title: Amphibian Melanophore Technology as a Functional Screen for Antagonists of G-Protein Coupled 7-Transmembrane Receptors.
abstract: Xenopus laevis melanophores stably expressing 7-transmembrane G-protein-coupled receptors were established and evaluated, either as a primary screening utility for antagonists of the human calcium receptor, or as a screen to assign function to binding inhibitors of human cannabinoid receptors. Stably or transiently expressing melanophores responded selectively to respective effectors 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 cannabinoid (CB(2)) receptor. The results were consistent with changes in cAMP content of hCB(2)-transfected human embryonic kidney (HEK) cells challenged with the same CB(2)-binding antagonists. A stable melanophore cell line expressing the human calcium receptor was used to screen a compound collection directly for functional antagonists, several of which were confirmed as antagonists in secondary screens by stimulating parathyroid hormone (PTH) secretion from bovine parathyroid cells. The percentage of hits in this cell-based screen was reasonably low (1.2%), indicating minimal interference due to toxic effects and validating melanophores as a primary screening modality. Also described is the development of a novel procedure for cryopreservation and reconstitution of cells retaining functional human receptors. ()
authors.affiliation: Department of Bone and Cartilage Biology, SmithKline Beecham Pharmaceuticals, King of Prussia, PA.
authors.initials: ME
authors.lastname: Nuttall
authors.initials: JC
authors.lastname: Lee
authors.initials: PR
authors.lastname: Murdock
authors.initials: AM
authors.lastname: Badger
authors.initials: FL
authors.lastname: Wang
authors.initials: JT
authors.lastname: Laydon
authors.initials: GA
authors.lastname: Hofmann
authors.initials: GR
authors.lastname: Pettman
authors.initials: JA
authors.lastname: Lee
authors.initials: A
authors.lastname: Parihar
authors.lastname: Van Wagenen BC
authors.initials: J
authors.lastname: Fox
authors.initials: M
authors.lastname: Gowen
authors.initials: RK
authors.lastname: Johnson
authors.initials: MR
authors.lastname: Mattern