Zinc oxide nanoparticles disrupt development and function of the olfactory sensory system impairing olfaction-mediated behaviour in zebrafish.

Zinc (Zn) is an essential metal present in numerous enzymes throughout the body, playing a vital role in animal and human health. However, the increasing use of zinc oxide nanomaterials (ZnONPs) in a diverse range of products has raised concerns regarding their potential impacts on health and the environment. Despite these concerns, the toxicity of ZnONP exposure on animal health remain poorly understood. To help address this knowledge gap, we have developed a highly sensitive oxidative stress (OS) biosensor zebrafish capable of detecting cell/tissue-specific OS responses to low doses of various oxidative stressors, including Zn, in a live fish embryo. Using live-imaging analysis with this biosensor zebrafish embryo, we discovered that the olfactory sensory neurons in the brain are especially sensitive to ZnOP exposure. Furthermore, through studies monitoring neutrophil migration and neuronal activation in the embryonic brain and via behaviour analysis, we have found that sub-lethal doses of ZnONPs (ranging from 0.033 to 1 mg/L nominal concentrations), which had no visible effect on embryo growth or morphology, cause significant localised inflammation, disrupting the neurophysiology of olfactory brain tissues and ultimately impaired olfaction-mediated behaviour. Collectively, these findings establish a potent and important effect mechanism for ZnONP toxicity, indicating the olfactory sensory system as the primary target for ZnONPs as an environmental toxicant in aquatic environments. Our result also highlights that even low doses of ZnONPs can have detrimental effects on the olfactory sensory system, surpassing previous expectations. The importance of olfaction in environment sensing, sex behaviours and overall fitness across species raises concerns about the potential impact of ZnONPs on olfaction-mediated brain function and behaviour in animals and humans. Our study emphasises the need for greater consideration of the potential risks associated with these nanomaterials.

Application of Transgenic Zebrafish Models for Studying the Effects of Estrogenic Endocrine Disrupting Chemicals on Embryonic Brain Development.

Endocrine disrupting chemicals (EDCs) are environmental pollutants that mimic hormones and/or disrupt their function. Estrogenic EDCs (eEDCs) interfere with endogenous estrogen signalling pathway(s) and laboratory animal and human epidemiological studies have provided evidence for a causal link between exposure to them during embryonic/early life and neurological impairments. However, our understanding of the molecular and cellular mechanism(s) underlying eEDCs exposure effects on brain development, tissue architecture and function and behaviour are limited. Transgenic (TG) zebrafish models offer new approach methodologies (NAMs) to help identify the modes of action (MoAs) of EDCs and their associated impacts on tissue development and function. Estrogen biosensor TG zebrafish models have been applied to study eEDC interactions and resulting transcriptional activation (via a fluorescent reporter expression) across the entire body of the developing zebrafish embryo, including in real time. These estrogen biosensor TG zebrafish models are starting to deepen our understanding of the spatiotemporal actions of eEDCs and their resulting impacts on neurological development, brain function and behaviour. In this review, we first investigate the links between early life exposure to eEDCs and neurodevelopmental alterations in model organisms (rodents and zebrafish) and humans. We then present examples of the application of estrogen biosensor and other TG zebrafish models for elucidating the mechanism(s) underlying neurodevelopmental toxicities of eEDCs. In particular we illustrate the utility of combining estrogen biosensor zebrafish models with other TG zebrafish models for understanding the effects of eEDCs on the brain, spanning cellular processes, brain circuitry, neurophysiology and behaviour. Finally, we discuss the future prospects of TG zebrafish models as experimental models for studying more complex scenarios for exposure to contaminant mixtures on neurological development and function.

Estrogens regulate early embryonic development of the olfactory sensory system via estrogen-responsive glia.

