Heterotrimeric G Proteins Research Articles

Role of a heterotrimeric G-protein, Gi2, in the corticogenesis: possible involvement in periventricular nodular heterotopia and intellectual disability.

We analyzed the role of a heterotrimeric G-protein, Gi2, in the development of the cerebral cortex. Acute knockdown of the α-subunit (Gαi2) with in utero electroporation caused delayed radial migration of excitatory neurons during corticogenesis, perhaps because of impaired morphology. The migration phenotype was rescued by an RNAi-resistant version of Gαi2. On the other hand, silencing of Gαi2 did not affect axon elongation, dendritic arbor formation or neurogenesis at ventricular zone invivo. When behavior analyses were conducted with acute Gαi2-knockdown mice, they showed defects in social interaction, novelty recognition and active avoidance learning as well as increased anxiety. Subsequently, using whole-exome sequencing analysis, we identified a de novo heterozygous missense mutation (c.680C>T; p.Ala227Val) in the GNAI2 gene encoding Gαi2 in an individual with periventricular nodular heterotopia and intellectual disability. Collectively, the phenotypes in the knockdown experiments suggest a role of Gαi2 in the brain development, and impairment of its function might cause defects in neuronal functions which lead to neurodevelopmental disorders.

Kinetics of recruitment and allosteric activation of ARHGEF25 isoforms by the heterotrimeric G-protein Gαq

Rho GTPases are master regulators of the eukaryotic cytoskeleton. The activation of Rho GTPases is governed by Rho guanine nucleotide exchange factors (GEFs). Three RhoGEF isoforms are produced by the gene ARHGEF25; p63RhoGEF580, GEFT and a recently discovered longer isoform of 619 amino acids (p63RhoGEF619). The subcellular distribution of p63RhoGEF580 and p63RhoGEF619 is strikingly different in unstimulated cells, p63RhoGEF580 is located at the plasma membrane and p63RhoGEF619 is confined to the cytoplasm. Interestingly, we find that both P63RhoGEF580 and p63RhoGEF619 activate RhoGTPases to a similar extent after stimulation of Gαq coupled GPCRs. Furthermore, we show that p63RhoGEF619 relocates to the plasma membrane upon activation of Gαq coupled GPCRs, resembling the well-known activation mechanism of RhoGEFs activated by Gα12/13. Synthetic recruitment of p63RhoGEF619 to the plasma membrane increases RhoGEF activity towards RhoA, but full activation requires allosteric activation via Gαq. Together, these findings reveal a dual role for Gαq in RhoGEF activation, as it both recruits and allosterically activates cytosolic ARHGEF25 isoforms.

Modeling of phototransduction processes in the photoreceptor disk membranes by the Monte Carlo method

The Monte Carlo method was used to model the diffusion behaviors of functionally important proteins of the phototransduction system in retinal rod outer segment disk membranes. The results expand our knowledge of the mechanisms of inactivation of the main phototransduction heterotrimeric GTP-binding protein transducin.

Autophagy-mediated catabolism of visual transduction proteins prevents retinal degeneration

ABSTRACTAutophagy is a lysosomal degradation pathway critical to preventing the accumulation of cytotoxic proteins. Deletion of the essential autophagy gene Atg5 from the rod photoreceptors of the retina (atg5Δrod mouse) results in the accumulation of the phototransduction protein transducin and the degeneration of these neurons. The purpose of this study is to test the hypothesis that autophagic degradation of visual transduction proteins prevents retinal degeneration. Targeted deletion of both Gnat1 (a gene encoding the α subunit of the heterotrimeric G-protein transducin) and Atg5 in the rod photoreceptors resulted in a significantly decreased rate of rod cell degeneration as compared to the atg5Δrod mouse retina, and considerable preservation of photoreceptors. Supporting this we used a novel technique to immunoprecipitate green fluorescent protein (GFP)-tagged autophagosomes from the retinas of the GFP-LC3 mice and demonstrated that the visual transduction proteins transducin and ARR/arrestin are associated with autophagosome-specific proteins. Altogether, this study shows that degradation of phototransduction proteins by autophagy is necessary to prevent retinal degeneration. In addition, we demonstrate a simple and easily reproducible immunoisolation technique for enrichment of autophagosomes from the GFP-LC3 mouse retina, providing a novel application to the study of autophagosome contents across different organs and specific cell types in vivo.

Function of heterotrimeric G-protein γ subunit RGG1 in providing salinity stress tolerance in rice by elevating detoxification of ROS.

The present study provides evidence of a unique function of RGG1 in providing salinity stress tolerance in transgenic rice without affecting yield. It also provides a good example for signal transduction from the external environment to inside for enhanced agricultural production that withstands the extreme climatic conditions and ensures food security. The role of heterotrimeric G-proteins functioning as signalling molecules has not been studied as extensively in plants as in animals. Recently, their importance in plant stress signalling has been emerging. In this study, the function of rice G-protein γ subunit (RGG1) in the promotion of salinity tolerance in rice (Oryza sativa L. cv. IR64) was investigated. The overexpression of RGG1 driven by the CaMV35S promoter in transgenic rice conferred high salinity tolerance even in the presence of 200mM NaCl. Transcript levels of antioxidative genes, i.e., CAT, APX, and GR, and their enzyme activities increased in salinity-stressed transgenic rice plants suggesting a better antioxidant system to cope the oxidative-damages caused by salinity stress. The RGG1-induced signalling events that conferred tolerance to salinity was mediated by increased gene expression of the enzymes that scavenged reactive oxygen species. In salinity-stressed RGG1 transgenic lines, the transcript levels of RGG2, RGB, RGA, DEP1, and GS3 also increased in addition to RGG1. These observations suggest that most likely the stoichiometry of the G-protein complex was not disturbed under stress. Agronomic parameters, endogenous sugar content (glucose and fructose) and hormones (GA3, zeatin and IAA) were also higher in the transgenic plants compared with the wild-type plants. A BiFC assay confirmed the interaction of RGG1 with different stress-responsive proteins which play active roles in signalling and prevention of aggregation of proteins under stress-induced perturbation. The present study will help in understanding the G-protein-mediated stress tolerance in plants.

Connecting G protein signaling to chemoattractant-mediated cell polarity and cytoskeletal reorganization

ABSTRACTThe directional movement toward extracellular chemical gradients, a process called chemotaxis, is an important property of cells. Central to eukaryotic chemotaxis is the molecular mechanism by which chemoattractant-mediated activation of G-protein coupled receptors (GPCRs) induces symmetry breaking in the activated downstream signaling pathways. Studies with mainly Dictyostelium and mammalian neutrophils as experimental systems have shown that chemotaxis is mediated by a complex network of signaling pathways. Recently, several labs have used extensive and efficient proteomic approaches to further unravel this dynamic signaling network. Together these studies showed the critical role of the interplay between heterotrimeric G-protein subunits and monomeric G proteins in regulating cytoskeletal rearrangements during chemotaxis. Here we highlight how these proteomic studies have provided greater insight into the mechanisms by which the heterotrimeric G protein cycle is regulated, how heterotrimeric G proteins-induced symmetry breaking is mediated through small G protein signaling, and how symmetry breaking in G protein signaling subsequently induces cytoskeleton rearrangements and cell migration.

GNB5 mutation causes a novel neuropsychiatric disorder featuring attention deficit hyperactivity disorder, severely impaired language development and normal cognition.

BackgroundNeuropsychiatric disorders are common forms of disability in humans. Despite recent progress in deciphering the genetics of these disorders, their phenotypic complexity continues to be a major challenge. Mendelian neuropsychiatric disorders are rare but their study has the potential to unravel novel mechanisms that are relevant to their complex counterparts.ResultsIn an extended consanguineous family, we identified a novel neuropsychiatric phenotype characterized by severe speech impairment, variable expressivity of attention deficit hyperactivity disorder (ADHD), and motor delay. We identified the disease locus through linkage analysis on 15q21.2, and exome sequencing revealed a novel missense variant in GNB5. GNB5 encodes an atypical β subunit of the heterotrimeric GTP-binding proteins (Gβ5). Gβ5 is enriched in the central nervous system where it forms constitutive complexes with members of the regulator of G protein signaling family of proteins to modulate neurotransmitter signaling that affects a number of neurobehavioral outcomes. Here, we show that the S81L mutant form of Gβ5 has significantly impaired activity in terminating responses that are elicited by dopamine.ConclusionsWe demonstrate that these deficits originate from the impaired expression of the mutant Gβ5 protein, resulting in the decreased ability to stabilize regulator of G protein signaling complexes. Our data suggest that this novel neuropsychiatric phenotype is the human equivalent of Gnb5 deficiency in mice, which manifest motor deficits and hyperactivity, and highlight a critical role of Gβ5 in normal behavior as well as language and motor development in humans.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-016-1061-6) contains supplementary material, which is available to authorized users.

G proteins diverge and conquer

Evolution In plants, signaling through G (heterotrimeric GTP-binding) proteins involves either canonical Gα proteins, which are structurally and functionally similar to animal Gα proteins, or extra-large Gα (XLG) proteins. Urano et al. found that plant canonical Gα proteins evolved slowly, like their animal counterparts, and were involved in developmental processes. In contrast, XLG proteins rapidly diversified early in the land plant lineage and mediated stress responses. Thus, diversification of plant XLG proteins coincided with the colonization of land and may have helped plants to withstand the new stresses encountered in this environment. Sci. Signal. 9 , ra93 (2016).

Gα and regulator of G-protein signaling (RGS) protein pairs maintain functional compatibility and conserved interaction interfaces throughout evolution despite frequent loss of RGS proteins in plants.

Signaling pathways regulated by heterotrimeric G-proteins exist in all eukaryotes. The regulator of G-protein signaling (RGS) proteins are key interactors and critical modulators of the Gα protein of the heterotrimer. However, while G-proteins are widespread in plants, RGS proteins have been reported to be missing from the entire monocot lineage, with two exceptions. A single amino acid substitution-based adaptive coevolution of the Gα:RGS proteins was proposed to enable the loss of RGS in monocots. We used a combination of evolutionary and biochemical analyses and homology modeling of the Gα and RGS proteins to address their expansion and its potential effects on the G-protein cycle in plants. Our results show that RGS proteins are widely distributed in the monocot lineage, despite their frequent loss. There is no support for the adaptive coevolution of the Gα:RGS protein pair based on single amino acid substitutions. RGS proteins interact with, and affect the activity of, Gα proteins from species with or without endogenous RGS. This cross-functional compatibility expands between the metazoan and plant kingdoms, illustrating striking conservation of their interaction interface. We propose that additional proteins or alternative mechanisms may exist which compensate for the loss of RGS in certain plant species.

Development of the main olfactory system and main olfactory epithelium-dependent male mating behavior are altered in Go-deficient mice

In mammals, initial detection of olfactory stimuli is mediated by sensory neurons in the main olfactory epithelium (MOE) and the vomeronasal organ (VNO). The heterotrimeric GTP-binding protein Go is widely expressed in the MOE and VNO of mice. Early studies indicated that Go expression in VNO sensory neurons is critical for directing social and sexual behaviors in female mice [Oboti L, et al. (2014) BMC Biol 12:31]. However, the physiological functions of Go in the MOE have remained poorly defined. Here, we examined the role of Go in the MOE using mice lacking the α subunit of Go Development of the olfactory bulb (OB) was perturbed in mutant mice as a result of reduced neurogenesis and increased cell death. The balance between cell types of OB interneurons was altered in mutant mice, with an increase in the number of tyrosine hydroxylase-positive interneurons at the expense of calbindin-positive interneurons. Sexual behavior toward female mice and preference for female urine odors by olfactory sensory neurons in the MOE were abolished in mutant male mice. Our data suggest that Go signaling is essential for the structural and functional integrity of the MOE and for specification of OB interneurons, which in turn are required for the transmission of pheromone signals and the initiation of mating behavior with the opposite sex.

Two new competing pathways establish the threshold for cyclin-B–Cdk1 activation at the meiotic G2/M transition

Extracellular ligands control biological phenomena. Cells distinguish physiological stimuli from weak noise stimuli by establishing a ligand-concentration threshold. Hormonal control of the meiotic G2/M transition in oocytes is essential for reproduction. However, the mechanism for threshold establishment is unclear. In starfish oocytes, maturation-inducing hormones activate the PI3K–Akt pathway through the Gβγ complex of heterotrimeric G-proteins. Akt directly phosphorylates both Cdc25 phosphatase and Myt1 kinase, resulting in activation of cyclin-B–Cdk1, which then induces meiotic G2/M transition. Here, we show that cyclin-B–Cdk1 is partially activated after subthreshold hormonal stimuli, but this triggers negative feedback, resulting in dephosphorylation of Akt sites on Cdc25 and Myt1, thereby canceling the signal. We also identified phosphatase activity towards Akt substrates that exists independent of stimuli. In contrast to these negative regulatory activities, an atypical Gβγ-dependent pathway enhances PI3K–Akt-dependent phosphorylation. Based on these findings, we propose a model for threshold establishment in which hormonal dose-dependent competition between these new pathways establishes a threshold; the atypical Gβγ-pathway becomes predominant over Cdk-dependent negative feedback when the stimulus exceeds this threshold. Our findings provide a regulatory connection between cell cycle and signal transduction machineries.

Regulation of Polycystin-1 Function by Calmodulin Binding.

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a common genetic disease that leads to progressive renal cyst growth and loss of renal function, and is caused by mutations in the genes encoding polycystin-1 (PC1) and polycystin-2 (PC2), respectively. The PC1/PC2 complex localizes to primary cilia and can act as a flow-dependent calcium channel in addition to numerous other signaling functions. The exact functions of the polycystins, their regulation and the purpose of the PC1/PC2 channel are still poorly understood. PC1 is an integral membrane protein with a large extracytoplasmic N-terminal domain and a short, ~200 amino acid C-terminal cytoplasmic tail. Most proteins that interact with PC1 have been found to bind via the cytoplasmic tail. Here we report that the PC1 tail has homology to the regulatory domain of myosin heavy chain including a conserved calmodulin-binding motif. This motif binds to CaM in a calcium-dependent manner. Disruption of the CaM-binding motif in PC1 does not affect PC2 binding, cilia targeting, or signaling via heterotrimeric G-proteins or STAT3. However, disruption of CaM binding inhibits the PC1/PC2 calcium channel activity and the flow-dependent calcium response in kidney epithelial cells. Furthermore, expression of CaM-binding mutant PC1 disrupts cellular energy metabolism. These results suggest that critical functions of PC1 are regulated by its ability to sense cytosolic calcium levels via binding to CaM.

Sporophyte formation and life cycle completion in moss requires heterotrimeric G-proteins

In this study, we report the functional characterization of heterotrimeric G-proteins from a nonvascular plant, the moss Physcomitrella patens. In plants, G-proteins have been characterized from only a few angiosperms to date, where their involvement has been shown during regulation of multiple signaling and developmental pathways affecting overall plant fitness. In addition to its unparalleled evolutionary position in the plant lineages, the P. patens genome also codes for a unique assortment of G-protein components, which includes two copies of Gβ and Gγ genes, but no canonical Gα Instead, a single gene encoding an extra-large Gα (XLG) protein exists in the P. patens genome. Here, we demonstrate that in P. patens the canonical Gα is biochemically and functionally replaced by an XLG protein, which works in the same genetic pathway as one of the Gβ proteins to control its development. Furthermore, the specific G-protein subunits in P. patens are essential for its life cycle completion. Deletion of the genomic locus of PpXLG or PpGβ2 results in smaller, slower growing gametophores. Normal reproductive structures develop on these gametophores, but they are unable to form any sporophyte, the only diploid stage in the moss life cycle. Finally, the mutant phenotypes of ΔPpXLG and ΔPpGβ2 can be complemented by the homologous genes from Arabidopsis, AtXLG2 and AtAGB1, respectively, suggesting an overall conservation of their function throughout the plant evolution.

Canon Fodder-A Case for Contrarian Science.

