No abstract

Free fatty acids (FFAs) play important physiological roles in many tissues as an energy source and as signaling molecules in various cellular processes. Elevated levels of circulating FFAs are associated with obesity, dyslipidemia, and diabetes. Here we show that GPR84, a previously orphan G protein-coupled receptor, functions as a receptor for medium-chain FFAs with carbon chain lengths of 9 -14. Medium-chain FFAs elicit calcium mobilization, inhibit 3,5-cyclic AMP production, and stimulate [ 35 S]guanosine 5-O-(3-thiotriphosphate) binding in a GPR84-dependent manner. The activation of GPR84 by medium-chain FFAs couples primarily to a pertussis toxin-sensitive G i/o pathway. In addition, we show that GPR84 is selectively expressed in leukocytes and markedly induced in monocytes/macrophages upon activation by lipopolysaccharide. Furthermore, we demonstrate that medium-chain FFAs amplify lipopolysaccharidestimulated production of the proinflammatory cytokine interleukin-12 p40 through GPR84. Our results indicate a role for GPR84 in directly linking fatty acid metabolism to immunological regulation.G protein-coupled receptors (GPCRs) 3 constitute one of the largest gene families yet identified (1, 2). It has been estimated that more than half of all modern drugs target these receptors (3, 4). GPCRs contain seven transmembrane domains and are activated by a wide variety of ligand types, including light, ions, amino acids, nucleotides, lipids, peptides, and proteins. In addition to about 250 characterized receptors, over 100 human genes encode proteins that belong to this family of receptors but for which ligands and functions remain to be determined (1). These orphan receptors are expected to play important roles in the regulation of a diversity of physiological functions.In the past decade an increasing number of GPCRs have been deorphanized. Many of the identified ligands are metabolic intermediates, including succinate (ligand for GPR91) (5), ␣-ketoglutarate (ligand for GPR99) (5), fatty acids (ligands for GPR40/41/43/120) (6 -10), ketone body (ligand for HM74a) (11), bile acids (ligands for BG37) (12), and kynurenic acid (ligand for GPR35) (13). We have built a library of biochemical intermediates to test their ability to activate orphan GPCRs. In this report, we have identified medium-chain FFAs as ligands for GPR84. Short-chain and long-chain saturated and unsaturated FFAs, previously shown to activate GPR40/41/43/120 (6 -10), are inactive at GPR84. GPR84 is an orphan GPCR originally isolated using an expressed sequence tag data mining strategy (14) and as a gene differentially expressed in granulocytes (15). No close homologs of GPR84 could be identified, although GPR84 is distantly related to monoamine receptors. Expression analysis revealed significant induction of GPR84 in monocytes/macrophages upon lipopolysaccharide (LPS) stimulation, suggesting that medium-chain FFAs may regulate inflammatory responses through activation of GPR84. EXPERIMENTAL PROCEDURESCloning and Cell Culture-Human and mouse GPR84 ...

Transient receptor potential canonical (TRPC) proteins form nonselective cation channels that play physiological roles in a wide variety of cells. Despite growing evidence supporting the therapeutic potential of TRPC6 inhibition in treating pathological cardiac and renal conditions, mechanistic understanding of TRPC6 function and modulation remains obscure. Here we report cryo-EM structures of TRPC6 in both antagonist-bound and agonist-bound states. The structures reveal two novel recognition sites for the small-molecule modulators corroborated by mutagenesis data. The antagonist binds to a cytoplasm-facing pocket formed by S1-S4 and the TRP helix, whereas the agonist wedges at the subunit interface between S6 and the pore helix. Conformational changes upon ligand binding illuminate a mechanistic rationale for understanding TRPC6 modulation. Furthermore, structural and mutagenesis analyses suggest several disease-related mutations enhance channel activity by disrupting interfacial interactions. Our results provide principles of drug action that may facilitate future design of small molecules to ameliorate TRPC6-mediated diseases.

Redox regulation of DNA-binding proteins through the reversible oxidation of key cysteine sulfhydryl groups has been demonstrated to occur in vitro for a range of transcription factors. The direct redox regulation of DNA binding has not been described in vivo, possibly because most protein thiol groups are strongly buffered against oxidation by the highly reduced intracellular environment mediated by glutathione, thioredoxin, and associated pathways. For this reason, only accessible protein thiol groups with high thiol-disulfide oxidation potentials are likely to be responsive to intracellular redox changes. In this article, we demonstrate that zinc finger DNA-binding proteins, in particular members of the Sp-1 family, appear to contain such redox-sensitive -SH groups. These proteins displayed a higher sensitivity to redox regulation than other redox-responsive factors both in vitro and in vivo. This effect was reflected in the hyperoxidative repression of transcription from promoters with essential Sp-1 binding sites, including the simian virus 40 early region, glycolytic enzyme, and dihydrofolate reductase genes. Promoter analyses implicated the Sp-1 sites in this repression. Non-Sp-1-dependent redox-regulated genes including metallothionein and heme oxygenase were induced by the same hyperoxic stress. The studies demonstrate that cellular redox changes can directly regulate gene expression in vivo by determining the level of occupancy of strategically positioned GC-binding sites.There is compelling evidence for both direct and indirect pathways for the regulation of gene expression by changes in cellular redox state. Hypoxic and hyperoxic stresses can activate or repress the transcription of certain genes by pathways that probably involve protein kinases (5,16,18,24,28,52,68,70,82,84). The response to severe oxidative stress may involve an additional effect in which redox-sensitive factors can be directly activated or inactivated through the oxidation of sulfhydryl residues. The binding of factors AP-1, Sp-1, Egr-1, NF-B, v-rel, c-myb, E2, IRE-BP, p53, and USF to nucleic acid is reduced or lost when critical cysteine residues are oxidized or alkylated (1, 3, 4, 33-35, 37, 48, 53, 60, 66, 78, 89). HoxB5, a member of the mammalian homeodomain gene family, is an example of a factor that is activated by oxidation (30). In the case of AP-1, a cellular DNA-repair protein that may regulate the redox equilibrium has been described previously (90). Oxidative inactivation of USF has been shown to correlate directly with transcriptional activity in an in vitro assay (60). It has been proposed that the reactive cysteines may constitute redox-sulfhydryl switches which directly regulate gene expression (35,60). In support of this, factor Sp-1 in rat liver appears to become progressively oxidized during aging, resulting in the reversible loss of binding activity by an in vitro assay (3). To date, there have been no reports to demonstrate that oxidizing agents or redox stresses can directly mediate transcription factor bindi...

We report that the asthma drugs cromolyn disodium and nedocromil sodium are potent G-protein-coupled receptor 35 (GPR35) agonists. We utilized calcium flux and inositol phosphate accumulation assays to examine the pharmacology of these asthma drugs on the human, mouse and rat GPR35. The compounds were more potent on the human GPR35 than on mouse and rat receptors. In contrast, zaprinast, a known GPR35 agonist, was more potent on mouse and rat GPR35 than the human ortholog. We show by quantitative PCR that GPR35 is expressed in human mast cells, human basophils and human eosinophils. We also demonstrate that GPR35 mRNA is upregulated upon challenge with IgE antibodies. We show that, unlike zaprinast, a potent phosphodiesterase 5 (PDE5) inhibitor, cromolyn disodium and nedocromil sodium lack inhibitory activity towards PDE5. These findings suggest that GPR35 may play an important role in mast cell biology and be a potential target for the treatment of asthma.

The transcription rates of glycolytic enzyme genes are coordinately induced when cells are exposed to low oxygen tension. This effect has been described in many cell types and is not restricted to species or phyla. In mammalian cells, there are 11 distinct glycolytic enzymes, at least 9 of which are induced by hypoxia. Recent reports described a role for the hypoxia-inducible factor-1 (HIF-1) in the transcriptional activation of lactate dehydrogenase A, aldolase-A, phosphoglycerate kinase, and enolase-1 genes. It is not known whether the HIF-1 factor acts exclusively to regulate these genes during hypoxia, or how the other genes of the pathway are regulated. In this paper, we describe analyses of the musclespecific pyruvate kinase-M and ␤-enolase promoters that implicate additional mechanisms for the regulation of glycolytic enzyme gene transcription by hypoxia. Transient transcription of a reporter gene directed by either promoter was activated when transfected muscle cells were exposed to hypoxia. Neither of these promoters contain HIF-1 binding sites. Instead, the hypoxia response was localized to a conserved GC-rich element positioned immediately upstream of a GATAA site in the proximal promoter regions of both genes. The GC element was essential for both basal and hypoxia-induced expression and bound the transcription factors Sp1 and Sp3. Hypoxia caused the progressive depletion of Sp3 determined by DNA binding studies and Western analyses, whereas Sp1 protein levels remained unchanged. Overexpression of Sp3 repressed expression of ␤-enolase promoters. It is concluded that hypoxia activates these glycolytic enzyme gene promoters by down-regulating Sp3, thereby removing the associated transcriptional repression.

No abstract

No abstract