PLC1 mediated Cycloastragenol-induced stomatal movement by regulating the production of NO in Arabidopsis thaliana.

BACKGROUND: Astragalus grows mainly in drought areas. Cycloastragenol (CAG) is a tetracyclic triterpenoid allelochemical extracted from traditional Chinese medicine Astragalus root. Phospholipase C (PLC) and Gα-submit of the heterotrimeric G-protein (GPA1) are involved in many biotic or abiotic stresses. Nitric oxide (NO) is a crucial gas signal molecule in plants. RESULTS: In this study, using the seedlings of Arabidopsis thaliana (A. thaliana), the results showed that low concentrations of CAG induced stomatal closure, and high concentrations inhibited stomatal closure. 30 µmol·L-1 CAG significantly increased the relative expression levels of PLC1 and GPA1 and the activities of PLC and GTP hydrolysis. The stomatal aperture of plc1, gpa1, and plc1/gpa1 was higher than that of WT under CAG treatment. CAG increased the fluorescence intensity of NO in guard cells. Exogenous application of c-PTIO to WT significantly induced stomatal aperture under CAG treatment. CAG significantly increased the relative expression levels of NIA1 and NOA1. Mutants of noa1, nia1, and nia2 showed that NO production was mainly from NOA1 and NIA1 by CAG treatment. The fluorescence intensity of NO in guard cells of plc1, gpa1, and plc1/gpa1 was lower than WT, indicating that PLC1 and GPA1 were involved in the NO production in guard cells. There was no significant difference in the gene expression of PLC1 in WT, nia1, and noa1 under CAG treatment. The gene expression levels of NIA1 and NOA1 in plc1, gpa1, and plc1/gpa1 were significantly lower than WT, indicating that PLC1 and GPA1 were positively regulating NO production by regulating the expression of NIA1 and NOA1 under CAG treatment. CONCLUSIONS: These results suggested that the NO accumulation was essential to induce stomatal closure under CAG treatment, and GPA1 and PLC1 acted upstream of NO.

High-throughput chemogenetic drug screening reveals PKC-RhoA/PKN as a targetable signaling vulnerability in GNAQ-driven uveal melanoma.

Uveal melanoma (UM) is the most prevalent cancer of the eye in adults, driven by activating mutation of GNAQ/GNA11; however, there are limited therapies against UM and metastatic UM (mUM). Here, we perform a high-throughput chemogenetic drug screen in GNAQ-mutant UM contrasted with BRAF-mutant cutaneous melanoma, defining the druggable landscape of these distinct melanoma subtypes. Across all compounds, darovasertib demonstrates the highest preferential activity against UM. Our investigation reveals that darovasertib potently inhibits PKC as well as PKN/PRK, an AGC kinase family that is part of the "dark kinome." We find that downstream of the Gαq-RhoA signaling axis, PKN converges with ROCK to control FAK, a mediator of non-canonical Gαq-driven signaling. Strikingly, darovasertib synergizes with FAK inhibitors to halt UM growth and promote cytotoxic cell death in vitro and in preclinical metastatic mouse models, thus exposing a signaling vulnerability that can be exploited as a multimodal precision therapy against mUM.

Assessment of biased agonism at the A3 adenosine receptor using ß-arrestin and miniGαi recruitment assays.

The A3 adenosine receptor (A3AR) is a G protein-coupled receptor that is involved in a wide variety of physiological and pathological processes, such as cancer. However, the use of compounds pharmacologically targeting this receptor remains limited in clinical practice, despite extensive efforts for compound synthesis. Moreover, the possible occurrence of biased agonism further complicates the interpretation of the functional characteristics of compounds. Hence the need for simple assays, which are comparable in terms of the used cell lines and read-out technique. We previously established a stable ß-arrestin 2 (ßarr2) bioassay, employing a simple, luminescent read-out via functional complementation of a split nanoluciferase enzyme. Here, we developed a complementary, new bioassay in which coupling of an engineered miniGαi protein to activated A3AR is monitored using a similar approach. Application of both bioassays for the concurrent determination of the potencies and efficacies of a set of 19 N6-substituted adenosine analogues not only allowed for the characterization of structure-activity relationships, but also for the quantification of biased agonism. Although a broad distribution in potency and efficacy values was obtained within the test panel, no significant bias was observed toward either the ßarr2 or miniGαi pathway.

Genetic analyses uncover pleiotropic compensatory roles for Drosophila Nucleobindin-1 in inositol trisphosphate-mediated intracellular calcium homeostasis.

