Effective information transfer from the α1b-adrenoceptor to Gα11 requires both β/γ interactions and an aromatic group four amino acids from the C terminus of the G protein

Co-expression of the alpha(1b)-adrenoreceptor and Galpha(11) in cells derived from a Galpha(q)/Galpha(11) knock-out mouse allows agonist-mediated elevation of intracellular Ca2+ levels that is transduced by beta/gamma released from the G protein a subunit. Mutation of Tyr(356) of Galpha(11) to Phe, within a receptor contact domain, had little effect on function but this was reduced greatly by alteration to Ser and virtually eliminated by conversion to Asp. This pattern was replicated following incorporation of each form of Ga-11 into fusion proteins with the alpha(1b)-adrenoreceptor. Following a [S-35] guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) binding assay, immunoprecipitation of the wild type alpha(1b)-adrenoreceptor-Galpha(11) fusion protein indicated that the agonist phenylephrine stimulated guanine nucleotide exchange on Galpha(11) more than 30-fold. Information transfer by agonist was controlled in residue 356 Galpha(11) mutants with rank order Tyr > Phe > Trp > Ile > Ala = Gln = Arg > Ser > Asp, although these alterations did not alter the binding affinity of either phenylephrine or an antagonist ligand. Mutation of a beta/gamma contact interface in the alpha(1b)-adrenoreceptor-Tyr(356) Gat, fusion protein did not alter ligand binding affinity but did reduce greatly beta/gamma binding and phenylephrine stimulation of [S-35]GTPgammas binding. It also prevented agonist elevation of intracellular Ca2+ levels, as did a mutation in Galpha(11) that prevents G protein subunit dissociation. These results indicate that a bulky aromatic group is required four amino acids from the C terminus of Galpha(11) to maximize information transfer from an agonist-occupied receptor and disprove the hypothesis that tyrosine phosphorylation of this residue is required for G protein activation (Umemori, H., Inoue, T., Kume, S., Seldyama, N., Nagao, M, Itoh, H., Nakanishi, S., Mikoshiba, K, and Yamamoto, T. (1997) Science 276,1878-1881). This is distinct from Galpha(i1) where hydrophobicity of the amino acid is the key determinant at this location. They also further demonstrate a key role for the IVY complex in enhancing receptor to G protein a subunit information transfer.