The molecular basis of opioid receptor mechanisms was studied in reconstitution experiments using purified or membrane-bound opioid receptors and purified GTP-binding proteins (G-proteins).
μ-Opioid receptor exclusively purified from rat brains was reconstituted with G-proteins in lipid vesicles. The μ-agonist stimulated the G-protein activity in both G, or G
o-reconstituted vesicles. The stoichiometry revealed that one molecule of μ-receptor is functionally coupled to plural numbers of G, or G
o molecules and that μ-preceptor exists in at least two different subtypes, μ
i and μ
o, separately coupled to G
i and G
o, respectively. In addition, when the μ-receptor was phosphorylated by cAMP-dependent protein kinase, the μ-agonist-stimulation of G-protein activity disappeared, while the guanine nucleotide-sensitivity of agonist binding was unchanged. These findings suggest that there are independent domains in the receptor which are related to functional coupling to G-protein and to the agonist-binding modulation by G-protein.
κ-Opioid receptor agonist inhibited the G-protein activity in guinea pig cerebellar membranes. Further experiments revealed that the
κ-opioid receptor is functionally coupled to an inhibition of phospholipase C activity via an inhibition of G
i-activity. Such a receptor-mediated inhibition of G-protein activity may be the first demonstration of a signal transduction mechanism. The
δ-opioid receptor agonist showed no effect on G-protein activity in guinea pig striatal and rat cortical membranes, while it stimulated it in NG108-15 cells. In all these membranes, the δ-agonist binding was markedly reduced by GTPγS in the presence of MgCl
2. These findings suggest that δ-receptors in the brain might be coupled to G-protein without signal transduction.
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