FUNCTIONAL RECONSTITUTION OF A HIGHLY PURIFIED MU-OPIOID RECEPTOR PROTEIN WITH PURIFIED G-PROTEINS IN LIPOSOMES

A mu-opioid receptor protein (mu-ORP) purified to homogeneity from bov ine striatal membranes has been functionally reconstituted in liposome s with highly purified heterotrimeric guanine nucleotide regulatory pr oteins (G proteins). A mixture of bovine brain G proteins, predominant ly G(oA), was used for most of the experiments, but some experiments w ere performed with individual pure G proteins, G(oA), G(oB), G(i1), an d G(i2). LOW K-m GTPase was stimulated up to 150% by mu-opioid recepto r agonists when both mu-ORP and a G protein (either the brain G protei n mixture or a single heterotrimeric G protein) were present in the li posomes. Stimulation by a selective mu-agonist was concentration depen dent and was reversed by the antagonist (-)-naloxone, but not by its i nactive enantiomer, (+)-naloxone. The mu selectivity of mu-ORP was dem onstrated by the inability of delta and kappa agonists to stimulate GT Pase in this system. High-affinity gamma-agonist binding was also rest ored by reconstitution with the brain G protein mixture and with each of the four pure G(i) and G(o) proteins studied. The binding of mu ago nists is sensitive to inhibition by GTP gamma S and by sodium.