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Characterization of G-Protein alpha Subunits in the Gq Class: Expression in Murine Tissues and in Stromal and Hematopoietic Cell Lines
Abstract
Murine G alpha 14 and G alpha 15 cDNAs encode distinct alpha subunits of heterotrimeric guanine nucleotide-binding proteins (G proteins). These alpha subunits are related to members of the Gq class and share certain sequence characteristics with G alpha q, G alpha 11, and G alpha 16, such as the absence of a pertussis toxin ADP-ribosylation site. G alpha 11 and G alpha q are ubiquitously expressed among murine tissues but G alpha 14 is predominantly expressed in spleen, lung, kidney, and testis whereas G alpha 15 is primarily restricted to hematopoietic lineages. Among hematopoietic cell lines, G alpha 11 mRNA is found in all cell lines tested, G alpha q is expressed widely but is not found in most T-cell lines, G alpha 15 is predominantly expressed in myeloid and B-cell lineages, and G alpha 14 is expressed in bone marrow adherent (stromal) cells, certain early myeloid cells, and progenitor B cells. Polyclonal antisera produced from synthetic peptides that correspond to two regions of G alpha 15 react with a protein of 42 kDa expressed in B-cell membranes and in Escherichia coli transformed with G alpha 15 cDNA. The expression patterns that were observed in mouse tissues and cell lines indicate that each of the alpha subunits in the Gq class may be involved in pertussis toxin-insensitive signal-transduction pathways that are fundamental to hematopoietic cell differentiation and function.
... Highly specific tissue distribution was also observed for G15 and G14 belonging to the PLC-activating Gq/11 class of G-proteins. While G11 and Gq are widely distributed, G14 is more expressed in the thyroid 19 , spleen, lung, kidney, and testis 20,21 . G15 is primarily restricted to hematopoietic lineages 20 and epithelial cells, which are characterized by a high rate of cell turnover 22,23 . ...
... While G11 and Gq are widely distributed, G14 is more expressed in the thyroid 19 , spleen, lung, kidney, and testis 20,21 . G15 is primarily restricted to hematopoietic lineages 20 and epithelial cells, which are characterized by a high rate of cell turnover 22,23 . 33 . ...
... Tissue-specific distribution of several Gq/11 class G proteins is well described 20,22 . Therefore, distinct activation of individual Gαq/11 by pathway-specific compounds leading to tissue-specific activation of muscarinic receptors is expected. ...
Selective activation of individual subtypes of muscarinic receptors is a promising way to safely alleviate a wide range of pathological conditions in the central nervous system and the periphery as well. The flexible G-protein interface of muscarinic receptors allows them to interact with several G-proteins with various efficacy, potency, and kinetics. Agonists biased to the particular G-protein mediated pathway may result in selectivity among muscarinic subtypes and, due to the non-uniform expression of individual G-protein alpha subunits, possibly achieve tissue specificity. Here, we demonstrate that novel tetrahydropyridine-based agonists exert specific signalling profiles in coupling with individual G-protein α subunits. These signalling profiles profoundly differ from the reference agonist carbachol. Moreover, coupling with individual Gα induced by these novel agonists varies among subtypes of muscarinic receptors which may lead to subtype selectivity. Thus, the novel tetrahydropyridine-based agonist can contribute to the elucidation of the mechanism of pathway-specific activation of muscarinic receptors and serve as a starting point for the development of desired selective muscarinic agonists.
... There are four families of G proteins that are classified according to their α subunit: G s , G i , G q and G 12/13 where G s and G i participate in cAMP-dependent pathways, G q activates phospholipase C-dependent pathways, and G 12/13 activates a small family of GTPases [3][4][5]. In addition, the G q family is further divided into four G q proteins due to the use of four different α polypeptides, αq, α11, α14, and α15/16 [6]. The G α15/16 polypeptide has a restricted tissue expression and is predominantly expressed in myeloid and B-cell lineages while G αq and G α11 are ubiquitously expressed [6]. ...
