title: A new high affinity binding site for suppressor of cytokine signaling-3 on the erythropoietin receptor.
abstract: Erythropoietin (Epo) is a hematopoietic cytokine that is crucial for the differentiation and proliferation of erythroid progenitor cells. Epo acts on its target cells by inducing homodimerization of the erythropoietin receptor (EpoR), thereby triggering intracellular signaling cascades. The EpoR encompasses eight tyrosine motifs on its cytoplasmic tail that have been shown to recruit a number of regulatory proteins. Recently, the feedback inhibitor suppressor of cytokine signaling-3 (SOCS-3), also referred to as cytokine-inducible SH2-containing protein 3 (CIS-3), has been shown to act on Epo signaling by both binding to the EpoR and the EpoR-associated Janus kinase 2 (Jak2) [Sasaki, A., Yasukawa, H., Shouda, T., Kitamura, T., Dikic, I. & Yoshimura, A. (2000) J. Biol. Chem 275, 29338-29347]. In this study tyrosine 401 was identified as a binding site for SOCS-3 on the EpoR. Here we show that human SOCS-3 binds to pY401 with a Kd of 9.5 microm while another EpoR tyrosine motif, pY429pY431, can also interact with SOCS-3 but with a ninefold higher affinity than we found for the previously reported motif pY401. In addition, SOCS-3 binds the double phosphorylated motif pY429pY431 more potently than the respective singly phosphorylated tyrosines indicating a synergistic effect of these two tyrosine residues with respect to SOCS-3 binding. Surface plasmon resonance analysis, together with peptide precipitation assays and model structures of the SH2 domain of SOCS-3 complexed with EpoR peptides, provide evidence for pY429pY431 being a new high affinity binding site for SOCS-3 on the EpoR.
authors.affiliation: Bayer Pharma Research Center, Wuppertal, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Hörtner
authors.forename: Ulrich
authors.initials: U
authors.lastname: Nielsch
authors.forename: Lorenz M
authors.initials: LM
authors.lastname: Mayr
authors.forename: Peter C
authors.initials: PC
authors.lastname: Heinrich
authors.forename: Serge
authors.initials: S
authors.lastname: Haan
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D015374:Biosensing Techniques; D019556:COS Cells; D002460:Cell Line; D003001:Cloning, Molecular; D004926:Escherichia coli; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010766:Phosphorylation; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D017467:Receptors, Erythropoietin / Q000737:chemistry / Q000378:metabolism*; D012097:Repressor Proteins; D016415:Sequence Alignment; D000071223:Suppressor of Cytokine Signaling 3 Protein; D050826:Suppressor of Cytokine Signaling Proteins; D014157:Transcription Factors; D014443:Tyrosine / Q000378:metabolism; D018909:src Homology Domains
chemical_list: D011506:Proteins; D017467:Receptors, Erythropoietin; D012097:Repressor Proteins; C106644:SOCS3 protein, human; D000071223:Suppressor of Cytokine Signaling 3 Protein; D050826:Suppressor of Cytokine Signaling Proteins; D014157:Transcription Factors; D014443:Tyrosine