The leptin/leptin receptor system shows strong similarities to the long-chain cytokine interleukin-6 (IL-6) and granulocyte colony-stimulating factor cytokine/receptor systems. The IL-6 family cytokines interact with their receptors through three different binding sites I-III. The leptin structure was superposed on the crystal structures of several long-chain cytokines, and a series of leptin mutants was generated focusing on binding sites I-III. The effect of the mutations on leptin receptor (LR) signaling and on binding to the membrane proximal cytokine receptor homology domain (CRH2) of the LR was determined. Mutations in binding site I at the C terminus of helix D show a modest effect on signaling and do not affect binding to CRH2. Binding site II is composed of residues at the surface of helices A and C. Mutations in this site impair binding to CRH2 but have only limited effect on signaling. Site III mutations around the N terminus of helix D impair receptor activation without affecting binding to CRH2. We identified an S120A/T121A mutant in binding site III, which lacks any signaling capacity, but which still binds to CRH2 with wild type affinity. This leptin mutant behaves as a potent leptin antagonist both in vitro and in vivo.

Background: TLR4 signaling requires unknown interactions between TIR domains of TLR4 and its adapters. Results: We identify three binding sites in the TLR4 TIR domain that are important for TLR4 interactions. Conclusion: Two binding sites in TLR4 are important for adapter binding and NF-B activation. Significance: This work provides new insights in the first steps of TLR activation.

SOCS (suppressors of cytokine signaling) proteins are negative regulators of cytokine signaling that function primarily at the receptor level. Remarkably, in vitro and in vivo observations revealed both inhibitory and stimulatory effects of SOCS2 on growth hormone signaling, suggesting an additional regulatory level. In this study, we examined the possibility of direct crossmodulation between SOCS proteins and found that SOCS2 could interfere with the inhibitory actions of other SOCS proteins in growth hormone, interferon, and leptin signaling. This SOCS2 effect was SOCS box-dependent, required recruitment of the elongin BC complex, and coincided with degradation of target SOCS proteins. Detailed mammalian protein-protein interaction trap (MAPPIT) analysis indicated that SOCS2 can interact with all members of the SOCS family. SOCS2 may thus function as a molecular bridge between a ubiquitin-protein isopeptide ligase complex and SOCS proteins, targeting them for proteasomal turnover. We furthermore extended these observations to SOCS6 and SOCS7. Our findings point to a unique regulatory role for SOCS2, SOCS6, and SOCS7 within the SOCS family and provide an explanation for the unexpected phenotypes observed in SOCS2 and SOCS6 transgenic mice.Cytokine signaling is typically a transient event, implying rapid and finely tuned attenuation. Receptor binding leads to rapid activation of receptor-associated members of the JAK 3 family. Subsequent phosphorylation of tyrosine residues in the receptor tails enables recruitment of downstream signaling molecules whereby STATs play a prominent role. Activated STATs translocate to the nucleus, where they control cytokineregulated gene transcription. Negative control occurs at many levels and involves receptor down-regulation, protein-tyrosine phosphatases, protein inhibitors of activated STATs, and members of the SOCS (suppressors of cytokine signaling) protein family.The SOCS family consists of eight different members (SOCS1-7 and CIS (cytokine-inducible SH2 domain-containing protein)) characterized by conserved structural features. All SOCS proteins consist of a central SH2 domain flanked by a variable N-terminal region and a conserved C-terminal SOCS box (1, 2). The SH2 domain can inhibit STAT activation by direct competition for the phosphorylated receptor recruitment sites (3-8). SOCS1 and SOCS3 carry an additional kinase inhibitory region (KIR) in their N-terminal domains that acts as a pseudosubstrate for the JAK kinase, thereby blocking signaling (5). The SOCS box was shown to act as an interaction domain for the elongin BC complex (9, 10), which, in turn, is a component of an ubiquitin-protein isopeptide ligase (E3) complex (11). This way, the SOCS box can control protein turnover by marking target proteins for proteasomal degradation (12). However, the significance of the interaction between SOCS proteins and the elongin BC complex is not totally clarified, as some reports propose that elongin association targets SOCS molecules for proteasomal degradation (10,(12)(13)...