Nucleobindin-1 is an EF-hand calcium-binding protein with a distinctive profile, predominantly localized to the Golgi in insect and wide-ranging vertebrate cell types, alike. Its putative involvements in intracellular calcium (Ca2+) homeostasis have never been phenotypically characterized in any model organism. We have analyzed an adult-viable mutant that completely disrupts the G protein α-subunit binding and activating (GBA) motif of Drosophila Nucleobindin-1 (dmNUCB1). Such disruption does not manifest any obvious fitness-related, morphological/developmental, or behavioral abnormalities. A single copy of this mutation or the knockdown of dmnucb1 in restricted sets of cells variously rescues pleiotropic mutant phenotypes arising from impaired inositol 1,4,5-trisphosphate receptor (IP3R) activity (in turn depleting cytoplasmic Ca2+ levels across diverse tissue types). Additionally, altered dmNUCB1 expression or function considerably reverses lifespan and mobility improvements effected by IP3R mutants, in a Drosophila model of amyotrophic lateral sclerosis. Homology modeling-based analyses further predict a high degree of conformational conservation in Drosophila, of biochemically validated structural determinants in the GBA motif that specify in vertebrates, the unconventional Ca2+-regulated interaction of NUCB1 with Gαi subunits. The broad implications of our findings are hypothetically discussed, regarding potential roles for NUCB1 in GBA-mediated, Golgi-associated Ca2+ signaling, in health and disease.

The guanine nucleotide-binding protein α subunit protein ChGnaq positively regulates Hsc70 transcription in Crassostrea hongkongensis.

Gnaq, one of Guanine nucleotide-binding protein α subunits, was isolated from cellular nucleus extracts of oyster Crassostrea hongkongensis gills with biotin-labeled ChHsc70 promoter by means of DNA-affinity purification, and preliminarily identified with mass spectrometry analysis. ChGnaq mRNA depletion by RNAi technique led to clear reduction in ChHsc70 mRNA expression of C. hongkongensis hemocytes. Correspondently, ChGnaq over-expression in heterologous HEK293T cells correlated with elevated expression activation of ChHsc70 promoter. Quantitative real time PCR analysis showed that both ChHsc70 and ChGnaq transcriptions were responsive to external physical/chemical stresses by heat, CdCl2 and NP. This suggested a plausible association between ChHsc70 and ChGnaq in the stress-induced genetic regulatory pathway. This study discovered a positively regulatory role of ChGnaq in controlling ChHsc70 transcription of C. hongkongensis, and conduced to a better understanding of the regulatory mechanisms in control of Hsc70 transcription.

Combinatorial effects of quercetin and sex-steroids on fluid and electrolytes' (Na+, Cl-, HCO3-) secretory mechanisms in the uterus of ovariectomised female Sprague-Dawley rats.

Dysregulation of uterine fluid environment could impair successful reproduction and this could be due to the effect of environmental estrogens. Therefore, in this study, effect of quercetin, an environmental estrogen on uterine fluid and electrolytes concentrations were investigated under sex-steroid influence. Ovariectomised adult female Sprague-Dawley rats were given 10, 50 or 100mg/kg/day quercetin subcutaneously with 17-ß estradiol (E) for seven days or three days E, then three days E plus progesterone (P) (E+P) treatment. Uterine fluid secretion rate, Na+, Cl- and HCO3- concentrations were determined by in-vivo perfusion. Following sacrifice, uteri were harvested and levels of the proteins of interest were identified by Western blotting and Realtime PCR. Distribution of these proteins in the uterus was observed by immunofluorescence. Levels of uterine cAMP were measured by enzyme-linked immunoassay (EIA). Administration of quercetin at increasing doses increased uterine fluid secretion rate, Na+, Cl- and HCO3- concentrations, but to the levels lesser than that of E. In concordant, levels of CFTR, SLC4A4, ENaC (α, ß and γ), Na+/K+-ATPase, GPα/ß, AC and cAMP in the uterus increased following increased in the doses of quercetin. Co-administration of quercetin with E caused uterine fluid secretion rate, Na+, Cl- and HCO3- concentrations to decrease. In concordant, uterine CFTR, SLC26A6, SLC4A4, ENaC (α, ß and γ), Na+/K+-ATPase, GPα/ß, AC and cAMP decreased. Greatest effects were observed following co-administration of 10mg/kg/day quercetin with E. Co-administration of quercetin with E+P caused uterine fluid Na+ and HCO3- concentrations to increase but no changes in fluid secretion rate and Cl- concentration were observed. Co-administration of high dose quercetin (100 mg/kg/day) with E+P caused uterine CFTR, SLC26A6, AC, GPα/ß and ENaC (α, ß and γ) to increase. Quercetin-induced changes in the uterine fluid secretion rate and electrolytes concentrations could potentially affect the uterine reproductive functions under female sex-steroid influence.

