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

Iris Levin, Jacob Cohen, Lia Supino-Rosin, Akihiko Yoshimura, Stephanie S. Watowich, Drorit Neumann

Research output: Contribution to journalArticle

25 Citations (Scopus)

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 mild 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 1251-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 or 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.

Original languageEnglish
Pages (from-to)164-170
Number of pages7
JournalFEBS Letters
Volume427
Issue number2
DOIs
Publication statusPublished - 1998 May 8
Externally publishedYes

Fingerprint

Erythropoietin Receptors
Erythropoietin
COS Cells
Cell Surface Receptors
Endocytosis
Amino Acids

Keywords

  • Endocytosis
  • Erythropoietin
  • Erythropoietin receptor

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Identification of a cytoplasmic motif in the erythropoietin receptor required for receptor internalization. / Levin, Iris; Cohen, Jacob; Supino-Rosin, Lia; Yoshimura, Akihiko; Watowich, Stephanie S.; Neumann, Drorit.

In: FEBS Letters, Vol. 427, No. 2, 08.05.1998, p. 164-170.

Research output: Contribution to journalArticle

Levin, Iris ; Cohen, Jacob ; Supino-Rosin, Lia ; Yoshimura, Akihiko ; Watowich, Stephanie S. ; Neumann, Drorit. / Identification of a cytoplasmic motif in the erythropoietin receptor required for receptor internalization. In: FEBS Letters. 1998 ; Vol. 427, No. 2. pp. 164-170.
@article{e22d8febeb32443fb8735ade2ead4cc3,
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 mild 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 1251-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 or 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.",
keywords = "Endocytosis, Erythropoietin, Erythropoietin receptor",
author = "Iris Levin and Jacob Cohen and Lia Supino-Rosin and Akihiko Yoshimura and Watowich, {Stephanie S.} and Drorit Neumann",
year = "1998",
month = "5",
day = "8",
doi = "10.1016/S0014-5793(98)00414-1",
language = "English",
volume = "427",
pages = "164--170",
journal = "FEBS Letters",
issn = "0014-5793",
publisher = "Elsevier",
number = "2",

}

TY - JOUR

T1 - Identification of a cytoplasmic motif in the erythropoietin receptor required for receptor internalization

AU - Levin, Iris

AU - Cohen, Jacob

AU - Supino-Rosin, Lia

AU - Yoshimura, Akihiko

AU - Watowich, Stephanie S.

AU - Neumann, Drorit

PY - 1998/5/8

Y1 - 1998/5/8

N2 - 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 mild 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 1251-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 or 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.

AB - 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 mild 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 1251-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 or 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.

KW - Endocytosis

KW - Erythropoietin

KW - Erythropoietin receptor

UR - http://www.scopus.com/inward/record.url?scp=0032496370&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0032496370&partnerID=8YFLogxK

U2 - 10.1016/S0014-5793(98)00414-1

DO - 10.1016/S0014-5793(98)00414-1

M3 - Article

C2 - 9607304

AN - SCOPUS:0032496370

VL - 427

SP - 164

EP - 170

JO - FEBS Letters

JF - FEBS Letters

SN - 0014-5793

IS - 2

ER -