Reconstitution of Rat Brain μ Opioid Receptors with Purified Guanine Nucleotide-Binding Regulatory Proteins, Gi and Go

Reconstitution of purified μ opioid receptors with purified guanine nucleotide-binding regulatory proteins (G proteins) was investigated. μ opioid receptors were purified by 6-succinylmorphine AF-Amino TOYOPEARL 650M affinity chromatography and by PBE isoelectric chromatography. The purified μ 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 μ receptors. When purified μ receptors were reconstituted with purified Gi, the G protein that mediates the inhibition of adenylate cyclase, the displacement of [3H]naloxone (a μ opioid antagonist) binding by [D-Ala2,MePhe4,Gly-ol$^{5}$< /latex>]enkephalin (a μ opioid agonist) was increased 215-fold; this increase was abolished by adding 100 μ M (guanosine 5′-[γ -thio]triphosphate. Similar increases in agonist displacement of [3H]naloxone binding (33-fold) and its abolition by guanosine 5′-[γ -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 δ opioid agonist; where Pen is penicillamine) nor U-69,593 (a κ opioid agonist) showed displacement of the [3H]naloxone binding. In addition, the μ agonist stimulated both [3H]guanosine 5′-[β,γ -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 μ receptor led to the binding of [3H]guanosine 5′-[β,γ -imido]triphosphate to 2.5 mol of Gi or to 1.37 mol of Go. These results suggest that the purified μ opioid receptor is functionally coupled to Gi and Go in the reconstituted phospholipid vesicles.