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

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.