title: Cytosolic lysine residues enhance anterograde transport and activation of the erythropoietin receptor.
abstract: Lysine residues are key residues in many cellular processes, in part due to their ability to accept a wide variety of post-translational modifications. In the present study, we identify the EPO-R [EPO (erythropoietin) receptor] cytosolic lysine residues as enhancers of receptor function. EPO-R drives survival, proliferation and differentiation of erythroid progenitor cells via binding of its ligand EPO. We mutated the five EPO-R cytosolic lysine residues to arginine residues (5KR EPO-R), eliminating putative lysine-dependent modifications. Overexpressed 5KR EPO-R displayed impaired ubiquitination and improved stability compared with wt (wild-type) EPO-R. Unexpectedly, fusion proteins consisting of VSVGtsO45 (vesicular stomatitis virus glycoprotein temperature-sensitive folding mutant) with wt or 5KR EPO-R cytosolic domains demonstrated delayed glycan maturation kinetics upon substitution of the lysine residues. Moreover, VSVG-wt EPO-R, but not VSVG-5KR EPO-R, displayed endoplasmic reticulum-associated ubiquitination. Despite similar cell-surface EPO-binding levels of both receptors and the lack of EPO-induced ubiquitination by 5KR EPO-R, the lysine-less mutant produced weaker receptor activation and signalling than the wt receptor. We thus propose that EPO-R cytosolic lysine residues enhance receptor function, most probably through ubiquitination and/or other post-translational modifications.
authors.affiliation: Cell and Developmental Biology, Sackler Faculty of Medicine, Tel Aviv University, Ramat-Aviv, Tel-Aviv 69978, Israel.
authors.forename: Liron
authors.initials: L
authors.lastname: Yosha
authors.forename: Orly
authors.initials: O
authors.lastname: Ravid
authors.forename: Nathalie
authors.initials: N
authors.lastname: Ben-Califa
authors.forename: Drorit
authors.initials: D
authors.lastname: Neumann
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006801:Humans; D008239:Lysine / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D051379:Mice; D008954:Models, Biological; D050505:Mutant Proteins / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000235:genetics / Q000502:physiology; D021381:Protein Transport / Q000235:genetics; D017467:Receptors, Erythropoietin / Q000819:agonists* / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D050505:Mutant Proteins; D017467:Receptors, Erythropoietin; D008239:Lysine