Non-classical testosterone signaling in spermatogenic GC-2 cells is mediated through ZIP9 interacting with Gnα11.

Although classical and non-classical signaling of testosterone has been documented in several investigations, the nature of the receptor involved in the non-classical pathway remains a source of controversy. While some investigators favor the exclusive participation of the cytosolic/nuclear androgen receptor (AR) in both pathways, others propose a membrane-bound receptor as the mediator of the non-classical testosterone signaling. Evidence is provided here that in the spermatogenic cell line GC-2 the non-classical signaling pathway of testosterone, characterized through the activation of Erk1/2 and transcription factors like CREB or ATF-1, is not mediated through the classical nuclear androgen receptor (AR) but rather by a membrane-associated receptor. This receptor is ZIP9, a Zn(2+) transporter from the family of the ZRT, IRT-like proteins (ZRT=zinc-regulated transporter; IRT=iron-regulated transporter), which directly interacts with the G-protein Gnα11. siRNA-induced abrogation of the expression of either of these two proteins, whose close contacts are demonstrated by an in situ proximity assay, completely prevents all non-classical signaling effects of testosterone addressed. In contrast, silencing of AR expression does not influence the same signaling events. The identification of ZIP9/Gnα11 interactions as the mediators of the non-classical testosterone signaling cascade in spermatogenic GC-2 cells might help to supplement our knowledge concerning the role of testosterone in male fertility and reproduction.

Analgesic effect of the neuropeptide cortistatin in murine models of arthritic inflammatory pain.

OBJECTIVE: To investigate the role of the antiinflammatory neuropeptide cortistatin in chronic pain evoked by joint inflammation. METHODS: Thermal and mechanical hyperalgesia was evoked in mouse knee joints by intraplantar injection of tumor necrosis factor α and intraarticular infusion of Freund's complete adjuvant, and the analgesic effects of cortistatin, administered centrally, peripherally, and systemically, were assessed. In addition, the effects of cortistatin on the production of nociceptive peptides and the activation of pain signaling were assayed in dorsal root ganglion cultures and in inflammatory pain models. The role of endogenous cortistatin in pain sensitization and perpetuation of chronic inflammatory states was evaluated in cortistatin-deficient mice. Finally, the effect of noxious/inflammatory stimuli in the production of cortistatin by the peripheral nociceptive system was assayed in vitro and in vivo. RESULTS: Expression of cortistatin was observed in peptidergic nociceptors of the peripheral nociceptive system, and endogenous cortistatin was found to participate in the tuning of pain sensitization, especially in pathologic inflammatory conditions. Results showed that cortistatin acted both peripherally and centrally to reduce the tactile allodynia and heat hyperalgesia evoked by arthritis and peripheral tissue inflammation in mice, via mechanisms that were independent of its antiinflammatory action. These mechanisms involved direct action on nociceptive neurons and regulation of central sensitization. The analgesic effects of cortistatin in murine arthritic pain were linked to binding of the neuropeptide to somatostatin and ghrelin receptors, activation of the G protein subunit Gαi , impairment of ERK signaling, and decreased production of calcitonin gene-related peptide in primary nociceptors. CONCLUSION: These findings indicate that cortistatin is an antiinflammatory factor with potent analgesic effects that may offer a new approach to pain therapy in pathologic inflammatory states, including osteoarthritis and rheumatoid arthritis.

Substrate specificity of Pasteurella multocida toxin for α subunits of heterotrimeric G proteins.

