Functional cross-talk between cytokine receptors revealed by activating mutations in the extracellular domain of the beta-subunit of the GM-CSF receptor

Several reports have suggested an interaction between the erythropoietin receptor (EpoR) and the shared signaling subunit (hbeta(c)) of the human granulocyte macrophage-colony stimulating factor (GM-CSF), interleukin (IL)-3, and IL-5 receptors, although the functional consequences of this interaction are unclear. We previously showed that in vivo expression of constitutively active extracellular (EC) mutants of hbeta(c) induces erythrocytosis and Epo independence of erythroid colony-forming units (CFU-E). This occurs despite an apparent requirement of these mutants for the GM-CSF receptor alpha-subunit (GMRalpha), which is not expressed in CFU-E. Here, we show that coexpression of hbeta(c) EC mutants and EpoR in BaF-B03 cells, which lack GMRalpha, results in factor-independent proliferation and JAK2 activation. Mutant receptors that cannot activate JAK2 fail to produce a functional interaction. As there is no detectable phosphorylation of hbeta(c). on intracellular tyrosine residues, EpoR displays constitutive tyrosine phosphorylation. These observations suggest that JAK2 activation mediates cross-talk between EC mutants of hbeta(c) and EpoR. The implications of these data are discussed as are our findings that activated hbeta(c) mutants can functionally interact with certain other cytokine receptors.

Cytokine-related genes identified from the RIKEN full-length mouse cDNA data set

To identify novel cytokine-related genes, we searched the set of 60,770 annotated RIKEN mouse cDNA clones (FANTOM2 clones), using keywords such as cytokine itself or cytokine names (such as interferon, interleukin, epidermal growth factor, fibroblast growth factor, and transforming growth factor). This search produced 108 known cytokines and cytokine-related products such as cytokine receptors, cytokine-associated genes, or their products (enhancers, accessory proteins, cytokine-induced genes). We found 15 clusters of FANTOM2 clones that are candidates for novel cytokine-related genes. These encoded products with strong sequence similarity to guanylate-binding protein (GBP-5), interleukin-1 receptor-associated kinase 2 (IRAK-2), interleukin 20 receptor alpha isoform 3, a member of the interferon-inducible proteins of the Ifi 200 cluster, four members of the membrane-associated family 1-8 of interferon-inducible proteins, one p27-like protein, and a hypothetical protein containing a Toll/Interleukin receptor domain. All four clones representing novel candidates of gene products from the family contain a novel highly conserved cross-species domain. Clones similar to growth factor-related products included transforming growth factor beta-inducible early growth response protein 2 (TIEG-2), TGFbeta-induced factor 2, integrin beta-like 1, latent TGF-binding protein 4S, and FGF receptor 4B. We performed a detailed sequence analysis of the candidate novel genes to elucidate their likely functional properties.

Flexibility revealed by the 1.85 angstrom crystal structure of the beta sliding-clamp subunit of Escherichia coli DNA polymerase III

The beta subunit of the Escherichia coli replicative DNA polymerase III holoenzyme is the sliding clamp that interacts with the alpha (polymerase) subunit to maintain the high processivity of the enzyme. The beta protein is a ring-shaped dimer of 40.6 kDa subunits whose structure has previously been determined at a resolution of 2.5 Angstrom [Kong et al. (1992), Cell, 69, 425-437]. Here, the construction of a new plasmid that directs overproduction of beta to very high levels and a simple procedure for large-scale purification of the protein are described. Crystals grown under slightly modified conditions diffracted to beyond 1.9 Angstrom at 100 K at a synchrotron source. The structure of the beta dimer solved at 1.85 Angstrom resolution shows some differences from that reported previously. In particular, it was possible at this resolution to identify residues that differed in position between the two subunits in the unit cell; side chains of these and some other residues were found to occupy alternate conformations. This suggests that these residues are likely to be relatively mobile in solution. Some implications of this flexibility for the function of beta are discussed.