Functional Reconstitution of Purified G_i and G_o with μ‐Opioid Receptors in Guinea Pig Striatal Membranes Pretreated with Micromolar Concentrations of N‐Ethylmaleimide

Abstract: Functional coupling between μ‐opioid receptors and GTP‐binding regulatory proteins (G proteins) was investigated in reconstituted membranes of the guinea pig striatum. Selective μ‐opioid agonists stimulated low‐K_m GTPase in striatal membranes, in a Na⁺‐dependent manner. The same μ‐opioid agonist {[D‐Ala²,N‐Me‐Phe⁴,Gly⁵‐ol]‐enkephalin (DAGO)} caused no stimulation when the membranes were exposed to islet‐activating protein (IAP; pertussis toxin). There was also no DAGO stimulation in preparations pretreated with a lower concentration (5 μM) of N‐ethylmaleimide (NEM), which abolished the ADP‐ribosylation of purified G_i (the G protein that mediates inhibition of adenylate cyclase) and G_o (a G protein of unknown function purified from bovine brain) by IAP. In addition, as the NEM treatment caused no change in the μ‐agonist binding, NEM could probably substitute for IAP in inactivating native G proteins, without exhibiting effects on the receptor binding in membranes. The μ‐agonist stimulation of low‐K_m GTPase activity in NEM‐treated membranes was recovered by reconstitution with purified G_i, or G_o. The μ‐agonist stimulation of low‐K_m GTPase was additive when G_i and G_o were simultaneously reconstituted in NEM‐treated membranes in amounts of 0.5 pmol/assay, which was required for maximal recovery, in either reconstitution experiment. The present findings provide the first evidence that the μ‐opioid receptor may exist in at least two different forms, separately coupled to G_i or G_o.