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Identification of a Novel Site within G Protein Subunits Important for Specificity of Receptor-G Protein Interaction

Laboratory for Molecular Pharmacology, the Panum Institute, University of Copenhagen, Copenhagen, Denmark (A.H., M.M.R.); 7TM Pharma A/S, Hørsholm, Denmark (R.J., T.M.F., E.K.); Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, Scotland, United Kingdom (R.J.W., G.M.)

Several domains of G protein subunits are implicated in the control of receptor-G protein coupling specificity. Among these are the extreme N-and C-termini, the 4/6-loops, and the loop linking the N-terminal -helix to the 1-strand of the ras-like domain. In this study, we illustrate that single-point mutations of a highly conserved glycine residue within the linker I region of the G_q subunit confers upon the mutant G_q the ability to be activated by G_i- and G_s -coupled receptors, as evidenced by guanosine 5'-O-(3-[³⁵S]thio)triphosphate binding and inositol phosphate turnover assays. The mutations did not affect expression of G_q proteins nor their ability to stimulate phospholipase C. It is noteworthy that both mutant and wild-type G_q proteins are indistinguishable in their ability to reconstitute a functional Gq-PLC-calcium signaling pathway when cotransfected with the G_q-coupled neurokinin 1 or muscarinic M3 receptor into mouse embryonic fibroblasts derived from G_q/11 knockout mice. On a three-dimensional model of the receptor-G protein complex, the highly conserved linker I region connecting the helical and the GTPase domain of the G protein is inaccessible to the intracellular surface of the receptors. Our data indicate that receptor-G protein coupling specificity is not exclusively governed by direct receptor-G protein interaction and that it even bypasses the requirement of the extreme C terminus of G, a well accepted receptor recognition domain, suggesting a novel allosteric mechanism for G protein-coupled receptor-G protein selectivity.

Address correspondence to: Evi Kostenis, 7TM Pharma A/S, Fremtidsvej 3, 2970 Hørsholm, Denmark. E-mail: ek{at}7tm.com

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