title: Activation of the erythropoietin receptor is not required for internalization of bound erythropoietin.
abstract: Erythropoietin (EPO) is required for the survival and expansion of red blood cell progenitor cells and supports continued differentiation of these committed progenitors to mature red blood cells. After binding to its cognate receptor, EPO promotes receptor homodimerization, activation of receptor-associated JAK2, subsequent receptor tyrosine phosphorylation, and transduction of signal. EPO is also internalized and degraded in lysosomes. The contribution of EPO-induced receptor internalization to modulation of EPO signals has not been determined. To examine this question, we generated a panel of hematopoietic cell lines containing progressively truncated isoforms of the erythropoietin receptor (EPO-R) and determined the rate and extent of EPO internalization and receptor downregulation. We demonstrated that a membrane-proximal domain of the cytoplasmic tail of the EPO-R was the minimal region required for EPO-induced receptor internalization. This cytoplasmic domain is also the minimal domain required for activation of JAK2, a cytosolic tyrosine kinase essential for the function of the EPO-R. However, neither EPO activation of cytosolic JAK2 tyrosine kinase activity nor tyrosine phosphorylation of the EPO-R cytoplasmic tail was required for EPO-induced receptor downregulation. Both functional and nonfunctional cell surface receptor isoforms were internalized equally. These results suggest that, for downregulation of cell surface ligand occupied EPO-R and possibly for signaling receptors of the cytokine receptor superfamily in general, internalization of cell surface ligand occupied receptors may follow a pathway distinct from signaling receptors of the receptor tyrosine kinase (RTK) family.
authors.affiliation: Departments of Medicine and Cell Biology, Washington University School of Medicine, St Louis, MO 63110, USA.
authors.forename: D L
authors.initials: DL
authors.lastname: Beckman
authors.forename: L L
authors.initials: LL
authors.lastname: Lin
authors.forename: M E
authors.initials: ME
authors.lastname: Quinones
authors.forename: G D
authors.initials: GD
authors.lastname: Longmore
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D019281:Dimerization; D004705:Endocytosis / Q000502:physiology*; D004789:Enzyme Activation; D004921:Erythropoietin / Q000378:metabolism*; D006412:Hematopoietic Stem Cells / Q000378:metabolism; D053614:Janus Kinase 2; D051379:Mice; D005810:Multigene Family; D010766:Phosphorylation; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins; D017467:Receptors, Erythropoietin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion; D015398:Signal Transduction / Q000502:physiology*; D013329:Structure-Activity Relationship
chemical_list: D011518:Proto-Oncogene Proteins; D017467:Receptors, Erythropoietin; D004921:Erythropoietin; D011505:Protein-Tyrosine Kinases; C507926:Jak2 protein, mouse; D053614:Janus Kinase 2