Reconstitution of the solubilized mu-opioid receptor coupled to a GTP-binding protein.
A mu-opioid receptor-GTP binding protein (mu-opioid receptor-G-protein) complex from the 7315c cell was solubilized with CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate) and reconstituted into phospholipid vesicles. Pretreatment of the tissue with either [3H]etorphine or morphine greatly improved recovery of the receptor and maintained it in a GTP-sensitive state. GTP sensitivity was consistent with the hypothesis that a receptor-G-protein complex had been obtained. Other evidence consistent with this hypothesis was that recovery of the solubilized, prelabelled receptor was decreased by approximately 70% by pretreatment of 7315c cells with pertussis toxin. The reconstituted receptor was mu-selective: DAGO (Tyr-D-Ala-Gly-Met-Phe- NH(CH2)2OH), but not ICI 174864 or U50488-H, displaced [3H]etorphine binding with high affinity. The affinity of the reconstituted receptor for [3H]etorphine (1.25 +/- 0.20 nM) was similar to that observed for the membrane-associated receptor (0.53 +/- 0.25 nM). GTP gamma S decreased this affinity 3-fold without changing the number of binding sites. The potencies of GTP gamma S and GTP in diminishing [3H]etorphine binding were similar in the membrane and vesicle preparations, but were 10-fold lower than the potencies observed in diminishing binding to the solubilized receptor. The ability to reconstitute a functional mu-opioid receptor-G-protein complex will facilitate further study of the structure and function of the receptor and the specific identification of the associated GTP-binding protein(s).
Frey
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Uniformed Services University of the Health Sciences, Bethesda, MD 20814-4799.
Gosse
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Cote
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D000262:Adenylyl Cyclases / Q000032:analysis / Q000302:isolation & purification; D005048:Etorphine / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism*; D007700:Kinetics; D010743:Phospholipids / Q000032:analysis / Q000302:isolation & purification; D011957:Receptors, Opioid / Q000302:isolation & purification / Q000378:metabolism*; D017450:Receptors, Opioid, mu; D014407:Tumor Cells, Cultured / Q000648:ultrastructure
D010743:Phospholipids; D011957:Receptors, Opioid; D017450:Receptors, Opioid, mu; D005048:Etorphine; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases