Identification of erythropoietin receptors in isolated membranes from Friend virus infected mice spleen cells

It has been shown that radiolabeled erythropoietin (epo) specifically binds to homogeneous epo-responsive spleen cells from mice infected with the anemic variant of the Friend virus. We now report that membranes isolated from these cells retain the ability to specifically bind epo. Spleen cells were swollen in ice-cold 10 mM Tris-Cl pH 7.4 containing 0.2 mM phenylmethyl sulfonylfluoride for 5 minutes, after which the cells were homogenized and centrifuged to remove nuclear fraction. Membranes were collected by centrifuging the supernatant at 75,000 g for 90 min. Utilizing 3H-epo labeled at the terminal sialic acids of the carbohydrate moieties or 125I-epo labeled at the tyrosine residues, it was shown that the isolated membranes contained specific epo binding sites. About 90% of the binding could be inhibited by the presence of unlabeled epo whereas no inhibition was seen with other glycoproteins and growth factors. Equilibrium could be reached in approximately 2.5 hr at 25 degrees C or 1.5 hr at 37 degrees C. Binding could be saturated at an epo concentration of about 3 nM, Scatchard analysis indicated a single class of receptors with a Kd of approximately 1.5 nM.