Regulation of Erythropoietin Receptor Endocytosis and Signaling
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Erythropoietin (Epo) and its receptor (EpoR) play an essential role in the survival, proliferation and differentiation of precursor red blood cells into mature erythrocytes. EpoR lacks intrinsic enzymatic activity essential to mediate downstream signaling cascades, instead, it associates with Janus tyrosine kinase 2 (JAK2), which upon Epo binding is auto-phosphorylated and activated. Activated JAK2 phosphorylates many of the tyrosines in the EpoR which recruit SH2-domain containing proteins that will carry on the signaling event. EpoR -/- and JAK2 -/- mice die during embryogenesis due to the absence of definitive erythropoiesis. Once activated, the EpoR is internalized and degraded through unidentified mechanisms. Sustained EpoR signaling and aberrant JAK2 activation are observed in hematological malignancies. Here we show that cell-surface EpoR is internalized via clathrin-mediated endocytosis. Ligand-dependent EpoR internalization requires both JAK2 kinase activity and EpoR cytoplasmic tyrosines. In addition, phosphorylated Y429, Y431, and Y479 in the EpoR cytoplasmic domain binds p85 subunit of phosphoinositide 3-kinase (PI3-kinase) upon Epo stimulation and individually is sufficient to mediate Epo-dependent EpoR internalization. Knockdown of p85 alpha and p85 beta but not inhibition of PI3-kinase activity dramatically impaired EpoR internalization, indicating that p85 alpha and p85 beta may recruit proteins in the endocytic machinery upon Epo stimulation. We carried on a candidate siRNA screen for endocytic proteins involved in EpoR endocytosis. c-Cbl, a E3 ubiquitin ligase associated with p85, was identified to be essential in mediating EpoR internalization. Ligase-deficient c-Cbl mutants dramatically diminished ligand-induced EpoR internalization. Consistent with this finding, c-Cbl knockout animals exhibit erythroid hyperplasia. We further demonstrate that ubiquitination of the EpoR itself is not required for internalization but may be important for endosomal sorting and degradation, and K428 in the EpoR cytoplasmic domain may be the primary ubiquitination site. Further studies will uncover the role of c-Cbl in EpoR down-regulation. These results provide the first characterization of EpoR downregulation mechanisms.