Reconstitution of rat brain mu opioid receptors with purified guanine nucleotide-binding regulatory proteins, Gi and Go.
Reconstitution of purified mu opioid receptors with purified guanine nucleotide-binding regulatory proteins (G proteins) was investigated. mu opioid receptors were purified by 6-succinylmorphine AF-AminoTOYOPEARL 650M affinity chromatography and by PBE isoelectric chromatography. The purified mu opioid receptor (pI 5.6) migrated as a single Mr 58,000 polypeptide by NaDodSO4/PAGE, a value identical to that obtained by affinity cross-linking purified mu receptors. When purified mu receptors were reconstituted with purified Gi, the G protein that mediates the inhibition of adenylate cyclase, the displacement of [3H]naloxone (a mu opioid antagonist) binding by [D-Ala2,MePhe4,Gly-ol5]enkephalin (a mu opioid agonist) was increased 215-fold; this increase was abolished by adding 100 microM (guanosine 5'-[gamma-thio]triphosphate. Similar increases in agonist displacement of [3H]naloxone binding (33-fold) and its abolition by guanosine 5'-[gamma-thio]triphosphate were observed with Go, the G protein of unknown function, but not with the v-Ki-ras protein p21. In reconstituted preparations with Gi or Go, neither [D-Pen2,D-Pen5]enkephalin (a delta opioid agonist; where Pen is penicillamine) nor U-69,593 (a kappa opioid agonist) showed displacement of the [3H]naloxone binding. In addition, the mu agonist stimulated both [3H]guanosine 5'-[beta,gamma-imido]triphosphate binding (in exchange for GDP) and the low-Km GTPase in such reconstituted preparations, with Gi and Go but not with the v-Ki-ras protein p21, in a naloxone-reversible manner. The stoichiometry was such that the stimulation of 1 mol of mu receptor led to the binding of [3H]guanosine 5'-[beta,gamma-imido]triphosphate to 2.5 mol of Gi or to 1.37 mol of Go. These results suggest that the purified mu opioid receptor is functionally coupled to Gi and Go in the reconstituted phospholipid vesicles.
Ueda
H
H
Department of Pharmacology, Faculty of Pharmaceutical Sciences, Kyoto University, Japan.
Harada
H
H
Nozaki
M
M
Katada
T
T
Ui
M
M
Satoh
M
M
Takagi
H
H
D000818:Animals; D001923:Brain Chemistry; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism; D006165:Guanylyl Imidodiphosphate / Q000378:metabolism; D007700:Kinetics; D008297:Male; D008970:Molecular Weight; D051381:Rats; D011919:Rats, Inbred Strains; D011957:Receptors, Opioid / Q000378:metabolism*; D017450:Receptors, Opioid, mu; D012995:Solubility
D011957:Receptors, Opioid; D017450:Receptors, Opioid, mu; D006153:Guanosine Diphosphate; D006165:Guanylyl Imidodiphosphate; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins