We have continued our investigations of the basic mechanisms of receptor (GPCR) activation of G-proteins using a combination of surface plasmon resonance (SPR) measurement of GPCR-G-protein binding and in vitro assay of GPCR-catalyzed guanine nucleotide turnover. We have previously reported that functionally active rhodopsin, the prototype GPCR, can be immobilized for SPR measurement via capture through extracellular carbohydrate binding to immobilized concanavalin A (conA). These investigations have demonstrated that the G-protein alpha and beta-gamma subunit dimer bind independently and synergistically to the receptor. Further, the kinetics of beta-gamma dimer interaction with rhodopsin were strikingly altered by the composition of the gamma subunit chain. Our current investigations have focused on the alpha subunit interaction. We have examined the properties of homogeneously myristoylated alpha-i1 and alpha-o as substrates for receptor-catalyzed guanine nucleotide exchange by bovine rhodopsin in comparison with the endogenous retinal G-protein, transducin (Gt). All of these G-alpha subunits are members of the structurally-related alpha-i family, but they display distinct endogenous GTP exchange rates, beta-gamma dimer affinities, and receptor specificities in addition to cellular expression. We find that rhodopsin will catalyze guanine nucleotide exchange on all three alpha subunits with nearly identical kinetics in vitro. This interaction is absolutely dependent on the presence of myristate modification. However, examined by SPR, the alpha-i1 and alpha-o show dramatically decreased dissociation rates from conA-immobilized rhodopsin in comparison to alpha-t. Whereas alpha-t combined with the retinal beta1-gamma1 dissociates with half-times under 5 sec, the half-lives of alpha-i1 and alpha-o in the presence of beta1-gamma1 are on the order of 1000 sec. Detailed kinetic examination of the rhodopsin-catalyzed nucleotide exchange on alpha-t suggest that the currently held mechanism of GTP-assisted dissociation of activated alpha from receptor may not explain these data. As an initial test of this, we have constructed point mutations of alpha-i1 at amino acid residues that undergo changes in guanine nucleotide interaction between GDP-bound and GTP-bound conformations of alpha. Initial data obtained by Ryba and Hoon (NIDCR) for gustducin alpha suggested that G203A mutations would not exchange GDP. Our data for alpha-i1 show that the mutant spontaneously exchanges GDP similar to wild-type, but is impaired in rhodopsin-catalyzed exchange. Mutation of R208A leads to an alpha that exchanges GDP similar to wild-type, but shows diminished affinity for GTP. SPR studies find that G203A alpha-i1 binds to rhodopsin with a prolonged life-time, independent of exogenous guanine nucleotide. Together, these data suggest that GTP is not required for dissociation of activated alpha from receptor; rather, the receptor catalyzes the activation and dissociation of an un-liganded alpha subunit, that subsequently binds GTP. We have also continued examination of the unique properties of the family3 GPCR structures, examining mutant constructs of metabotropic glutamate receptors (mGluR1) and calcium-sensing receptors (CaR) as well as a gold-fish arginine receptor (Arg-R). Previously we have reported that the seven-transmembrane helix bundle (7TM) of the human CaR (t903-rhoC) without the amino-terminal calcium binding domain (ECD) can be activated by three allosterically interacting sites for divalent cations, polyvalent organic cations (poly-Arg, spermine) and the synthetic ligand NPS 568. Mutation of all five acidic residues in the second extracellular loop of t903-RhoC abrogated the NPS568, but not PolyArg synergy of calcium activation. We have addressed the importance of the second extracellular loop and other loci of acidic residues in the extracelluar sequences of the hCaR 7TM in the context of the full-length structure. The 5-alanine mutant homolog was found to be strongly activating. Of particular interest, mutation of a single residue (E767A) displays a high intrinsic activity, and abrogates the steeply cooperative activation of the hCaR by calcium. These studies amplify on our initial observations of the interacting allosteric sites within the 7TM core of the hCaR, suggesting that the calcium-binding ECD interacts with the 7TM core through a contact at this residue. Further, these data strongly imply that the ECD may be an inhibitory constraint on the activity of the 7TM core. We have set out to test this suggestion by independently expressing the ECD and 7TM core structures for mGluR1, hCaR, and Arg-R. Currently, we have high level expression of the mGluR1 and hCaR 7TM cores and baculoviral constructs for secreted ECD proteins from mGluR1 and Arg-R. When our complement of molecular reagents is completed, we will undertake the isolation to homogeneity of the three ECD structures, and we will examine the ligand binding and 7TM core regulating properties of these constructs. The Arg-R ECD is currently expressed with sufficient yield to entertain obtaining a crystal structure for this protein, which would be of immense interest to compare with the available structure of the rat mGluR1 ECD. In this past year we have initiated a new project in collaboration with Dr. Susan Sullivan, NIDCD. This project seeks to identify the tastant compounds recognized by the entire repertoire of human genes encoding bitter taste receptors. Dr. Sullivan has identified 23 candidate genes from the human genetic databases which have high similarity to the known mouse and rat bitter taste receptors but which are not olfactory receptors. At present we have constructed 11 baculoviral vectors for expressing these, and we have tested five of them as directing the expression of a cell-surface localized receptor. In parallel, Dr. Dennis Drayna, also of NIDCD, has identified the human gene locus encoding the phenylthiocarbimide (PTC) trait. His studies revealed five allelic variants (2 tasting, 3 non-tasting) within the human population. We are currently constructing baculoviral vectors for the expression of these gene products also. In our laboratory, we have developed a novel screening strategy to identify the ligands for these receptors. We have constructed baculoviral vectors for the taste-enriched G-beta-3 and gamma-13 subunits, the myeloid alpha-15/16 proteins and jellyfish aquorin. By simultaneous infection of Sf9 cells with these and viruses encoding the taste receptors, we expect to re-direct the G-protein signaling pathways initiated by the tastants to the production of chemiluminescence. Our initial tests of this have utilized several well-characterized receptors (5-HT1A, 5-HT2C and GRP-R) to confirm that the strategy can succeed. Further, infection with the mouse bitter taste receptor T2R5 succeeds in producing chemiluminescent Sf9 cells in response to cyclohexamide. We also are pursuing a strategy of in vitro assay for these receptors utilizing the in situ reconstitution approach we have developed for the 5-HT and bombesin receptors. We have piloted this strategy with the mouse T2R5, and we have demonstrated that cyclohexamide stimulates the binding of GTP-S to the retinal G-protein. With this latter approach we plan to obtain rigorous quantitative information about the structure-activity profiles for those receptors for which we identify ligands. Further, we will employ the in situ reconstitution to characterize the molecular impact of the allelic variants of the bitter taste receptors (PTC and others).
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