Heterotrimeric G Proteins Research Articles

A live cell assay of GPCR coupling allows identification of optogenetic tools for controlling Go and Gi signaling

BackgroundAnimal opsins are light-sensitive G-protein-coupled receptors (GPCRs) that enable optogenetic control over the major heterotrimeric G-protein signaling pathways in animal cells. As such, opsins have potential applications in both biomedical research and therapy. Selecting the opsin with the best balance of activity and selectivity for a given application requires knowing their ability to couple to a full range of relevant Gα subunits. We present the GsX assay, a set of tools based on chimeric Gs subunits that transduce coupling of opsins to diverse G proteins into increases in cAMP levels, measured with a real-time reporter in living cells. We use this assay to compare coupling to Gi/o/t across a panel of natural and chimeric opsins selected for potential application in gene therapy for retinal degeneration.ResultsOf the opsins tested, wild-type human rod opsin had the highest activity for chimeric Gs proxies for Gi and Gt (Gsi and Gst) and was matched in Go proxy (Gso) activity only by a human rod opsin/scallop opsin chimera. Rod opsin drove roughly equivalent responses via Gsi, Gso, and Gst, while cone opsins showed much lower activities with Gso than Gsi or Gst, and a human rod opsin/amphioxus opsin chimera demonstrated higher activity with Gso than with Gsi or Gst. We failed to detect activity for opsin chimeras bearing three intracellular fragments of mGluR6, and observed unexpectedly complex response profiles for scallop and amphioxus opsins thought to be specialized for Go.ConclusionsThese results identify rod opsin as the most potent non-selective Gi/o/t-coupled opsin, long-wave sensitive cone opsin as the best for selectively activating Gi/t over Go, and a rod opsin/amphioxus opsin chimera as the best choice for selectively activating Go over Gi/t.

Increased store-operated Ca2+ entry mediated by GNB5 and STIM1.

Recent human genetic studies have shown that Gβ5 is related to various clinical symptoms, such as sinus bradycardia, cognitive disability, and attention deficit hyperactivity disorder. Although the calcium signaling cascade is closely associated with a heterotrimeric G-protein, the function of Gβ5 in calcium signaling and its relevance to clinical symptoms remain unknown. In this study, we investigated the in vitro changes of store-operated calcium entry (SOCE) with exogenous expression of Gβ5. The cells expressing Gβ5 had enhanced SOCE after depletion of calcium ion inside the endoplasmic reticulum. Gβ5 also augmented Stim1- and Orai1-dependent SOCE. An ORAI1 loss-of-function mutant did not show inhibition of Gβ5-induced SOCE, and a STIM1-ERM truncation mutant showed no enhancement of SOCE. These results suggested a novel role of GNB5 and Stim1, and provided insight into the regulatory mechanism of SOCE.

Correction.

The Plant JournalVolume 92, Issue 6 p. 1246-1246 CorrectionFree Access Correction This article corrects the following: The role of PLDα1 in providing specificity to signal-response coupling by heterotrimeric G-protein components in Arabidopsis Swarup Roy Choudhury, Sona Pandey, Volume 86Issue 1The Plant Journal pages: 50-61 First Published online: April 7, 2016 First published: 06 December 2017 https://doi.org/10.1111/tpj.13735AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat In the article “The role of PLDα1 in providing specificity to signal-response coupling by heterotrimeric G-protein components in Arabidopsis” by Swarup Roy Choudhury and Sona Pandey (2016), the authors would like to amend an incorrect grant number referenced in the Acknowledgement section. The correct grant number should read as follows: Research in the Pandey laboratory is supported by NIFA/AFRI (2015-67013-22964) and NSF (IOS-1157942) grants to SP. Reference Choudhury, S.R. and Pandey, S. (2016) The role of PLDα1 in providing specificity to signal-response coupling by heterotrimeric G-protein components in Arabidopsis. Plant J., 86, 50– 61. Volume92, Issue6December 2017Pages 1246-1246 ReferencesRelatedInformation

Light, stress, sex and carbon – The photoreceptor ENVOY as a central checkpoint in the physiology of Trichoderma reesei

Trichoderma reesei represents one of the most prolific producers of homologous and heterologous proteins. Discovery of the photoreceptor ENV1 as a regulator of cellulase gene expression initiated analysis of light response pathways and their physiological relevance for T. reesei. The function of ENV1 in regulation of plant cell wall degrading enzymes is conserved in Neurospora crassa. ENV1 emerged as a central checkpoint for integration of nutrient sensing, light response and development. This photoreceptor exerts its function by influencing transcript abundance and feedback cycles of the alpha subunits of the heterotrimeric G-protein pathway and impacts regulation of the beta and gamma subunits via mutual regulation with the phosducin PhLP1. The output of regulation by ENV1 is in part mediated by the cAMP pathway and likely aimed at cellulose recognition. Lack of ENV1 causes deregulation of the pheromone system and female sterility in light. A regulatory interconnection with VEL1 and influence on other regulators of secondary metabolism like YPR2 as well as polyketide synthase encoding genes indicates a function in secondary metabolism. The function of ENV1 in integrating light response with signaling of osmotic and oxidative stress is evolutionary conserved in Hypocreales and distinct from other sordariomycetes including N. crassa.

G-Protein Gα13 Functions with Abl Kinase to Regulate Actin Cytoskeletal Reorganization

Heterotrimeric G-proteins are essential cellular signal transducers. One of the G-proteins, Gα13, is critical for actin cytoskeletal reorganization, cell migration, cell proliferation, and apoptosis. Previously, we have shown that Gα13 is essential for both G-protein-coupled receptor and receptor tyrosine kinase-induced actin cytoskeletal reorganization such as dynamic dorsal ruffle turnover and cell migration. However, the mechanism by which Gα13 signals to actin cytoskeletal reorganization is not completely understood. Here we show that Gα13 directly interacts with Abl tyrosine kinase, which is a critical regulator of actin cytoskeleton. This interaction is critical for Gα13-induced dorsal ruffle turnover, endothelial cell remodeling, and cell migration. Our data uncover a new molecular signaling pathway by which Gα13 controls actin cytoskeletal reorganization.

G-Protein Dependent Signal Transduction and Ubiquitination in Dictyostelium.

Signal transduction through G-protein-coupled receptors (GPCRs) is central for the regulation of virtually all cellular functions, and it has been widely implicated in human diseases. These receptors activate a common molecular switch that is represented by the heterotrimeric G-protein generating a number of second messengers (cAMP, cGMP, DAG, IP3, Ca2+ etc.), leading to a plethora of diverse cellular responses. Spatiotemporal regulation of signals generated by a given GPCR is crucial for proper signalling and is accomplished by a series of biochemical modifications. Over the past few years, it has become evident that many signalling proteins also undergo ubiquitination, a posttranslational modification that typically leads to protein degradation, but also mediates processes such as protein-protein interaction and protein subcellular localization. The social amoeba Dictyostelium discoideum has proven to be an excellent model to investigate signal transduction triggered by GPCR activation, as cAMP signalling via GPCR is a major regulator of chemotaxis, cell differentiation, and multicellular morphogenesis. Ubiquitin ligases have been recently involved in these processes. In the present review, we will summarize the most significant pathways activated upon GPCRs stimulation and discuss the role played by ubiquitination in Dictyostelium cells.

The balance between Gαi-Cdc42/Rac and Gα12/13-RhoA pathways determines endothelial barrier regulation by sphingosine-1-phosphate.

The bioactive sphingosine-1-phosphatephosphate (S1P) is present in plasma, bound to carrier proteins, and involved in many physiological processes, including angiogenesis, inflammatory responses, and vascular stabilization. S1P can bind to several G-protein-coupled receptors (GPCRs) activating a number of different signaling networks. At present, the dynamics and relative importance of signaling events activated immediately downstream of GPCR activation are unclear. To examine these, we used a set of fluorescence resonance energy transfer-based biosensors for different RhoGTPases (Rac1, RhoA/B/C, and Cdc42) as well as for heterotrimeric G-proteins in a series of live-cell imaging experiments in primary human endothelial cells. These experiments were accompanied by biochemical GTPase activity assays and transendothelial resistance measurements. We show that S1P promotes cell spreading and endothelial barrier function through S1PR1-Gαi-Rac1 and S1PR1-Gαi-Cdc42 pathways. In parallel, a S1PR2-Gα12/13-RhoA pathway is activated that can induce cell contraction and loss of barrier function, but only if Gαi-mediated signaling is suppressed. Our results suggest that Gαq activity is not involved in S1P-mediated regulation of barrier integrity. Moreover, we show that early activation of RhoA by S1P inactivates Rac1 but not Cdc42, and vice versa. Together, our data show that the rapid S1P-induced increase in endothelial integrity is mediated by a S1PR1-Gαi-Cdc42 pathway.

