A METHOD FOR EVALUATING THE EFFECTS OF LIGANDS UPON G(S) PROTEIN-COUPLED RECEPTORS USING A RECOMBINANT MELANOPHORE-BASED BIOASSAY

As an increasing number of medically important receptors that couple to stimulatory guanine nucleotide (Gs) proteins are isolated and cloned, there is an equally escalating need for methods to rapidly and reproducibly evaluate potential ligands for their properties as agonists or antagonists. Recently, a bioassay that can quickly and accurately determine the effects of numerous chemicals on a β1-like adrenergic receptor (AR) endogenous to melanophores derived from Xenopus laevis was developed. Here, the general utility of the melanophore-based pigment dispersion assay is demonstrated by employing it to evaluate the effects of drugs on a human β2 AR. Melanophores were both transiently and stably transfected with a plasmid encoding a β2 AR. Stimulation of recombinant cells expressing the β2 AR, but not wild-type cells, with β2-selective agonists induced pigment dispersion and concomitant elevations in intracellular cAMP. Using a microtiter plate reader, it was straightforward to construct reproducible dose-response curves and rapidly determine rank-order potency and EC50 and IC50 values for agonists and antagonists, respectively. The demonstration of functional expression of a human β2 AR in the melanophore-based bioassay suggests that the system may be used for the rapid pharmacological characterization of ligands upon any specific Gs-linked receptor for which a cDNA clone is available. © 1992.