signals to effector proteins inside cells. Seven-helix receptors are found in slime molds, so the genes for these proteins originated in early eukaryotes more than 1 billion years ago. Four percent of the genes of the nematode Caenorhabditis elegans (790) encode seven-helix receptors, the largest family of proteins in the organism. In mammals, olfactory cells alone use 500 to 1000 different seven-helix receptors to discriminate odorant molecules (see Fig. 27-1). Other cells are estimated to express another 375 seven-helix receptors to respond to light, amino acids, peptide and protein hormones, catecholamines, and lipids. The chemical ligand remains to be determined for about 40% of these 375 receptors, which are termed orphan receptors. A majority of medically useful drugs bind seven-helix receptors.

Seven hydrophobic sequences traverse the plasma membrane as α-helices (Fig. 24-2). The topology is the same as that of bacteriorhodopsin (see Fig. 7-8), but this might be an example of convergent evolution. Comparative analysis of amino acid sequences suggests that all seven-helix receptors have the same arrangement of helices. For example, the minimum length of sequences connecting the helices is compatible only with the helices being arranged sequentially from I to VII in a serpentine fashion as they cross the lipid bilayer. The N-terminus is outside the cell and varies from 7 to 6000 residues. Some of the larger N-terminal domains participate in ligand binding. The C-terminal segment of the polypeptide is in the cytoplasm and varies in length from 12 to more than 350 residues. Seven-helix receptors are shown throughout this book as individual proteins, but multiple lines of evidence show that many seven-helix receptors function as dimers or larger oligomers, allowing for cross talk between the subunits.

Figure 24-2 structure of seven-helix receptors. A, Model of the generic seven-helix receptor, used throughout this text. The structure is based on the structure of bacteriorhodopsin and the amino acid sequence of a typical vertebrate seven-helix receptor. Seven hydrophobic segments cross the membrane as α-helices. Oligosaccharides are blue. The C-terminal cytoplasmic tail is anchored to the lipid bilayer by two fatty acids covalently bound to a pair of adjacent cysteines. B, Atomic structure of bovine rhodopsin, the receptor for photons in the retina. Covalently bound retinal pigment is green. Oligosaccharides on extracellular loops are blue.

(PDB file: 1F88. Reference: Palczewski K, Kumasaka T, Hori T, et al: Crystal structure of rhodopsin: A G protein–coupled receptor. Science 289:739–745, 2000.)

Most seven-helix receptors are activated by binding a soluble chemical ligand, but some interesting variations exist. Biochemical and mutagenesis experiments indicate that most small ligands bind in a central pocket among the extracellular ends of the helices. Residues lining this pocket are highly variable between receptors, providing specificity for each receptor to bind a particular ligand. The light-absorbing pigment 11- cis retinal, of the photoreceptor protein rhodopsin, is the best-characterized “ligand.” 11-cis retinal is unusual in that it is bound covalently to the receptor (Fig. 24-2B) and is activated by absorbing a photon (see Fig. 27-2). In other respects, it is a good model for other ligands (Fig. 24-3). Neurotransmitters, such as norepinephrine, and drugs also bind between the helices about one third of the way across the membrane. Peptide hormones bind deep in the helical pocket but probably also interact with residues that are more exposed on the cell surface. Receptors for some large ligands (pituitary glycoprotein hormones, such as luteinizing hormone, follicle-stimulating hormone, and thyroid-stimulating hormone) and some small ligands (glutamate, g-amino butyric acid, calcium) bind with high affinity to extracellular N-terminal domains of their seven-helix receptor. The N-terminal domain with bound ligand then stimulates the transmembrane domain of the receptor. The blood-clotting enzyme thrombin activates its receptor on platelets by proteolysis of the receptor rather than by direct binding (see Fig. 30-14). The N-terminal peptide cleaved from the receptor disso-ciates and activates other receptors; what is left of the newly truncated N-terminus folds back and activates its own receptor.

Figure 24-3 activation and adaptation of a seven-helix receptor. A, Ligand binding shifts the equilibrium from the resting conformation toward the active conformation. B, The active receptor promotes dissociation of guanosine diphosphate from the α-subunit of multiple trimeric G-proteins, allowing GTP to bind. Typically, this dissociates G_a from G_bg, each of which activates downstream effectors that produce, for example, the second messengers cAMP and diacylglycerol (DAG). cAMP and DAG activate PKA and PKC, which phosphorylate active receptors on their C-terminus. C, This attracts arrestin, putting the receptor into the inactive adapted state. PKA, protein kinase A; PKC, protein kinase C. (PDB file for arrestin: 1CF1.)