Estrogens are well-known to regulate development of sexual dimorphism of the brain; however, their role in embryonic brain development prior to sex-differentiation is unclear. Using estrogen biosensor zebrafish models, we found that estrogen activity in the embryonic brain occurs from early neurogenesis specifically in a type of glia in the olfactory bulb (OB), which we name estrogen-responsive olfactory bulb (EROB) cells. In response to estrogen, EROB cells overlay the outermost layer of the OB and interact tightly with olfactory sensory neurons at the olfactory glomeruli. Inhibiting estrogen activity using an estrogen receptor antagonist, ICI182,780 (ICI), and/or EROB cell ablation impedes olfactory glomerular development, including the topological organisation of olfactory glomeruli and inhibitory synaptogenesis in the OB. Furthermore, activation of estrogen signalling inhibits both intrinsic and olfaction-dependent neuronal activity in the OB, whereas ICI or EROB cell ablation results in the opposite effect on neuronal excitability. Altering the estrogen signalling disrupts olfaction-mediated behaviour in later larval stage. We propose that estrogens act on glia to regulate development of OB circuits, thereby modulating the local excitability in the OB and olfaction-mediated behaviour.

Functional brain imaging in larval zebrafish for characterising the effects of seizurogenic compounds acting via a range of pharmacological mechanisms.

BACKGROUND AND PURPOSE: Functional brain imaging using genetically encoded Ca2+ sensors in larval zebrafish is being developed for studying seizures and epilepsy as a more ethical alternative to rodent models. Despite this, few data have been generated on pharmacological mechanisms of action other than GABAA antagonism. Assessing larval responsiveness across multiple mechanisms is vital to test the translational power of this approach, as well as assessing its validity for detecting unwanted drug-induced seizures and testing antiepileptic drug efficacy. EXPERIMENTAL APPROACH: Using light-sheet imaging, we systematically analysed the responsiveness of 4 days post fertilisation (dpf; which are not considered protected under European animal experiment legislation) transgenic larval zebrafish to treatment with 57 compounds spanning more than 12 drug classes with a link to seizure generation in mammals, alongside eight compounds with no such link. KEY RESULTS: We show 4dpf zebrafish are responsive to a wide range of mechanisms implicated in seizure generation, with cerebellar circuitry activated regardless of the initiating pharmacology. Analysis of functional connectivity revealed compounds targeting cholinergic and monoaminergic reuptake, in particular, showed phenotypic consistency broadly mapping onto what is known about neurotransmitter-specific circuitry in the larval zebrafish brain. Many seizure-associated compounds also exhibited altered whole brain functional connectivity compared with controls. CONCLUSIONS AND IMPLICATIONS: This work represents a significant step forward in understanding the translational power of 4dpf larval zebrafish for use in neuropharmacological studies and for studying the events driving transition from small-scale pharmacological activation of local circuits, to the large network-wide abnormal synchronous activity associated with seizures.

New insights into organ-specific oxidative stress mechanisms using a novel biosensor zebrafish.

BACKGROUND: Reactive oxygen species (ROS) arise as a result from, and are essential in, numerous cellular processes. ROS, however, are highly reactive and if left unneutralised by endogenous antioxidant systems, can result in extensive cellular damage and/or pathogenesis. In addition, exposure to a wide range of environmental stressors can also result in surplus ROS production leading to oxidative stress (OS) and downstream tissue toxicity. OBJECTIVES: Our aim was to produce a stable transgenic zebrafish line, unrestricted by tissue-specific gene regulation, which was capable of providing a whole organismal, real-time read-out of tissue-specific OS following exposure to a wide range of OS-inducing environmental contaminants and conditions. This model could, therefore, serve as a sensitive and specific mechanistic in vivo biomarker for all environmental conditions that result in OS. METHODS: To achieve this aim, we exploited the pivotal role of the electrophile response element (EpRE) as a globally-acting master regulator of the cellular response to OS. To test tissue specificity and quantitative capacity, we selected a range of chemical contaminants known to induce OS in specific organs or tissues, and assessed dose-responsiveness in each using microscopic measures of mCherry fluorescence intensity. RESULTS: We produced the first stable transgenic zebrafish line Tg (3EpRE:hsp70:mCherry) with high sensitivity for the detection of cellular RedOx imbalances, in vivo in near-real time. We applied this new model to quantify OS after exposure to a range of environmental conditions with high resolution and provided quantification both of compound- and tissue-specific ROS-induced toxicity. DISCUSSION: Our model has an extremely diverse range of potential applications not only for biomonitoring of toxicants in aqueous environments, but also in biomedicine for identifying ROS-mediated mechanisms involved in the progression of a number of important human diseases, including cancer.

