Identification of a cytoplasmic motif in the erythropoietin receptor required for receptor internalization

FEBS Letters

Watowich, Stephanie S¹  Levin, Iris²  Supino-Rosin, Lia²  Yoshimura, Akihiko³  Cohen, Jacob²  Neumann, Drorit² 

[1] Department of Immunology, The University of Texas, M.D. Anderson Cancer Center, Houston, TX 77030, USA;Department of Cell Biology and Histology, Sackler Faculty of Medicine, Tel-Aviv University, Ramat Aviv 69978, Israel;Institute of Life Science, Kurume University, 2432-3 Aikawa-machi, Kurume 839, Japan

关键词: Erythropoietin;    Erythropoietin receptor;    Endocytosis;    EPO;    erythropoietin;    EPO-R;    erythropoietin receptor;    Endo H;    endoglycosidase H;    ER;    endoplasmic reticulum;   

DOI  :  10.1016/S0014-5793(98)00414-1

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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 (Δ281–300), 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.

【 授权许可】

Unknown