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CELLULAR AND MOLECULAR

Comparison of the Relative Efficacy and Potency of µ-Opioid Agonists to Activate G_i/o Proteins Containing a Pertussis Toxin-Insensitive Mutation

Department of Pharmacology, University of Michigan Medical School, Ann Arbor, Michigan

Pertussis toxin (PTX)-insensitive mutants of G_i/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 G_i/o subtypes, as measured by the degree of stimulation of [³⁵S]guanosine 5'-O-(3-thio)triphosphate (GTPS) binding. Binding of [³⁵S]GTPS to endogenous G_i/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 G 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 G_i3 and G_o and lowest with G_i1 and G_i2, 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 G_i/o subtypes. Highest relative efficacy to stimulate [³⁵S]GTPS binding was seen with G_i3. Consequently, reported observations of agonist-directed trafficking at µ-opioid receptors most likely involve non-PTX-sensitive G protein mechanisms.

Address correspondence to: Dr. John R. Traynor, Department of Pharmacology, 1301 MSRB III, 1150 W. Medical Center Dr., Ann Arbor, MI 48109-0632. E-mail: jtraynor{at}umich.edu

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