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CELLULAR AND MOLECULAR

Estimation of Agonist Activity at G Protein-Coupled Receptors: Analysis of M₂ Muscarinic Receptor Signaling through G_i/o,G_s, and G₁₅

Department of Physical Sciences, Chapman University, Orange, California (M.T.G., S.L.); and Department of Pharmacology, School of Medicine, University of California, Irvine, Irvine, California (K.W.F., F.J.E.)

We developed novel methods for analyzing the concentration-response curve of an agonist to estimate the product of observed affinity and intrinsic efficacy, expressed relative to that of a standard agonist. This parameter, termed intrinsic relative activity (RA_i), is most applicable for the analysis of responses at G protein-coupled receptors. RA_i is equivalent to the potency ratios that agonists would exhibit in a hypothetical, highly sensitive assay in which all agonists behave as full agonists, even those with little intrinsic efficacy. We investigated muscarinic responses at the M₂ receptor, including stimulation of phosphoinositide hydrolysis through G₁₅ in HEK 293T cells, inhibition of cAMP accumulation through G_i in Chinese hamster ovary (CHO) cells, and stimulation of cAMP accumulation through G_s in CHO cells treated with pertussis toxin. The RA_i values of carbachol, oxotremorine-M, and the enantiomers of aceclidine were approximately the same in the three assay systems. In contrast, the activity of 4-[[N-[3-chlorophenyl]carbamoy]oxy-2-butynyl]trimethylammonium chloride (McN-A-343) was 10-fold greater at M₂ receptors coupled to G₁₅ in HEK 293T cells compared with M₂ receptors coupled to G_i in the same cells or in CHO cells. Our results show that the RA_i estimate is a useful measure for quantifying agonist activity across different assay systems and for detecting agonist directed signaling.

Address correspondence to: Dr. Frederick J. Ehlert, Department of Pharmacology, University of California, Irvine, Irvine, CA 92697-4625. E-mail: fjehlert{at}uci.edu

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