Insertion of an NPVY sequence into the cytosolic domain of the erythropoietin receptor selectively affects erythropoietin-mediated signalling and function.
EPO (erythropoietin), the major hormone regulating erythropoiesis, functions via activation of its cell-surface receptor (EPO-R) present on erythroid progenitor cells. One of the most striking properties of EPO-R is its low expression on the cell surface, as opposed to its high intracellular levels. The low cell-surface expression of EPO-R may thus limit the efficacy of EPO that is routinely used to treat primary and secondary anaemia. In a recent study [Nahari, Barzilay, Hirschberg and Neumann (2008) Biochem. J. 410, 409-416] we have shown that insertion of an NPVY sequence into the intracellular domain of EPO-R increases its cell-surface expression. In the present study we demonstrate that this NPVY EPO-R insert has a selective effect on EPO-mediated downstream signalling in Ba/F3 cells expressing this receptor (NPVY-EPO-R). This is monitored by increased phosphorylation of the NPVY-EPO-R (on Tyr479), Akt, JAK2 (Janus kinase 2) and ERK1/2 (extracellular-signal-regulated kinase 1/2), but not STAT5 (signal transducer and activator of transcription 5), as compared with cells expressing wild-type EPO-R. This enhanced signalling is reflected in augmented proliferation at low EPO levels (0.05 units/ml) and protection against etoposide-induced apoptosis. Increased cell-surface levels of NPVY-EPO-R are most probably not sufficient to mediate these effects as the A234E-EPO-R mutant that is expressed at high cell-surface levels does not confer an augmented response to EPO. Taken together, we demonstrate that insertion of an NPVY sequence into the cytosolic domain of the EPO-R confers not only improved maturation, but also selectively affects EPO-mediated signalling resulting in an improved responsiveness to EPO reflected in cell proliferation and protection against apoptosis.
Liron
Tamar
T
Department of Cell and Developmental Biology, Tel-Aviv University, Ramat-Aviv, Israel.
Nahari
Tal
T
Souroujon
Miriam C
MC
Neumann
Drorit
D
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D049109:Cell Proliferation; D003600:Cytosol; D004921:Erythropoietin / Q000378:metabolism*; D006801:Humans; D051379:Mice; D009154:Mutation; D010766:Phosphorylation; D017467:Receptors, Erythropoietin / Q000032:analysis / Q000737:chemistry* / Q000235:genetics; D015398:Signal Transduction; D015854:Up-Regulation
D017467:Receptors, Erythropoietin; D004921:Erythropoietin