Effective information transfer from the alpha 1b-adrenoceptor to Galpha 11 requires both beta/gamma 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 Ca(2+) levels that is transduced by beta/gamma released from the G protein alpha 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 Galpha(11) into fusion proteins with the alpha(1b)-adrenoreceptor. Following a [(35)S]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) Galpha(11) fusion protein did not alter ligand binding affinity but did reduce greatly beta/gamma binding and phenylephrine stimulation of [(35)S]GTPgammaS binding. It also prevented agonist elevation of intracellular Ca(2+) 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., Sekiyama, 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 beta/gamma complex in enhancing receptor to G protein alpha subunit information transfer.
Liu
Sen
S
Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Scotland, United Kingdom.
Carrillo
Juan J
JJ
Pediani
John D
JD
Milligan
Graeme
G
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D020962:Heterotrimeric GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D009154:Mutation; D011233:Precipitin Tests; D018340:Receptors, Adrenergic, alpha-1 / Q000737:chemistry / Q000378:metabolism*
C547716:ADRA1B protein, human; C547717:Adra1b protein, mouse; D018340:Receptors, Adrenergic, alpha-1; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020962:Heterotrimeric GTP-Binding Proteins; D002118:Calcium