Amphibian melanophore technology as a functional screen for antagonists ofG-protein coupled 7-transmembrane receptors

Xenopus laevis melanophores stably expressing 7-transmembrane G-protein-cou pled receptors were established and evaluated, either as a primary screenin g utility for antagonists of the human calcium receptor, or as a screen to assign function to binding inhibitors of human cannabinoid receptors, Stabl y or transiently expressing melanophores responded selectively to respectiv e effecters of the human calcium, cannabinoid, and neurokinin-1 receptors, Several selective cannabinoid receptor-binding inhibitors of known potency were characterized as agonists or antagonists of the human peripheral canna binoid (CB2) receptor. The results were consistent with changes in cAMP con tent of hCB(2)-transfected human embryonic kidney (HEK) cells challenged wi th the same CB2-binding antagonists. A stable melanophore cell line express ing the human calcium receptor was used to screen a compound collection dir ectly for functional antagonists, several of which were confirmed as antago nists in secondary screens by stimulating parathyroid hormone (PTH) secreti on from bovine parathyroid cells. The percentage of hits in this cell-based screen was reasonably low (1.2%), indicating minimal interference due to t oxic effects and validating melanophores as a primary screening modality. A lso described is the development of a novel procedure for cryopreservation and reconstitution of cells retaining functional human receptors.