7TM motif of GPCRs based on the X-ray structure of rhodopsin is a relatively simple, single protein comprised of approximately 300-500 amino acids with discrete amino acid motifs in the transmembrane regions and on the C-
terminal extracellular loop, determining the ligand specificity of the receptor and those on the amino terminal designating G-protein interactions (63) Postgenomic alternative splicing can alter the composition of the GPCR to create isoforms that may be species, tissue, and disease-state dependent (54). RL interactions are thought to take place within a pocket in the 7TM motif that can be generically designated to lie between TMs III, IV, and VI. Much of the current knowledge related to the structure of GPCRs is based on the high resolution structure of crystalline bovine rhodop-sin, a 7TM protein (64), which has provided proof of concept that GPCR-like structures can be isolated, purified, and crystallized, and has stimulated efforts to solve the crystal structures of GPCRs (65).
The number of GPCRs present in the human genome, including orphan receptors, has been estimated to be 1000-2000 (66,67), with over 1000 of these coding for odorant and pheromone receptors.
GPCRs can be organized into six main families (66). There are approximately 150 GPCRs, 18 amine receptors, 50 peptide receptors, and 80 orphan GPCRs in family 1. This family also contains receptors for oderants with subfamilies: l a t hat includes rhodopsin, thrombin, and the adenosine A receptor, with a binding site localized within the 7TM motif; l b t hat includes receptors for peptides with the ligand-binding site in the extracellular loops, the N-terminal, and the superior regions of the TM motifs: and lc that comprises receptors for glycoproteins. Binding to this receptor class is mostly extracellular. Family 2 is morphologically, but not sequence, related to the lc family and consists of four GPCRs activated by hormones like glucagon, secretin, and VIP-PACAP. Family 3 contains four metabotropic glutamate receptors and three GABAb receptors. Family 4 is a pheromone (VN) family and family 5 is a group of GPCRs that includes "frizzled" and "smoothened," both involved in embryonic development. The sixth receptor family is a group of cAMP receptors that to date has only been identified in the slime mold, D. discoidium.
Interactions with G-proteins and other associated signaling molecules have the potential for considerable complexity (67) because there are four major G-protein families that interact with GPCRs: (1) G„ that activates adenylyl cyclase; (2)Gai/o that inhibits adeny-lyl cyclase and can also regulate ion channels and activation of cGMP PDE; (3) G„q that activates phospholipase C; and (4) Gal2 that regulates Na+/H+ exchange. G-proteins are het-eromers formed from 20 or more a-, 5 0-, and 10 7-isoforms that offer a considerable variety of potential functional G-proteins. Ras, Roe, and Rho are low molecular weight G-proteins involved in mitogenic signaling. Receptor-associated guanylyl cyclase activity is also regulated by G-proteins.
9.1.2 Other C-Protein-Associatedor -Modulating Proteins. The cyclic nucleotide phosphodiesterases (PDEs) responsible for hydro-lytic degradation of the cyclic nucleotides, cAMP and cGMP, exist in more than 15 isoforms (68), whereas the protein kinases, PKA and PKC, are responsible for protein phosphorylation, the GPCR kinases, GRK 1-6, are responsible for GPCR phosphorylation (69), and the protein phosphatases are responsible for dephosphorylation, the latter potentially numbering in excess of 300, significantly increasing the complexity of GPCR-associated signaling processes.
Superimposed on these signaling proteins are the calmodulins that mediate calcium modulation of receptor function, the j3-ar-involved in inactivation of phos-phorylated receptors (6 members), a group of 15 proteins termed RGS (regulators of G-protein signaling), RAMPs (15 receptor-activated modulating proteins), and a protein known as Sst2p that is involved in receptor desensitization.
The number of GPCRs present in the human genome, including orphan receptors, has been estimated to vary between 600 and 800. With 35 G-protein isoforms, 300 phospha-tases, and the various GPCR-associated signaling proteins described above, there is obviously considerable scope for complexity in cell signaling associated with the GPCR family.
As noted, there is a considerable body of data showing that GPCRs can form homo- and heteromeric forms (e.g., GABAb, adenosine A, and A9A, angiotensin, bradykinin, chemokine, dopamine, metabotropic glutamate, musca-
rinic, opioid, serotonin, and somatostatin), increasing the potential complexity of ligand-driven GPCR signaling processes and offering an opportunity to explore new targets in medicinal chemistry.
Applying an evolutionary trace method (ETM) to assess potential protein-protein interactions, functionally important residue clusters have been identified on transmembrane (TM) helices 5 and 6 in over 700 aligned GPCR sequences (44). Similar clusters have been found on TMs 2 and 3. TM 5 and 6 clusters were consistent with 5,6-contact and 5,6-domain swapped dimer formation. Additional application of ETM to 113 aligned G-protein sequences identified two functional sites: one associated with adenylyl cyclase, Ply, and regulator of G-protein signaling (RGS) binding, and the other extending from the ras-like to helical domain that seems to be associated with GPCR dimer binding. From such findings, it was concluded that GPCR dimeriza-tion and heterodimerization occur in all members of the GPCR superfamily and its subfamilies.
From these findings, potential new approaches to ligand design include the following: (l)antagonists that can act by inhibiting dimer formation, e.g., transmembrane peptide mimics; (2) bivalent compounds/binary conjugates; and (3) compounds targeting the GPCR-G-protein interface.
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