Estrogenic Mechanisms and Cardiac Responses Following Early Life Exposure to Bisphenol A (BPA) and Its Metabolite 4-Methyl-2,4-bis( p-hydroxyphenyl)pent-1-ene (MBP) in Zebrafish.

Environmental exposure to Bisphenol A (BPA) has been associated with a range of adverse health effects, including on the cardiovascular system in humans. Lack of agreement on its mechanism(s) of action likely stem from comparisons between in vivo and in vitro test systems and potential multiple effects pathways. In rodents, in vivo, metabolic activation of BPA produces 4-methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene (MBP), which is reported to be up to 1000 times more potent as an estrogen than BPA. We investigated the estrogenic effects and estrogen receptor signaling pathway(s) of BPA and MBP following early life exposure using a transgenic, estrogen responsive (ERE-TG) zebrafish and a targeted morpholino approach to knockdown the three fish estrogen receptor (ER) subtypes. The functional consequences of BPA exposure on the cardiovascular system of zebrafish larvae were also examined. The heart atrioventricular valves and the bulbus arteriosus were primary target tissues for both BPA and MBP in the ERE-TG zebrafish, and MBP was approximately 1000-fold more potent than BPA as an estrogen in these tissues. Estrogen receptor knockdown with morpholinos indicated that the estrogenic responses in the heart for both BPA and MBP were mediated via an estrogen receptor 1 (esr1) dependent pathway. At the highest BPA concentration tested (2500 µg/L), alterations in the atrial:ventricular beat ratio indicated a functional impact on the heart of 5 days post fertilization (dpf) larvae, and there was also a significantly reduced heart rate in these larvae at 14 dpf. Our findings indicate that some of the reported adverse effects on heart function associated with BPA exposure (in mammals) may act through an estrogenic mechanism, but that fish are unlikely to be susceptible to adverse effects on heart development for environmentally relevant exposures.

Early life exposure to ethinylestradiol enhances subsequent responses to environmental estrogens measured in a novel transgenic zebrafish.

Estrogen plays fundamental roles in a range of developmental processes and exposure to estrogen mimicking chemicals has been associated with various adverse health effects in both wildlife and human populations. Estrogenic chemicals are found commonly as mixtures in the environment and can have additive effects, however risk analysis is typically conducted for single-chemicals with little, or no, consideration given for an animal's exposure history. Here we developed a transgenic zebrafish with a photoconvertable fluorophore (Kaede, green to red on UV light exposure) in a skin pigment-free mutant element (ERE)-Kaede-Casper model and applied it to quantify tissue-specific fluorescence biosensor responses for combinations of estrogen exposures during early life using fluorescence microscopy and image analysis. We identify windows of tissue-specific sensitivity to ethinylestradiol (EE2) for exposure during early-life (0-5 dpf) and illustrate that exposure to estrogen (EE2) during 0-48 hpf enhances responsiveness (sensitivity) to different environmental estrogens (EE2, genistein and bisphenol A) for subsequent exposures during development. Our findings illustrate the importance of an organism's stage of development and estrogen exposure history for assessments on, and possible health risks associated with, estrogen exposure.

High-Content and Semi-Automated Quantification of Responses to Estrogenic Chemicals Using a Novel Translucent Transgenic Zebrafish.

