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.