The Journal of biological chemistry
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Vanilloid receptor 1 (TRPV1), a membrane-associated cation channel, is activated by the pungent vanilloid from chili peppers, capsaicin, and the ultra potent vanilloid from Euphorbia resinifera, resiniferatoxin (RTX), as well as by physical stimuli (heat and protons) and proposed endogenous ligands (anandamide, N-arachidonyldopamine, N-oleoyldopamine, and products of lipoxygenase). Only limited information is available in TRPV1 on the residues that contribute to vanilloid activation. Interestingly, rabbits have been suggested to be insensitive to capsaicin and have been shown to lack detectable [(3)H]RTX binding in membranes prepared from their dorsal root ganglia. ⋯ We also show that these residues differentially affect ligand recognition as well as the assays of functional response versus ligand binding. Furthermore, these residues account for the reported pharmacological differences of RTX, PPAHV (phorbol 12-phenyl-acetate 13-acetate 20-homovanillate) and capsazepine between human and rat TRPV1. Based on our data we propose a model of the TM3/4 region of TRPV1 bound to capsaicin or RTX that may aid in the development of potent TRPV1 antagonists with utility in the treatment of sensory disorders.
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Toll-like receptors (TLRs) are sensors for the detection of invading infectious agents and can initiate innate immune responses. Because the innate immune system induces an appropriate defense against different pathogens, different TLR signaling domains may have unique properties that are responsible for eliciting distinctive responses to different types of pathogens. To test this hypothesis, we created ligand-regulated TLR chimeric receptors composed of the extracellular region of TLR4 and the transmembrane and cytoplasmic regions of other TLRs and expressed these chimeras in macrophages lacking endogenous TLR4. ⋯ Furthermore, the pair of TLR2+TLR6 chimera required approximately 10-fold less LPS to induce the same responses compared with the TLR1+TLR2 pair. Finally, LPS induced effective interferon-beta production and subsequent Stat1 phosphorylation in macrophages expressing full-length TLR4 but not other cell surface TLR chimeras. These results suggest that the functions of TLRs are diversified not only in their extracellular regions for ligand recognition but also in their transmembrane and cytoplasmic regions for subcellular localization and signaling properties.
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To identify binding domains in a ligand-gated ion channel for etomidate, an intravenous general anesthetic, we photolabeled nicotinic acetylcholine receptor (nAChR)-rich membranes from Torpedo electric organ with a photoactivatable analog, [(3)H]azietomidate. Based upon the inhibition of binding of the noncompetitive antagonist [(3)H]phencyclidine, azietomidate and etomidate bind with 10-fold higher affinity to nAChRs in the desensitized state (IC(50) = 70 microm) than in the closed channel state. In addition, both drugs between 0.1 and 1 mm produced a concentration-dependent enhancement of [(3)H]ACh equilibrium binding affinity, but they inhibited binding at higher concentrations. ⋯ Within the acetylcholine binding sites, [(3)H]azietomidate photolabeled alphaTyr-93, alphaTyr-190, and alphaTyr-198 in the site at the alpha-gamma interface and deltaAsp-59 (but not the homologous position, gammaGlu-57). Increasing [(3)H]azietomidate concentration from 1.8 to 150 microm increased the efficiency of incorporation into amino acids within the ion channel by 10-fold and in the ACh sites by 100-fold, consistent with higher affinity binding within the ion channel. The state dependence and subunit selectivity of [(3)H]azietomidate photolabeling are discussed in terms of the structures of the nAChR transmembrane and extracellular domains.
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Toll-like receptor 4 (TLR4) and TLR2 agonists from bacterial origin require acylated saturated fatty acids in their molecules. Previously, we reported that TLR4 activation is reciprocally modulated by saturated and polyunsaturated fatty acids in macrophages. However, it is not known whether fatty acids can modulate the activation of TLR2 or other TLRs for which respective ligands do not require acylated fatty acids. ⋯ Together, these results demonstrate that lauric acid activates TLR2 dimers as well as TLR4 for which respective bacterial agonists require acylated fatty acids, whereas DHA inhibits the activation of all TLRs tested. Thus, responsiveness of different cell types and tissues to saturated fatty acids would depend on the expression of TLR4 or TLR2 with either TLR1 or TLR6. These results also suggest that inflammatory responses induced by the activation of TLRs can be differentially modulated by types of dietary fatty acids.
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Gab1 is a scaffolding/docking protein that has been suggested to play a role in signal transduction downstream of certain plasma membrane receptors, including platelet-derived growth factor (PDGF) receptors. We found that PDGF induced a rapid Gab1 phosphorylation, which depended on the recruitment of Grb2, indicating that Grb2 acts as a bridge between Gab1 and the PDGF beta-receptor. PDGF also enhanced the binding of Gab1 to the phosphatase SHP-2, but not to p85. ⋯ These effects of PDGF on the actin cytoskeleton were not altered by the inhibition of p38 or Erk, but could be blocked by a dominant-negative form of Rac (Asn(17)). Finally, Gab1-deficient fibroblasts showed a decreased chemotactic response toward gradients of PDGF as compared with wild-type cells. In conclusion, Gab1 plays a selective role in the regulation of the mitogen-activated protein kinases Erk and p38 downstream of the PDGF beta-receptor, and contributes to cytoskeletal reorganization and chemotaxis in response to PDGF.