Structural Requirements of the Extracellular to Transmembrane Domain Junction for Erythropoietin Receptor Function

Kubatzky, Katharina F.; Liu, Wei; Goldgraben, Kerri; Simmerling, Carlos; Smith, Steven O.; Constantinescu, Stefan N.

doi:10.1074/jbc.m411251200

Cited by 41 publications

(40 citation statements)

References 51 publications

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“…Our results differ in many respects from those of Kubatzky et al (28), who also performed cysteine-scanning mutagenesis to study the dimerization interface of the EpoR juxtamembrane and transmembrane domains, and identified the same three constitutively active mutants L223C, L226C, and I227C. Using selected lines of infected Ba/F3 cells they found that these mutants all grow at the same high rate in the absence of Epo (28). Our studies employed unselected populations of EpoR-expressing cells to eliminate bias in clonal selection, and we demonstrated significant differences in the ability of these activating mutants to mediate cytokine-independent growth even though they all expressed the same number of cell surface EpoRs.…”

Section: Discussioncontrasting

confidence: 88%

“…These subtle differences in the biological activities of the various EpoR mutants, unnoticed in Ref. 28, suggest these mutants assume distinct activated conformations. Further structural analysis of synthetic EpoR transmembrane domains bearing these mutations will provide crucial structural details relevant to the optimal receptor conformation for activation.…”

Section: Discussionmentioning

confidence: 98%

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Active Conformation of the Erythropoietin Receptor

Lü

Gross

Lodish

2006

Journal of Biological Chemistry

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Section: Discussioncontrasting

confidence: 88%

Section: Discussionmentioning

confidence: 98%

Active Conformation of the Erythropoietin Receptor

Lü

Gross

Lodish

2006

Journal of Biological Chemistry

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“…5A, SI Methods, and SI Table 1). It is worth noting that Ba/F3 cells expressing the chimeric EPO-Rs maintained their ability to proliferate when EPO was the sole added growth factor (data not shown), suggesting that the chimeras meet basic structural requirements for ligand activation (36). EPO activation of these cells resulted in Tyr phosphorylation of both EPO-R and STAT5, indicating the functionality of these EPO-Rs (SI Fig.…”

Section: Extracellular Segments Of Sepo-r Confer Enhanced Maturationmentioning

confidence: 92%

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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“…[3][4][5] The creation of disulfide bonds is critical for the activation of the receptors because elimination of the cysteines abrogated the cytokine independent growth. 4,6,7 Here we report and analyze a novel class of noncysteine mutations in cytokine type I receptors in ALL leading to ligand independent activation.…”

Section: Introductionmentioning

confidence: 99%

Novel activating mutations lacking cysteine in type I cytokine receptors in acute lymphoblastic leukemia

Shochat

Tal

Gryshkova

et al. 2014

Blood

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Key Points• Two distinct regions of transmembrane somatic mutations in type I cytokine receptors IL7R and CRLF2 exist in acute lymphoblastic leukemias.• Noncysteine transmembrane mutations cause functional receptor dimerization and activation transforming pro-B cells.Gain-of-function somatic mutations introducing cysteines to either the extracellular or to the transmembrane domain (TMD) in interleukin-7 receptor a (IL7R) or cytokine receptorlike factor 2 (CRLF2) have been described in acute lymphoblastic leukemias. Here we report noncysteine in-frame mutations in IL7R and CRLF2 located in a region of the TMD closer to the cytosolic domain. Biochemical and functional assays showed that these are activating mutations conferring cytokine-independent growth of progenitor lymphoid cells in vitro and are transforming in vivo. Protein fragment complementation assays suggest that despite the absence of cysteines, the mechanism of activation is through ligand-independent dimerization. Mutagenesis experiments and ConSurf calculations suggest that the mutations stabilize the homodimeric conformation, positioning the cytosolic kinases in predefined orientation to each other, thereby inducing spontaneous receptor activation independently of external signals. Hence, type I cytokine receptors may be activated in leukemia through 2 types of transmembrane somatic dimerizing mutations. (Blood. 2014;124(1):106-110)

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Structural Requirements of the Extracellular to Transmembrane Domain Junction for Erythropoietin Receptor Function

Cited by 41 publications

References 51 publications

Active Conformation of the Erythropoietin Receptor

Active Conformation of the Erythropoietin Receptor

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

Novel activating mutations lacking cysteine in type I cytokine receptors in acute lymphoblastic leukemia

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