Structural basis for pharmacological modulation of the TRPC6 channel.

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.

Identification of Human Islet Amyloid Polypeptide as a BACE2 Substrate.

Pancreatic amyloid formation by islet amyloid polypeptide (IAPP) is a hallmark pathological feature of type 2 diabetes. IAPP is stored in the secretory granules of pancreatic beta-cells and co-secreted with insulin to maintain glucose homeostasis. IAPP is innocuous under homeostatic conditions but imbalances in production or processing of IAPP may result in homodimer formation leading to the rapid production of cytotoxic oligomers and amyloid fibrils. The consequence is beta-cell dysfunction and the accumulation of proteinaceous plaques in and around pancreatic islets. Beta-site APP-cleaving enzyme 2, BACE2, is an aspartyl protease commonly associated with BACE1, a related homolog responsible for amyloid processing in the brain and strongly implicated in Alzheimer's disease. Herein, we identify two distinct sites of the mature human IAPP sequence that are susceptible to BACE2-mediated proteolytic activity. The result of proteolysis is modulation of human IAPP fibrillation and human IAPP protein degradation. These results suggest a potential therapeutic role for BACE2 in type 2 diabetes-associated hyperamylinaemia.

Trefoil Factor 3 (TFF3) Is Regulated by Food Intake, Improves Glucose Tolerance and Induces Mucinous Metaplasia.

Trefoil factor 3 (TFF3), also called intestinal trefoil factor or Itf, is a 59 amino acid peptide found as a homodimer predominantly along the gastrointestinal tract and in serum. TFF3 expression is elevated during gastrointestinal adenoma progression and has been shown to promote mucosal wound healing. Here we show that in contrast to other trefoil factor family members, TFF1 and TFF2, TFF3 is highly expressed in mouse duodenum, jejunum and ileum and that its expression is regulated by food intake. Overexpression of TFF3 using a recombinant adeno-associated virus (AAV) vector, or daily administration of recombinant TFF3 protein in vivo improved glucose tolerance in a diet-induced obesity mouse model. Body weight, fasting insulin, triglyceride, cholesterol and leptin levels were not affected by TFF3 treatment. Induction of mucinous metaplasia was observed in mice with AAV-mediated TFF3 overexpression, however, no such adverse histological effect was seen after the administration of recombinant TFF3 protein. Altogether these results suggest that the therapeutic potential of targeting TFF3 to treat T2D may be limited.

GPR35 is a target of the loop diuretic drugs bumetanide and furosemide.

We report that the loop diuretic drugs bumetanide and furosemide used in the treatment of hypertension are GPR35 agonists. We utilized calcium flux, inositol phosphate accumulation, and dynamic redistribution assays to examine the pharmacology of these compounds on the human, mouse and rat GPR35. While potent on human GPR35, neither bumetanide nor furosemide were active against mouse or rat GPR35. Furthermore, the Na(+)-Cl(-) cotransporter inhibi- tors chlorothiazide and hydrochlorothiazide were inactive against GPR35 in all three species. We also demonstrate that GPR35 is expressed in human skin where it has been shown that loop diuretics inhibit histamine-induced flare and itch response. These findings suggest that GPR35 may play an important role in skin cell biology and be a potential target for the treatment of a variety of immune disorders.

Glucagon receptor antagonist-mediated improvements in glycemic control are dependent on functional pancreatic GLP-1 receptor.

