Site-Specific Antibodies Directed Against G Protein β and γ Subunits: Effects on α and βγ Subunit Interaction

Little is known about the specific domains of G protein β and γ subunits which interact with each other and with the α subunit. We used site-specific anti-peptide antibodies directed against β and γ subunits to investigate domains on β and γ subunits involved in α subunit interaction. Antibodies included four against the transducin (G_t) β subunit (residues 1-10 = MS, 127-136 = KT, 256-265 = RA, and 330-340 = SW) and two against the γ subunit (residues 2-12 = PV and 58-68 = PE). All antisera, when affinity-purified on peptide columns, yielded antibodies capable of recognizing the denatured cognate subunit on immunoblots, but only RA, SW, PV, and PE recognized native βγ_t subunits. Affinity purification of MS and KT antisera on columns of immobilized native G_t yielded antibodies capable of recognizing native βγ_t subunits. The functional effects of each antibody preparation on α_t, -βγ_t, interaction were assessed by assaying the ability of the preparations to immunoprecipitate βγ_t subunits in the presence of excess a subunits and by testing the inhibition of βγ_t, -dependent ADP-ribosylation of α_t, -subunits catalyzed by pertussis toxin. On the basis of the results, we conclude that the domains on βγ_t, which may be directly involved in α_t-βγ_t interaction include the extreme amino terminus, residues 127-136 and 256-265 of β_t and the carboxyl terminus of γ_t. The carboxyl-terminal region of β_t, and the amino terminus of γ_t, are less likely to be directly involved in α_t, -βγ_t, interaction.