G-CSF is a polypeptide growth factor used in treatment following chemotherapy. G-CSF regulates granulopoiesis and acts on its target cells by inducing homodimerization of the G-CSFR, thereby activating intracellular signaling cascades. The G-CSFR encompasses four tyrosine motifs on its cytoplasmic tail that have been shown to recruit a number of regulatory proteins. Suppressor of cytokine signaling 3 (SOCS-3), also referred to as cytokine-inducible Src homolgy 2-containing protein 3, is a member of a recently discovered family of feedback inhibitors that have been shown to inhibit the Janus kinase/STAT pathway. In this study, we demonstrate that human SOCS-3 is rapidly induced by G-CSF in polymorphonuclear neutrophils as well as in the myeloid precursor cell line U937 and that SOCS-3 negatively regulates G-CSFR-mediated STAT activation. Most importantly, we show that SOCS-3 is recruited to the G-CSFR in a phosphorylation-dependent manner and we identify phosphotyrosine (pY)729 as the major recruitment site for SOCS-3. Furthermore, we demonstrate that SOCS-3 directly binds to this pY motif. Surface plasmon resonance analysis reveals a dissociation constant (KD) for this interaction of around 2.8 μM. These findings strongly suggest that the recruitment of SOCS-3 to pY729 is important for the modulation of G-CSFR-mediated signal transduction by SOCS-3.

Human atria and ventricles show specific gene expression profiles. Our data provide the basis of a comprehensive understanding of chamber-specific gene expression in diseased human hearts and will support the identification of therapeutic targets in the treatment of arrhythmia and heart failure.

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. Chem275, 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 µm 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.

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