A new discrete dynamic model of ABA-induced stomatal closure predicts key feedback loops.

Stomata, microscopic pores in leaf surfaces through which water loss and carbon dioxide uptake occur, are closed in response to drought by the phytohormone abscisic acid (ABA). This process is vital for drought tolerance and has been the topic of extensive experimental investigation in the last decades. Although a core signaling chain has been elucidated consisting of ABA binding to receptors, which alleviates negative regulation by protein phosphatases 2C (PP2Cs) of the protein kinase OPEN STOMATA 1 (OST1) and ultimately results in activation of anion channels, osmotic water loss, and stomatal closure, over 70 additional components have been identified, yet their relationships with each other and the core components are poorly elucidated. We integrated and processed hundreds of disparate observations regarding ABA signal transduction responses underlying stomatal closure into a network of 84 nodes and 156 edges and, as a result, established those relationships, including identification of a 36-node, strongly connected (feedback-rich) component as well as its in- and out-components. The network’s domination by a feedback-rich component may reflect a general feature of rapid signaling events. We developed a discrete dynamic model of this network and elucidated the effects of ABA plus knockout or constitutive activity of 79 nodes on both the outcome of the system (closure) and the status of all internal nodes. The model, with more than 1024 system states, is far from fully determined by the available data, yet model results agree with existing experiments in 82 cases and disagree in only 17 cases, a validation rate of 75%. Our results reveal nodes that could be engineered to impact stomatal closure in a controlled fashion and also provide over 140 novel predictions for which experimental data are currently lacking. Noting the paucity of wet-bench data regarding combinatorial effects of ABA and internal node activation, we experimentally confirmed several predictions of the model with regard to reactive oxygen species, cytosolic Ca2+ (Ca2+c), and heterotrimeric G-protein signaling. We analyzed dynamics-determining positive and negative feedback loops, thereby elucidating the attractor (dynamic behavior) repertoire of the system and the groups of nodes that determine each attractor. Based on this analysis, we predict the likely presence of a previously unrecognized feedback mechanism dependent on Ca2+c. This mechanism would provide model agreement with 10 additional experimental observations, for a validation rate of 85%. Our research underscores the importance of feedback regulation in generating robust and adaptable biological responses. The high validation rate of our model illustrates the advantages of discrete dynamic modeling for complex, nonlinear systems common in biology.

Characterization of a spectrally diverse set of fluorescent proteins as FRET acceptors for mTurquoise2

The performance of Förster Resonance Energy Transfer (FRET) biosensors depends on brightness and photostability, which are dependent on the characteristics of the fluorescent proteins that are employed. Yellow fluorescent protein (YFP) is often used as an acceptor but YFP is prone to photobleaching and pH changes. In this study, we evaluated the properties of a diverse set of acceptor fluorescent proteins in combination with the optimized CFP variant mTurquoise2 as the donor. To determine the theoretical performance of acceptors, the Förster radius was determined. The practical performance was determined by measuring FRET efficiency and photostability of tandem fusion proteins in mammalian cells. Our results show that mNeonGreen is the most efficient acceptor for mTurquoise2 and that the photostability is better than SYFP2. The non-fluorescent YFP variant sREACh is an efficient acceptor, which is useful in lifetime-based FRET experiments. Among the orange and red fluorescent proteins, mCherry and mScarlet-I are the best performing acceptors. Several new pairs were applied in a multimolecular FRET based sensor for detecting activation of a heterotrimeric G-protein by G-protein coupled receptors. Overall, the sensor with mNeonGreen as acceptor and mTurquoise2 as donor showed the highest dynamic range in ratiometric FRET imaging experiments with the G-protein sensor.

Alteration of cell wall xylan acetylation triggers defense responses that counterbalance the immune deficiencies of plants impaired in the β-subunit of the heterotrimeric G-protein.

Arabidopsis heterotrimeric G-protein complex modulates pathogen-associated molecular pattern-triggered immunity (PTI) and disease resistance responses to different types of pathogens. It also plays a role in plant cell wall integrity as mutants impaired in the Gβ- (agb1-2) or Gγ-subunits have an altered wall composition compared with wild-type plants. Here we performed a mutant screen to identify suppressors of agb1-2 (sgb) that restore susceptibility to pathogens to wild-type levels. Out of the four sgb mutants (sgb10-sgb13) identified, sgb11 is a new mutant allele of ESKIMO1 (ESK1), which encodes a plant-specific polysaccharide O-acetyltransferase involved in xylan acetylation. Null alleles (sgb11/esk1-7) of ESK1 restore to wild-type levels the enhanced susceptibility of agb1-2 to the necrotrophic fungus Plectosphaerella cucumerina BMM (PcBMM), but not to the bacterium Pseudomonas syringae pv. tomato DC3000 or to the oomycete Hyaloperonospora arabidopsidis. The enhanced resistance to PcBMM of the agb1-2 esk1-7 double mutant was not the result of the re-activation of deficient PTI responses in agb1-2. Alteration of cell wall xylan acetylation caused by ESK1 impairment was accompanied by an enhanced accumulation of abscisic acid, the constitutive expression of genes encoding antibiotic peptides and enzymes involved in the biosynthesis of tryptophan-derived metabolites, and the accumulation of disease resistance-related secondary metabolites and different osmolites. These esk1-mediated responses counterbalance the defective PTI and PcBMM susceptibility of agb1-2 plants, and explain the enhanced drought resistance of esk1 plants. These results suggest that a deficient PTI-mediated resistance is partially compensated by the activation of specific cell-wall-triggered immune responses.

Recently duplicated plant heterotrimeric Gα proteins with subtle biochemical differences influence specific outcomes of signal-response coupling

Heterotrimeric G-proteins, comprising Gα, Gβ, and Gγ subunits, regulate key signaling processes in eukaryotes. The Gα subunit determines the status of signaling by switching between inactive GDP-bound and active GTP-bound forms. Unlike animal systems, in which multiple Gα proteins with variable biochemical properties exist, plants have fewer, highly similar Gα subunits that have resulted from recent genome duplications. These proteins exhibit subtle differences in their GTP-binding, GDP/GTP-exchange, and GTP-hydrolysis activities, but the extent to which these differences contribute to affect plant signaling and development remains unknown. To evaluate this, we expressed native and engineered Gα proteins from soybean in an Arabidopsis Gα-null background and studied their effects on modulating a range of developmental and hormonal signaling phenotypes. Our results indicated that inherent biochemical differences in these highly similar Gα proteins are biologically relevant, and some proteins are more flexible than others in influencing the outcomes of specific signals. These observations suggest that alterations in the rate of the G-protein cycle itself may contribute to the specificity of response regulation in plants by affecting the duration of active signaling and/or by the formation of distinct protein-protein complexes. In species such as Arabidopsis having a single canonical Gα, this rate could be affected by regulatory proteins in the presence of specific signals, whereas in plants with multiple Gα proteins, an even more complex regulation may exist, which likely contributes to the specificity of signal-response coupling.

Heterotrimeric G-proteins are required for FLT3-ITD autophosphorylation and leukemogenic function but are dispensable in normal hematopoiesis

Heterotrimeric G-proteins are required for FLT3-ITD autophosphorylation and leukemogenic function but are dispensable in normal hematopoiesis

Microtubules and heterotrimeric G-protein regulate ABA-induced stomatal movement synergistically

Stomata, formed by a pair of specialized guard cells, are present on the epidermis of most plant leaves. The regulation of stomatal apertures controls the exchange of moisture and gas between plants and the environment, and balances the carbon dioxide (CO2) entry into the plant for photosynthesis with water loss via transpiration. G-protein, a ubiquitous signaling molecule in eukaryotic cells, is an indispensable participator in the mechanism of stomatal movement. It was widely recognized that G-protein α subunit (GPA1) can regulate a majority of signals during stomatal movement, such as Ca2+, reactive oxygen species (ROS), H2O2, ethylene, and so on. On the other hand, guard cell microtubule (MT) dynamics play a comparably critical role in regulating stomatal movement, and MTs rearrangement has been observed in stomatal responses to a variety of stimuli, like light, dark, nitric oxide (NO). Both heterotrimeric G-protein and MTs are essential for abscisic acid (ABA) signaling in guard cells, but whether and how these two signaling components work together is poorly understood.Here, the interplay between MTs and G-protein during ABA-induced stomatal movement was investigated in Arabidopsis thaliana .It was found that the heterotrimeric G-protein α subunit mutant, gpa1 , had a significantly higher water loss ratio than the wild type under the same conditions.Stomatal bioassay analyses revealed that guard cell sensitivity to ABA was attenuated in gpa1 , and the mutant′s ABA-insensitive phenotype could be partially restored by Oryzalin, an MT-specific inhibitor. However, exogenously applied Ca2+ chelator BAPTA-AM together with Oryzalin impaired the inhibition of stomatal opening by ABA. These results suggest that both MTs dynamics and Ca2+ flux may participate in the process of G-protein-mediated regulation of ABA-induced stomatal movement. Confocal microscopic images showed that after ABA treatment, MTs arrays in wild type underwent rapid disruption from radial arrays to disassembled fragments, whereas those in gpa1 largely remained bundled.A similar behavior was observed in BAPTA-AM plus ABA treatment, which is consistent with the stomatal bioassay results. In other words, G protein can control MTs rearrangement by adjusting the Ca2+ level in ABA-induced stomatal movement.In addition, non-invasive micro-test (NMT) demonstrated that the ABA-induced transmembrane Ca2+ flux was abolished in gpa1 guard cells, but Oryzalin enhanced the ABA response; Ca2+ efflux was suppressed dramatically when Oryzalin was added in gpa1. Therefore, our data clearly indicate that it exists a possitive crosstalking among G-protein, MTs and Ca2+ during ABA-induced stomatal movement. In this signal pathway, both MTs and Ca2+ act downstream of G-protein. Intracellular Ca2+ of guard cells relays signal information from G-protein to MTs; in turn, the changes of MTs dynamics in guard cells also have a feedback to Ca2+ flux. Finally the ABA signalling transduction results in stomatal movement, as well as the regulation of transpirational water loss and drought resistance.

Relationship between GNAS1 T393C polymorphism and aseptic loosening after total hip arthroplasty

BackgroundAseptic loosening is a main cause for revision surgery after total hip arthroplasty (THA) and there is no reliable marker for the early detection of patients at high risk. This study has been performed to validate association of the T393C polymorphism (rs7121) in the GNAS1 gene, encoding for the alpha-subunit of heterotrimeric G-protein Gs, with risk for and time to aseptic loosening after THA, which has been demonstrated in our previous study.Methods231 patients with primary THA and 234 patients suffering from aseptic loosening were genotyped for dependency on GNAS1 genotypes and analyzed.ResultsGenotyping revealed almost similar minor allele frequencies of 0.49 and 0.46, respectively. Consistently, genotype distributions of both groups were not significantly different (p = 0.572). Neither gender nor GNAS1 genotype showed a statistically significant association with time to loosening (p = 0.501 and p = 0.840). Stratification by gender, as performed in our previous study, was not able to show a significant genotype-dependent difference in time (female p = 0.313; male p = 0.584) as well as median time to aseptic loosening (female p = 0.353; male p = 0.868).ConclusionThis study was not able to confirm the results of our preliminary study. An association of the GNAS1 T393C polymorphisms with risk for and time to aseptic loosening after THA is unlikely.

The G-Protein β Subunit AGB1 Promotes Hypocotyl Elongation through Inhibiting Transcription Activation Function of BBX21 in Arabidopsis

