IMMUNOPRECIPITATION OF OPIOID RECEPTOR-G(0)-PROTEIN COMPLEXES USING SPECIFIC GTP-BINDING-PROTEIN ANTISERA

Solubilization of opioid receptors from rat cortical membranes that re tained high-affinity guanine nucleotide-sensitive agonist binding was achieved using 10 mM CHAPS. We report the nature of the interactions o f mu and delta opioid receptors with the guanine nucleotide-binding pr otein G(0) by immunoprecipitation of CHAPS extracts with selective G(0 ) alpha-subunit protein antisera. Antiserum IM1 raised against amino a cids 22-35 of G(0) alpha selectively co-immunoprecipitated G(0) alpha- mu and G(0) alpha-delta opioid receptor complexes detected in the immu noprecipitates by specific [H-3][D-Ala(2),N-Me-Phe(4),Gly(5)-ol]enkeph alin and [H-3][D-Ser(2), Leu(5),Thr(6)]enkephalin binding respectively . By contrast, antisera directed against the C-terminal decapeptide (O C2) and the N-terminal hexadecapeptide (ON1) of isoforms of G(0) alpha were unable to immunoprecipitate solubilized opioid receptor-G(0) com plexes, although both were able to immunoprecipitate solubilized G(0) alpha and have been shown to reduce the affinity of [D-ala(2)-D-leu(5) ]enkephalin for opioid receptors in rat cortical membranes [Georgoussi , Carr and Milligan (1993) Mol. Pharmacol. 44, 62-69]. These findings demonstrate that CHAPS-solubilized mu and delta opioid receptors from rat cortical membranes form stable complexes with one or more variants of G(0).