Characterization of the domains of the erythropoietin receptor necessary for induction of cell growth and differentiation.

To define the cytoplasmic region(s) of the erythropoietin receptor (EpoR) necessary for promotion of growth and induction of differentiation, mutated EpoR cDNAs containing truncations and conversions of tyrosine residues to phenylalanines were generated. Constructs were introduced into IL-3 dependent Ba/F3 cells by electroporation, and individual transfectants were propagated in methylcellulose-containing medium in the presence of erythropoietin (Epo). Truncated receptor at amino acid 350 was partially deficient in promoting cellular growth and the receptor lacking the box 2 region was not capable of inducing cellular growth. When a large number of cells were screened in Epo-containing liquid medium, clones arose in which Epo-dependent growth was due to activation of the endogenous EpoR gene. Analysis of the degree of Epo-dependent differentiation of the transfectants, based on the steady-state levels of beta major-globin mRNA, showed that the carboxyl terminal 133 amino acids and tyrosyl residues located at positions 429/431 and 460/464 were not necessary for the induction of differentiation. Examination of clones from diverse origins demonstrated that an inverse relationship existed between the rate of Epo-induced cellular replication and the degree of Epo-induced differentiation.