Identification of a cytoplasmic motif in the erythropoietin receptor required for receptor internalization.
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.
Levin
I
I
Department of Cell Biology and Histology, Sackler Faculty of Medicine, Tel-Aviv University, Ramat Aviv, Israel.
Cohen
J
J
Supino-Rosin
L
L
Yoshimura
A
A
Watowich
S S
SS
Neumann
D
D
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
D017467:Receptors, Erythropoietin; D004921:Erythropoietin