Pasteurella multocida is the causative agent of a number of epizootic and zoonotic diseases. Its major virulence factor associated with atrophic rhinitis in animals and dermonecrosis in bite wounds is P. multocida toxin (PMT). PMT stimulates signal transduction pathways downstream of heterotrimeric G proteins, leading to effects such as mitogenicity, blockade of apoptosis, or inhibition of osteoblast differentiation. On the basis of Gα(i2), it was demonstrated that the toxin deamidates an essential glutamine residue of the Gα(i2) subunit, leading to constitutive activation of the G protein. Here, we studied the specificity of PMT for its G-protein targets by mass spectrometric analyses and by utilizing a monoclonal antibody, which recognizes specifically G proteins deamidated by PMT. The studies revealed deamidation of 3 of 4 families of heterotrimeric G proteins (Gα(q/11), Gα(i1,2,3), and Gα(12/13) of mouse or human origin) by PMT but not by a catalytic inactive toxin mutant. With the use of G-protein fragments and chimeras of responsive or unresponsive G proteins, the structural basis for the discrimination of heterotrimeric G proteins was studied. Our results elucidate substrate specificity of PMT on the molecular level and provide evidence for the underlying structural reasons of substrate discrimination.

Expression, purification and preliminary NMR characterization of isotopically labeled wild-type human heterotrimeric G protein αi1.

Molecular-level investigation of proteins is increasingly important to researchers trying to understand the mechanisms of signal transmission. Heterotrimeric G proteins control the activation of many critical signal transmission cascades and are also implicated in numerous diseases. As part of a longer-term investigation of intramolecular motions in RGS and Gα proteins in their apo and complexed forms, we have successfully developed a protocol for preparing milligram quantities of highly purified, isotopically labeled wild-type human Gα(i1) (hGα(i1)) subunit for NMR studies. High levels of expression in Escherichia coli can be attributed to the use of the SUMO fusion protein system, a bacterial strain that produces rare codons, supplementation of minimal medium with small quantities of isotopically labeled rich medium and a lowered induction temperature. Purification of hGα(i1) utilized affinity and size exclusion chromatography, and protein activity was confirmed using fluorescence-based GTP-binding studies. Preliminary NMR analysis of hGα(i1) has shown that high-quality spectra can be obtained at near-physiological temperatures, whereas lower temperature spectra display numerous weak and broadened peaks, providing preliminary evidence for widespread µs-ms timescale exchange. In an effort to further optimize the NMR spectra we prepared a truncated form of hGα(i1) (hGα(i1)-Δ31) in which the 31-residue unstructured N-terminus was removed. This resulted in further improvements in spectral quality by eliminating high-intensity peaks that obscured resonances from structured segments of the protein. We plan to use hGα(i1)-Δ31 in future investigations of protein dynamics by NMR spectroscopy to gain insight into the role of these motions in RGS/Gα binding selectivity.

Inhibition of heterotrimeric G protein signaling by a small molecule acting on Galpha subunit.

The simultaneous activation of many distinct G protein-coupled receptors (GPCRs) and heterotrimeric G proteins play a major role in various pathological conditions. Pan-inhibition of GPCR signaling by small molecules thus represents a novel strategy to treat various diseases. To better understand such therapeutic approach, we have characterized the biomolecular target of BIM-46187, a small molecule pan-inhibitor of GPCR signaling. Combining bioluminescence and fluorescence resonance energy transfer techniques in living cells as well as in reconstituted receptor-G protein complexes, we observed that, by direct binding to the Galpha subunit, BIM-46187 prevents the conformational changes of the receptor-G protein complex associated with GPCR activation. Such a binding prevents the proper interaction of receptors with the G protein heterotrimer and inhibits the agonist-promoted GDP/GTP exchange. These observations bring further evidence that inhibiting G protein activation through direct binding to the Galpha subunit is feasible and should constitute a new strategy for therapeutic intervention.

G protein ßγ subunits: central mediators of G protein-coupled receptor signaling.

G protein betagamma subunits are central participants in G protein-coupled receptor signaling pathways. They interact with receptors, G protein alpha subunits and downstream targets to coordinate multiple, different GPCR functions. Much is known about the biology of Gbetagamma subunits but mysteries remain. Here, we will review what is known about general aspects of structure and function of Gbetagamma as well as discuss emerging mechanisms for regulation of Gbetagamma signaling. Recent data suggest that Gbetagamma is a potential therapeutic drug target. Thus, a thorough understanding of the molecular and physiological functions of Gbetagamma has significant implications.

Functional human 5-HT6 receptor assay for high throughput screening of chemical ligands and binding proteins.

