Proteasomes Regulate the Duration of Erythropoietin Receptor Activation by Controlling Down-regulation of Cell Surface Receptors

Verdier, Frédérique; Walrafen, Pierre; Hubert, Nathalie; Chrétien, Stany; Gisselbrecht, Sylvie; Lacombe, Catherine; Mayeux, Patrick

doi:10.1074/jbc.275.24.18375

Cited by 81 publications

(60 citation statements)

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“…Similarly, overexpression of mEPO-R in UT7 cells led to replacement of the endogenous human EPO-R on the cell surface, suggesting that chaperones and/or auxiliary molecules may be a limiting factor for surface deposition (43). In that respect, overexpression of exogenous EPO-Rs in other cell lines, including Ba/F3 cells, still results in its low surface expression (4,44). Improved glycan processing and cell-surface expression of sEPO-R and EPO-R derivatives containing sEPO-R extracellular sequences in both Ba/F3 and in BOSC-23T cells suggest that the role of these domains extends beyond a particular cell type.…”

Section: Er Exit and Intracellular Traffickingmentioning

confidence: 99%

An extracellular region of the erythropoietin receptor of the subterranean blind mole rat Spalax enhances receptor maturation

Ravid¹,

Shams²,

Califa³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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Erythropoietic functions of erythropoietin (EPO) are mediated by its receptor (EPO-R), which is present on the cell surface of erythroid progenitors and induced by hypoxia. We focused on EPO-R from Spalax galili (sEPO-R), one of the four Israeli species of the subterranean blind mole rat, Spalax ehrenbergi superspecies, as a special natural animal model of high tolerance to hypoxia. Led by the intriguing observation that most of the mouse EPO-R (mEPO-R) is retained in the endoplasmic reticulum (ER), we hypothesized that sEPO-R is expressed at higher levels on the cell surface, thus maximizing the response to elevated EPO, which has been reported in this species. Indeed, we found increased cell-surface levels of sEPO-R as compared with mEPO-R by using flow cytometry analysis of BOSC cells transiently expressing HA-tagged EPO-Rs (full length or truncated). We then postulated that unique extracellular sEPO-R sequence features contribute to its processing and cell-surface expression. To map these domains of the sEPO-R that augment receptor maturation, we generated EPO-R derivatives in which parts of the extracellular region of mEPO-R were replaced with the corresponding fragments of sEPO-R. We found that an extracellular portion of sEPO-R, harboring the N-glycosylation site, conferred enhanced maturation and increased transport to the cell surface of the respective chimeric receptor.Taken together, we demonstrate higher surface expression of sEPO-R, attributed at least in part to increased ER exit, mediated by an extracellular region of this receptor. We speculate that these sEPO-R sequence features play a role in the adaptation of Spalax to extreme hypoxia.signal transduction ͉ intracellular trafficking ͉ hypoxia ͉ glycosylation E rythropoietin (EPO) promotes proliferation and differentiation of erythroid cell precursors via activation of its cellsurface receptor (EPO-R) (1, 2). EPO-R is a member of the cytokine-receptor superfamily, characterized by the presence of four conserved Cys residues and a ''WSXWS'' motif in its extracellular domain. The lack of enzymatic activity in the intracellular domain of these receptors necessitates complex formation of ligand-bound receptors with other signaling partners [i.e., Janus kinases (JAKs)] (3) to initiate signaling cascades. EPO-R is synthesized in the endoplasmic reticulum (ER) as a major 64-kDa form with a single endoglycosidase H (Endo H)-sensitive high-mannose oligosaccharide and as a nonglycosylated 62-kDa form. Forty to 60% of the 64-kDa EPO-R molecules undergo further glycan maturation to generate the 66-kDa Endo H-resistant EPO-R (4).In contrast to other type 1 cytokine receptors (5-7), a most striking property of the EPO-R is its low expression on the cell surface (4, 8-10) despite its high intracellular levels. The majority of newly synthesized EPO-R remains sequestered intracellularly and is rapidly degraded (t 1/2 Ϸ 45 min) (11). These metabolic features are similar in both transfected (4) and fetal liver cells that endogenously express the EPO-R (12), suppor...

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Section: Er Exit and Intracellular Traffickingmentioning

confidence: 99%

An extracellular region of the erythropoietin receptor of the subterranean blind mole rat Spalax enhances receptor maturation

Ravid¹,

Shams²,

Califa³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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“…The activation of EpoR and subsequent interactions of these receptors with intracellular proteins are more important than their increased expression (Verdier et al, 2000). Therefore, we were incubated with (0, 50µmol/L) Rg1, after 6 h, the cells were further stimulated with or without 4μM Epo for 5min.…”

Section: Discussionmentioning

confidence: 99%

Ginsenoside Rg1 Induces Apoptosis through Inhibition of the EpoR-Mediated JAK2/STAT5 Signalling Pathway in the TF-1/Epo Human Leukemia Cell Line

Li¹,

Wei²,

Zuo³

et al. 2014

Asian Pacific Journal of Cancer Prevention

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Ginsenoside Rg1 is one effective anticancer and antioxidant constituent of total saponins of Panax ginseng (TSPG), which has been shown to have various pharmacological effects. Our previous study demonstrated that Rg1 had anti-tumor activity in K562 leukemia cells. The aim of this study was designed to investigate whether Rg1 could induce apoptosis in TF-1/Epo cells and further to explore the underlying molecular mechanisms. Here we found that Rg1 could inhibit TF-1/Epo cell proliferation and induce cell apoptosis in vitro in a concentration and time dependent manner. It also suppressed the expression of EpoR on the surface membrane and inhibited JAK2/STAT5 pathway activity. Rg1 induced up-regulation of Bax, cleaved caspase-3 and C-PAPR protein and down-regulation of Bcl-2 and AG490, a JAK2 specific inhibitor, could enhance the effects of Rg1. Our studies showed that EpoR-mediated JAK2/STAT5 signaling played a key role in Rg1-induced apoptosis in TF-1/Epo cells. These results may provide new insights of Rg1 protective roles in the prevention and treatment of leukemia.

