A method for evaluating the effects of ligands upon Gs protein-coupled receptors using a recombinant melanophore-based bioassay

Anal Biochem. 1992 Nov 1;206(2):315-22. doi: 10.1016/0003-2697(92)90372-e.

Abstract

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 beta 1-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 beta 2 AR. Melanophores were both transiently and stably transfected with a plasmid encoding a beta 2 AR. Stimulation of recombinant cells expressing the beta 2 AR, but not wild-type cells, with beta 2-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 beta 2 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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Biological Assay / methods
  • Cells, Cultured
  • Cyclic AMP / metabolism
  • GTP-Binding Proteins / metabolism*
  • Kinetics
  • Ligands
  • Melanocyte-Stimulating Hormones / pharmacology*
  • Melanophores / cytology
  • Melanophores / metabolism*
  • Metaproterenol / pharmacology*
  • Plasmids
  • Receptors, Adrenergic, beta / drug effects
  • Receptors, Adrenergic, beta / metabolism*
  • Receptors, Adrenergic, beta / radiation effects
  • Recombinant Proteins / drug effects
  • Recombinant Proteins / metabolism
  • Transfection
  • Xenopus laevis

Substances

  • Ligands
  • Receptors, Adrenergic, beta
  • Recombinant Proteins
  • Metaproterenol
  • Melanocyte-Stimulating Hormones
  • Cyclic AMP
  • GTP-Binding Proteins