title: Dimer- and oligomerization of the erythropoietin receptor by disulfide bond formation and significance of the region near the WSXWS motif in intracellular transport.
abstract: The receptor for erythropoietin (EpoR), a primary regulator of erythropoiesis, belongs to the cytokine receptor family. Although the mechanisms of signal transduction through the receptors of this family are largely unknown, increasing numbers of the receptors have been shown to form a hetero- or homodimer. To address the possibility that the EpoR dimerizes, we made a truncated mutant receptor that lacks most of the cytoplasmic domain and expressed it either alone or with the wild-type receptor in an IL-3-dependent cell line, DA-3. Dimerization of the receptor was demonstrated by a coimmunoprecipitation using an antiserum against the cytoplasmic domain of the receptor. Epo stimulation did not have a detectable effect on dimerization. Coimmunoprecipitation experiments in COS-7 cells further revealed that only the extracellular domain of the receptor is required for dimerization. The "WSXWS" motif, conserved in the cytokine receptor family, was shown to not be required for dimerization. Diagonal two-dimensional gel analysis of the EpoR expressed in DA-3 transfectants showed that a substantial portion of the receptor forms dimers or oligomers with disulfide bonds. Western blot analysis, using an antiphosphotyrosine antibody, revealed that a portion of these dimers or oligomers become tyrosine phosphorylated after Epo stimulation, thus suggesting that these forms are expressed on the cell surface and activated by Epo stimulation.
authors.affiliation: Department of Biochemistry, St. Jude Children's Research Hospital, Memphis, Tennessee 38101.
authors.forename: O
authors.initials: O
authors.lastname: Miura
authors.forename: J N
authors.initials: JN
authors.lastname: Ihle
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D017124:Conserved Sequence; D004220:Disulfides / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D046911:Macromolecular Substances; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017467:Receptors, Erythropoietin / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D014162:Transfection
chemical_list: D000906:Antibodies; D004220:Disulfides; D046911:Macromolecular Substances; D017467:Receptors, Erythropoietin; D011994:Recombinant Proteins