title: A transplanted NPVY sequence in the cytosolic domain of the erythropoietin receptor enhances maturation.
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.
authors.affiliation: Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel-Aviv University, Ramat-Aviv 69978, Israel.
authors.forename: Tal
authors.initials: T
authors.lastname: Nahari
authors.forename: Eran
authors.initials: E
authors.lastname: Barzilay
authors.forename: Koret
authors.initials: K
authors.lastname: Hirschberg
authors.forename: Drorit
authors.initials: D
authors.lastname: Neumann
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D001692:Biological Transport; D019921:Biotinylation; D019556:COS Cells; D002522:Chlorocebus aethiops; D003600:Cytosol / Q000502:physiology*; D017931:DNA Primers; D000882:Haplorhini; D006801:Humans; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D010446:Peptide Fragments; D011817:Rabbits; D017467:Receptors, Erythropoietin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D000906:Antibodies; D017931:DNA Primers; D010446:Peptide Fragments; D017467:Receptors, Erythropoietin; D011994:Recombinant Proteins; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase