Mutations in the WSAWSE and cytosolic domains of the erythropoietin receptor affect signal transduction and ligand binding and internalization.
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.
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Department of Molecular and Cell Biology, Pennsylvania State University, University Park 16802.
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Wojchowski
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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*
D003470:Culture Media; D008024:Ligands; D017467:Receptors, Erythropoietin; D004921:Erythropoietin