A melanophore-based screening assay for erythropoietin receptors

A rapid, functional assay in frog melanophore cells for the erythropoietin receptor (EPOR), a member of the cytokine receptor family, is demonstrated. A chimeric receptor that comprised the extracellular portion of the murine EPOR and the transmembrane and intracellular domains of the human epiderma l growth factor receptor (EGFR) was subcloned into the expression vector pJ G3,6, When the full-length EGFR was expressed in melanophores, EGF but not EPO mediated pigment dispersion in a time- and dose-dependent manner with a n EC50 Of 12.6 +/- 2.9 pM, However, when the chimeric EPOR/EGFR was express ed, EPO but not EGF stimulated pigment dispersion in a time- and dose-depen dent manner with an EC50 Of 380 +/- 107 pM, Neither EGF nor EPO had any eff ect on pigment dispersion in wild-type melanophores, EGF- and EPO-mediated pigment dispersion was blocked by the bis-indolylmaleimide protein kinase C inhibitor Ro 31-8220, This study extends the use of the melanophore-based bioassay to include cytokine receptors in addition to G protein- and tyrosi ne kinase-coupled receptors, It represents a potentially powerful method fo r screening of combinatorial libraries to identify novel small molecule ago nists and antagonists to this clinically important class of binding sites a s well as performing studies of functional ligand-receptor interactions.