Gαq Proteins Research Articles

OR20-05 Regulation of Mouse Uterine Contractility by Regulator of G-Protein Signalling 2

INTRODUCTION: Uterine contractions in labour are produced by activation of receptors such as FP (PGF2α) and OXTR (oxytocin). Such receptors signal via Gαq proteins that can be selectively turned off by the regulator of G-protein signaling 2 (RGS2). RGS2 expression can be upregulated by Gαs-coupled receptor signaling. PGE2 elicits various uterine effects, some via activation of Gαs-coupled receptors. In primary human myometrial cells, PGE2 enhances RGS2 expression that attenuates subsequent oxytocin-stimulated calcium responses associated with contraction. RGS2 expression is higher in preterm non-labour versus labour human myometrium, suggesting that RGS2 promotes quiescence in the pregnant uterus. To directly study whether RGS2 dampens uterine contraction, we analyzed the ex vivo contractility of pregnant mouse uteri. We hypothesize that loss of RGS2 expression enhances oxytocin and PGF2α-stimulated uterine contractility. METHODS: Wildtype (WT) and RGS2 knockout (KO) female mice (8-14 weeks) were time-mated and euthanized on days 15-20 of pregnancy or during active labour (n=5-11 per group). Uteri were snap frozen for RNA/protein expression analysis by qPCR and western blot. Uterine sections from d18-19 were dissected into longitudinal strips for ex vivo contractility measurement. Tissues were submerged in Krebs-Henseleit buffer and tied to force transducers to measure isometric force development. Tissues were treated with or without 1μM PGE2 for 2 hours. In parallel, pretreated and non-pretreated tissues were exposed to increasing concentrations of oxytocin or PGF2α (0.1-10uM). All tissues were treated with 100mM KCl to determine the maximum contraction at the end of the experiment. Data were analyzed by ANOVA (Bonferroni post-hoc), or Student’s t-test, as appropriate. RESULTS: RGS2 expression in WT uteri sharply decreases at day 19 (p<0.05), remaining low during labour. OXTR and FP receptor expression increase at day 19 (p<0.01 and p<0.05, respectively) and are sustained through labour. Compared to WT, uteri from KO mice produced larger oxytocin-stimulated contractile amplitudes (p<0.05), and more frequent PGF2α-stimulated contractions (p<0.05). PGE2 pretreatment enhanced RGS2 expression in WT uteri, which reduced the peak amplitudes and integrals elicited by subsequent oxytocin treatment, compared to non-pretreated uteri (p<0.05). CONCLUSION: RGS2 may play a pro-quiescent role in pregnancy, since RGS2 loss enhances uterine contractility. Concomitant upregulation of pro-contractile receptors and RGS2 downregulation may facilitate labour activation. This mechanisms may be involved in the etiology of spontaneous preterm labour; loss of RGS2 expression observed in human preterm labour may allow for early contractile activation. Understanding RGS2’s role in quiescence is important to developing more effective strategies for managing preterm labour.

GPCR-induced calcium transients trigger nuclear actin assembly for chromatin dynamics

Although the properties of the actin cytoskeleton in the cytoplasm are well characterized, the regulation and function of nuclear actin filaments are only recently emerging. We previously demonstrated serum-induced, transient assembly of filamentous actin within somatic cell nuclei. However, the extracellular cues, cell surface receptors as well as underlying signaling mechanisms have been unclear. Here we demonstrate that physiological ligands for G protein-coupled receptors (GPCRs) promote nuclear F-actin assembly via heterotrimeric Gαq proteins. Signal-induced nuclear actin responses require calcium release from the endoplasmic reticulum (ER) targeting the ER-associated formin INF2 at the inner nuclear membrane (INM). Notably, calcium signaling promotes the polymerization of linear actin filaments emanating from the INM towards the nuclear interior. We show that GPCR and calcium elevations trigger nuclear actin-dependent alterations in chromatin organization, uncovering a general cellular mechanism by which physiological ligands and calcium promote nuclear F-actin assembly for rapid responses towards chromatin dynamics.