Cannon fodder are expendable soldiers deployed as “food” for enemy cannon fire when there is no hope of ultimately prevailing: generals dispose of “worthless” soldiers by sacrificing them to the cannon. Here, I adopt the term Canon fodder to describe contrarian scientific concepts dismissed because they do not conform to conventional wisdom: reviewers dispose of “unworthy” science by sacrificing it to the canon. It is a rare opportunity to be invited to opine to one’s peers on a topic of choice. Actually, my peers are my chosen topic. Specifically, peer review. Twenty-five years ago, I eschewed job security as an interventional cardiologist in favor of academic research. Consequently, I needed NIH R01 funding. After my initial attempts failed spectacularly, one of my mentors (it was Jeff Robbins or Rick Walsh, or perhaps both) provided surprising guidance: I should compose my grant application with 4 specific aims, then discard the most exciting aim and submit what remained. This would avoid provocation in favor of the conventional and acceptable. It was sage advice from highly successful senior investigators (I learned this the hard way, by initially disregarding it and suffering the consequences). I envisioned testing the neurohormonal hypothesis of heart failure1 by expressing in cardiac myocytes a signaling molecule common to neurohormonal pathways postulated to mediate pathological hypertrophy. I would use the then-new mouse α-myosin heavy chain (α-MHC) promoter2 to drive cardiac-specific expression of the α subunit of heterotrimeric G-protein, Gq, the common proximal transducer for angtiotensin II (type 1), endothelin, and norepinephrine (α-adrenergic) signaling.3 I reasoned that cardiomyocyte-specific activation of Gq signaling would circumvent hemodynamic effects (eg, hypertension) of Gq-coupled neurohormones. If the planned Gαq transgenic mice developed hypertrophy and heart failure, then cardiomyocytes were autonomously responding to neurohormonal signaling, thus proving the neurohormonal hypothesis. My first postdoctoral …

Manduca Contactin Regulates Amyloid Precursor Protein-Dependent Neuronal Migration.

Amyloid precursor protein (APP) was originally identified as the source of β-amyloid peptides that accumulate in Alzheimer's disease (AD), but it also has been implicated in the control of multiple aspects of neuronal motility. APP belongs to an evolutionarily conserved family of transmembrane proteins that can interact with a variety of adapter and signaling molecules. Recently, we showed that both APP and its insect ortholog [APPL (APP-Like)] directly bind the heterotrimeric G-protein Goα, supporting the model that APP can function as an unconventional Goα-coupled receptor. We also adapted a well characterized assay of neuronal migration in the hawkmoth, Manduca sexta, to show that APPL-Goα signaling restricts ectopic growth within the developing nervous system, analogous to the role postulated for APP family proteins in controlling migration within the mammalian cortex. Using this assay, we have now identified Manduca Contactin (MsContactin) as an endogenous ligand for APPL, consistent with previous work showing that Contactins interact with APP family proteins in other systems. Using antisense-based knockdown protocols and fusion proteins targeting both proteins, we have shown that MsContactin is selectively expressed by glial cells that ensheath the migratory neurons (expressing APPL), and that MsContactin-APPL interactions normally prevent inappropriate migration and outgrowth. These results provide new evidence that Contactins can function as authentic ligands for APP family proteins that regulate APP-dependent responses in the developing nervous system. They also support the model that misregulated Contactin-APP interactions might provoke aberrant activation of Goα and its effectors, thereby contributing to the neurodegenerative sequelae that typify AD. Members of the amyloid precursor protein (APP) family participate in many aspects of neuronal development, but the ligands that normally activate APP signaling have remained controversial. This research provides new evidence that members of the Contactin family function as authentic ligands for APP and its orthologs, and that this evolutionarily conserved class of membrane-attached proteins regulates key aspects of APP-dependent migration and outgrowth in the embryonic nervous system. By defining the normal role of Contactin-APP signaling during development, these studies also provide the framework for investigating how the misregulation of Contactin-APP interactions might contribute to neuronal dysfunction in the context of both normal aging and neurodegenerative conditions, including Alzheimer's disease.

Blunted apoptosis of erythrocytes in mice deficient in the heterotrimeric G-protein subunit Gαi2

