DELTA-OPIOID-RECEPTOR-MEDIATED INHIBITION OF ADENYLATE-CYCLASE IS TRANSDUCED SPECIFICALLY BY THE GUANINE-NUCLEOTIDE-BINDING PROTEIN GI2

Mouse neuroblastoma x rat glioma hybrid cells (NG108-15) express an opioid receptor of the δ subclass which both stimulates high-affinity GTPase activity and inhibits adenylate cyclase by interacting with a pertussis-toxin-sensitive guanine-nucleotide-binding protein(s) (G-protein). Four such G-proteins have now been identified outwith photoreceptor-containing tissues. We have generated anti-peptide antisera against synthetic peptides which correspond to the C-terminal decapeptides of the α-subunit of each of these G-proteins and also to the stimulatory G-protein of the adenylate cyclase cascade (G(s)). Using these antisera, we demonstrate the expression of three pertussis-toxin-sensitive G-proteins in these cells, which correspond to the products of the G(i)2, G(i)3 and G(o) genes, as well as G(s). G(i)1, however, is not expressed in detectable amounts. IgG fractions from each of these antisera and from normal rabbit serum were used to attempt to interfere with the interaction of the opioid receptor with the G-protein system by assessing ligand stimulation of high-affinity GTPase activity, inhibition of adenylate cyclase activity and conversion of the receptor to a state which displays reduced affinity for agonists. The IgG fraction from the antiserum (AS7) which specifically identifies G(i)2 in these cells attenuated the effects of the opioid receptor. This effect was complete and was not mimicked by any of the other antisera. We conclude that the δ-opioid receptor of these cells interacts directly and specifically with G(i)2 to cause inhibition of adenylate cyclase, and that G(i)2 represents the true G(i) of the adenylate cyclase cascade. The ability to measure alterations in agonist affinity for receptors following the use of specific antisera against a range of G-proteins implies that such techniques should be applicable to investigations of the molecular identity of the G-protein(s) which interacts with any receptor.