The human cytomegalovirus-encoded G protein–coupled receptor UL33 exhibits oncomodulatory properties

Herpesviruses can rewire cellular signaling in host cells by expressing viral G protein-coupled receptors (GPCRs). These viral receptors exhibit homology to human chemokine receptors, but some display constitutive activity and promiscuous G protein coupling. Human cytomegalovirus (HCMV) has been detected in multiple cancers, including glioblastoma, and its genome encodes four GPCRs. One of these receptors, US28, is expressed in glioblastoma and possesses constitutive activity and oncomodulatory properties. UL33, another HCMV-encoded GPCR, also displays constitutive signaling via Gαq, Gαi, and Gαs proteins. However, little is known about the nature and functional effects of UL33-driven signaling. Here, we assessed UL33's signaling repertoire and oncomodulatory potential. UL33 activated multiple proliferative, angiogenic, and inflammatory signaling pathways in HEK293T and U251 glioblastoma cells. Notably, upon infection, UL33 contributed to HCMV-mediated STAT3 activation. Moreover, UL33 increased spheroid growth in vitro and accelerated tumor growth in different in vivo tumor models, including an orthotopic glioblastoma xenograft model. UL33-mediated signaling was similar to that stimulated by US28; however, UL33-induced tumor growth was delayed. Additionally, the spatiotemporal expression of the two receptors only partially overlapped in HCMV-infected glioblastoma cells. In conclusion, our results unveil that UL33 has broad signaling capacity and provide mechanistic insight into its functional effects. UL33, like US28, exhibits oncomodulatory properties, elicited via constitutive activation of multiple signaling pathways. UL33 and US28 might contribute to HCMV's oncomodulatory effects through complementing and converging cellular signaling, and hence UL33 may represent a promising drug target in HCMV-associated malignancies.

Conformational plasticity of the intracellular cavity of GPCR−G-protein complexes leads to G-protein promiscuity and selectivity

While the dynamics of the intracellular surface in agonist-stimulated GPCRs is well studied, the impact of GPCR dynamics on G-protein selectivity remains unclear. Here, we combine molecular dynamics simulations with live-cell FRET and secondary messenger measurements, for 21 GPCR-G-protein combinations, to advance a dynamic model of the GPCR-G-protein interface. Our data show C terminus peptides of Gαs, Gαi, and Gαq proteins assume a small ensemble of unique orientations when coupled to their cognate GPCRs, similar to the variations observed in 3D structures of GPCR-G-protein complexes. The noncognate G proteins interface with latent intracellular GPCR cavities but dissociate due to weak and unstable interactions. Three predicted mutations in β2-adrenergic receptor stabilize binding of noncognate Gαq protein in its latent cavity, allowing promiscuous signaling through both Gαs and Gαq in a dose-dependent manner. This demonstrates that latent GPCR cavities can be evolved, by design or nature, to tune G-protein selectivity, giving insights to pluridimensional GPCR signaling.

Mechanisms of G Protein‐Selectivity in Muscarinic Acetylcholine Receptor Family

Muscarinic acetylcholine receptors are integral membrane proteins that regulate physiological activity. Acetylcholine is a neurotransmitter that binds to the extracellular side of these G protein coupled receptors (GPCRs) activating the Gα subunit of the trimeric G protein inside the cell. There are 5 different muscarinic receptor subtypes (M1–M5), where the same ligand acetylcholine causes M2 and M4 receptors to preferentially couple to the Gαi subunit and the receptors M1, M3, and M5 to preferentially couple to the Gαq subunit. This selectivity of Gα subunits mediates different secondary messenger pathways in the cell: Gαi being responsible for deactivating adenyl cyclase activity and Gαq being responsible for activating phospholipase C. We are using computational biophysical methods to probe the mechanisms of G protein selectivity of this muscarinic receptor family. We have built structural models for the M1 and M2 receptors coupled to Gαi or Gαq proteins and used molecular dynamics simulations to obtain relaxed structures of these complexes, so that the thermodynamic analysis of the receptor G protein interactions can be conducted. The analyses are beginning to show that thermodynamically, some complexes are more stable than others in agreement with corresponding biochemical experiments. In addition, some receptor ‐ G protein interactions may be kinetically controlled. Overall, these studies are beginning to provide us with the missing mechanistic insight into the G protein selectivity of the muscarinic receptors.Support or Funding InformationWe acknowledge the CSUPERB New Investigator Grant. We thank the RISE (GM063787) program for their student support.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

LPA1 receptor activation induces PKCα nuclear translocation in glioblastoma cells.

