Pulmonary edema resulting from high pulmonary venous pressure (PVP) is a major cause of morbidity and mortality in heart failure (HF) patients, but current treatment options demonstrate substantial limitations. Recent evidence from rodent lungs suggests that PVP-induced edema is driven by activation of pulmonary capillary endothelial transient receptor potential vanilloid 4 (TRPV4) channels. To examine the therapeutic potential of this mechanism, we evaluated TRPV4 expression in human congestive HF lungs and developed small-molecule TRPV4 channel blockers for testing in animal models of HF. TRPV4 immunolabeling of human lung sections demonstrated expression of TRPV4 in the pulmonary vasculature that was enhanced in sections from HF patients compared to controls. GSK2193874 was identified as a selective, orally active TRPV4 blocker that inhibits Ca(2+) influx through recombinant TRPV4 channels and native endothelial TRPV4 currents. In isolated rodent and canine lungs, TRPV4 blockade prevented the increased vascular permeability and resultant pulmonary edema associated with elevated PVP. Furthermore, in both acute and chronic HF models, GSK2193874 pretreatment inhibited the formation of pulmonary edema and enhanced arterial oxygenation. Finally, GSK2193874 treatment resolved pulmonary edema already established by myocardial infarction in mice. These findings identify a crucial role for TRPV4 in the formation of HF-induced pulmonary edema and suggest that TRPV4 blockade is a potential therapeutic strategy for HF patients.

Previous studies have demonstrated that non-visual arrestins function as adaptors in clathrin-mediated endocytosis to promote agonist-induced internalization of the ␤ 2 -adrenergic receptor (␤ 2 AR). Here, we characterized the effects of arrestins and other modulators of clathrin-mediated endocytosis on down-regulation of the ␤ 2 AR. In COS-1 and HeLa cells, non-visual arrestins promote agonist-induced internalization and down-regulation of the ␤ 2 AR, whereas dynamin-K44A, a dominant-negative mutant of dynamin that inhibits clathrin-mediated endocytosis, attenuates ␤ 2 AR internalization and down-regulation. In HEK293 cells, dynamin-K44A profoundly inhibits agonist-induced internalization and down-regulation of the ␤ 2 AR, suggesting that receptor internalization is critical for down-regulation in these cells. Moreover, a dominantnegative mutant of ␤-arrestin, ␤-arrestin-(319 -418), also inhibits both agonist-induced receptor internalization and down-regulation. Immunofluorescence microscopy analysis reveals that the ␤ 2 AR is trafficked to lysosomes in HEK293 cells, where presumably degradation of the receptor occurs. These studies demonstrate that down-regulation of the ␤ 2 AR is in part due to trafficking of the ␤ 2 AR via the clathrin-coated pit endosomal pathway to lysosomes.Stimulation of G protein-coupled receptors (GPRs) 1 leads to adaptive changes that serve to modulate the responsiveness of a cell to further stimulation (1). Three temporally distinct mechanisms of agonist-promoted regulation of GPRs have been identified: desensitization, internalization, and down-regulation. Desensitization often occurs within seconds of stimulation and for many GPRs is mediated by phosphorylation of the receptor by a specific G protein-coupled receptor kinase. This phosphorylation promotes the binding of an arrestin to the receptor, which sterically prevents subsequent coupling to G proteins. Agonist stimulation also promotes rapid (minutes) internalization of many GPRs into an intracellular endosomal pool, thereby effectively removing the receptor from the cell surface. Finally, prolonged receptor stimulation (hours) results in receptor down-regulation, defined by an overall decrease in receptor number.The ␤ 2 -adrenergic receptor (␤ 2 AR) has served as an important model for elucidating the molecular mechanisms involved in GPR regulation. The ␤ 2 AR is rapidly desensitized after agonist stimulation, in part a consequence of phosphorylation of carboxyl-terminal serines and threonines by G protein-coupled receptor kinase 2 (2). This phosphorylation promotes the binding of an arrestin (either ␤-arrestin or arrestin 3), which directly inhibits the ability of the receptor to couple to G s (3)(4)(5). Recent studies have suggested that ␤-arrestin and arrestin 3 are not only involved in the initial stages of desensitization of the ␤ 2 AR but that they are important components in receptor internalization (6, 7). Since both ␤-arrestin and arrestin 3 can specifically bind to clathrin, the major structural protein of clathrin-...

To date, the visualization of ␤ 2 -adrenergic receptor (␤ 2 AR) trafficking has been largely limited to immunocytochemical analyses of acute internalization events of epitope-tagged receptors in various transfection systems. The development of a ␤ 2 AR conjugated with green fluorescent protein (␤ 2 AR-GFP) provides the opportunity for a more extensive optical analysis of ␤ 2 AR sequestration, down-regulation, and recycling in cells. Here we demonstrate that stable expression of ␤ 2 AR-GFP in HeLa cells enables a detailed temporal and spatial analysis of these events. Time-dependent colocalization of ␤ 2 AR-GFP with rhodamine-labeled transferrin and rhodamine-labeled dextran following agonist exposure demonstrates receptor distribution to early endosomes (sequestration) and lysosomes (down-regulation), respectively. The observed temporal distribution of ␤ 2 AR-GFP was consistent with measures of receptor sequestration and down-regulation generated by radioligand-receptor binding assays. Cells stimulated with different ␤-agonists revealed time courses of ␤ 2 AR-GFP redistribution reflective of the intrinsic activity of each agonist.

