The use of stimulus-biased assay systems to detect agonist-specific receptor active states: Implications for the trafficking of receptor stimulus by agonists

The quantitative comparison of the relative potency of agonists is a standa rd method of receptor and agonist classification. If agonist potency ratios do not correspond in two given tissues, this is used as presumptive data t o conclude that the receptors in those two tissues are different. This arti cle presents data to show that a single receptor can demonstrate varying ag onist potency ratios in different host cells. These data are described in t erms of the production of more than one agonist-selective receptor active s tate and the interaction of these different active states with multiple G p roteins in the membrane to produce cellular response. Stable host human emb ryonic kidney 293 cells with enhanced quantities of the respective G alpha -protein were created. Wild-type and G alpha -subunit enriched cells were t hen transiently transfected with human calcitonin receptor type 2 (hCTR2). Binding did not detect differences in the G protein-enriched cells versus w ild-type cells. In contrast, functional studies did show differences betwee n the host cell lines and G alpha -subunit enriched cell lines. The relativ e potency of eight calcitonin agonists was measured in studies of calcium f luorescence in transfected cells containing human calcitonin receptor type 2 by comparing pEC(50) (-log molar concentration producing half-maximal res ponse) values. In G alphas-enriched cells, the relative order of potency of the agonists changed. The host-cell dependent differences in potency ratio s ranged from 2-fold to more than 46-fold. This finding is not consistent w ith 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 consis tent with the idea that these different agonists produce arrays of active s tates that differentially use G proteins. This idea is discussed in terms o f the design of stimulus-bias assay systems to detect agonist-selective rec eptor active states with resulting potential for increased selectivity of a gonists.