Lysophosphatidic acid (LPA) is a ubiquitous lysophospholipid that induces a wide range of cellular processes such as wound healing, differentiation, proliferation, migration, and survival. LPA signaling is increased in a number of cancers. In Glioblastoma (GBM), the most aggressive brain tumor, autotaxin the enzyme that produces LPA and its receptor LPA1 are overexpressed. LPA1 is preferentially couple to Gαq proteins in these tumors that in turn activates PKCs. PKCs are involved in many cellular processes including proliferation and metastasis. In this study, we aimed to determine if a classical PKC (α isozyme), could be activated through LPA1 in GBM cell lines and if this activation impacts on cell number. We found that LPA1 induces PKCα translocation to the nucleus, but not to the cell membrane after LPA treatment and the cell number diminished when LPA1/PKCα signaling was blocked, suggesting a relevant role of LPA1 and PKCα in GBM growth.

Gβγ signaling to the chemotactic effector P-REX1 and mammalian cell migration is directly regulated by Gαq and Gα13 proteins

G protein-coupled receptors stimulate Rho guanine nucleotide exchange factors that promote mammalian cell migration. Rac and Rho GTPases exert opposing effects on cell morphology and are stimulated downstream of Gβγ and Gα12/13 or Gαq, respectively. These Gα subunits might in turn favor Rho pathways by preventing Gβγ signaling to Rac. Here, we investigated whether Gβγ signaling to phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchange factor 1 (P-REX1), a key Gβγ chemotactic effector, is directly controlled by Rho-activating Gα subunits. We show that pharmacological inhibition of Gαq makes P-REX1 activation by Gq/Gi-coupled lysophosphatidic acid receptors more effective. Moreover, chemogenetic control of Gi and Gq by designer receptors exclusively activated by designer drugs (DREADDs) confirmed that Gi differentially activates P-REX1. GTPase-deficient GαqQL and Gα13QL variants formed stable complexes with Gβγ, impairing its interaction with P-REX1. The N-terminal regions of these variants were essential for stable interaction with Gβγ. Pulldown assays revealed that chimeric Gα13-i2QL interacts with Gβγ unlike to Gαi2-13QL, the reciprocal chimera, which similarly to Gαi2QL could not interact with Gβγ. Moreover, Gβγ was part of tetrameric Gβγ-GαqQL-RGS2 and Gβγ-Gα13-i2QL-RGS4 complexes, whereas Gα13QL dissociated from Gβγ to interact with the PDZ-RhoGEF-RGS domain. Consistent with an integrated response, Gβγ and AKT kinase were associated with active SDF-1/CXCL12-stimulated P-REX1. This pathway was inhibited by GαqQL and Gα13QL, which also prevented CXCR4-dependent cell migration. We conclude that a coordinated mechanism prioritizes Gαq- and Gα13-mediated signaling to Rho over a Gβγ-dependent Rac pathway, attributed to heterotrimeric Gi proteins.

Atypical activation of the G protein Gαq by the oncogenic mutation Q209P

The causative role of G protein-coupled receptor (GPCR) pathway mutations in uveal melanoma (UM) has been well-established. Nearly all UMs bear an activating mutation in a GPCR pathway mediated by G proteins of the Gq/11 family, driving tumor initiation and possibly metastatic progression. Thus, targeting this pathway holds therapeutic promise for managing UM. However, direct targeting of oncogenic Gαq/11 mutants, present in ∼90% of UMs, is complicated by the belief that these mutants structurally resemble active Gαq/11 WT. This notion is solidly founded on previous studies characterizing Gα mutants in which a conserved catalytic glutamine (Gln-209 in Gαq) is replaced by leucine, which leads to GTPase function deficiency and constitutive activation. Whereas Q209L accounts for approximately half of GNAQ mutations in UM, Q209P is as frequent as Q209L and also promotes oncogenesis, but has not been characterized at the molecular level. Here, we characterized the biochemical and signaling properties of Gαq Q209P and found that it is also GTPase-deficient and activates downstream signaling as efficiently as Gαq Q209L. However, Gαq Q209P had distinct molecular and functional features, including in the switch II region of Gαq Q209P, which adopted a conformation different from that of Gαq Q209L or active WT Gαq, resulting in altered binding to effectors, Gβγ, and regulators of G-protein signaling (RGS) proteins. Our findings reveal that the molecular properties of Gαq Q209P are fundamentally different from those in other active Gαq proteins and could be leveraged as a specific vulnerability for the ∼20% of UMs bearing this mutation.

Dampening of neurotransmitter action: molecular similarity within the melatonin structure.

