A transplanted NPVY sequence in the cytosolic domain of the erythropoietin receptor enhances maturation

Tal Nahari, Eran Barzilay, Koret Hirschberg, Drorit Neumann*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

Abstract

Activation of the EPO-R [EPO (erythropoietin) receptor] by its ligand EPO promotes erythropoiesis. Low cell surface EPO-R levels are traditionally attributed to inefficient folding mediated by the receptor extracellular domain. In the present study, we addressed the role of the EPO-R intracellular domain in exit from the ER (endoplasmic reticulum) and surface expression. A fusion protein between the thermo-reversible folding mutant of VSVG (vesicular-stomatitis-virus glycoprotein) (VSVGtsO45) and the EPO-R cytosolic domain [VSVG-WT (wild-type)] displayed delayed intracellular trafficking as compared with the parental VSVGtsO45, suggesting that the EPO-R cytosolic domain can hamper ER exit. Although NPXY-based motifs were originally associated with clathrin binding and endocytosis, they may also function in other contexts of the secretory pathway. A fusion protein between VSVGtsO45 and the cytosolic portion of EPO-R containing an NPVY insert (VSVG-NPVY) displayed enhanced glycan maturation and surface expression as compared with VSVG-WT. Notably, the NPVY insert also conferred improved maturation and augmented cell surface EPO-R. Our findings highlight three major concepts: (i) the EPO-R cytosolic domain is involved in ER exit of the receptor. (ii) Sequence motifs that participate in endocytosis can also modulate transport along the secretory pathway. (iii) VSVG-fusion proteins may be employed to screen for intracellular sequences that regulate transport.

Original languageEnglish
Pages (from-to)409-416
Number of pages8
JournalBiochemical Journal
Volume410
Issue number2
DOIs
StatePublished - 1 Mar 2008

Keywords

  • Cytosolic domain
  • Endoplasmic reticulum
  • Erythropoietin receptor
  • Janus kinase
  • NPXY motif
  • Vesicular-stomatitis-virus glycoprotein (VSVG)

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