title: Identification of a cytoplasmic motif in the erythropoietin receptor required for receptor internalization.
abstract: Erythropoietin (EPO) promotes the viability, proliferation and differentiation of mammalian erythroid progenitor cells via its specific cell surface receptor. The EPO receptor (EPO-R) is a member of the cytokine receptor superfamily and is comprised of one identified subunit which homodimerizes upon ligand binding. To study the role of the intracellular domain of the EPO-R in the endocytosis of EPO, we compared the rate and extent of 125I-EPO endocytosis by wild type (wt) EPO-R and five cytoplasmically truncated EPO-Rs: 1-251 EPO-R, 1-257 EPO-R, 1-267 EPO-R, 1-276 EPO-R and 1-306 EPO-R which contain 4, 10, 20, 29 or 59 amino acids of the cytoplasmic domain, respectively. We also studied an EPO-R mutant (PB) which lacks amino acid residues 281-300 of the cytoplasmic domain. The experiments were conducted in COS 7 cells transfected with the EPO-R cDNAs and in Ba/F3 cells stably expressing the wt EPO-R, 1-251 or 1-257 EPO-R. Cells expressing wt EPO-R, PB EPO-R (delta281-300), 1-276 EPO-R or 1-306 EPO-R internalized approximately 50% of 125I-EPO bound to the cell surface, while cells expressing 1-251, 1-257 or 1-267 EPO-R internalized only 25% of the bound 125I-EPO. The steady-state expression levels of these latter receptors on the cell surface were typically 2-5-fold higher than wt 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 labeling 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 degraded there. The half-life of both receptors was essentially similar and was 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.
authors.affiliation: Department of Cell Biology and Histology, Sackler Faculty of Medicine, Tel-Aviv University, Ramat Aviv, Israel.
authors.forename: I
authors.initials: I
authors.lastname: Levin
authors.forename: J
authors.initials: J
authors.lastname: Cohen
authors.forename: L
authors.initials: L
authors.lastname: Supino-Rosin
authors.forename: A
authors.initials: A
authors.lastname: Yoshimura
authors.forename: S S
authors.initials: SS
authors.lastname: Watowich
authors.forename: D
authors.initials: D
authors.lastname: Neumann
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001402:B-Lymphocytes; D019556:COS Cells; D002460:Cell Line; D003593:Cytoplasm / Q000737:chemistry*; D004705:Endocytosis / Q000502:physiology*; D004921:Erythropoietin / Q000378:metabolism; D008969:Molecular Sequence Data; D017467:Receptors, Erythropoietin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D014162:Transfection
chemical_list: D017467:Receptors, Erythropoietin; D004921:Erythropoietin