Rapid embryogenesis, together with genetic similarities with mammals, and the desire to reduce mammalian testing, are major incentives for using the zebrafish model in chemical screening and testing. Transgenic zebrafish, engineered for identifying target gene expression through expression of fluorophores, have considerable potential for both high-content and high-throughput testing of chemicals for endocrine activity. Here we generated an estrogen responsive transgenic zebrafish model in a pigment-free "Casper" phenotype, facilitating identification of target tissues and quantification of these responses in whole intact fish. Using the ERE-GFP-Casper model we show chemical type and concentration dependence for green fluorescent protein (GFP) induction and both spatial and temporal responses for different environmental estrogens tested. We also developed a semiautomated (ArrayScan) imaging and image analysis system that we applied to quantify whole body fluorescence responses for a range of different estrogenic chemicals in the new transgenic zebrafish model. The zebrafish model developed provides a sensitive and highly integrative system for identifying estrogenic chemicals, their target tissues and effect concentrations for exposures in real time and across different life stages. It thus has application for chemical screening to better direct health effects analysis of environmental estrogens and for investigating the functional roles of estrogens in vertebrates.

Biosensor zebrafish provide new insights into potential health effects of environmental estrogens.

BACKGROUND: Environmental estrogens alter hormone signaling in the body that can induce reproductive abnormalities in both humans and wildlife. Available testing systems for estrogens are focused on specific systems such as reproduction. Crucially, however, the potential for significant health impacts of environmental estrogen exposures on a variety of body systems may have been overlooked. OBJECTIVE: Our aim was to develop and apply a sensitive transgenic zebrafish model to assess real-time effects of environmental estrogens on signaling mechanisms in a whole body system for use in integrated health assessments. METHODS: We created a novel transgenic biosensor zebrafish containing an estrogen-inducible promoter derived with multiple tandem estrogen responsive elements (EREs) and a Gal4ff-UAS system for enhanced response sensitivity. RESULTS: Using our novel estrogen-responsive transgenic (TG) zebrafish, we identified target tissues for environmental estrogens; these tissues have very high sensitivity even at environmentally relevant concentrations. Exposure of the TG fish to estrogenic endocrine-disrupting chemicals (EDCs) induced specific expression of green fluorescent protein (GFP) in a wide variety of tissues including the liver, heart, skeletal muscle, otic vesicle, forebrain, lateral line, and ganglions, most of which have not been established previously as targets for estrogens in fish. Furthermore, we found that different EDCs induced GFP expression with different tissue response patterns and time trajectories, suggesting different potential health effects. CONCLUSION: We have developed a powerful new model for understanding toxicological effects, mechanisms, and health impacts of environmental estrogens in vertebrates.

Solute carrier family 3 member 2 (Slc3a2) controls yolk syncytial layer (YSL) formation by regulating microtubule networks in the zebrafish embryo.

The yolk syncytial layer (YSL) in the zebrafish embryo is a multinucleated syncytium essential for embryo development, but the molecular mechanisms underlying YSL formation remain largely unknown. Here we show that zebrafish solute carrier family 3 member 2 (Slc3a2) is expressed specifically in the YSL and that slc3a2 knockdown causes severe YSL defects including clustering of the yolk syncytial nuclei and enhanced cell fusion, accompanied by disruption of microtubule networks. Expression of a constitutively active RhoA mimics the YSL phenotypes caused by slc3a2 knockdown, whereas attenuation of RhoA or ROCK activity rescues the slc3a2-knockdown phenotypes. Furthermore, slc3a2 knockdown significantly reduces tyrosine phosphorylation of c-Src, and overexpression of a constitutively active Src restores the slc3a2-knockdown phenotypes. Our data demonstrate a signaling pathway regulating YSL formation in which Slc3a2 inhibits the RhoA/ROCK pathway via phosphorylation of c-Src to modulate YSL microtubule dynamics. This work illuminates processes at a very early stage of zebrafish embryogenesis and more generally informs the mechanism of cell dynamics during syncytium formation.