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“…3), after Epo binding, the heterodimeric EpoR complex may be efficiently internalized without either the binding/activation/phosphorylation of JAK2 or the ubiquitination process, thus not involving the protein kinase cascade/network [41,48]. The internalization process of the Epo-EpoR complex probably occurs either by conformational changes induced by Epo binding or by caveolae/ clathrin-dependent pathway(s).…”

Section: Trying To Assemble the Jigsaw Puzzle Pieces For Breast Tissuementioning

confidence: 99%

“…Analyzing the ubiquitination, internalization, and degradation model of Epo and its receptor in hematopoietic cells [41][42][43], at least two mechanisms may justify the confounding results obtained in breast cells and tissues. Let's put together the "scattered" pieces of Epo and EpoR findings in breast through (a) JAK-dependent ubiquitination/ degradation and (b) JAK-independent internalizationrecycling mechanisms.…”

Section: Trying To Assemble the Jigsaw Puzzle Pieces For Breast Tissuementioning

confidence: 99%

“…In conjunction with 764 The Jigsaw Puzzle of Epo in Breast Cancer other kinases, JAK2 phosphorylates several tyrosine residues in the EpoR, creating docking sites for the SH2 domains of several signal transduction proteins (e.g., STAT5 and other signaling proteins become phosphorylated and activated, promote intracellular signaling, move to the nucleus, and activate gene expression) [33]. The JAK2-induced tyrosine-phosphorylated form of EpoR is rapidly ubiquitinated [44], an important step both for the proteasomal degradation of its intracellular domain (preventing further signal transduction) and for the targeting and routing of Epo and EpoR to lysosomes [41,43]. The proteasomal cleaved EpoR (still complexed with Epo protein) may be internalized (probably by a clathrin/caveolae-dependent mechanism) and may follow two fates: (a) lysosome targeting for the final proteolytic degradation of both Epo and cleaved EpoR [42] and (b) endosome formation for the extracellular secretion of the soluble form of cleaved EpoR and Epo.…”

Section: Trying To Assemble the Jigsaw Puzzle Pieces For Breast Tissuementioning

confidence: 99%

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Erythropoietin and Its Receptor in Breast Cancer: Putting Together the Pieces of the Puzzle

Mannello

Tonti

2008

The Oncologist

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The expression of erythropoietin (Epo) and the Epo receptor (EpoR) has been detected in healthy tissue as well as in a variety of human cancers, including breast. Functional Epo/EpoR signaling in cancer cells, which contributes to disease initiation/progression, is not completely straightforward and is difficult to reconcile with the clinical practice of preventing/treating anemia in cancer patients with recombinant Epo. Preclinical and clinical investigations have provided contrasting results, ranging from a beneficial role that improves the patient's overall survival to a negative impact that promotes tumor growth progression. A careful gathering of Epo/EpoR biomolecular information enabled us to assemble an unexpected jigsaw puzzle which, via distinct JAK-dependent and JAK-independent mechanisms and different internalization/recycling as well as ubiquitination/degradation pathways, could explain most of the controversies of preclinical and clinical studies. However, until the mechanisms of the contrasting literature data are resolved, this new point of view may shed light on the Epo/EpoR paracrine/autocrine system and function, providing a basis for further studies in order to achieve the highest possible benefit for cancer patients. The Oncologist 2008;13:761-768 STATE OF THE ARTErythropoietin (Epo) is a glycoprotein hormone that serves as the primary regulator of erythropoiesis (by stimulating growth, preventing apoptosis, and inducing the differentiation of RBC precursors) [1]; it also has been localized in several nonhematopoietic tissues and cells. In humans Epo mRNA encodes a protein with 193 amino acids (aa). After cleavage of the signal peptide and post-translation modification, the mature protein consists of a 165-aa structure; while the O-linked sugar has no important function, the Nlinked sugars are necessary for stability of the Epo molecule in the circulation. Erythropoietin receptor (EpoR) is a type-1, single-transmembrane receptor that is expressed in several forms in erythropoietic progenitor cells, including a full-length form (484 aa), a truncated form (303 aa), and a soluble form (115 aa). The truncated and soluble forms contain the extracellular Epo-binding domain, but alternative splicing of transcripts truncates the cytoplasmic or trans-

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Proteasomes Regulate the Duration of Erythropoietin Receptor Activation by Controlling Down-regulation of Cell Surface Receptors

Cited by 81 publications

References 50 publications

An extracellular region of the erythropoietin receptor of the subterranean blind mole rat Spalax enhances receptor maturation

An extracellular region of the erythropoietin receptor of the subterranean blind mole rat Spalax enhances receptor maturation

Ginsenoside Rg1 Induces Apoptosis through Inhibition of the EpoR-Mediated JAK2/STAT5 Signalling Pathway in the TF-1/Epo Human Leukemia Cell Line

Erythropoietin and Its Receptor in Breast Cancer: Putting Together the Pieces of the Puzzle

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