Increased cell surface expression of <i>C</i>‐terminal truncated erythropoietin receptors in polycythemia

Motohashi, Tsutomu; Nakamura, Yukio; Osawa, Mitsujiro; Hiroyama, Takashi; Iwama, Atsushi; Shibuya, Akira; Nakauchi, Hiromitsu

doi:10.1034/j.1600-0609.2001.t01-1-00446.x

Cited by 14 publications

(9 citation statements)

References 24 publications

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“…ingly, these mutations have been shown to lead to the accumulation of truncated EPOR at the plasma membrane, which is thought to underlie increased downstream signaling (24,25). Perhaps most notably, many of the disease-associated truncations eliminate Pro 419 (and Pro 426 ), which is predicted to cause a failure in the negative regulation of EPOR via VHL and a consequential prolongation of cell surface EPOR expression.…”

Section: Discussionmentioning

confidence: 99%

Oxygen-dependent Regulation of Erythropoietin Receptor Turnover and Signaling

Heir

Srikumar

Bikopoulos

et al. 2016

Journal of Biological Chemistry

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von Hippel-Lindau (VHL) disease is a rare familial cancer predisposition syndrome caused by a loss or mutation in a single gene, VHL, but it exhibits a wide phenotypic variability that can be categorized into distinct subtypes. The phenotypic variability has been largely argued to be attributable to the extent of deregulation of the ␣ subunit of hypoxia-inducible factor ␣, a well established target of VHL E3 ubiquitin ligase, ECV (Elongins/ Cul2/VHL). Here, we show that erythropoietin receptor (EPOR) is hydroxylated on proline 419 and 426 via prolyl hydroxylase 3. EPOR hydroxylation is required for binding to the ␤ domain of VHL and polyubiquitylation via ECV, leading to increased EPOR turnover. In addition, several type-specific VHL diseasecausing mutants, including those that have retained proper binding and regulation of hypoxia-inducible factor ␣, showed a severe defect in binding prolyl hydroxylated EPOR peptides. These results identify EPOR as the second bona fide hydroxylation-dependent substrate of VHL that potentially influences oxygen homeostasis and contributes to the complex genotypephenotype correlation in VHL disease.von Hippel-Lindau (VHL) 2 disease is a rare familial cancer predisposition syndrome characterized by the development of tumors in several organ systems, including the central nervous system and retinal hemangioblastoma (HAB), which represent the two cardinal features of VHL disease, as well as endolymphatic sac tumor of the inner ear, pancreatic cystadenoma, benign cystadenoma of epididymis, adnexal papillary cystadenoma of probable mesonephric origin, clear-cell renal cell carcinoma (RCC), paragangliomas, and pheochromocytoma (PHEO) (1). Some VHL patients also develop polycythemia with or without the other stigmata of VHL disease. VHL disease is subcategorized into type 1, characterized by HAB and RCC without PHEO; type 2A, characterized by PHEO and HAB without RCC; type 2B, characterized by PHEO and HAB with RCC; type 2C, characterized by exclusive development of PHEO; and type 3, which is characterized by the development of polycythemia without an increased cancer predisposition (2). Notably, despite the phenotypic heterogeneity, VHL disease is caused by a mutation or loss of a single gene, VHL.VHL is the substrate recognizing subunit of a multiprotein E3 ubiquitin ligase ECV (Elongins BC/Cul2/VHL) that targets the ␣ subunit of hypoxia-inducible factor (HIF␣) for ubiquitylation under normal oxygen tension (3). HIF␣ is hydroxylated on conserved prolines within the oxygen-dependent degradation domain (ODD) by a family of prolyl hydroxylase enzymes (PHD1-3) in the presence of 2-oxoglutarate, iron, and oxygen (4 -7). VHL binds exclusively to prolyl hydroxylated HIF␣. Thus, under hypoxia, the unmodified HIF␣ escapes the recognition by VHL and heterodimerizes with the constitutively expressed HIF␤ to form an active transcription factor, which transactivates numerous hypoxia-inducible genes involved in various adaptive processes to hypoxia such as anaerobic metabolism, angiogenesis, and er...

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Section: Discussionmentioning

confidence: 99%

Oxygen-dependent Regulation of Erythropoietin Receptor Turnover and Signaling

Heir

Srikumar

Bikopoulos

et al. 2016

Journal of Biological Chemistry

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“…These findings imply that (i) the extracellular domain of sEPO-R overrides putative cytosolic motifs (in mEPO-R or sEPO-R) that may confer retention/degradation in early compartments of the secretory pathway (10,35), or (ii) the cytosolic segment of sEPO-R is inert with respect to receptor maturation. Truncated mEPO-R lacking the cytosolic region was found at higher levels at the cell surface (13), although the majority of the receptor is retained intracellularly (38). In addition to its role in endocytosis of surface receptor (38,39), the EPO-R intracellular domain acts in several pathways that determine surface expression, including binding to auxiliary molecules (10) and directing to ER-associated degradation (35).…”

Section: Er Exit and Intracellular Traffickingmentioning

confidence: 99%

“…These metabolic features are similar in both transfected (4) and fetal liver cells that endogenously express the EPO-R (12), supporting the idea that structural features of the receptor molecule regulate its surface expression. Low surface-expression levels of EPO-R have been attributed, among other factors (13,14), to poor folding of its extracellular domain (15). Hence, targeted mutations in the extracellular domain of EPO-R either facilitate (15) or inhibit (16-18) surface expression of the receptor.…”

mentioning

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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“…The first construct, Stop1, lacks residues after S409 and corresponds to the most extensive EpoR truncation identified from PFCP patients. 28 The second construct, Stop2, lacks residues after L427 and is analogous to the minimal EpoR truncation identified from PFCP patients ( Figure 7A). The ability of ␥2A cells stably expressing these receptors to internalize EpoR was tested by our flow cytometry internalization assay.…”

Section: Org Frommentioning

confidence: 99%