Abstract

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 G_α15 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 G_α15 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.