Microtubules regulate migratory polarity through Rho/ROCK signaling in T cells.

BACKGROUND: Migrating leukocytes normally have a polarized morphology with an actin-rich lamellipodium at the front and a uropod at the rear. Microtubules (MTs) are required for persistent migration and chemotaxis, but how they affect cell polarity is not known. METHODOLOGY/PRINCIPAL FINDINGS: Here we report that T cells treated with nocodazole to disrupt MTs are unable to form a stable uropod or lamellipodium, and instead often move by membrane blebbing with reduced migratory persistence. However, uropod-localized receptors and ezrin/radixin/moesin proteins still cluster in nocodazole-treated cells, indicating that MTs are required specifically for uropod stability. Nocodazole stimulates RhoA activity, and inhibition of the RhoA target ROCK allows nocodazole-treated cells to re-establish lamellipodia and uropods and persistent migratory polarity. ROCK inhibition decreases nocodazole-induced membrane blebbing and stabilizes MTs. The myosin inhibitor blebbistatin also stabilizes MTs, indicating that RhoA/ROCK act through myosin II to destabilize MTs. CONCLUSIONS/SIGNIFICANCE: Our results indicate that RhoA/ROCK signaling normally contributes to migration by affecting both actomyosin contractility and MT stability. We propose that regulation of MT stability and RhoA/ROCK activity is a mechanism to alter T-cell migratory behavior from lamellipodium-based persistent migration to bleb-based migration with frequent turning.

T-cell receptor- and CD28-induced Vav1 activity is required for the accumulation of primed T cells into antigenic tissue.

Localization of primed T cells to antigenic tissue is essential for the development of effective immunity. Together with tissue-selective homing molecules, T-cell receptor (TCR)- and CD28-mediated signals have been shown to promote transendothelial migration of specific T cells into nonlymphoid antigen-rich tissue. However, the cellular and molecular requirements for T-cell accumulation to target tissue following their recruitment are largely undefined. The guanine nucleotide exchange factor (GEF) Vav1 has an integral role in coupling TCR and CD28 to signaling pathways that regulate T-cell activation and migration. Here, we have investigated the contribution of TCR- and CD28-induced Vav1 activity to the trafficking and localization of primed HY-specific CD4(+) T cells to antigenic sites. Severe migratory defects displayed by Vav1(-/-) T cells in vitro were fully compensated by a combination of shear flow and chemokines, leading to normal recruitment of Vav1(-/-) T cells in vivo. In contrast, Vav1(-/-) T-cell retention into antigen-rich tissue was severely impaired, reflecting T cells' inability to engage in sustained TCR- and CD28-mediated interactions with tissue-resident antigen-presenting cells (APCs). This novel function of APC-induced, and TCR- and CD28-mediated Vav1 activity in the regulation of effector T-cell immunity highlights its potential as a therapeutic target in T cell-mediated tissue damage.

T cell receptor-induced phosphoinositide-3-kinase p110delta activity is required for T cell localization to antigenic tissue in mice.

The establishment of T cell-mediated inflammation requires the migration of primed T lymphocytes from the blood stream and their retention in antigenic sites. While naive T lymphocyte recirculation in the lymph and blood is constitutively regulated and occurs in the absence of inflammation, the recruitment of primed T cells to nonlymphoid tissue and their retention at the site are enhanced by various inflammatory signals, including TCR engagement by antigen-displaying endothelium and resident antigen-presenting cells. In this study, we investigated whether signals downstream of TCR ligation mediated by the phosphoinositide-3-kinase (PI3K) subunit p110delta contributed to the regulation of these events. T lymphocytes from mice expressing catalytically inactive p110delta displayed normal constitutive trafficking and migratory responses to nonspecific stimuli. However, these cells lost susceptibility to TCR-induced migration and failed to localize efficiently to antigenic tissue. Importantly, we showed that antigen-induced T cell trafficking and subsequent inflammation was abrogated by selective pharmacological inhibition of PI3K p110delta activity. These observations suggest that pharmacological targeting of p110delta activity is a viable strategy for the therapy of T cell-mediated pathology.