Putative functions of the heterotrimeric G-protein subunit Gαi2-dependent signaling include ion channel regulation, cell differentiation, proliferation and apoptosis. Erythrocytes may, similar to apoptosis of nucleated cells, undergo eryptosis, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine (PS) exposure. Eryptosis may be triggered by increased cytosolic Ca2+ activity and ceramide. In the present study, we show that Gαi2 is expressed in both murine and human erythrocytes and further examined the survival of erythrocytes drawn from Gαi2-deficient mice (Gαi2−/−) and corresponding wild-type mice (Gαi2+/+). Our data show that plasma erythropoietin levels, erythrocyte maturation markers, erythrocyte counts, hematocrit and hemoglobin concentration were similar in Gαi2−/− and Gαi2+/+ mice but the mean corpuscular volume was significantly larger in Gαi2−/− mice. Spontaneous PS exposure of circulating Gαi2−/− erythrocytes was significantly lower than that of circulating Gαi2+/+ erythrocytes. PS exposure was significantly lower in Gαi2−/− than in Gαi2+/+ erythrocytes following ex vivo exposure to hyperosmotic shock, bacterial sphingomyelinase or C6 ceramide. Erythrocyte Gαi2 deficiency further attenuated hyperosmotic shock-induced increase of cytosolic Ca2+ activity and cell shrinkage. Moreover, Gαi2−/− erythrocytes were more resistant to osmosensitive hemolysis as compared to Gαi2+/+ erythrocytes. In conclusion, Gαi2 deficiency in erythrocytes confers partial protection against suicidal cell death.

Heterotrimeric G-proteins facilitate resistance to plant pathogenic viruses in Arabidopsis thaliana (L.) Heynh

ABSTRACTHeterotrimeric G-proteins, consisting of Gα, Gβ and Gγ subunits, are important signal transducers in eukaryotes. In plants, G-protein-mediated signaling contributes to defense against a range of fungal and bacterial pathogens. Here we studied response of G-protein-deficient mutants to ssRNA viruses representing 2 different families: Cucumber mosaic virus (CMV) (Bromoviridae) and Turnip mosaic virus (TuMV) (Potyviridae). We found that development of spreading necrosis on infected plants was suppressed in the Gβ-deficient mutant (agb1-2) compared to wild type and Gα-deficient mutant (gpa1-4). In accordance, ion leakage caused by viral infection was also significantly reduced in agb1-2 compared to wild type and gpa1-4. Nevertheless, both viruses replicated better in agb1-2 plants, while gpa1-4 was similar to wild type. Analysis of pathogenesis-related genes showed that Gβ negatively regulated salicylic acid, jasmonic acid and abscisic acid marker genes during CMV and TuMV infections. Interestingly, analysis of salicylic acid deficient transgenic plants indicated that salicylic acid did not affect resistance against these viruses and did not influence the Gβ-mediated defense response. We conclude that heterotrimeric G-proteins play a positive role in defense against viral pathogens probably by promoting cell death.

Abstract 4572: Simultaneous activation of C-X-C chemokine receptor 4 and cannabinoid receptor 2 leads to decreased Rho-induced prostate cancer metastasis

Abstract BACKGROUND: The binding of the homeostatic chemokine SDF-1 to the G -protein-coupled receptor C-X-C Chemokine Receptor 4 (CXCR4) induces cell migration via heterotrimeric G-proteins of the Gi family. Additionally, activated CXCR4 can stimulate the small GTPase RhoA through Gα13 which is required for directional cell migration, contributing to the metastatic potential of cancer cells. Recently, GPCR hetero-oligomerization has emerged as a means by which new signaling entities can be created, yet how receptor heteromers affect receptor pharmacology remains largely unknown. Here we report that agonist-bound CXCR4 is able to form a heteromer with agonist-bound cannabinoid receptor 2 (CB2) at the cell membrane of prostate cancer (PCa) cells and that receptor interaction inhibits the activation of the cytoskeleton regulator RhoA. EXPERIMENTAL METHODS: To confirm CXCR4-CB2 interactions on the cell membrane, we utilized an in situ proximity ligation assay to detect heterodimer formation on the surface PC-3 cells treated with SDF-1 alone or, simultaneous treatment with SDF-1 and the CB2 agonist AM1241. In order to establish that RhoA activity decreased upon heterodimerization of CXCR4 and CB2, we performed RhoA-GTP pulldown assays in PC-3 cells. We then tested the effects of CXCR4/CB2 heterodimerization on actin cytoskeletal reorganization by performing immunocytochemistry using an anti-Phalloidin antibody to detect F-actin. The ROCK kinase inhibitor Y-27632 was used as positive control of RhoA inhibition in these studies. Next, we assessed the effects of receptor heterodimerization on cellular adhesion to extracellular matrix components as the ability of cancer cells to adhere to the endothelium is critical of transendothelial migration during tumor cell intravasation and extravasation. Intuitively, alterations in adhesion capabilities should affect the ability of PCa cells to migrate through an endothelial cell monolayer. Thus we performed transendothelial migration assays. RESULTS: The proximity ligation assay revealed that CXCR4 and CB2 physically interacted on the cell surface of prostate cancer cells. Furthermore, receptor heterodimerization diminished SDF-1 induced actin cytoskeleton reorganization as characterized by membrane ruffling in a similar manner to the ROCK kinase inhibitor Y-27632. Heterodimer formation, stimulated by simultaneous treatment of cells with SDF-1 and AM1241 also attenuated PCa adhesion and transendothelial migration. CONCLUSION The attenuation of RhoA has multiple functional consequences leading to a less metastatic phenotype. Thus induction of CXCR4-CB2 heterodimerization is a viable pharmacological approach to targeting the CXCR4-RhoA axis as a means of preventing the metastatic dissemination of PCa cells. Citation Format: Kisha A. Scarlett, Christopher J. Coke, Imani Fennell, Cimona V. Hinton. Simultaneous activation of C-X-C chemokine receptor 4 and cannabinoid receptor 2 leads to decreased Rho-induced prostate cancer metastasis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4572.

