Functional reconstruction of purified Gi and Go with mu-opioid receptors in guinea pig striatal membranes pretreated with micromolar concentrations of N-ethylmaleimide.
Functional coupling between mu-opioid receptors and GTP-binding regulatory proteins (G proteins) was investigated in reconstituted membranes of the guinea pig striatum. Selective mu-opioid agonists stimulated low-Km GTPase in striatal membranes, in a Na(+)-dependent manner. The same mu-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 microM) of N-ethylmaleimide (NEM), which abolished the ADP-ribosylation of purified Gi (the G protein that mediates inhibition of adenylate cyclase) and Go (a G protein of unknown function purified from bovine brain) by IAP. In addition, as the NEM treatment caused no change in the mu-agonist binding, NEM could probably substitute for IAP in inactivating native G proteins, without exhibiting effects on the receptor binding in membranes. The mu-agonist stimulation of low-Km GTPase activity in NEM-treated membranes was recovered by reconstitution with purified Gi or Go. The mu-agonist stimulation of low-Km GTPase was additive when Gi and Go 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 mu-opioid receptor may exist in at least two different forms, separately coupled to Gi or Go.
Ueda
H
H
Department of Pharmacology, Faculty of Pharmaceutical Sciences, Kyoto University, Japan.
Misawa
H
H
Katada
T
T
Ui
M
M
Takagi
H
H
Satoh
M
M
D037361:Adenylate Cyclase Toxin; D000818:Animals; D005033:Ethylmaleimide / Q000494:pharmacology*; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006168:Guinea Pigs; D008566:Membranes / Q000378:metabolism; D009994:Osmolar Concentration; D037342:Pertussis Toxin; D011957:Receptors, Opioid / Q000378:metabolism*; D017450:Receptors, Opioid, mu; D010566:Virulence Factors, Bordetella / Q000494:pharmacology; D014793:Visual Cortex / Q000378:metabolism*
D037361:Adenylate Cyclase Toxin; D011957:Receptors, Opioid; D017450:Receptors, Opioid, mu; D010566:Virulence Factors, Bordetella; D037342:Pertussis Toxin; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D005033:Ethylmaleimide