Specific binding of erythropoietin to spleen cells infected with the anemia strain of Friend virus.

Abstract

Tritiated erythropoietin with full biological activity was prepared, and a relatively homogeneous population of enriched progenitor cells that respond to the hormone was generated by infection of mice with the Friend virus that produces anemia. These cells, obtained from the spleens of infected mice, develop into mature erythroblasts and erythrocytes in the presence of erythropoietin. The binding of erythropoietin to these target cells was measured; 62% of the binding was inhibited by excess unlabeled erythropoietin, but no inhibition occurred with albumin, serum or a variety of growth factors and glycoproteins. Apparent equilibrium was reached by 2 h at 37.degree. C and by 3.5-4 h at 10.degree. C. The extent of specific binding increased linearly with cell concentration. In binding experiments at 10.degree. C, apparent saturation of specific binding occurred at .simeq. 8.7 nM. Scatchard analysis showed a single class of binding sites. The dissociation constant is 5.2 nM with an average of 660 binding sites per cell. At 0.06 nM, wheres most of the cells are induced to terminally differentiate in vitro, an average of only 8 erythropoietin molecules bound per cell. Erythropoietin probably attaches to specific binding sites, which are most likely receptors since they manifest high affinity and specificity, and the biologic effect of the hormone may be produced by attachment of a very small number of erythropoietin molecules.