Construction, expression, and mitogenic activity of Epo receptor forms used to study Epo and SCF coinduced FDC2 cell proliferation. (A) In experiments aimed at testing possible mechanisms of Epo and SCF cosignaling, two minimal Epo receptor forms were constructed, expressed in FDC2 cells, and assayed initially for their activities in mediating ligand-induced mitogenesis in derived cell lines. In the construct ER-Bx1, 178 of a total of 236 cytoplasmic residues of the wt-ER have been deleted, including the conserved box 2 domain and eight (phospho)tyrosine sites for effector recruitment. The construct EE483 is a chimeric receptor form in which the extracellular domain of the Epo receptor was replaced by that of the human EGF receptor. (B) The mitogenic activity of the above receptor forms in FDC2-derived cell lines (FDC2, FDC2–wt-ER, FDC2–ER-Bx1, FDC2-EE483) was assayed based on cytokine-stimulated rates of [³H] thymidine incorporation. To account for any minor differences in plating, values were normalized versus maximal responsiveness to IL-3 in each cell line. Northern blotting of Epo and EE483 transcripts served to show apparent equivalence in receptor expression (B.J., August 1996, data not shown) and for the wt-ER and ER-Bx1 this has been confirmed through [¹²⁵I] Epo equilibrium binding assays (400 to 600 receptors per cell).19 25