... In addition, the G q family is further divided into four G q proteins due to the use of four different α polypeptides, αq, α11, α14, and α15/16 [6]. The G α15/16 polypeptide has a restricted tissue expression and is predominantly expressed in myeloid and B-cell lineages while G αq and G α11 are ubiquitously expressed [6]. ...
CCR6 is a chemokine receptor highly implicated in inflammatory diseases and could be a potential therapeutic target; however, no therapeutic agents targeting CCR6 have progressed into clinical evaluation. Development of a high-throughput screening assay for CCR6 should facilitate the identification of novel compounds against CCR6. To develop a cell-based assay, RBL-2H3 cells were transfected with plasmids encoding β-hexosaminidase and CCR6. Intracellular calcium mobilization of transfected cells was measured with a fluorescent substrate using the activity of released hexosaminidase as readout of the assay. This stable, transfected cell showed a specific signal to the background ratio of 19.1 with low variability of the signal along the time. The assay was validated and optimized for high-throughput screening. The cell-based calcium mobilization assay responded to the specific CCR6 ligand, CCL20, in a dose-dependent manner with an EC50 value of 10.72 nM. Furthermore, the assay was deemed robust and reproducible with a Z’ factor of 0.63 and a signal window of 7.75. We have established a cell-based high-throughput calcium mobilization assay for CCR6 receptor. This assay monitors calcium mobilization, due to CCR6h activation by CCL20, using hexosaminidase activity as readout. This assay was proved to be robust, easy to automate and could be used as method for screening of CCR6 modulators.
... Furthermore, once activated, Gα15 is poorly constrained by β-arrestin and RGS desensitization 6,7 . Possibly related to these signaling peculiarities, Gα15 expression is highly restricted, primarily to hematopoietic cells 8 . Gα15 may have evolved by providing a sensitive and potent signaling node in host cells dedicated to respond to a rapidly changing microenvironment. ...
... Gα15 de novo expression distinguishes transformed pancreas from other cancers. In mice 8 and humans 4 , normal tissues predominately express GNA15 in hematopoietic cells and skin progenitor cells. Systemic data extracted from the Genotype-Tissue Expression (GTEx) Project confirms these findings and includes two additional epithelia, esophagus mucosa and vagina (Supp. ...
The GNA15 gene is ectopically expressed in human pancreatic ductal adenocarcinoma cancer cells. The encoded Gα15 protein can promiscuously redirect GPCR signaling toward pathways with oncogenic potential. We sought to describe the distribution of GNA15 in adenocarcinoma from human pancreatic specimens and to analyze the mechanism driving abnormal expression and the consequences on signaling and clinical follow-up. We detected GNA15 expression in pre-neoplastic pancreatic lesions and throughout progression. The analysis of biological data sets, primary and xenografted human tumor samples, and clinical follow-up shows that elevated expression is associated with poor prognosis for GNA15, but not any other GNA gene. Demethylation of the 5′ GNA15 promoter region was associated with ectopic expression of Gα15 in pancreatic neoplastic cells, but not in adjacent dysplastic or non-transformed tissue. Down-modulation of Gα15 by shRNA or CRISPR/Cas9 affected oncogenic signaling, and reduced adenocarcimoma cell motility and invasiveness. We conclude that de novo expression of wild-type GNA15 characterizes transformed pancreatic cells. The methylation pattern of GNA15 changes in preneoplastic lesions coincident with the release a transcriptional blockade that allows ectopic expression to persist throughout PDAC progression. Elevated GNA15 mRNA correlates with poor prognosis. In addition, ectopic Gα15 signaling provides an unprecedented mechanism in the early steps of pancreas carcinogenesis distinct from classical G protein oncogenic mutations described previously in GNAS and GNAQ/GNA11.
