Previously, we had constructed an ensemble of chimeric G-alpha subunits using G-alpha-i1 as the backbone and introducing sequence from G-alpha-q to test for the sequences within G-alpha conferring receptor-selectivity. These studies revealed that the receptor contact surface of the G-alpha subunit comprises an extensive region of at least 50 amino acid residues of the carboxyl terminus of the G-alpha-q. To determine if these results apply to other G-protein types and to examine for additional sequence(s) specifying selective contact, we have constructed a similar set of chimeric structures introducing sequence from the G-alpha-s protein into G-alpha-i1 and testing for interaction with the beta2-adrenergic receptor (b2AR). We were able in these studies to replace the majority of the sequence of G-alpha-i1 with G-alpha-s derived sequence and still obtain expression of functional G-alpha protein. These studies with the beta2-adrenergic receptor and G-alphapi1/s chimeras recapitulated the results found previously for G-alpha-i1/q chimeras and the 5HT2c receptor. In addition, comparison of selectivity for G-alpha-i1/C-terminal-s and G-alphai1Nterminal/G-alpha-s constructs eliminates all other G-alpha-s structure save the N-terminal alpha helix and C-terminal 53 residues as providing receptor-selective contact for b2AR. In a result we were unable to examine with the alpha-i1/q chimeras, we find that the N-terminal alpha helix of G-alpha-s provides the dominant binding interaction for b2AR (Gutierrez et al, manuscript in preparation). In a collaboration with Dr. Reinhard Grisshammer, NINDS, we have examined the Gq interactions with the NTS1 neurotensin receptor incorportated into apolipoprotein A1 scaffolded nanodiscs. These receptor preparations have been characterized hydrodynamically to be assemblies of a single NTS1 protein per HDL particle. We examined the contribution of phospholipid composition to the functional interaction of NTS1 with Gq, finding that acidic phospholipid (palmitoyl-oleyl-phosphatidylglycerate, POPG) supported 160-fold enhanced activation of Gq compared with basic phosphatidylcholine (POPC) or mixtures of POPC/POPG (Ingaka et al., manuscript in revision).

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National Institute on Deafness and Other Communication Disorders
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Inagaki, Sayaka; Ghirlando, Rodolfo; White, Jim F et al. (2012) Modulation of the interaction between neurotensin receptor NTS1 and Gq protein by lipid. J Mol Biol 417:95-111
Lyon, Angeline M; Tesmer, Valerie M; Dhamsania, Vishan D et al. (2011) An autoinhibitory helix in the C-terminal region of phospholipase C-? mediates G?q activation. Nat Struct Mol Biol 18:999-1005