Kinase-independent functions for Itk in TCR-induced regulation of Vav and the actin cytoskeleton.

The Tec family kinase Itk is an important regulator of Ca(2+) mobilization and is required for in vivo responses to Th2-inducing agents. Recent data also implicate Itk in TCR-induced regulation of the actin cytoskeleton. We have evaluated the requirements for Itk function in TCR-induced actin polarization. Reduction of Itk expression via small interfering RNA treatment of the Jurkat human T lymphoma cell line or human peripheral blood T cells disrupted TCR-induced actin polarization, a defect that correlated with decreased recruitment of the Vav guanine nucleotide exchange factor to the site of Ag contact. Vav localization and actin polarization could be rescued by re-expression of either wild-type or kinase-inactive murine Itk but not by Itk containing mutations affecting the pleckstrin homology or Src homology 2 domains. Additionally, we find that Itk is constitutively associated with Vav. Loss of Itk expression did not alter gross patterns of Vav tyrosine phosphorylation but appeared to disrupt the interactions of Vav with SLP-76. Expression of membrane-targeted Vav, Vav-CAAX, can rescue the small interfering RNA to Itk-induced phenotype, implicating the alteration in Vav localization as directly contributing to the actin polarization defect. These data suggest a kinase-independent scaffolding function for Itk in the regulation of Vav localization and TCR-induced actin polarization.

Requirement for Tec kinases in chemokine-induced migration and activation of Cdc42 and Rac.

Cell polarization and migration in response to chemokines is essential for proper development of the immune system and activation of immune responses. Recent studies of chemokine signaling have revealed a critical role for PI3-Kinase, which is required for polarized membrane association of pleckstrin homology (PH) domain-containing proteins and activation of Rho family GTPases that are essential for cell polarization and actin reorganization. Additional data argue that tyrosine kinases are also important for chemokine-induced Rac activation. However, how and which kinases participate in these pathways remain unclear. We demonstrate here that the Tec kinases Itk and Rlk play an important role in chemokine signaling in T lymphocytes. Chemokine stimulation induced transient membrane association of Itk and phosphorylation of both Itk and Rlk, and purified T cells from Rlk(-/-)Itk(-/-) mice exhibited defective migration to multiple chemokines in vitro and decreased homing to lymph nodes upon transfer to wt mice. Expression of a dominant-negative Itk impaired SDF-1alpha-induced migration, cell polarization, and activation of Rac and Cdc42. Thus, Tec kinases are critical components of signaling pathways required for actin polarization downstream from both antigen and chemokine receptors in T cells.

Improved generation of C57BL/6J mouse embryonic stem cells in a defined serum-free media.

C57BL/6 is a well-characterized mouse strain that is used extensively for immunological and neurological research. The establishment of C57BL/6 ES cell lines has facilitated the study of gene-altered mice in a pure genetic background-however, relatively few such lines exist. Using a defined media supplement, knockout serum replacement (KSR) with knockout DMEM (KSR-KDMEM), we find that we can readily establish ES cell lines from blastocysts of C57BL/6J mice. Six lines were established, all of which were karyotypically normal and could be maintained in the undifferentiated state on mouse embryonic fibroblast (MEF) feeders. One line was further tested and found to be karyotypically stable and germline competent, both prior to manipulation and after gene targeting. For this cell line, efficiencies of cell cloning and chimera generation were greater when maintained in KSR-KDMEM. Our work suggests that the use of defined serum-free media may facilitate the generation of ES cells from inbred mouse strains.