Seven-helix receptors exist in an equilibrium between two conformations: a resting state and an activated state with the ability to catalyze the exchange of nucleotide bound to trimeric G-proteins (Fig. 24-3). Without bound ligand, the resting state is strongly favored. Ligand binding to the receptor (or the isomerization of retinal after absorbing light) initiates signal transduction by shifting the equilibrium to the active state. Activation involves movement of at least two transmembrane helices, but the structural details of this conformational change are not yet well defined. In any event, activation must rearrange the cytoplasmic ends of the helices and the loops connecting them to create a binding site for a target G-protein.

Active receptors transfer the signal to the cyto-plasm by activating trimeric G-proteins. Cytoplasmic loops of active receptors catalyze the dissociation of guanosine diphosphate bound (EDP) to an inactive Ga subunit. GTP then binds and activates Ga (see Fig. 25-9). A single active seven-helix receptor can amplify the signal by activating up to 100 G-proteins. After dissociating from the receptor and each other, both Ga-GTP and Gbg stimulate downstream effector proteins, further amplifying the signal (see Fig. 27-3 for an example of amplification).

Most seven-helix receptors adapt to sustained stimulation. In the short term, phosphorylation of the C-terminal tail of the receptor provides negative feedback that inactivates receptors with ligands still bound (Fig. 24-3C). Along one pathway, second messengers—produced in response to receptor activation—stimulate protein kinases, including cyclic adenosine monophosphate (cAMP)–activated protein kinase A and protein kinase C (see Fig. 25-4). These kinases phosphorylate the C-terminal tails of active receptors, inhibiting interactions with G-proteins. This mechanism allows for cross talk between receptors, as activation of one class of receptors can inactivate other receptors. A second pathway uses Gbg subunits released in response to receptor stimulation to activate protein kinases specific for the receptors themselves, called G-protein-coupled receptor kinases. These kinases phosphorylate serines or threonines on the C-terminal tails of active (but not inactive) receptors.

Phosphorylation of receptor tails creates a binding site for arrestin, a protein with multiple functions. First, arrestin blocks interactions of the receptor with G-proteins, terminating signaling through the main pathway downstream of most seven-helix receptors. In some cases, arrestin initiates a new signal through the MAP kinase pathway (see Figs. 27-6 and 27-7 for two other pathways). Arrestin also promotes the removal of seven-helix receptors from the plasma membrane by endocytosis in clathrin-coated vesicles, a longer-term mechanism that turns down the response of a cell to continuous stimulation. Some internalized receptors recycle to the plasma membrane, but others modified by ubiquitin are directed to lysosomes for destruction. Chapter 27 covers three dramatic examples of seven-helix receptor adaptation.

Mutations of more than 30 seven-helix receptors have been linked to human diseases (Table 24-1). More than 600 mutations are known to inactivate seven-helix receptors in humans by every conceivable means from failure to synthesize the full length protein to reduced affinity for ligands to failure to activate G-proteins. These inherited loss of function mutations are recessive. For example, loss of function mutations in rhodopsin cause retinitis pigmentosa, a degeneration of photoreceptor cells. Another example is severe obesity associated with loss of function mutations of a seven-helix receptor that participates in the neural circuits controlling eating. More than a hundred different mutations produce receptors that are constitutively active without ligand. Particular mutations of rhodopsin cause night blindness, and mutations in a calcium receptor cause dysfunction of the parathyroid gland. The physiology of these activating mutations is complicated, because cells use feedback mechanisms to compensate for the continually active receptors.

Table 24-1 SEVEN-HELIX RECEPTORS AND DISEASE

Receptor Tyrosine Kinases

Many polypeptide growth factors activate cells by binding plasma membrane receptors with cytoplasmic protein tyrosine kinase activity (Fig. 24-4). Ligand binding to extracellular domains allows the cytoplasmic kinase domains of pairs receptors to activate each other and to phosphorylate each other and downstream proteins that control cellular proliferation and differentiation. Mammals have 20 families of receptor tyrosine kinases with distinct structural features.