... GNA15 is a member of the GNA gene family, which is crucial in regulating cell proliferation and apoptosis. It is expressed in highly specific cell types, such as hematopoietic [52] and epithelial cells [53], during certain stages of differentiation. GNA15 has been identified as highly expressed in small intestinal neuroendocrine neoplasia [49] and pancreatic ductal adenocarcinoma [54], correlating with poor survival. ...
Purpose
This study aims to explore the contribution of differentially expressed programmed cell death genes (DEPCDGs) to the heterogeneity of serous ovarian cancer (SOC) through single-cell RNA sequencing (scRNA-seq) and assess their potential as predictors for clinical prognosis.
Methods
SOC scRNA-seq data were extracted from the Gene Expression Omnibus database, and the principal component analysis was used for cell clustering. Bulk RNA-seq data were employed to analyze SOC-associated immune cell subsets key genes. CIBERSORT and single-sample gene set enrichment analysis (ssGSEA) were utilized to calculate immune cell scores. Prognostic models and nomograms were developed through univariate and multivariate Cox analyses.
Results
Our analysis revealed that 48 DEPCDGs are significantly correlated with apoptotic signaling and oxidative stress pathways and identified seven key DEPCDGs (CASP3, GADD45B, GNA15, GZMB, IL1B, ISG20, and RHOB) through survival analysis. Furthermore, eight distinct cell subtypes were characterized using scRNA-seq. It was found that G protein subunit alpha 15 (GNA15) exhibited low expression across these subtypes and a strong association with immune cells. Based on the DEGs identified by the GNA15 high- and low-expression groups, a prognostic model comprising eight genes with significant prognostic value was constructed, effectively predicting patient overall survival. Additionally, a nomogram incorporating the RS signature, age, grade, and stage was developed and validated using two large SOC datasets.
Conclusion
GNA15 emerged as an independent and excellent prognostic marker for SOC patients. This study provides valuable insights into the prognostic potential of DEPCDGs in SOC, presenting new avenues for personalized treatment strategies.
... Here, using three muscarinic agonists, we demonstrate that other G protein families can also functionally interact with the M 3 R and activate canonical second messenger signaling pathways. From the data collected in this study, a relatively preserved rank order of Gq>>Gi>Gs>G13 across assays was observed, although in vivo functional rank order is likely to be determined by G protein expression differences, which can vary dramatically between cell and tissue types (El-Haibi et al., 2013;Wilkie et al., 1991). We demonstrate functional G protein association to the M 3 R in a variety of assays, including radioligand binding, purified protein, and multiple signaling assays in live cells. ...
The M3 muscarinic acetylcholine receptor (M3R) is a G protein-coupled receptor (GPCR) that regulates important physiologic processes, including vascular tone, bronchoconstriction, and insulin secretion. It is expressed on a wide variety of cell types, including pancreatic beta, smooth muscle, neuronal, and immune cells. Agonist binding to the M3R is thought to initiate intracellular signaling events primarily through the heterotrimeric G protein Gq. However, reports differ on the ability of M3R to couple to other G proteins beyond Gq. Using members from the four primary G protein families (Gq, Gi, Gs, and G13) in radioligand binding, GTP turnover experiments, and cellular signaling assays, including live cell G protein dissociation and second messenger assessment of cAMP and inositol trisphosphate, we show that other G protein families, particularly Gi and Gs, can also interact with the human M3R. We further show that these interactions are productive as assessed by amplification of classic second messenger signaling events. Our findings demonstrate that the M3R is more promiscuous with respect to G protein interactions than previously appreciated. SIGNIFICANCE STATEMENT: The study reveals that the human M3 muscarinic acetylcholine receptor (M3R), known for its pivotal roles in diverse physiological processes, not only activates intracellular signaling via Gq as previously known but also functionally interacts with other G protein families such as Gi and Gs, expanding our understanding of its versatility in mediating cellular responses. These findings signify a broader and more complex regulatory network governed by M3R and have implications for therapeutic targeting.
