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Journal of Biomolecular Screening
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A Melanophore-Based Screening Assay for Erythropoietin Receptors

Michael D. Carrithers

Department of Neurology, Yale University School of Medicine, New Haven, CT 06520-8024

Louis A. Marotti

Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06520-8024

Akihiko Yoshimura

Institute of Life Science, Kurume University, Aikawamachi, Japan

Michael R. Lerner

Department of Dermatology, University of Texas Southwestern Medical Center, Dallas, TX 75235-9069

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 epidermal growth factor receptor (EGFR) was subcloned into the expression vector pJG3.6. When the full-length EGFR was expressed in melanophores, EGF but not EPO mediated pigment dispersion in a time- and dose-dependent manner with an EC50 of 12.6 ± 2.9 pM. However, when the chimeric EPOR/EGFR was expressed, EPO but not EGF stimulated pigment dispersion in a time- and dose-dependent manner with an EC50 of 380 ± 107 pM. Neither EGF nor EPO had any effect on pigment dispersion in wild-type melanophores. EGF- and EPO-mediated pigment dispersion was blocked by the bisindolylmaleimide 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 tyrosine kinase-coupled receptors. It represents a potentially powerful method for screening of combinatorial libraries to identify novel small molecule agonists and antagonists to this clinically important class of binding sites as well as performing studies of functional ligand-receptor interactions.

Journal of Biomolecular Screening, Vol. 4, No. 1, 9-14 (1999)
DOI: 10.1177/108705719900400103
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