Melatonin initiates physiologic and therapeutic responses in various tissues through binding to poorly defined MT receptors regulated by G-proteins and purine nucleotides. Melatonin's interaction with other G-protein regulated receptors, including those of serotonin, is unclear. This study explores the potential for the interaction of melatonin with nucleotide and receptor ligand structures. The study uses a computational program to investigate relative molecular similarity by the comparative superimposition and quantitative fitting of molecular structures to adenine and guanine nucleotide templates. A minimum energy melatonin conformer replicates the nucleotide fits of ligand structures that regulate Gαi and Gαq proteins via serotonin, dopamine, opioid, α-adrenoceptor, and muscarinic receptor classes. The same conformer also replicates the nucleotide fits of ligand structures regulating K+ and Ca2+ ion channels. The acyl-methoxy distance within the melatonin conformer matches a carbonyl-hydroxyl distance in guanine nucleotide. Molecular similarity within the melatonin and ligand structures relates to the established effects of melatonin on cell receptors regulated by purine nucleotides in cell signal transduction processes. Pharmacologic receptor promiscuity may contribute to the widespread effects of melatonin.

Abstract 968: Targeting FAK inhibits YAP-dependent tumor growth in uveal melanoma

Abstract Activating mutations in GNAQ and GNA11 (known as GNAQ oncogenes), which encode constitutively active Gαq proteins, occur in ~90% of uveal melanoma (UM) and 4% of skin cutaneous melanoma (SKCM) respectively. UM is the most common primary cancer of the eye in adults, diagnosed in about 2,500 adults in the US every year. Approximately 50% of UM patients develop liver metastasis within 5-10 years after diagnosis, independently of the successful treatment of the primary lesions. There are effective therapeutic strategies for primary UM lesions, but UM metastatic disease is refractory to current chemotherapies and immune checkpoint inhibitors, and most patients with UM metastasis die within a year. The MEK inhibitor selumetinib has been recently approved for UM treatment, but MEK inhibition has nearly no impact on UM patient overall survival. To date, there are no effective treatment options for metastatic UM. We used bioinformatics approaches to identify systems vulnerabilities that can be exploited for UM treatment. Using the TCGA UM dataset (N=80) and SKCM samples that do not harbor Gαq genomic alterations (N=209) as control, (i) we first identified genes that are highly overexpressed in UM. We validated this approach by confirming that the genes over- and under-expressed in UM and Gαq-altered SKCM are highly overlapping (hyper-geometric p&amp;lt;1E-198 and p&amp;lt;1E-232 respectively). ii) We then filtered the genes for those whose inactivation leads to better patient survival in UM based on TCGA survival data. (iii) We finally used large datasets of gene essentiality and drug response screens in cancer cells to identify genes that are predicted to reduce cell viability when targeted in Gαq-expressing tumors. By filtering these genes for those that can be targeted by approved and experimental drugs (N=756), we identified 7 candidate druggable genes. Among them, the top was PTK2, which encodes a non- receptor tyrosine kinase known as FAK (focal adhesion kinase). By dissecting the Gαq signaling pathway, we found that Gαq actives FAK (p379-FAK) through a Trio-RhoA signaling pathway, independent of PLC-β regulated second messenger systems. RNA-seq of UM cells treated with a FAK inhibitor (FAKi, VS-4718) revealed that YAP gene-expression signatures were highly sensitive to FAKi. Of interest, YAP is a key regulator of cancer growth, and we have shown that YAP is highly activated by Gαq in UM, contributing to tumor growth (Cancer Cell. 2014). We found FAK regulates YAP activation by Gαq through a mechanism that involves YAP tyrosine phosphorylation on Y357. Interestingly, CRISPR-Cas9 KO of FAK and clinically relevant FAKi showed strong inhibition of UM growth in vitro and in vivo, concomitant with repression of YAP-regulated gene programs. Overall, our computational biology approach revealed that FAK may represent a novel precision therapeutic target human diseases initiated by aberrant Gαq signaling, including UM, the first identified Gαq-driven human malignancy. Citation Format: Xiaodong Feng, Damiano Rigiracciolo, Joo-Sang Lee, Huwate Yeerna, Nadia Arang, Simone Lubrano, David D. Schlaepfer, Pablo Tamayo, Eytan Ruppin, J. Silvio Gutkind. Targeting FAK inhibits YAP-dependent tumor growth in uveal melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 968.

TGR5 contributes to hepatic cystogenesis in rodents with polycystic liver diseases through cyclic adenosine monophosphate/Gαs signaling.

TGR5 contributes to hepatic cystogenesis by increasing cAMP and enhancing cholangiocyte proliferation; our data suggest that a TGR5 antagonist alone or concurrently with somatostatin receptor agonists represents a potential therapeutic approach in polycystic liver disease. (Hepatology 2017;66:1197-1218).

Native metabotropic glutamate receptor 4 depresses synaptic transmission through an unusual Gαq transduction pathway

In cerebellar cortex, mGlu4 receptors located on parallel fibers play an essential role in normal motor function, but the molecular mechanisms involved are not yet completely understood. Using a strategy combining biochemical and electrophysiological approaches in the rodent cerebellum, we demonstrate that presynaptic mGlu4 receptors control synaptic transmission through an atypical activation of Gαq proteins. First, the Gαq subunit, PLC and PKC signaling proteins present in cerebellar extracts are retained on affinity chromatography columns grafted with different sequences of the cytoplasmic domain of mGlu4 receptor. The i2 loop and the C terminal domain were used as baits, two domains that are known to play a pivotal role in coupling selectivity and efficacy. Second, in situ proximity ligation assays show that native mGlu4 receptors and Gαq subunits are in close physical proximity in cerebellar cortical slices. Finally, electrophysiological experiments demonstrate that the molecular mechanisms underlying mGlu4 receptor-mediated inhibition of transmitter release at cerebellar Parallel Fiber (PF) – Molecular Layer Interneuron (MLI) synapses involves the Gαq-PLC signaling pathway.Taken together, our results provide compelling evidence that, in the rodent cerebellar cortex, mGlu4 receptors act by coupling to the Gαq protein and PLC effector system to reduce glutamate synaptic transmission.

ARF6 Is an Actionable Node that Orchestrates Oncogenic GNAQ Signaling in Uveal Melanoma

Activating mutations in Gαq proteins, which form the α subunit of certain heterotrimeric G proteins, drive uveal melanoma oncogenesis by triggering multiple downstream signaling pathways, including PLC/PKC, Rho/Rac, and YAP. Here we show that the small GTPase ARF6 acts as a proximal node of oncogenic Gαq signaling to induce all of these downstream pathways as well as β-catenin signaling. ARF6 activates these diverse pathways through a common mechanism: the trafficking of GNAQ and β-catenin from the plasma membrane to cytoplasmic vesicles and the nucleus, respectively. Blocking ARF6 with a small-molecule inhibitor reduces uveal melanoma cell proliferation and tumorigenesis in a mouse model, confirming the functional relevance of this pathway and suggesting a therapeutic strategy for Gα-mediated diseases.

Minimal contribution of ERK1/2-MAPK signalling towards the maintenance of oncogenic GNAQQ209P-driven uveal melanomas in zebrafish.

Mutations affecting Gαq proteins are pervasive in uveal melanoma (UM), suggesting they ‘drive’ UM pathogenesis. The ERK1/2-MAPK pathway is critical for cutaneous melanoma development and consequently an important therapeutic target. Defining the contribution of ERK1/2-MAPK signalling to UM development has been hampered by the lack of an informative animal model that spontaneously develops UM. Towards this end, we engineered transgenic zebrafish to express oncogenic GNAQQ209P in the melanocyte lineage. This resulted in hyperplasia of uveal melanocytes, but with no evidence of malignant progression, nor perturbation of skin melanocytes. Combining expression of oncogenic GNAQQ209P with p53 inactivation resulted in earlier onset and even more extensive hyperplasia of uveal melanocytes that progressed to UM. Immunohistochemistry revealed only weak immunoreactivity to phosphorylated (p)ERK1/2 in established uveal tumours—in contrast to strong immunoreactivity in oncogenic RAS-driven skin lesions—but ubiquitous positive staining for nuclear Yes-associated protein (YAP). Moreover, no changes were observed in pERK1/2 levels upon transient knockdown of GNAQ or phospholipase C-beta (PLC-β) inhibition in the majority of human UM cell lines we tested harbouring GNAQ mutations. In summary, our findings demonstrate a weak correlation between oncogenic GNAQQ209P mutation and sustained ERK1/2-MAPK activation, implying that ERK1/2 signalling is unlikely to be instrumental in the maintenance of GNAQQ209P-driven UMs.

G protein-coupled receptors: signalling and regulation by lipid agonists for improved glucose homoeostasis.

G protein-coupled receptors (GPCRs) play a pivotal role in cell signalling, controlling many processes such as immunity, growth, cellular differentiation, neurological pathways and hormone secretions. Fatty acid agonists are increasingly recognised as having a key role in the regulation of glucose homoeostasis via stimulation of islet and gastrointestinal GPCRs. Downstream cell signalling results in modulation of the biosynthesis, secretion, proliferation and anti-apoptotic pathways of islet and enteroendocrine cells. GPR40 and GPR120 are activated by long-chain fatty acids (>C12) with both receptors coupling to the Gαq subunit that activates the Ca(2+)-dependent pathway. GPR41 and GPR43 are stimulated by short-chain fatty acids (C2-C5), and activation results in binding to Gαi that inhibits the adenylyl cyclase pathway attenuating cAMP production. In addition, GPR43 also couples to the Gαq subunit augmenting intracellular Ca(2+) and activating phospholipase C. GPR55 is specific for cannabinoid endogenous agonists (endocannabinoids) and non-cannabinoid fatty acids, which couples to Gα12/13 and Gαq proteins, leading to enhancing intracellular Ca(2+), extracellular signal-regulated kinase 1/2 (ERK) phosphorylation and Rho kinase. GPR119 is activated by fatty acid ethanolamides and binds to Gαs utilising the adenylate cyclase pathway, which is dependent upon protein kinase A. Current research indicates that GPCR therapies may be approved for clinical use in the near future. This review focuses on the recent advances in preclinical diabetes research in the signalling and regulation of GPCRs on islet and enteroendocrine cells involved in glucose homoeostasis.

ARMS/Kidins220 and synembryn-B levels regulate NGF-mediated secretion.

Proper development of the nervous system requires a temporally and spatially orchestrated set of events including differentiation, synapse formation and neurotransmission. Nerve growth factor (NGF) acting through the TrkA neurotrophin receptor (also known as NTRK1) regulates many of these events. However, the molecular mechanisms responsible for NGF-regulated secretion are not completely understood. Here, we describe a new signaling pathway involving TrkA, ARMS (also known as Kidins220), synembryn-B and Rac1 in NGF-mediated secretion in PC12 cells. Whereas overexpression of ARMS blocked NGF-mediated secretion, without affecting basal secretion, a decrease in ARMS resulted in potentiation. Similar effects were observed with synembryn-B, a protein that interacts directly with ARMS. Downstream of ARMS and synembryn-B are Gαq and Trio proteins, which modulate the activity of Rac1 in response to NGF. Expression of dominant-negative Rac1 rescued the secretion defects of cells overexpressing ARMS or synembryn-B. Thus, this neurotrophin pathway represents a new mechanism responsible for NGF-regulated secretion.

Imipramine administration induces changes in the phosphorylation of FAK and PYK2 and modulates signaling pathways related to their activity

BackgroundAntidepressants can modify neuronal functioning by affecting many levels of signal transduction pathways that are involved in neuroplasticity. We investigated whether the phosphorylation status of focal adhesion kinase (FAK/PTK2) and its homolog, PYK2/PTK2B, and their complex with the downstream effectors (Src kinase, p130Cas, and paxillin) are affected by administration of the antidepressant drug, imipramine. The treatment influence on the levels of ERK1/2 kinases and their phosphorylated forms (pERK1/2) or the Gαq, Gα11 and Gα12 proteins were also assessed. MethodsRats were injected with imipramine (10mg/kg, twice daily) for 21days. The levels of proteins investigated in their prefrontal cortices were measured by Western blotting. ResultsImipramine induced contrasting changes in the phosphorylation of FAK and PYK2 at Tyr397 and Tyr402, respectively. The decreased FAK phosphorylation and increased PYK2 phosphorylation were reflected by changes in the levels of their complex with Src and p130Cas, which was observed predominantly after chronic imipramine treatment. Similarly only chronic imipramine decreased the Gαq expression while Gα11 and Gα12 proteins were untouched. Acute and chronic treatment with imipramine elevated ERK1 and ERK2 total protein levels, whereas only the pERK1 was significantly affected by the drug. ConclusionThe enhanced activation of PYK2 observed here could function as compensation for FAK inhibition. General significanceThese data demonstrate that treatment with imipramine, which is a routine in counteracting depressive disorders, enhances the phosphorylation of PYK2, a non-receptor kinase instrumental in promoting synaptic plasticity. This effect documents as yet not considered target in the mechanism of imipramine action.