... Gαq, encoded by the GNAQ gene, belongs to the Gq/11 subfamily of heterotrimeric G proteins, and it is ubiquitously expressed in mammalian cells (Wilkie et al., 1991). The Gαq links GPCRs to activate Phospholipase C (PLC) that hydrolyzes membrane phospholipids phosphatidylinositol bisphosphate, PIP2 to release inositol trisphosphate (IP3) and diacylglycerol (DAG) -two important second messengers causing Ca 2+ mobilization and protein kinase C (PKC) activation (Ho et al., 2009). ...
Decidualization, denoting the transformation of endometrial stromal cells into specialized decidual cells, is a prerequisite for normal embryo implantation and a successful pregnancy in human. Here, we demonstrated that knockout of Gαq lead to an aberrantly enhanced inflammatory state during decidualization. Furthermore, we showed that deficiency of Gαq resulted in over-activation of nuclear factor (NF)-κB signaling, due to the decreased expression of NFκBIA, which encode the IκB protein and is the negative regulator for NF-κB. Mechanistically, Gαq deficiency decreased the Protein kinase D (PKD, also called PKCμ) phosphorylation levels, leading to attenuated HDAC5 phosphorylation and thus its nuclear export. Aberrantly high level of nuclear HDAC5 retarded histone acetylation to inhibit the induced NFκBIA transcription during decidualization. Consistently, pharmacological activation of the PKD/PKCμ or inhibition of the HDAC5 restored the inflammatory state and proper decidual response. Finally, we disclosed that over-active inflammatory state in Gαq-deficient decidua deferred the blastocyst hatching and adhesion in vitro, and the decidual expression of Gαq was significantly lower in women with recurrent pregnancy loss compared with normal pregnancy. In brief, we showed here that Gαq as a key regulator of the inflammatory cytokine's expression and decidual homeostasis in response to differentiation cues, which is required for successful implantation and early pregnancy.
... The Gαq family of G proteins comprises four family members. The ubiquitously expressed Gαq and Gα11, Gα14 mainly expressed in liver, lung and kidney, and Gα15/16 (orthologues in mouse/human), specifically expressed in hematopoietic cells [86][87][88]. In this review we will focus on Gαq, the most widely studied member. ...
All processes in human physiology relies on homeostatic mechanisms which require the activation of specific control circuits to adapt the changes imposed by external stimuli. One of the critical modulators of homeostatic balance is autophagy, a catabolic process that is responsible of the destruction of long-lived proteins and organelles through a lysosome degradative pathway. Identification of the mechanism underlying autophagic flux is considered of great importance as both protective and detrimental functions are linked with deregulated autophagy. At the mechanistic and regulatory levels, autophagy is activated in response to diverse stress conditions (food deprivation, hyperthermia and hypoxia), even a novel perspective highlight the potential role of physical forces in autophagy modulation. To understand the crosstalk between all these controlling mechanisms could give us new clues about the specific contribution of autophagy in a wide range of diseases including vascular disorders, inflammation and cancer. Of note, any homeostatic control critically depends in at least two additional and poorly studied interdependent components: a receptor and its downstream effectors. Addressing the selective receptors involved in autophagy regulation is an open question and represents a new area of research in this field. G-protein coupled receptors (GPCRs) represent one of the largest and druggable targets membrane receptor protein superfamily. By exerting their action through G proteins, GPCRs play fundamental roles in the control of cellular homeostasis. Novel studies have shown Gαq, a subunit of heterotrimeric G proteins, as a core modulator of mTORC1 and autophagy, suggesting a fundamental contribution of Gαq-coupled GPCRs mechanisms in the control of this homeostatic feedback loop. To address how GPCR-G proteins machinery integrates the response to different stresses including oxidative conditions and mechanical stimuli, could provide deeper insight into new signaling pathways and open potential and novel therapeutic strategies in the modulation of different pathological conditions.
