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 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 GoA, was used for most of the experiments, but some experiments were performed with individual pure G proteins, GoA, GoB, Gi1, and Gi2. Low Km GTPase was stimulated up to 150% by mu-opioid receptor agonists when both mu-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 mu-agonist was concentration dependent and was reversed by the antagonist (-)-naloxone, but not by its inactive enantiomer, (+)-naloxone. The mu selectivity of mu-ORP was demonstrated by the inability of delta and kappa agonists to stimulate GTPase in this system. High-affinity mu-agonist binding was also restored by reconstitution with the brain G protein mixture and with each of the four pure Gi and G(o) proteins studied. The binding of mu agonists is sensitive to inhibition by GTP gamma S and by sodium.
Fan
L Q
LQ
Department of Psychiatry, New York University Medical Center, NY 10016, USA.
Gioannini
T L
TL
Wolinsky
T
T
Hiller
J M
JM
Simon
E J
EJ
D000818:Animals; D001921:Brain / Q000378:metabolism; D002417:Cattle; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D004745:Enkephalins / Q000378:metabolism / Q000494:pharmacology; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D008081:Liposomes / Q000378:metabolism*; D009270:Naloxone / Q000494:pharmacology; D009294:Narcotics / Q000494:pharmacology; D017450:Receptors, Opioid, mu / Q000819:agonists / Q000302:isolation & purification / Q000378:metabolism*
D004745:Enkephalins; D008081:Liposomes; D009294:Narcotics; D017450:Receptors, Opioid, mu; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D009270:Naloxone; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins