A new high affinity binding site for suppressor of cytokine signaling-3 on the erythropoietin receptor.

in European Journal of Biochemistry (2002), 269(10), 2516-26

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 ... [more ▼]

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. [less ▲]

The molecular and genetic analysis of mouse development.

in European Journal of Biochemistry (1992), 204(1), 5-11

This review describes some recent advances in the molecular-genetic analysis of mouse development. Reversed genetics and gene assignment have been used to isolate genes affected in developmental mutations ... [more ▼]

This review describes some recent advances in the molecular-genetic analysis of mouse development. Reversed genetics and gene assignment have been used to isolate genes affected in developmental mutations. The establishment of a high-density molecular-genetic map promises to facilitate cloning of additional genes with developmental functions. Based on molecular, biochemical or other biological criteria many mouse genes that code for transcriptional regulators, growth-factor-like molecules and their receptors have been isolated. The role of these genes during development can be analysed in vivo after producing targeted mutations. Mutations can be generated by homologous recombination in the genome of embryonic stem cells and can then be introduced into the mouse germ line by means of germ-line chimaeras. Additional approaches employing stem cells to identify and mutate putative developmental genes are coming into use. [less ▲]