Immunoprecipitation of Adenylate Cyclase with an Antibody to a Carboxyl-Terminal Peptide from Gsα

An antibody (RM) raised against the carboxyl-terminal decapeptide of the α subunit of the stimulatory guanine nucleotide regulatory protein (Gsα) has been used to study the interaction of Gsα with bovine brain adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1]. RM antibody immunoprecipitated about 60% of the solubilized adenylate cyclase preactivated with either GTP-γ-S or A1F₄−. In contrast, RM antibody immunoprecipitated about 5% of the adenylate cyclase not preactivated (control) and 15% of the adenylate cyclase pretreated with forskolin. Adenylate cyclase solubilized from control membranes or GTP-γ-S preactivated membranes was partially purified by using forskolin-agarose affinity chromatography. The amount of Gsα protein in the partially purified preparations was determined by immunoblotting with RM antibody. There was 3-fold more Gsα detected in partially purified adenylate cyclase from preactivated membranes than in the partially purified adenylate cyclase from control membranes. Partially purified adenylate cyclase from preactivated membranes was immunoprecipitated with RM antibody and the amount of adenylate cyclase activity immunoprecipitated (65% of total) corresponded to the amount of Gsα protein immunoprecipitated. Only 15% of the partially purified adenylate cyclase from control membranes was immunoprecipitated. The presence of other G proteins in the partially purified preparations of adenylate cyclase was investigated by using specific antisera that detect Goα, Giα, and Gβ. The Gβ protein was the only subunit detected in the partially purified preparations of adenylate cyclase and the amount of Gβ was about the same in adenylate cyclase from preactivated membranes and from control membranes. Examination of the RM antibody immunoprecipitates from control and GTP-7-S preactivated solubilized membranes failed to detect any of these G proteins. These results indicate that the complex between the adenylate cyclase catalytic subunit and the activated Gsα protein can be isolated by immunoprecipitation with an anti-Gsα antibody. There does not appear to be a specific association of Goα, Giα, or Gβ with the preactivated complex of the catalytic subunit and the activated Gsα subunit.