... In addition, G15 and G16, which belong to the Gq/11 family, can couple promiscuously to a wide variety of GPCRs, and therefore can theoretically link Gs-, Gi/o-and G12/13-coupled receptors to PLCB activity (Offermanns and Simon, 1995). This could, in turn, expand functional consequences of ARCND2 variants to a group of cell types and tissues expressing G15 and G16 (Wilkie et al., 1991). Free Gβγ subunits from Gi/o-coupled receptors can also stimulate PLCB activity (Smrcka, 2008), although our assays did not test whether the ARCND2 variants could also interfere with Gβγ-mediated phospholipase activity. ...
Auriculocondylar syndrome 2 (ARCND2) is a rare autosomal dominant craniofacial malformation syndrome linked to multiple genetic variants in the coding sequence of phospholipase C β4 (PLCB4). PLCB4 is a direct signaling effector of the endothelin receptor type A (EDNRA)-Gq/11 pathway, which establishes the identity of neural crest cells (NCCs) that form lower jaw and middle ear structures. However, the functional consequences of PLCB4 variants on EDNRA signaling is not known. Here, we show, using multiple signaling reporter assays, that known PLCB4 variants resulting from missense mutations exert a dominant-negative interference over EDNRA signaling. In addition, using CRISPR/Cas9, we find that F0 mouse embryos modeling one PLCB4 variant have facial defects recapitulating those observed in hypomorphic Ednra mouse models, including a bone that we identify as an atavistic change in the posterior palate/oral cavity. Remarkably, we have identified a similar osseous phenotype in a child with ARCND2. Our results identify the disease mechanism of ARCND2, demonstrate that the PLCB4 variants cause craniofacial differences and illustrate how minor changes in signaling within NCCs may have driven evolutionary changes in jaw structure and function.
This article has an associated First Person interview with the first author of the paper.
... Jusqu'à présent, aucune publication n'a montré un couplage de Gα 15 avec le hMC4R. De plus, cette protéine G est surtout retrouvée au niveau de lignées hématopoïétiques et ne serait présente qu'à l'état de traces au niveau du cerveau (Wilkie et al., 1991). ...
Le sous-type 4 des récepteurs aux mélanocortines (MC4R) est une cible thérapeutique pour le traitement des troubles de l’homéostasie énergétique. Aucun traitement efficace n’est pour l’instant sur le marché. Dans ce contexte, une banque de toxines animales synthétiques a été criblée, aboutissant à 286 hits. Au vue de ce chiffre exceptionnel, cinq des toxines les plus originales au niveau séquence ont été sélectionnées et caractérisées pharmacologiquement. N-TRTX-Preg1a de l’araignée Poecilotheria regalis et N-BUTX-Ptr1a du scorpion Parabuthus transvaalicus sont les premières toxines agonistes du sous-type 1 des récepteurs aux mélanocortines (MC1R). Ce récepteur stimule la pigmentation de la peau et joue un rôle clé dans la prévention du mélanome. Les deux toxines forment chacune un nouveau groupe au sein de leur famille de toxines, respectivement les knottin et les toxines CSαβ. L’étude des conotoxines N-CTX-Can1a et N-CTX-Mil1a, a été abandonnée du fait de la complexité de leur synthèse et de leur faible affinité. N-CTX-Ltg1a, issue du cône Conus lithoglyphus, est une toxine prometteuse. Sa séquence ne ressemble à aucune famille connue de toxine et elle présente une affinité nanomolaire sur MC4R. Avec 4 cystéines, elle peut être oxydée de trois façons différentes, rendant son étude complexe. Nous avons simplifié progressivement cette toxine pour aboutir à un peptide linéaire simple à produire et plus affin d’un facteur 3 que la toxine de départ avec une sélectivité d’un facteur 3 à 13 par rapport aux autres MCRs. La Hunger-Toxin (HT1-0) antagonise l’activation des voies de signalisation Gs, G15 et β-arrestine du MC4R. Des études de relation structure-activité ont permis de délimiter les zones importantes dans l’interaction avec MC4R et serviront à l’optimisation future du peptide. Prochainement, la confirmation in vivo de l’effet anorexigène pourrait ainsi ouvrir la voie vers un nouveau traitement de la cachexie.