The GNA13-RhoA signaling axis suppresses expression of tumor protective Kallikreins

Gα13 (encoded by GNA13 gene) is the alpha subunit of a heterotrimeric G-protein that mediates signaling through specific G-protein-coupled receptors (GPCRs). Increased GNA13 expression has been observed in metastatic breast cancer cells. Recently, we have shown that enhanced GNA13 signaling in MCF-10a cells, a benign breast cancer cell line increased its invasiveness. Previous studies have reported that Kallikrein-related peptidases (KLKs 1–15) are down-regulated in breast tumors and may have a tumor protective function. However, the mechanisms that lead to the down-regulation of KLK genes in breast cancer are yet to be elucidated. We found that enhanced GNA13 signaling represses KLK gene expression in breast cancer, and undertook examination of the mechanisms involved. A microarray analysis revealed down-regulation of several members of the Kallikrein-related peptidases (KLK) gene family, namely KLK5, KLK6, KLK7, KLK8 and KLK10, in MCF-10a lines with enhanced GNA13 protein expression. Using real-time PCR and promoter analysis, we identified that the mRNA expression and promoter activities of these KLKs are suppressed upon enforced expression of GNA13 in MCF-10a cells. Using Rhotekin pull-down assays, we identified that GNA13 suppressed Rho-A activation and protein levels in MCF-10a cells. Blocking Rho-A activation using C3-toxin or by inhibiting its down-stream effector, Rho-associated kinase (ROCK), reduced the above-mentioned KLK mRNAs in MCF-10A cells. Importantly, in a metastatic breast cancer cell line MDA-MB-157, knock down of GNA13 alone was sufficient to induce the expression KLK mRNAs. Taken together, our findings suggested that enhanced GNA13 signaling down-regulates KLK gene transcription. The ability of enhanced GNA13 signaling to suppress KLK gene expression appears at least in part due to the ability of enhanced GNA13 signaling to negatively impact Rho/ROCK-signaling.

Quantitative Single-Cell Analysis of Signaling Pathways Activated Immediately Downstream of Histamine Receptor Subtypes

Genetically encoded biosensors based on Förster resonance energy transfer (FRET) can visualize responses of individual cells in real time. Here, we evaluated whether FRET-based biosensors provide sufficient contrast and specificity to measure activity of G-protein-coupled receptors. The four histamine receptor subtypes (H1R, H2R, H3R, and H4R) respond to the ligand histamine by activating three canonical heterotrimeric G-protein-mediated signaling pathways with a reported high degree of specificity. Using FRET-based biosensors, we demonstrate that H1R activates Gαq. We also observed that H1R activates Gαi, albeit at a 10-fold lower potency. In addition to increasing cAMP levels, most likely via Gαs, we found that the H2R induces Gαq-mediated calcium release. The H3R and H4R activated Gαi with high specificity and a high potency. We demonstrate that a number of FRET sensors provide sufficient contrast to: 1) analyze the specificity of the histamine receptor subtypes for different heterotrimeric G-protein families with single-cell resolution, 2) probe for antagonist specificity, and 3) allow the measurement of single-cell concentration-response curves.