Figure 24-4 receptor tyrosine kinases. Domain architecture of nine of the 20 families of receptor (R) tyrosine kinases, with ribbon models of several domains. The globular domain of the EphB2 receptor is a b sandwich with a ligand-binding site that includes the exposed loop on the front of this model (PDB file: 1IGY). The extracellular part of the insulin-like growth factor consists of two similar β-helical domains connected by cysteine-rich domains (PDB file: 1IGR). The cytoplasmic kinase domain from the insulin receptor is similar to most known kinases (PDB file: 1IRK). Kinase inserts and C-terminal extensions contain tyrosine phosphorylation sites. Receptor names: EphR, receptor for ephrin, membrane-bound ligands in the nervous system, the largest class of receptor tyrosine kinases; PDGFR, platelet-derived growth factor receptor; FGFR, fibroblast growth factor receptor; VEGFR, vascular endothelial growth factor; Met, receptor for hepatocyte growth factor; TrkA, receptor for nerve growth factor; RET, a cadherin adhesion receptor; Axl, receptor for the growth factor Gas6; EGFR, epidermal growth factor receptor. Domain names: Ig, immunoglobulin; F3, fibronectin-III; CAD, cadherin.

(References: Hubbard SR, Till JH: Protein tyrosine kinase structure and function. Annu Rev Biochem 69:373–398, 2000; Garrett TP, McKern NM, Lou M, et al: Crystal structure of the first three domains of the type-1 insulin-like growth factor receptor. Nature 394:395–399, 1998; and Hubbard SR, Wei L, Ellis L, Hendrickson WA: Crystal structure of the tyrosine kinase domain of the human insulin receptor. Nature 372:746–754, 1994.)

The growth factors that activate receptor tyrosine kinases regulate development and differentiation. For example, epidermal growth factor (EGF) stimulates proliferation and differentiation of epithelial cells. Platelet-derived growth factor stimulates growth of smooth muscle cells, glial cells, and fibroblasts (see Fig. 32-11). Some growth factors were discovered by biochemical purification of proteins that stimulate cellular growth or differentiation. EGF was discovered with a bioassay, as it causes the eyelids of newborn mice to open prematurely. A homolog of the EGF receptor, HER2/ErbB2, was discovered as the normal version of a cancer-causing viral oncogene. Other ligands and receptors were discovered as genes in flies or worms required for development. The Drosophila sevenless gene encodes a receptor tyrosine kinase that is related to insulin receptor. Mutations in the sevenless gene result in failure to develop photoreceptor cell number 7 in the fly’s eye.

Receptor tyrosine kinases consist of an extracellular ligand-binding domain connected to a cytoplasmic tyrosine kinase domain by a single transmembrane helix (Fig. 24-4). The ligand binding is mediated by immunoglobulin domains, fibronectin III domains (see Fig. 3-13), cadherin domains (see Fig. 30-5), and less familiar domains such as β-helical and cysteine-rich domains. This domain architecture illustrates that genes for receptor tyrosine kinases were assembled from sequences for familiar domains followed by divergence to allow for interactions with diverse ligands.

Ligand binding activates all well-characterized receptor tyrosine kinases by bringing together a pair of kinase domains on the cytoplasmic face of the membrane. Dimeric ligands such as platelet-derived growth factor recruit a pair of receptors from the pool of subunits diffusing in the plane of the membrane and connect them physically. This induced dimerization juxtaposes two kinase domains in the cytoplasm. An EGF monomer binds a receptor and induces a conformational change that favors the formation of a receptor dimer but is not a physical part of the connection between the subunits (Fig. 24-5). Insulin binding induces a conformational change in a preformed receptor dimer held together by a disulfide bond. The conformational change brings together the kinase domains (see Fig. 27-7).

Figure 24-5 subunit dimerization mechanism for activating the egf receptor tyrosine kinase. In the absence of EGF, intramolecular interactions preclude dimerization. EGF binding changes the conformation of the extracellular domains allowing dimerization of two receptors, bringing together two cytoplasmic kinase domains. Transphosphorylation activates both kinases and creates phosphotyrosine bind-ing sites for SH2 and PTB domains of downstream signal transduction proteins.

(PDB files: 2AHX and 2A91. Reference: Burgess AW, Cho HS, Eigenbrot C, et al: An open-and-shut case? Recent insights into the activation of EGF/ErbB receptors. Mol Cell 12:541–552, 2003.)

The juxtaposition of kinase domains allows the partners to activate each other by direct interaction and by phosphorylating each other on tyrosine residues. In most cases, phosphorylation of tyrosines on the activation loop of the catalytic domain refolds the loop into an active conformation (see Fig. 25-3D), although this is not required for the EGF receptor. In all cases, the paired kinases phosphorylate tyrosines on inserts and C-terminal extensions of the kinase domain, creating phosphotyrosine-binding sites for downstream effector and adapter proteins with SH2 and PTB domains (Fig. 24-5; also see Figs. 27-6

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