Gephyrin is an essential and instructive molecule for the formation of inhibitory synapses. Gephyrin binds directly to the large cytoplasmic loop located between transmembrane helices three and four of the -subunit of the glycine receptor and to microtubules, thus promoting glycine receptor (GlyR) anchoring to the cytoskeleton and clustering in the postsynaptic membrane. Besides its structural role, gephyrin is involved in the biosynthesis of the molybdenum cofactor that is essential for all molybdenum-dependent enzymes in mammals. Gephyrin can be divided into an N-terminal trimeric G domain and a C-terminal E domain, which are connected by a central linker region. Here we have studied the in vitro interaction of gephyrin and its domains with the large cytoplasmic loop of the GlyR -subunit (GlyR-loop). Binding of gephyrin to the GlyR is exclusively mediated by the E domain, and the binding site was mapped to one of its sub-domains (residues 496 -654). By using isothermal titration calorimetry, a high affinity (K d ؍ 0.2-0.4 M) and low affinity (K d ؍ 11-30 M) binding site for the GlyR-loop was found on holo-gephyrin and the E domain, respectively, with a binding stoichiometry of two GlyR-loops per E domain in both cases. Binding of the GlyR-loop does not change the oligomeric state of either full-length gephyrin or the isolated E domain.Efficient transmission of synaptic signals in the central nervous system is dependent on high local concentrations of neurotransmitter ion channels in the postsynaptic membrane of excitatory and inhibitory synapses. Glycine is an important inhibitory neurotransmitter, and its postsynaptic receptors are an essential component of inhibitory nerve terminals (1-3). Glycine receptors (GlyRs) 1 are pentameric anion channels generally consisting of two different subunits with an ␣ 3  2 composition (4). Crucial for proper functioning of GlyRs is their clustering that generates a high packing density of neuroreceptors in the postsynaptic membrane. Gephyrin, a highly expressed neuronal protein, was found to co-localize with glycine (5, 6) and GABA A receptors (7,8). Gephyrin is crucial for the clustering of both types of inhibitory neuroreceptors (9) and anchors these receptors to the subsynaptic cytoskeleton. A direct binding of gephyrin to microtubules was shown (10), which is possibly mediated by a "tau" motif (11). So far no direct interaction with microfilaments has been demonstrated, but pharmacological studies indicate a function of actin filaments in determining the cluster size (12). Therefore, it is not surprising that binding of gephyrin to key regulators of microfilament dynamics such as profilin I, neuronal profilin IIa, and microfilament adaptors of the Mena/VASP family including neuronal Mena has been reported recently (13). The binding site of gephyrin on GlyR has been mapped to a stretch of 18 amino acids located within the large cytoplasmic loop connecting transmembrane helices three and four of the GlyR -subunit (GlyR-loop) (14), but the binding site on ge...
IL-18 and IL-18 binding protein (IL-18BP) are two newly described opponents in the cytokine network. Local concentrations of these two players may determine biological functions of IL-18 in the context of inflammation, infection, and cancer. As IL-18 appears to be involved in the pathogenesis of Crohn’s disease and may modulate tumor growth, we investigated the IL-18/IL-18BPa system in the human colon carcinoma/epithelial cell line DLD-1. In this study, we report that IFN-γ induces expression and release of IL-18BPa from DLD-1 cells. mRNA induction and secretion of IL-18BPa immunoreactivity were associated with an activity that significantly impaired release of IFN-γ by IL-12/IL-18-stimulated PBMC. Inducibility of IL-18BPa by IFN-γ was also observed in LoVo, Caco-2, and HCT116 human colon carcinoma cell lines and in the human keratinocyte cell line HaCaT. Induction of IL-18BPa in colon carcinoma/epithelial cell lines was suppressed by coincubation with sodium butyrate. IFN-γ-mediated IL-18BPa and its suppression by sodium butyrate were confirmed in organ cultures of intestinal colonic biopsy specimens. In contrast, sodium butyrate did not modulate expression of IL-18. The present data suggest that IFN-γ may limit biological functions of IL-18 at sites of colonic immune activation by inducing IL-18BPa production. Down-regulation of IL-18BPa by sodium butyrate suggests that reinforcement of local IL-18 activity may contribute to actions of this short-chain fatty acid in the colonic microenvironment.
The present data imply that IFN--activated synoviocytes mediate a negative feedback loop via IL-18BPa, which may limit IL-18 biological activity in arthritis.
Introduction Interleukin (IL)-18 is a pro-inflammatory member of the IL-1 cytokine family that is constitutively expressed primarily in monocytes/macrophages, neutrophils and epithelial cells [1][2][3]. This cellular pattern of expression suggests a key role of Abstract Interleukin (IL)-18, formerly known as interferon (IFN)-␥-inducing factor, is a crucial mediator of host defence and inflammation. Control of IL-18 bioactivity by its endogenous antagonist IL-18 binding protein (IL-18BP) is a major objective of immunoregulation. IL-18BP is strongly up-regulated by IFN-␥, thereby establishing a negative feedback mechanism detectable in cell culture and in vivo. Here we sought to investigate in D.L. Dexter (DLD) colon carcinoma cells molecular mechanisms of IL-18BP induction under the influence of IFN-␥. Mutational analysis revealed that a proximal ␥-activated sequence (GAS) at the IL-18BP promoter is of pivotal importance for expression by IFN-␥-activated cells. Use of siRNA underscored the essential role of the signal transducer and activator of transcription (STAT)-1 in this process. Indeed, electrophoretic mobility shift assay and chromatin immunoprecipitation analysis proved STAT1 binding to this particular GAS site. Maximal expression of IL-18BP was dependent on de novo protein synthesis but unaffected by silencing of interferon regulatory factor-1. Altogether, data presented herein indicate that direct action of STAT1 on the IL-18BP promoter at the proximal GAS element is key to IL-18BP expression by IFN-␥-stimulated DLD-1 colon carcinoma cells.
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