... Two types of OT-R have been identified in the CNS. They are metabotropic receptors, containing seven transmembrane domains, that act through protein-coupled G q or G i [31,32]. The OT-R is expressed in different areas of the CNS, for example, the OT-R coupled to protein G q is expressed in ventral tegmental area (VTA), nucleus accumbens (NAc), anterior cingulate cortex (ACC), central and basolateral amygdala (CeA and BLA, respectively), medial preoptic area (mPOA), and anterior and ventromedial hypothalamus, etc. [33][34][35]. ...
Oxytocin (OT) and vasopressin (AVP) are hypothalamic neuropeptides classically associated with their regulatory role in reproduction, water homeostasis, and social behaviors. Interestingly, this role has expanded in recent years and has positioned these neuropeptides as therapeutic targets for various neuropsychiatric diseases such as autism, addiction, schizophrenia, depression, and anxiety disorders. Due to the chemical-physical characteristics of these neuropeptides including short half-life, poor blood-brain barrier penetration, promiscuity for AVP and OT receptors (AVP-R, OT-R), novel ligands have been developed in recent decades. This review summarizes the role of OT and AVP in neuropsychiatric conditions, as well as the findings of different OT-R and AVP-R agonists and antagonists, used both at the preclinical and clinical level. Furthermore, we discuss their possible therapeutic potential for central nervous system (CNS) disorders.
... However, it is tempting to speculate that lower SERT levels in the frontal cortex may mask an up-regulation of the transporter turnover rate in male mice in this brain region, since apparent 5HT uptake rates are unchanged compared to wild-type (Table 1). In the midbrain we are mainly expressed in non-brain cell types (Wilkie et al., 1991). ...
The regulation of the serotonin transporter (SERT) by guanine nucleotide‐binding protein alpha (Gα) q was investigated using Gαq knockout mice. In the absence of Gαq, SERT‐mediated uptake of 5‐hydroxytryptamine (5HT) was enhanced in midbrain and frontal cortex synaptosomes, but only in female mice. The mechanisms underlying this sexual dimorphism were investigated using quantitative western blot analysis revealing brain region‐specific differences. In the frontal cortex, SERT protein expression was decreased in male knockout mice, seemingly explaining the sex‐dependent variation in SERT activity. The differential expression of Gαi1 in female mice contributes to the sex differences in the midbrain. In fact, Gαi1 levels inversely correlate with 5HT uptake rates across both sexes and genotypes. Likely due to differential SERT regulation as well as sex differences in the expression of tryptophan hydroxylase 2, Gαq knockout mice also displayed sex‐ and genotype‐dependent alterations in total 5HT tissue levels as determined by high‐performance liquid chromatography. Gαq inhibitors, YM‐254890 and BIM‐46187, differentially affected SERT activity in both, synaptosomes and cultured cells. YM‐254890 treatment mimicked the effect of Gαq knockout in the frontal cortex. BIM‐46187, which promotes the nucleotide‐free form of Gα proteins, substantially inhibited 5HT uptake, prompting us to hypothesise that Gαq interacts with SERT similarly as with G‐protein‐coupled receptors and inhibits SERT activity by modulating transport‐associated conformational changes. Taken together, our findings reveal a novel mechanism of SERT regulation and impact our understanding of sex differences in diseases associated with dysregulation of serotonin transmission, such as depression and anxiety.