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NOD2 is an intracellular pattern recognition receptor that assembles with receptor-interacting protein (RIP)-2 kinase in response to the presence of bacterial muramyl dipeptide (MDP) in the host cell cytoplasm, thereby inducing signals leading to the production of pro-inflammatory cytokines. The dysregulation of NOD2 signaling has been associated with various inflammatory disorders suggesting that small-molecule inhibitors of this signaling complex may have therapeutic utility. To identify inhibitors of the NOD2 signaling pathway, we utilized a cell-based screening approach and identified a benzimidazole diamide compound designated GSK669 that selectively inhibited an MDP-stimulated, NOD2-mediated IL-8 response without directly inhibiting RIP2 kinase activity. Moreover, GSK669 failed to inhibit cytokine production in response to the activation of Toll-like receptor (TLR)-2, tumor necrosis factor receptor (TNFR)-1 and closely related NOD1, all of which share common downstream components with the NOD2 signaling pathway. While the inhibitors blocked MDP-induced NOD2 responses, they failed to block signaling induced by NOD2 over-expression or single stranded RNA, suggesting specificity for the MDP-induced signaling complex and activator-dependent differences in NOD2 signaling. Investigation of structure-activity relationship allowed the identification of more potent analogs that maintained NOD2 selectivity. The largest boost in activity was achieved by N-methylation of the C2-ethyl amide group. These findings demonstrate that the NOD2 signaling pathway is amenable to modulation by small molecules that do not target RIP2 kinase activity. The compounds we identified should prove useful tools to investigate the importance of NOD2 in various inflammatory processes and may have potential clinical utility.

A persistent problem in early small-molecule drug discovery is the frequent lack of rank-order correlation between biochemical potencies derived from initial screens using purified proteins and the diminished potency and efficacy observed in subsequent disease-relevant cellular phenotypic assays. The introduction of the cellular thermal shift assay (CETSA) has bridged this gap by enabling assessment of drug target engagement directly in live cells based on ligand-induced changes in protein thermal stability. Initial success in applying CETSA across multiple drug target classes motivated our investigation into replacing the low-throughput, manually intensive Western blot readout with a quantitative, automated higher-throughput assay that would provide sufficient capacity to use CETSA as a primary hit qualification strategy. We introduce a high-throughput dose-response cellular thermal shift assay (HTDR-CETSA), a single-pot homogenous assay adapted for high-density microtiter plate format. The assay features titratable BacMam expression of full-length target proteins fused to the DiscoverX 42 amino acid ePL tag in HeLa suspension cells, facilitating enzyme fragment complementation-based chemiluminescent quantification of ligand-stabilized soluble protein. This simplified format can accommodate determination of full-dose CETSA curves for hundreds of individual compounds/analyst/day in replicates. HTDR-CETSA data generated for substrate site and alternate binding mode inhibitors of the histone-lysine N-methyltransferase SMYD3 in HeLa suspension cells demonstrate excellent correlation with rank-order potencies observed in cellular mechanistic assays and direct translation to target engagement of endogenous Smyd3 in cancer-relevant cell lines. We envision this workflow to be generically applicable to HTDR-CETSA screening spanning a wide variety of soluble intracellular protein target classes.

The Saccharomyces cerevisiae G protein ␣ subunit Gpa1p is involved in the response of both MATa and MAT␣ cells to pheromone. We mutagenized the GPA1 C terminus to characterize the receptor-interacting domain and to investigate the specificity of the interactions with the a-and ␣-factor receptors. The results are discussed with respect to a structural model of the Gpa1p C terminus that was based on the crystal structure of bovine transducin. Some mutants showed phenotypes different than the pheromone response and mating defects expected for mutations that affect receptor interactions, and therefore the mutations may affect other aspects of Gpa1p function. Most of the mutations that resulted in pheromone response and mating defects had similar effects in MATa and MAT␣ cells, suggesting that they affect the interactions with both receptors. Overexpression of the pheromone receptors increased the mating of some of the mutants tested but not the wild-type strain, consistent with defects in mutant Gpa1p-receptor interactions. The regions identified by the matingdefective mutants correlated well with the regions of mammalian G ␣ subunits implicated in receptor interactions. The strongest mating type-specific effects were seen for mutations to proline and a mutation of a glycine residue predicted to form a C-terminal ␤ turn. The analogous ␤ turn in mammalian G ␣ subunits undergoes a conformational change upon receptor interaction. We propose that the conformation of this region of Gpa1p differs during the interactions with the a-and ␣-factor receptors and that these mating type-specific mutations preclude the orientation necessary for interaction with one of the two receptors.G protein-mediated signaling involves heterotrimeric proteins that consist of ␣, ␤, and ␥ subunits. G proteins couple to membrane receptors of the rhodopsin/␤-adrenergic family of seven transmembrane-helix receptors (reviewed in reference 9). In the absence of a stimulus, the G protein ␣ subunit is bound to GDP and is complexed with the ␤␥ dimer. When a G protein-linked receptor is stimulated, the G ␣ subunit exchanges GTP for GDP and dissociates from G ␤␥ . After dissociation, G ␣ and/or G ␤␥ interact with targets that are effectors of cellular response pathways.Biochemical and genetic studies in mammalian systems have indicated that G ␣ C termini are involved in receptor interactions. Pertussis toxin modification of a cysteine residue 4 amino acids from the C terminus of G ␣t (transducin) results in reduced affinity for rhodopsin (58). Antibodies against C-terminal peptides of G ␣t and G ␣i2 block the interactions with rhodopsin and the ␣2-adrenergic receptor, respectively (6, 23, 51), and peptides corresponding to the C termini of G ␣t and G ␣s compete for binding to rhodopsin and the ␤-adrenergic receptor, respectively ( Fig. 1) (22, 44, 46). Most G ␣ mutations that result in receptor coupling defects cluster in G ␣ C termini (19,20,42,43,45,49,55).In the budding yeast Saccharomyces cerevisiae, a G proteinreceptor system is involved in the resp...