Functional Reconstitution of a Highly Purified μ-Opioid Receptor Protein with Purified G Proteins in Liposomes

A μ-opioid receptor protein (μ-ORP) purified to homogeneity from bovine striatal membranes has been functionally reconstituted in liposomes with highly purified heterotrimeric guanine nucleotide regulatory proteins (G proteins). A mixture of bovine brain G proteins, predominantly G oA , was used for most of the experiments, but some experiments were performed with individual pure G proteins, G oA , G oB , G i1 , and G i2 . Low K m GTPase was stimulated up to 150% by μ-opioid receptor agonists when both μ-ORP and a G protein (either the brain G protein mixture or a single heterotrimeric G protein) were present in the liposomes. Stimulation by a selective μ-agonist was concentration dependent and was reversed by the antagonist (-)-naloxone, but not by its inactive enantiomer, (+)-naloxone. The μ selectivity of μ-ORP was demonstrated by the inability of δ and κ agonists to stimulate GTPase in this system. High-affinity μ-agonist binding was also restored 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 μ agonists is sensitive to inhibition by GTPγS and by sodium.