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... Melanopsin was selected because it couples to the ubiquitous excitatory G q/11 pathway [20][21][22], meaning that the opsin/melanopsin chimeras could be utilised to light sensitise retinal cell types, such as bipolar cells, that do not natively express visual opsins. G q/11 second messengers are ubiquitous in the retina [23] and found throughout the body, with at least one or both of G q and G 11 found in every cell type screened so far [23][24][25][26]. By contrast, rhodopsin does not activate the G q/11 pathway in mammals [27] and the native G protein partner of rhodopsin, the rod isoform of transducin (G t [rod] ), is exclusively expressed in rod photoreceptors [28]. ...
Human opsin-based photopigments have great potential as light-sensitisers, but their requirement for phototransduction cascade-specific second messenger proteins may restrict their functionality in non-native cell types. In this study, eight chimeric human opsins were generated consisting of a backbone of either a rhodopsin (RHO) or long-wavelength-sensitive (LWS) opsin and intracellular domains from Gq/11-coupled human melanopsin. Rhodopsin/melanopsin chimeric opsins coupled to both Gi and Gq/11 pathways. Greater substitution of the intracellular surface with corresponding melanopsin domains generally showed greater Gq/11 activity with a decrease in Gi activation. Unlike melanopsin, rhodopsin and rhodopsin/melanopsin chimeras were dependent upon exogenous chromophore to function. By contrast, wild type LWS opsin and LWS/melanopsin chimeras showed only weak Gi activation in response to light, whilst Gq/11 pathway activation was not detected. Immunocytochemistry demonstrated that chimeric opsins with more intracellular domains of melanopsin were less likely to be trafficked to the plasma membrane. This study demonstrates the importance of Gα coupling efficiency to the speed of cellular responses and created human opsins with a unique combination of properties to expand the range of customised optogenetic biotools for basic research and translational therapies.
... G q and G 11 are the most ubiquitously expressed members of this family with 88.5% amino acid sequence identity (Astesano et al., 1999), whereas the expression of G 14 is more restricted and normally seen in specialised cells. G 15/16 , on the other hand, is limited to a subset of haematopoietic cells (Amatruda et al., 1991;Wilkie et al., 1991;Davignon et al., 1996;Mizuno and Itoh, 2009 (Mizuno and Itoh, 2009;Kozasa et al., 1993). G q, G 11 , (Lyon and Tesmer, 2013). ...
The heterotrimeric G-proteins are critically important intracellular signalling
molecules that regulate fundamental processes in cellular homeostasis. A
unique bacterial toxin, Pasteurella multocida toxin (PMT) modifies 3 of the 4
families of G-proteins (Gq, Gi, and G12) by deamidation which leads to a
plethora of downstream signalling. PMT induces drastic morphological
changes such as loss of adherence to the matrix, foci and stress fibre
formation in Swiss 3T3 and Vero cells. PMT is mitogenic for many cell types
but not all, and the work reported in this thesis aims to compare two cell
lines that respond differently to PMT.
Swiss 3T3 and Vero cells were treated with different concentrations of PMT
to determine the effects on cell proliferation, cytoskeletal reorganisation and
cell death. PMT induced prominent cytoskeletal changes, mitogenic and
anti-cell death responses in Swiss 3T3 cells while delayed cytoskeletal
changes with no evidence of mitogenicity and cell death were observed in
Vero cells. PMT modified G-proteins at different times in Swiss 3T3 and
Vero cells. The PMT-induced anti-cell death response in Swiss 3T3 cells
was dose-dependent while the delayed cytoskeletal response in Vero cells
was linked to the late PMT-mediated G12 activation. The amino acid
sequence of Gα12 differed in the two cell types – the G12 subunit in Vero
cells is missing N-terminal cysteine residue, which may have contributed to
the differences. Gq/11 signalling is active and sustained in Swiss 3T3 cells,
but not in Vero cells. G may have inhibited adenylyl cyclase activity so it is
unknown whether Gi signalling is active and sustained over the 3-day period
as the forskolin-stimulated cAMP level decreased in both serum-starved
untreated and PMT-treated cell lines. In summary, I have identified changes
in both the primary effect of PMT on the two cell types and also on
downstream signalling that is likely to reflect the differential cell response.