Evidence against dopamine D1/D2 receptor heteromers.

Hetero-oligomers of G-protein-coupled receptors have become the subject of intense investigation because their purported potential to manifest signaling and pharmacological properties that differ from the component receptors makes them highly attractive for the development of more selective pharmacological treatments. In particular, dopamine D1 and D2 receptors have been proposed to form hetero-oligomers that couple to Gαq proteins, and SKF83959 has been proposed to act as a biased agonist that selectively engages these receptor complexes to activate Gαq and thus phospholipase C. D1/D2 heteromers have been proposed as relevant to the pathophysiology and treatment of depression and schizophrenia. We used in vitro bioluminescence resonance energy transfer (BRET), ex vivo analyses of receptor localization and proximity in brain slices, and behavioral assays in mice to characterize signaling from these putative dimers/oligomers. We were unable to detect Gαq or Gα11 protein coupling to homomers or heteromers of D1 or D2 receptors using a variety of biosensors. SKF83959-induced locomotor and grooming behaviors were eliminated in D1 receptor knockout mice, verifying a key role for D1-like receptor activation. In contrast, SKF83959-induced motor responses were intact in D2 receptor and Gαq knockout mice, as well as in knock-in mice expressing a mutant Ala286-CaMKIIα, that cannot autophosphorylate to become active. Moreover, we found that in the shell of the nucleus accumbens, even in neurons in which D1 and D2 receptor promoters are both active, the receptor proteins are segregated and do not form complexes. These data are not compatible with SKF83959 signaling through Gαq or through a D1–D2 heteromer and challenge the existence of such a signaling complex in the adult animals that we used for our studies.

A Cell-Permeable Inhibitor to Trap Gαq Proteins in the Empty Pocket Conformation

In spite of the crucial role of heterotrimeric G proteins as molecular switches transmitting signals from G protein-coupled receptors, their selective manipulation with small molecule, cell-permeable inhibitors still remains an unmet challenge. Here, we report that the small molecule BIM-46187, previously classified as pan-G protein inhibitor, preferentially silences Gαq signaling in a cellular context-dependent manner. Investigations into its mode of action reveal that BIM traps Gαq in the empty pocket conformation by permitting GDP exit but interdicting GTP entry, a molecular mechanism not yet assigned to any other small molecule Gα inhibitor to date. Our data show that Gα proteins may be "frozen" pharmacologically in an intermediate conformation along their activation pathway and propose a pharmacological strategy to specifically silence Gα subclasses with cell-permeable inhibitors.

Functional activation of Gαq via serotonin2A (5-HT2A) and muscarinic acetylcholine M1 receptors assessed by guanosine-5׳-O-(3-[35S]thio)triphosphate ([35S]GTPγS) binding/immunoprecipitation in rat brain membranes

Functional coupling between serotonin2A (5-HT2A) receptors and Gαq proteins in native brain membranes has been sparsely reported thus far. In the present study, the guanosine-5׳-O-(3-[35S]thio)triphosphate ([35S]GTPγS) binding assay combined with immunoprecipitation using magnetic beads (Dynabeads Protein A) coated with anti-Gαq antibody was developed. Under experimental conditions optimised for assay constituents (GDP, MgCl2, and NaCl), for contents of membrane protein, anti-Gαq antibody, and Dynabeads Protein A, and for the incubation period, 5-HT stimulated specific [35S]GTPγS binding to Gαq in rat cerebral cortical membranes in a concentration-dependent and saturable manner, with a signal/noise ratio that was sufficiently high for further detailed pharmacological characterisation. This characterisation revealed an involvement of 5-HT2A receptors. Activation of Gαq proteins was also detectable by the addition of carbachol via muscarinic acetylcholine M1 receptors, (-)-epinephrine, and dopamine, but not by L-glutamate or (±)-baclofen. When 5-HT2A receptors and M1 receptors were stimulated simultaneously, there were non-additive effects, indicating that the two receptors were coupled to the same components of Gαq proteins in the rat cerebral cortex. This method will serve as an efficacious strategy for neurobiological investigations aimed at elucidating the physiological and pathological implications of signal transduction systems mediated via Gαq proteins coupled with 5-HT2A receptors and muscarinic acetylcholine M1 receptors.