Continuous identification and validation of novel drug targets require the development of rapid, reliable, and sensitive cell-based high-throughput screening (HTS) methods for proposed targets. Recently, the 5-HT(6) receptor (5-HT(6)R), a member of the class of recently discovered 5-HT receptors, has received considerable attention for its possible implications in depression, cognition, and anxiety. However, the cellular signaling mechanisms of 5-HT(6)R are poorly understood due to the lack of selective 5-HT(6)R ligands. In the present study, we examined functional coupling of the human 5-HT(6)R, 5-HT(7A)R, or 5-HT(7B)R with various Galpha-proteins (Galpha(15), Galpha(qs5), or Galpha(qG66Ds5)) to develop a reliable cell-based HTS method for 5-HT receptors. Among variable couplings between 5-HT receptors and G-proteins, we found that functional coupling of human 5-HT(6)R with Galpha(qG66Ds5) produced the highest levels of Ca(2+) signaling in HEK293 cells as measured by the fluorescence-based HTS plate reader, FDSS6000. After validation of this new 5-HT(6)R HTS system (Z'-factor = 0.56) in 96-well plates and characterization of the pharmacological profile of the 5-HT(6)R, we screened approximately 500 synthetic chemical compounds including butanamide and benzenesulfonamide derivatives. Based on this preliminary screening, we found that the butanamide derivative LSG11104 produced an IC(50) value of 6.3 microM. This compound will serve as a lead structure for further chemical modification to develop novel 5-HT(6)R ligands. Furthermore, we demonstrated that this HTS method can be utilized to identify proteins that modulate 5-HT(6)R function and present Fyn tyrosine kinase as an example, which is already known as a 5-HT(6)R interacting protein. Taken together, these results suggest that the 5-HT(6)R/Galpha(qG66Ds5) FDSS6000 system can be utilized to screen for selective 5-HT(6)R ligands and to examine any functional relationships between 5-HT(6)R and its binding proteins.

Heterotrimeric G-protein participation in Arabidopsis pollen germination through modulation of a plasmamembrane hyperpolarization-activated Ca2+-permeable channel.

The role of heterotrimeric G proteins in pollen germination and tube growth was investigated using Arabidopsis thaliana plants in which the gene (GPA) encoding the G-protein a subunit (Galpha) was null or overexpressed. Pollen germination, free cytosolic calcium concentration ([Ca(2+)](cyt)) and Ca(2+) channel activity in the plasma membrane (PM) of pollen cells were investigated. Results showed that, compared with pollen grains of the wild type (ecotype Wassilewskija, ws), in vitro germinated pollen of Galpha null mutants (gpa1-1 and gpa1-2) had lower germination percentages and shorter pollen tubes, while pollen from Galpha overexpression lines (wGalpha and cGalpha) had higher germination percentages and longer pollen tubes. Compared with ws pollen cells, [Ca(2+)](cyt) was lower in gpa1-1 and gpa1-2 and higher in wGalpha and cGalpha. In whole-cell patch clamp recordings, a hyperpolarization-activated Ca(2+)-permeable conductance was identified in the PM of pollen protoplasts. The conductance was suppressed by trivalent cations but insensitive to organic blockers; its permeability to divalent cations was Ba(2+) > Ca(2+) > Mg(2+) > Sr(2+) > Mn(2+). The activity of the Ca(2+)-permeable channel conductance was down-regulated in pollen protoplasts of gpa1-1 and gpa1-2, and up-regulated in wGalpha and cGalpha. The results suggest that Galpha may participate in pollen germination through modulation of the hyperpolarization-activated Ca(2+) channel in the PM of pollen cells.

Ligand screening system using fusion proteins of G protein-coupled receptors with G protein alpha subunits.

G protein-coupled receptors (GPCRs) constitute one of the largest families of genes in the human genome, and are the largest targets for drug development. Although a large number of GPCR genes have recently been identified, ligands have not yet been identified for many of them. Various assay systems have been employed to identify ligands for orphan GPCRs, but there is still no simple and general method to screen for ligands of such GPCRs, particularly of G(i)-coupled receptors. We have examined whether fusion proteins of GPCRs with G protein alpha subunit (Galpha) could be utilized for ligand screening and showed that the fusion proteins provide an effective method for the purpose. This article focuses on the followings: (1) characterization of GPCR genes and GPCRs, (2) identification of ligands for orphan GPCRs, (3) characterization of GPCR-Galpha fusion proteins, and (4) identification of ligands for orphan GPCRs using GPCR-Galpha fusion proteins.

Yeast-based screening to identify modulators of G-protein signaling using uncontrolled cell division cycle by overexpression of Stm1.

