Gi2 mediates alpha 2-adrenergic inhibition of adenylyl cyclase in platelet membranes: in situ identification with G alpha C-terminal antibodies

Abstract

A panel of antibodies to synthetic decapeptides corresponding to the C termini of guanine nucleotide-binding regulatory protein (G protein) alpha 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 alpha subunits expressed in Escherichia coli. Immunoblotting of human platelet membranes with these antibodies identified a variety of endogenous G proteins including Gs (stimulatory), Gi2 (inhibitory), Gi3, and Gx(z) (unknown function). Pretreatment of platelet membranes with C-terminal antibodies reactive with Gi2, but not with antibodies to Gi3 or Gx(z), blocked alpha 2-adrenergic inhibition of adenylyl cyclase. This identifies Gi2 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 and effectors in situ.