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 (Ep oR) necessary for promotion of growth and induction of differentiation , mutated EpoR cDNAs containing truncations and conversions of tyrosin e residues to phenylalanines were generated. Constructs mere introduce d into IL-3 dependent Ba/F3 cells by electroporation, and individual t ransfectants were propagated in methylcellulose-containing medium in t he presence of erythropoietin (Epo), Truncated receptor at amino acid 350 was partially deficient in promoting cellular growth and the recep tor lacking the box 2 region was not capable of inducing cellular grow th. When a large number of cells,were screened in Epo-containing liqui d medium, clones arose in which Epo-dependent growth was due to activa tion of the endogenous EpoR gene. Analysis of the degree of Epo-depend ent differentiation of the transfectants, based on the steady-state le vels 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. Examinat ion of clones from diverse origins demonstrated that an inverse relati onship existed between the rate of Epo-induced cellular replication an d the degree of Epo-induced differentiation. (C) 1998 Academic Press.