Hypocotyl development in Arabidopsis thaliana is regulated by light and endogenous hormonal cues, making it an ideal model to study the interplay between light and endogenous growth regulators. BBX21, a B-box (BBX)-like zinc-finger transcription factor, integrates light and abscisic acid signals to regulate hypocotyl elongation in Arabidopsis. Heterotrimeric G-proteins are pivotal regulators of plant development. The short hypocotyl phenotype of the G-protein β-subunit (AGB1) mutant (agb1-2) has been previously identified, but the precise role of AGB1 in hypocotyl elongation remains enigmatic. Here, we show that AGB1 directly interacts with BBX21, and the short hypocotyl phenotype of agb1-2 is partially suppressed in agb1-2bbx21-1 double mutant. BBX21 functions in the downstream of AGB1 and overexpression of BBX21 in agb1-2 causes a more pronounced reduction in hypocotyl length, indicating that AGB1 plays an oppositional role in relation to BBX21 during hypocotyl development. Furthermore, we demonstrate that the C-terminal region of BBX21 is important for both its intracellular localization and its transcriptional activation activity that is inhibited by interaction with AGB1. ChIP assays showed that BBX21 specifically associates with its own promoter and with those of BBX22, HY5, and GA2ox1. which is not altered in agb1-2. These data suggest that the AGB1-BBX21 interaction only affects the transcriptional activation activity of BBX21 but has no effect on its DNA binding ability. Taken together, our data demonstrate that AGB1 positively promotes hypocotyl elongation through repressing BBX21 activity.

The BEACH Protein LRBA Promotes the Localization of the Heterotrimeric G-protein Golf to Olfactory Cilia

BEACH domain proteins are involved in membrane protein traffic and human diseases, but their molecular mechanisms are not understood. The BEACH protein LRBA has been implicated in immune response and cell proliferation, and human LRBA mutations cause severe immune deficiency. Here, we report a first functional and molecular phenotype outside the immune system of LRBA-knockout mice: compromised olfaction, manifesting in reduced electro-olfactogram response amplitude, impaired food-finding efficiency, and smaller olfactory bulbs. LRBA is prominently expressed in olfactory and vomeronasal chemosensory neurons of wild-type mice. Olfactory impairment in the LRBA-KO is explained by markedly reduced concentrations (20–40% of wild-type levels) of all three subunits αolf, β1 and γ13 of the olfactory heterotrimeric G-protein, Golf, in the sensory cilia of olfactory neurons. In contrast, cilia morphology and the concentrations of many other proteins of olfactory cilia are not or only slightly affected. LRBA is also highly expressed in photoreceptor cells, another cell type with a specialized sensory cilium and heterotrimeric G-protein-based signalling; however, visual function appeared unimpaired by the LRBA-KO. To our knowledge, this is the first observation that a BEACH protein is required for the efficient subcellular localization of a lipid-anchored protein, and of a ciliary protein.

Inhibition of Transient Receptor Potential Melastatin 3 ion channels by G-protein βγ subunits.

Transient receptor potential melastatin 3 (TRPM3) channels are activated by heat, and chemical ligands such as pregnenolone sulphate (PregS) and CIM0216. Here, we show that activation of receptors coupled to heterotrimeric Gi/o proteins inhibits TRPM3 channels. This inhibition was alleviated by co-expression of proteins that bind the βγ subunits of heterotrimeric G-proteins (Gβγ). Co-expression of Gβγ, but not constitutively active Gαi or Gαo, inhibited TRPM3 currents. TRPM3 co-immunoprecipitated with Gβ, and purified Gβγ proteins applied to excised inside-out patches inhibited TRPM3 currents, indicating a direct effect. Baclofen and somatostatin, agonists of Gi-coupled receptors, inhibited Ca2+ signals induced by PregS and CIM0216 in mouse dorsal root ganglion (DRG) neurons. The GABAB receptor agonist baclofen also inhibited inward currents induced by CIM0216 in DRG neurons, and nocifensive responses elicited by this TRPM3 agonist in mice. Our data uncover a novel signaling mechanism regulating TRPM3 channels.

Plant G-protein β subunits positively regulate drought tolerance by elevating detoxification of ROS

Heterotrimeric guanine-nucleotide-binding proteins (G-proteins) consist of α, β and γ subunits and play important roles in response and tolerance to abiotic stresses in plants, but the function of the heterotrimeric G-protein β subunit in response to drought remains unclear. In the present study, the AGB1 mutants (agb1-2-1 and agb1-3-2) were more sensitive to drought than the wild-type. The overexpression of mulberry (Morus alba L.) G-protein β subunit in transgenic tobacco (Nicotiana tabacum L.) significantly enhanced the plants' drought tolerance. The transgenic tobacco plants had higher proline contents and peroxidase activities, and lower malonaldehyde and hydrogen peroxide contents and superoxide free radical accumulations under drought conditions. Additionally, transcript levels of the tobacco antioxidative genes, NtSOD and NtCAT, increased in drought-stressed transgenic tobacco plants. Thus, the heterotrimeric G-protein β subunits positively regulate drought tolerance in plants.

