title: Mutations in the WSAWSE and cytosolic domains of the erythropoietin receptor affect signal transduction and ligand binding and internalization.
abstract: The terminal development of erythroid progenitor cells is promoted in part through the interaction of erythropoietin (EPO) with its cell surface receptor. This receptor and a growing family of related cytokine receptors share homologous extracellular features, including a well-conserved WSXWS motif. To explore the functional significance of this motif in the murine EPO receptor, five WSAWSE mutants were prepared and their signal-transducing, ligand binding, and endocytotic properties were compared. EPO receptors mutated at tryptophan residues (W-232, W-235----G; W-235----G; W-235----F) failed to mediate EPO-induced growth or pp100 phosphorylation, while S-236----T and E-237----K mutants exhibited partial to full activity (50 to 100% of wild-type growth and induced phosphorylation). Ligand affinity was reduced for mutant receptors (two- to fivefold), yet expression at the cell surface for all receptors was nearly equivalent. Also, the ability of mutated receptors to internalize ligand was either markedly reduced or abolished (W-235----F), indicating a role for the WSAWSE region in hormone internalization. Interestingly, receptor forms lacking 97% of the cytosolic domain (no signal-transducing capacity; binding affinity reduced two- to threefold) internalized EPO efficiently. This and all WSAWSE receptor forms studied also mediated specific cross-linking of 125I-EPO to three accessory membrane proteins (M(r)s, 120,000, 105,000, and 93,000). These findings suggest that the WSAWSE domain of the EPO receptor is important for EPO-induced signal transduction and ligand internalization. In contrast, although the cytosolic domain is required for growth signaling, it appears nonessential for efficient endocytosis.
authors.affiliation: Department of Molecular and Cell Biology, Pennsylvania State University, University Park 16802.
authors.forename: D E
authors.initials: DE
authors.lastname: Quelle
authors.forename: F W
authors.initials: FW
authors.lastname: Quelle
authors.forename: D M
authors.initials: DM
authors.lastname: Wojchowski
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D003470:Culture Media; D003600:Cytosol / Q000378:metabolism; D004705:Endocytosis / Q000235:genetics; D004921:Erythropoietin / Q000378:metabolism*; D006031:Glycosylation; D008024:Ligands; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis; D017467:Receptors, Erythropoietin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics*
chemical_list: D003470:Culture Media; D008024:Ligands; D017467:Receptors, Erythropoietin; D004921:Erythropoietin