SITE-SPECIFIC ANTIBODIES DIRECTED AGAINST G-PROTEIN BETA-SUBUNIT AND GAMMA-SUBUNIT - EFFECTS ON ALPHA-SUBUNIT AND BETA-GAMMA-SUBUNIT INTERACTION

Little is known about the specific domains of G protein beta and gamma-subunits which interact with each other and with the a subunit. We used site-specific anti-peptide antibodies directed against beta and gamma-subunits to investigate domains on beta and gamma-subunits involved in alpha-subunit interaction. Antibodies included four against the transducin (G) beta-subunit (residues 1-10 = MS, 127-136 = KT, 256-265 = RA, and 330-340 = SW) and two against the gamma-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 beta-gamma(t) subunits. Affinity purification of MS and KT antisera on columns of immobilized native G(t) yielded antibodies capable of recognizing native beta-gamma(t), subunits. The functional effects of each antibody preparation on alpha(t)-beta-gamma(t) interaction were assessed by assaying the ability of the preparations to immunoprecipitate beta-gamma(t) subunits in the presence of excess alpha-subunits and by testing the inhibition of beta-gamma(t)-dependent ADP-ribosylation of alpha(t)-subunits catalyzed by pertussis toxin. On the basis of the results, we conclude that the domains on beta-gamma(t) which may be directly involved in alpha(t)-beta-gamma(t) interaction include the extreme amino terminus, residues 127-136 and 256-265 of beta(t), and the carboxyl terminus of gamma(t). The carboxyl-terminal region of beta(t) and the amino terminus of gamma(t) are less likely to be directly involved in alpha(t)-beta-gamma(t) interaction.