Stm1, a G-protein coupled receptor, which senses nutritional state drives cells to stop the proliferative cell cycle and enter meiosis under nutritionally deficient conditions in Schizosaccharomyces pombe. It was shown that overexpression of Stm1 led growth inhibition and uncontrolled mitotic haploidization presumably by the premature initiation of mitosis. Sty1 and Gpa2 seem to play important roles for Stm1 to deliver starvation signal to induce downstream function. Based on the observation that conversion of diploid to haploid by overexpression of Stm1 can be easily detected as pink or red colonies in the media containing low adenine, HTS drug screening system to identify modulators of GPCR was established and tested using 413 compounds. Four very potent modulators of GPCR including Biochanin A, which possess strong inhibitory activity against uncontrolled cell division, were identified in this screening. This study provides the yeast-based platform that allows robust cellular assays to identify novel modulators of G-protein signaling and MAP kinase pathway.

Acupuncture pretreatment protects heart from injury in rats with myocardial ischemia and reperfusion via inhibition of the beta(1)-adrenoceptor signaling pathway.

Our previous study showed that a cardioprotective effect was produced by pretreatment with acupuncture at bilateral Neiguan acupoints (PC6) and the effect of EA was diminished by propranolol, a nonspecific antagonist of beta-adrenoceptors (beta-ARs) which are the most powerful cardiac receptors, indicating an involvement of beta-ARs. The present study explored further the signaling mechanism underlying the cardioprotective effect of acupuncture pretreatment in rats subjected to myocardial ischemia and reperfusion (MIR). Myocardial ischemia was achieved by ligating the left anterior descending coronary artery and reperfusion by releasing the ligation. Adult rats were divided into three groups, namely, a normal control (NC) group, a group subjected to ischemia and reperfusion (IR) only, and a group given electro-acupuncture (EA) before IR. For EA, bilateral Neiguan points (PC6) of the rats were stimulated for 30 min once a day for 3 consecutive days. The ST segment of ECG, the ratio of infarct size over risk zone, and the contents of beta(1)-adrenoceptor (beta(1)-AR), Gsalpha protein and cAMP in ischemic myocardium were compared among the three groups. IR increased the elevation of ECG ST segment, myocardial infarct size, contents of beta(1)-AR, Gsalpha protein and cAMP. These effects were attenuated by EA pretreatment at bilateral Neiguan acupoints. In conclusion, the present results indicate that EA produces cardioprotective effect against IR which may be mediated via the beta(1)-AR-Gs-protein-cAMP pathway.

The Galpha protein controls a pH-dependent signal path to the induction of phytoalexin biosynthesis in Eschscholzia californica.

The function of a Galpha protein in the elicitation of phytoalexin (benzophenanthridine) biosynthesis was characterized in cultured cells of California poppy (Eschscholzia californica). Both the decrease of Galpha content via antisense transformation and the expression of recombinant anti-Galpha single-chain antibodies strongly impaired the induction of alkaloid biosynthesis by low elicitor concentrations. All transgenic cell types were deficient in two elicitor-triggered early signal events: activation of phospholipase A2 (PLA2) and efflux of vacuolar protons. The lacking H+ efflux could be restored (1) by adding lysophosphatidylcholine (LPC), a product of PLA2 activity, to vacuoles in situ and (2) by exposing intact cells to isotonic, near-neutral HEPES buffers. The latter treatment induced alkaloid biosynthesis in the absence of elicitor and in Galpha-deficient cells. We conclude that Galpha mediates the stimulation of PLA2 by low elicitor concentrations and that the resulting peak of LPC initiates a transient efflux of vacuolar protons. In this way, an acidic peak of the cytoplasmic pH is generated that causes the expression of enzymes of phytoalexin production independent of the hypersensitive response.

Chemical genetics reveals an RGS/G-protein role in the action of a compound.

We report here on a chemical genetic screen designed to address the mechanism of action of a small molecule. Small molecules that were active in models of urinary incontinence were tested on the nematode Caenorhabditis elegans, and the resulting phenotypes were used as readouts in a genetic screen to identify possible molecular targets. The mutations giving resistance to compound were found to affect members of the RGS protein/G-protein complex. Studies in mammalian systems confirmed that the small molecules inhibit muscarinic G-protein coupled receptor (GPCR) signaling involving G-alphaq (G-protein alpha subunit). Our studies suggest that the small molecules act at the level of the RGS/G-alphaq signaling complex, and define new mutations in both RGS and G-alphaq, including a unique hypo-adapation allele of G-alphaq. These findings suggest that therapeutics targeted to downstream components of GPCR signaling may be effective for treatment of diseases involving inappropriate receptor activation.