Abstract 1350: Direct pharmacological targeting of Gq/11 in uveal melanoma

Abstract Uveal (eye) melanoma is a highly aggressive cancer, in which almost half of patients develop distant metastases that are refractory to therapy. In particular, metastatic uveal melanoma has been clinically unresponsive to the immunotherapeutic agents that have shown success in skin tumors, making the need for novel therapeutic approaches to uveal melanoma all the more urgent. Unlike skin melanomas, which are driven by BRAF and NRAS mutations, uveal melanomas arise typically from mutations that result in constitutive activity of the alpha subunit of the heterotrimeric G-protein, Gq, or its paralog G11. The prevalence of constitutively active Gq/11 in uveal melanoma suggests a dependence of these tumors on Gq/11 activity that could be exploited therapeutically. To address this hypothesis, we are using a potent, bioavailable small molecule that binds to and inhibits Gq/11 to target constitutively active Gq/11 in uveal melanoma cells. This inhibitor functions by sequestering wild type or constitutively active Gq/11 in an inactive state. We first used inositol phosphate accumulation assays and confirmed inhibition of both wildtype and constitutively active Gq/11 by the inhibitor in all uveal melanoma cells. We then assayed the affect of Gq/11 inhibition on overall viability of uveal melanoma cells, and found that uveal melanoma cells with mutant Gq/11 were highly sensitive to the small molecule; whereas, uveal melanoma cells with wildtype Gq/11 showed no loss of viability, even at 1000-fold higher concentrations of inhibitor. In Gq-mutant uveal melanoma cells, Gq/11 inhibition caused cell cycle arrest in G1, and dysregulation of several cell cycle regulatory pathways. Inhibitor treatment also caused Gq/11-driven uveal melanoma cells to become more differentiated, as indicated by increased pigmentation, elevated expression of melanin synthesis and melanosome markers, changes in cell morphology and changes in melanocytic versus melanoma gene programs. None of these phenotypic changes were seen in BRAF-driven uveal melanoma cells treated with the Gq/11 inhibitor, demonstrating that the effects of this inhibitor were exquisitely dependent on the constitutively active Gq/11 oncogene. These results establish that Gq/11 is a druggable target in uveal melanoma cells, and show that Gq/11-mutant uveal melanoma cells are exquisitely sensitive to inhibition by small molecule inhibitors. We are currently transitioning these studies to animal models to establish drug efficacy and toxicity and explore treatment and delivery options. Citation Format: Michael D. Onken, Carol M. Makepeace, Shiqi Wang, Kevin M. Kaltenbronn, S. Michinobu Kanai, Tom J. Broekelmann, John A. Cooper, Kendall J. Blumer. Direct pharmacological targeting of Gq/11 in uveal melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1350. doi:10.1158/1538-7445.AM2017-1350

Genetic loci associated with heart rate variability and their effects on cardiac disease risk

Reduced cardiac vagal control reflected in low heart rate variability (HRV) is associated with greater risks for cardiac morbidity and mortality. In two-stage meta-analyses of genome-wide association studies for three HRV traits in up to 53,174 individuals of European ancestry, we detect 17 genome-wide significant SNPs in eight loci. HRV SNPs tag non-synonymous SNPs (in NDUFA11 and KIAA1755), expression quantitative trait loci (eQTLs) (influencing GNG11, RGS6 and NEO1), or are located in genes preferentially expressed in the sinoatrial node (GNG11, RGS6 and HCN4). Genetic risk scores account for 0.9 to 2.6% of the HRV variance. Significant genetic correlation is found for HRV with heart rate (−0.74<rg<−0.55) and blood pressure (−0.35<rg<−0.20). These findings provide clinically relevant biological insight into heritable variation in vagal heart rhythm regulation, with a key role for genetic variants (GNG11, RGS6) that influence G-protein heterotrimer action in GIRK-channel induced pacemaker membrane hyperpolarization.