Antagonism of the glucagon receptor (GCGR) is associated with increased circulating levels of glucagon-like peptide-1 (GLP-1). To investigate the contribution of GLP-1 to the antidiabetic actions of GCGR antagonism, we administered an anti-GCGR monoclonal antibody (mAb B) to wild-type mice and GLP-1 receptor knockout (GLP-1R KO) mice. Treatment of wild-type mice with mAb B lowered fasting blood glucose, improved glucose tolerance, and enhanced glucose-stimulated insulin secretion during an intraperitoneal glucose tolerance test (ipGTT). In contrast, treatment of GLP-1R KO mice with mAb B had little efficacy during an ipGTT. Furthermore, pretreatment with the GLP-1R antagonist exendin-(9-39) diminished the antihyperglycemic effects of mAb B in wild-type mice. To determine the mechanism whereby mAb B improves glucose tolerance, we generated a monoclonal antibody that specifically antagonizes the human GLP-1R. Using a human islet transplanted mouse model, we demonstrated that pancreatic islet GLP-1R signaling is required for the full efficacy of the GCGR antagonist. To identify the source of the elevated GLP-1 observed in GCGR mAb-treated mice, we measured active GLP-1 content in pancreas and intestine from db/db mice treated with anti-GCGR mAb for 8 wk. Elevated GLP-1 in GCGR mAb-treated mice was predominantly derived from increased pancreatic GLP-1 synthesis and processing. All together, these data show that pancreatic GLP-1 is a significant contributor to the glucose-lowering effects observed in response to GCGR antagonist treatment.

G-protein-coupled receptor 35 is a target of the asthma drugs cromolyn disodium and nedocromil sodium.

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.

Involvement of the neurotensin receptor 1 in the behavioral effects of two neurotensin agonists, NT-2 and NT69L: lack of hypothermic, antinociceptive and antipsychotic actions in receptor knockout mice.

Neurotensin (NT) is a neuropeptide implicated in the pathophysiology of schizophrenia and in mediating the efficacy of antipsychotic drugs. NT is also involved in the regulation of body temperature and pain sensitivity. Using neurotensin receptor 1 (NTR1) knockout (KO) and wild-type (WT) mice, these studies evaluated the involvement of NTR1 in the behavioral responses produced by peripheral administration of NT agonists (NT-2 and NT69L). Animals were characterized in paradigms designed to assess hypothermia, antinociception, and antipsychotic-like effects. Under basal conditions, there were no phenotypic differences between NTR1 KO and WT mice. In WT mice, both NTR1 agonists decreased core body temperature (active doses in mg/kg, i.p., for NT-2 and NT69L, respectively: 1 and 3), increased tail withdrawal latencies (1 and 3), produced decreased spontaneous climbing (0.1, 0.3, 1 and 1, 3, 10) and reversed apomorphine-induced climbing (0.3, 1 and 1, 3). In contrast, none of the effects of either agonist were present in KO mice. These results suggest that NTR1: (1) does not play a major role in the control of basal thermoregulation, nociception or psychomotor stimulation in mice (barring possible developmental plasticity), (2) does mediate these behavioral responses to NT agonists, and (3) may play a role in the potential antipsychotic effects of these agonists.

Medium-chain fatty acids as ligands for orphan G protein-coupled receptor GPR84.

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 [35S]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 lipopolysaccharide-stimulated 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.

Kynurenic acid as a ligand for orphan G protein-coupled receptor GPR35.

Local catabolism of the essential amino acid tryptophan is considered an important mechanism in regulating immunological and neurological responses. The kynurenine pathway is the main route for the non-protein metabolism of tryptophan. The intermediates of the kynurenine pathway are present at micromolar concentrations in blood and are regulated by inflammatory stimuli. Here we show that GPR35, a previously orphan G protein-coupled receptor, functions as a receptor for the kynurenine pathway intermediate kynurenic acid. Kynurenic acid elicits calcium mobilization and inositol phosphate production in a GPR35-dependent manner in the presence of G(qi/o) chimeric G proteins. Kynurenic acid stimulates [35S]guanosine 5'-O-(3-thiotriphosphate) binding in GPR35-expressing cells, an effect abolished by pertussis toxin treatment. Kynurenic acid also induces the internalization of GPR35. Expression analysis indicates that GPR35 is predominantly detected in immune cells and the gastrointestinal tract. Furthermore, we show that kynurenic acid inhibits lipopolysaccharide-induced tumor necrosis factor-alpha secretion in peripheral blood mononuclear cells. Our results suggest unexpected signaling functions for kynurenic acid through GPR35 activation.