[Molecular pharmacology of opioid receptor mechanisms]

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). mu-Opioid receptor exclusively purified from rat brains was reconstituted with G-proteins in lipid vesicles. The mu-agonist stimulated the G-protein activity in both G1 or Go-reconstituted vesicles. The stoichiometry revealed that one molecule of mu-receptor is functionally coupled to plural numbers of Gi or Go molecules and that mu-receptor exists in at least two different subtypes, mu i and mu o, separately coupled to Gi and Go, respectively. In addition, when the mu-receptor was phosphorylated by cAMP-dependent protein kinase, the mu-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. kappa-Opioid receptor agonist inhibited the G-protein activity in guinea pig cerebellar membranes. Further experiments revealed that the kappa-opioid receptor is functionally coupled to an inhibition of phospholipase C activity via an inhibition of Gi-activity. Such a receptor-mediated inhibition of G-protein activity may be the first demonstration of a signal transduction mechanism. The delta-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 delta-agonist binding was markedly reduced by GTP gamma S in the presence of MgCl2. These findings suggest that delta-receptors in the brain might be coupled to G-protein without signal transduction.