IDENTIFICATION OF A CYTOPLASMIC MOTIF IN THE ERYTHROPOIETIN RECEPTOR REQUIRED FOR RECEPTOR INTERNALIZATION

Erythropoietin (EPO) promotes the viability, proliferation and differe ntiation of mammalian erythroid progenitor cells via its specific cell surface receptor. The EPO receptor (EPO-R) is a member of the cytokin e receptor superfamily and is comprised of one identified subunit whic h homodimerizes upon ligand binding, To study the role of the intracel lular domain of the EPO-R in the endocytosis of EPO, we compared the r ate and extent of I-125-EPO endocytosis by wild type (wt) EPO-R and fi ve cytoplasmically truncated EPO-Rs: 1-251 EPO-R, 1-257 EPO-R, 1-267 E PO-R, 1-276 EPO-R and 1-306 EPO-R which contain 4, 10, 20, 29 or 59 am ino acids of the cytoplasmic domain, respectively. We also studied an EPO-R mutant (PB) which lacks amino acid residues 281-300 of the cytop lasmic domain. The experiments were conducted in COS 7 cells transfect ed with the EPO-R cDNAs and in Ba/F3 cells stably expressing the wt EP O-R, 1-251 or 1-257 EPO-R, Cells expressing wt EPO-R, PB EPO-R (Delta 281-300), 1-276 EPO-R or 1-306 EPO-R internalized approximately 50% of I-125-EPO bound to the cell surface, while cells expressing 1-251, 1- 257 or 1-267 EPO-R internalized only 25% of the bound I-125-EPO. The s teady-state expression levels of these latter receptors on the cell su rface mere typically 2-5-fold higher than vet EPO-R. Our data indicate that amino acid residues 267-276 (FEGLFTTHK) of the EPO-R cytoplasmic domain may have a role in receptor internalization. Metabolic labelin g experiments suggest that in transiently transfected COS 7 cells most of the wt EPO-R and 1-257 EPO-Rs do not exit the ER and may be degrad ed there, The half-life of both receptors was essentially similar and a-as in the range of 1 h, In Ba/F3 cells the mature Golgi processed 1- 257 EPO-R was more stable than the corresponding form of the wt EPO-R, possibly contributing to its higher cell surface expression. (C) 1998 Federation of European Biochemical Societies.