Pertussis toxin-insensitive activation of the heterotrimeric G-proteins Gi/Go by the NG108-15 G-protein activator.

A ligand-independent activator of heterotrimeric brain G-protein was partially purified from detergent-solubilized extracts of the neuroblastoma-glioma cell hybrid NG108-15. The G-protein activator (NG108-15 G-protein activator (NG-GPA)) increased [(35)S]guanosine 5'-O-(thiotriphosphate) ([(35)S]GTPgammaS) to purified brain G-protein in a magnesium-dependent manner and promoted GDP dissociation from Galpha(o). The NG-GPA also increased GTPgammaS binding to purified, recombinant Galpha(i2), Galpha(i3), and Galpha(o), but minimally altered nucleotide binding to purified transducin. The NG-GPA increased GTPgammaS binding to membrane-bound G-proteins and inhibited basal, forskolin- and hormone-stimulated adenylyl cyclase activity in DDT(1)-MF-2 cell membranes. In contrast to G-protein coupled receptor-mediated activation of heterotrimeric G-proteins in DDT(1)-MF-2 cell membrane preparations, the action of the NG-GPA was not altered by treatment of the cells with pertussis toxin. ADP-ribosylation of purified brain G-protein also failed to alter the increase in GTPgammaS binding elicited by the NG-GPA. Thus, the NG-GPA acts in a manner distinct from that of a G-protein coupled receptor and other recently described receptor-independent activators of G-protein signaling. These data indicate the presence of unexpected regulatory domains on G(i)/G(o) proteins and suggest the existence of pertussis toxin-insensitive modes of signal input to G(i)/G(o) signaling systems.

Beyond calcium: new signaling pathways for Tec family kinases.

The Tec kinases represent the second largest family of mammalian non-receptor tyrosine kinases and are distinguished by the presence of distinct proline-rich regions and pleckstrin homology domains that are required for proper regulation and activation. Best studied in lymphocyte and mast cells, these kinases are critical for the full activation of phospholipase-C gamma (PLC-gamma) and Ca(2+) mobilization downstream of antigen receptors. However, it has become increasingly clear that these kinases are activated downstream of many cell-surface receptors, including receptor tyrosine kinases, cytokine receptors, integrins and G-protein-coupled receptors. Evidence suggests that the Tec kinases influence a wide range of signaling pathways controlling activation of MAP kinases, actin reorganization, transcriptional regulation, cell survival and cellular transformation. Their impact on cellular physiology suggests that the Tec kinases help regulate multiple cellular processes beyond Ca(2+) mobilization.

Activator of G-protein signaling 1 blocks GIRK channel activation by a G-protein-coupled receptor: apparent disruption of receptor signaling complexes.

The Ras-related protein, activator of G-protein signaling 1 (AGS1) or Dexras1, interacts with G(i)/G(o)alpha and activates heterotrimeric G-protein signaling systems independent of a G-protein-coupled receptor (GPCR). As an initial approach to further define the cellular role of AGS1 in GPCR signaling, we determined the influence of AGS1 on the regulation of G(betagamma)-regulated inwardly rectifying K(+) channel (GIRK) current (I(ACh)) by M(2)-muscarinic receptor (M(2)-MR) in Xenopus oocytes. AGS1 expression inhibited receptor-mediated current activation by >80%. Mutation of a key residue (G31V) within the G(1) domain involved in nucleotide binding for Ras-related proteins eliminated the action of AGS1. The inhibition of I(ACh) was not overcome by increasing concentrations of the muscarinic agonist acetylcholine but was progressively lost upon injection of increasing amounts of M(2)-MR cRNA. These data suggest that AGS1 may antagonize GPCR signaling by altering the pool of heterotrimeric G-proteins available for receptor coupling and/or disruption of a preformed signaling complex. Such regulation would be of particular importance for those receptors that exist precoupled to heterotrimeric G-protein and for receptors operating within signaling complexes.