Abstract
Pertussis toxin (PTX)-insensitive mutants of Gαi/o proteins expressed in C6μ cells were used to examine the hypothesis that there are agonist-specific conformational states of the μ-opioid receptor with coupling preferences to different Gαi/o subtypes, as measured by the degree of stimulation of [35S]guanosine 5′-O-(3-thio)triphosphate (GTPγS) binding. Binding of [35S]GTPγS to endogenous Gαi/o proteins stimulated by the full μ-opioid agonist [d-Ala2,MePhe4,Gly5-ol]enkephalin (DAMGO) was completely blocked by overnight treatment with 100 ng/ml PTX. Treatment for 4 h with lower concentrations led to a PTX-dependent reduction in the maximal effect of DAMGO but no alteration in the potency of DAMGO or morphine nor in the relative maximal effect (relative efficacy) of the partial agonists morphine and buprenorphine compared with the full agonist DAMGO. Using PTX-insensitive Gα mutants in which the PTX-sensitive cysteine was replaced with isoleucine, the potency for a series of μ-opioid agonists was highest in cells expressing Gαi3 and Gαo and lowest with Gαi1 and Gαi2, with no significant change in the order of potency, namely, etorphine >> endomorphin-1 = DAMGO = endomorphin-2 = fentanyl = morphine >> meperidine. The order of agonist relative efficacy, etorphine = DAMGO = endomorphin-1 = endomorphin-2 = fentanyl ≥ morphine ≥ meperidine > buprenorphine ≥ nalbuphine, was also the same across all of the PTX-insensitive Gαi/o subtypes. Highest relative efficacy to stimulate [35S]GTPγS binding was seen with Gαi3. Consequently, reported observations of agonist-directed trafficking at μ-opioid receptors most likely involve non-PTX-sensitive Gα protein mechanisms.
Footnotes
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This work was supported by National Institute on Drug Abuse Grant DA 04087. T.D.G. was supported by the American Society for Pharmacology and Experimental Therapeutics Gerald J. Dalton/Vincent G. Zannoni Summer Undergraduate Research Fellowship.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.105.096818.
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ABBREVIATIONS: GPCR, G-protein-coupled receptor; PTX, pertussis toxin; DAMGO, [d-Ala2,MePhe4,Gly5-ol]enkephalin; C6μ, rat C6 glioma cells stably expressing the μ-opioid receptor; [35S]GTPγS, guanosine-5′-O-(3-[35S]thio)triphosphate; PTXi, PTX-insensitive; C351(2), 351Cys or 352Cys in Gαi/o proteins.
- Received October 7, 2005.
- Accepted January 23, 2006.
- The American Society for Pharmacology and Experimental Therapeutics
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