G(i2) mediates α₂-adrenergic inhibition of adenylyl cyclase in platelet membranes: In situ identification with G(α) C-terminal antibodies

A panel of antibodies to synthetic decapeptides corresponding to the C termini of guanine nucleotide-binding regulatory protein (G protein) α subunits has been generated in rabbits. The specificity of each antibody was assessed by ELISA for peptide binding and by immunoblotting for binding to defined, recombinant G(α) subunits expressed in Escherichia coli. Immunoblotting of human platelet membranes with these antibodies identified a variety of endogenous G proteins including G(s) (stimulatory), G(i2) (inhibitory), G(i3), and G(x(z)) (unknown function). Pretreatment of platelet membranes with C-terminal antibodies reactive with G(i2), but not with antibodies to G(i3) or G(x(z)), blocked α₂-adrenergic inhibition of adenylyl cyclase. This identifies G(i2) as the dominant mediator of cyclase inhibition in this pathway. This approach may provide a general means of identifying relevant functional interactions of G proteins with receptors an effectors in situ.