The Use of Stimulus-Biased Assay Systems to Detect Agonist-Specific Receptor Active States: Implications for the Trafficking of Receptor Stimulus by Agonists

Abstract

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.

Independent β-arrestin 2 and G protein-mediated pathways for angiotensin II activation of extracellular signal-regulated kinases 1 and 2

“…Thus, the receptor conformation induced by [Sar 1 ,Ile 4 ,Il 8 ]Ang II in the wild-type receptor, or by wild-type Ang II in the DRY͞AAY mutant receptor, which is competent to activate ERK1͞2 via ␤-arrestin 2, is not able to activate G proteins. Multiple active conformations of 7MSRs have been suggested previously, but have been difficult to document (29,30). The clear demonstration here of such functionally distinct active conformations, potentially coupled to distinct signaling pathways, gives credibility and impetus …”

The Effective Application of Biased Signaling to New Drug Discovery

“…Even modification of host cell background such as coexpression of Ga s protein in human embryonic kidney 293 cells has been shown to reverse calcitonin receptor agonist potency ratios for eel and porcine calcitonin (Watson et al, 2000). These effects strongly suggest that bias values measured in vitro may not always predict therapeutic biased signaling in vivo and bring into question how measured bias can be used to progress drug candidate molecules.…”

β-Arrestin-mediated activation of MAPK by inverse agonists reveals distinct active conformations for G protein-coupled receptors

“…This is reminiscent of the concept of ''trafficking of receptor stimulus'' (33,40) previously proposed to explain that ligands can activate diverse effectors with distinct rank order of potencies and͞or efficacies. However, results presented herein add a new level of complexity, showing that a ligand can have opposing efficacies on two effector systems engaged by the same receptor, indicating that the ''direction'' of response can be influenced by the effector considered.…”