A panel of antibodies to synthetic decapeptides corresponding to the C termini of guanine nucleotidebinding regulatory protein (G protein) a subunits has been generated in rabbits.

Many cell-surface receptors for hormones appear to exert their effects on target cells by interacting with specific guanine nucleotide binding regulatory proteins (G-proteins) which couple receptors to their second-messenger signal generation systems. A common intracellular second messenger, which is used by many hormones, is cyclic AMP. This is produced by adenylate cyclase, whose activity is controlled by two G-proteins, Gs which mediates stimulatory effects and Gi inhibitory effects on adenylate cyclase activity. In liver, the hormone glucagon increases intracellular cAMP concentrations by activating adenylate cyclase by a Gs-mediated process. This effect of glucagon is antagonised by the hormone insulin, although the molecular mechanism by which insulin elicits its actions is obscure. However, insulin receptors exhibit a tyrosyl kinase activity and appear to interact with G-proteins, perhaps by causing phosphorylation of them. In type I diabetes, circulating insulin levels are abnormally low, giving rise to gross perturbations of metabolism as well as to a variety of complications such as ionic disturbances, neuropathies of the nervous system, respiratory and cardiovascular aberrations and predisposition to infection. We show here that experimentally-induced type I diabetes leads to the loss of expression of Gi in rat liver. As it has been suggested that Gi may couple receptors to K+-channels as well as mediating the inhibition of adenylate cyclase, aberrations in the control of expression of this key regulatory protein in type I diabetes may be expected to lead to pleiotropic effects.

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Antibodies generated to a synthetic decapeptide, RMHLRQYELL, representing the carboxyl-terminus of Gs-ct have been characterized in immunoblots and functional studies. This antibody, designated RM, reacts exclusively with a doublet of proteins of 52 and 45 kDa in immunoblots of bovine brain and wild-type $49 murine lymphoma cell membranes. No such reactivity is seen in membranes from cyc-$49 cells, which lack Gs. RM blocks receptor-mediated activation of Gs and adenylyl cyclase in membranes from wild-type $49 cells. RM could also immunoprecipitate adenylyl cyclase activity in detergent extracts from GTP[),]S-or fluoride-preactivated bovine brain membranes; thus binding of cts to effector and carboxyl-terminal antibody was mutually compatible. Such experiments provide an approach for the elucidation of functionally relevant interactions of G-proteins with receptors and effectors in the membrane.