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) was observed in HC-D57 murine erythroleukemia cells deprived of erythropoietin (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 hmm-EPOR are more highly glycosylated forms of the EPOR, appear on the cell 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, correlating with a 10-fold increase in binding of 125I-labeled EPO. In addition, the EPO-dependent downregulation of 125I-EPO binding and disappearance 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 expressing 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 apparently resulted from additional N-linked glycosylation of a 62-kDa EPOR. (v) The hmm-EPOR turnover in HC-D57 cells was accelerated 12-fold in the presence of EPO (half-life changed from 3 hr to 15 min). (vi) Anti-phosphotyrosine antiserum detected an EPO-dependent phosphorylation of the 78-kDa EPOR. The kinetics of tyrosine phosphorylation of a 97-kDa protein correlated with the occupancy and internalization of hmm-EPOR. In summary, we suggest that the 78-kDa EPOR is directly involved in the initial biological actions of EPO.
Sawyer
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ST
Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN 37232-2287.
Hankins
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WD
D000818:Animals; D002451:Cell Compartmentation; D004705:Endocytosis; D004921:Erythropoietin / Q000172:deficiency / Q000378:metabolism*; D006023:Glycoproteins / Q000378:metabolism; D004915:Leukemia, Erythroblastic, Acute / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D017467:Receptors, Erythropoietin / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D014407:Tumor Cells, Cultured
D006023:Glycoproteins; D008565:Membrane Proteins; D017467:Receptors, Erythropoietin; D011994:Recombinant Proteins; D004921:Erythropoietin