THE FUNCTIONAL FORM OF THE ERYTHROPOIETIN RECEPTOR IS A 78-KDA PROTEIN - CORRELATION WITH CELL-SURFACE EXPRESSION, ENDOCYTOSIS, AND PHOSPHORYLATION

An abundant 70- to 78-kDa form of the erythropoietin receptor (EPOR) w as observed in HC-D57 murine erythroleukemia cells deprived of erythro poietin (EPO). In contrast to the 64- and 66-kDa EPOR proteins, these high molecular mass forms of EPOR (hmm-EPOR) correlated well with the number of binding sites and endocytosis of EPO. The hypothesis that hm m-EPOR are wore highly glycosylated forms of the EPOR, appear on the c ell surface, and represent at least one component of the biologically active EPOR was tested. Consistent findings were as follows. (i) Only hmm-EPOR increased following withdrawal of EPO from HC-D57 cells, corr elating with a 10-fold increase in binding of I-125-labeled EPO. In ad dition, the EPO-dependent downregulation of I-125-EPO binding and disa ppearance of hmm-EPOR occurred in parallel while the amount of 66-kDa EPOR did not change. (ii) The 78-kDa EPOR was detected in COS cells ex pressing EPOR cDNA. (iii) Probing of the intact surface of these cells with anti-NH2-terminal antibody recovered only the 78-kDa EPOR. (iv) Enzymatic deglycosylation and dephosphorylation showed that hmm-EPOR a pparently resulted from additional N-linked glycosylation of a 62-kDa EPOR. (v) The hmm-EPOR turnover in HC-D57 cells was accelerated 12-fol d in the presence of EPO (half-life changed from 3 hr to 15 min). (vi) Anti-phosphotyrosine antiserum detected an EPO-dependent phosphorylat ion of the 78-kDa EPOR. The kinetics of tyrosine phosphorylation of a 97-kDa protein correlated with the occupancy and internalization of hm m-EPOR. In summary, we suggest that the 78-kDa EPOR is directly involv ed in the initial biological actions of EPO.