The quantitative comparison of the relative potency of agonists is a standard method of receptor and agonist classification. If agonist potency ratios do not correspond in two given tissues, this is used as presumptive data to conclude that the receptors in those two tissues are different. This article presents data to show that a single receptor can demonstrate varying agonist potency ratios in different host cells. These data are described in terms of the production of more than one agonist-selective receptor active state and the interaction of these different active states with multiple G proteins in the membrane to produce cellular response. Stable host human embryonic kidney 293 cells with enhanced quantities of the respective Galpha-protein were created. Wild-type and Galpha-subunit enriched cells were then transiently transfected with human calcitonin receptor type 2 (hCTR2). Binding did not detect differences in the G protein-enriched cells versus wild-type cells. In contrast, functional studies did show differences between the host cell lines and Galpha-subunit enriched cell lines. The relative potency of eight calcitonin agonists was measured in studies of calcium fluorescence in transfected cells containing human calcitonin receptor type 2 by comparing pEC(50) (-log molar concentration producing half-maximal response) values. In Galphas-enriched cells, the relative order of potency of the agonists changed. The host-cell dependent differences in potency ratios ranged from 2-fold to more than 46-fold. This finding is not consistent with the idea that all of the agonists produce response in the same manner (i.e., through a common active state of the receptor). These data are consistent with the idea that these different agonists produce arrays of active states that differentially use G proteins. This idea is discussed in terms of the design of stimulus-bias assay systems to detect agonist-selective receptor active states with resulting potential for increased selectivity of agonists.

Human breast cell carcinoma MCF-7 cells were found to bind 125I-labeled rat amylin (rAmylin) and the peptide amylin antagonist radioligand 125I-AC512 with high affinity. This high affinity binding possessed characteristics unique to the already defined high affinity binding site for amylin in the rat nucleus accumbens [Mol. Pharmacol. 44:493-497 (1993); J. Pharmacol. Exp. Ther. 270:779-787 (1994); Eur. J. Pharmacol. 262:133-141 (1994)]. To further define this receptor, we report results of expression cloning studies from an MCF-7 cell library. We isolated two variants of a seven-transmembrane receptor that were identical to two previously described human calcitonin receptors (hCTR1 and hCTR2). These receptors were characterized by expression in different surrogate host cell systems. Transient expression of hCTR1 in COS cells yielded membranes that bound 125I-AC512 and 125I-salmon calcitonin with high affinity, but no high affinity binding was observed with 125I-human calcitonin (hCAL) or 125I-rAmylin. Stable expression of hCTR1 in HEK 293 cells produced similar data. In contrast, expression of hCTR2 in COS cells yielded membranes that bound 125I-AC512, 125I-hCAL, and 125I-rAmylin with high affinity. The agonists 125I-hCAL and 125I-rAmylin bound 65% and 1.5%, respectively, of the sites bound by the antagonist radioligand 125I-AC512 in this expression system. This pattern of binding was repeated in HEK 293 cells stably transfected with hCTR2 (125I-hCAL = 24.8% Bmax, 125I-rAmylin = 8% Bmax). In both expression systems, the agonists hCAL and rAmylin were much more potent in displacing their radioligand counterparts than was the antagonist radioligand 125I-AC512. For example, the pKi value for displacement of 125I-AC512 by rAmylin was 7.2 in HEK 293 cells but rose to 9.1 when displacing 125I-rAmylin. Finally, hCTR2 was expressed in baculovirus-infected Ti ni cells. In this system, only specific binding to the antagonist 125I-AC512 and agonist 125I-hCAL was observed; no binding to 125I-rAmylin could be detected. These data are discussed in terms of two working hypotheses. The first is that amylin is a weak agonist for hCTR2 and that this receptor is unrelated to the amylin receptor found in this cell line. The second is that hCTR2 couples to different G proteins for calcitonin and amylin function in different cells. At present, these data cannot be used to disprove conclusively either hypothesis.

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A series of 2'-alkylthio-2-(anilinomethyl)imidazolines were prepared to examine the effect of the alkyl group size, sulfur oxidation state, and phenyl ring substitution on ligand binding and agonism of alpha-adrenergic receptor subtypes alpha1a, alpha1b, alpha1d, alpha2a, and alpha2c. Binding at all receptor subtypes decreased for compounds in the sulfone oxidation state as compared to their sulfide analogues. While sulfides were generally potent, nonselective agonists, sulfones exhibited alpha1a subtype selectivity in a cell-based functional assay. Sulfone (32) was 250-7000-fold selective for alpha1a vs all other subtypes.