... The present study showed that the activation of IκB by TSH is dependent on G15. G15 is recognized as a hematopoietic-specific G protein subtype (19). However, in this study, we found that it is expressed in liver and thyroid tissues as well as by macrophages. ...
Thyroid-stimulating hormone (TSH) treatment activates IκB/NFκB (p65) and ERK, P38 in macrophages, but how these pathways are activated, and how they contribute to the pro-inflammatory effect of TSH on macrophages remain unknown. TSH receptor (TSHR) is coupled to four subfamilies of G proteins (Gs, Gi/o, Gq/11 and G12/13) for its downstream signalling. This study investigated the G protein subtypes responsible for the pro-inflammatory effect of TSH on macrophages. qPCR showed that Gi2, Gi3, Gas, Gq, G11, G12, G13, G15 are abundantly expressed by macrophages. The contribution of different G-protein pathways to the pro-inflammatory effect was studied by the corresponding inhibitors or siRNA interference. While TSH-induced IκB phosphorylation was not inhibited by Gs inhibitor NF449, Gi inhibitor pertussis toxin, Gq or G11 siRNA, it was blocked by phospholipase C inhibitor U73122 or G15 siRNA interference. TSH-induced ERK and P38 phosphorylation was blocked by G13 but not G12 siRNA interference. Interfering either G13 or G15 was able to block the pro-inflammatory effect of TSH on macrophages. The present study demonstrate that TSH activates macrophage inflammation by G13/ERK-P38/Rho GTPases and G15/PLC/PKCs/IκB pathways.
... Among these, Gs and Gi families regulate adenylyl cyclase activity, while Gq activates phospholipase Cβ, and G12/13 can activate small GTPase families [8]. The Gq family further can be divided into four members, such as Gq, G11, G14, and G15/16 [9,10], and their respective α subunits [11]. Every protein in G protein signaling is subjected to regulation that may alter the expression, localization, or activity. ...
Cell signaling plays critical role in health and disease. The normal functioning of body depends on the homeostasis of immunity players. One of the very important cell signaling participants is G protein-coupled receptor (GPCR). GPCRs transduce extracellular signals into target cell by binding to and activating different G proteins (G αβγ, families Gi, Gs, Gq/11, G12/13) leading to range of different functions. Abnormal GPCRs signaling leads to various abnormalities, including but not limited to, cancer, pain, cardiac problems, and asthma. Mutations, which lead to activation or inactivation of GPCR pathways, permanently alter the pathways controlled by these receptors. A large number of human cancer incidence is a consequence of genetic abnormalities in signaling pathways, which influence cell division. Some bacteria and pathogens may interfere with the GPCR signaling pathways for their survival and immune evasion. Inhibition of GPCR signaling by small inhibitors is a novel way to treat various pathological conditions. There are several types of GPCRs in human genome, which due to their central role in health and disease, are the target of many commercially available drugs. Importantly, GPCRs have huge impact on drug discovery and approximately 30% of current drug targets are GPCRs. There is a need of further studies to explore more the role of G protein and the GPCRs in human health and how certain mutations can lead to disease state. Such studies may be important to adjust the signaling pathways for health improvement.
... Notably, YM-254890 was less effective on GNAQ Q209L compared to the other Gαq variants. As the Gαq family has additional members, GNA14 and GNA15/16 [42,43], we tested the selectivity of YM-254890 acts on Gαq family members, using GNA14 Q205L [44] and GNA15 Q212L [45], constitutively active mutants corresponding to GNAQ/ 11 Q209L . For comparison, we also included the GNAS Q227L oncogene [46], a member of the G s family as a control. ...
Uveal melanoma (UM) is a currently untreatable form of melanoma with a 50% mortality rate. Characterization of the essential signaling pathways driving this cancer is critical to develop target therapies. Activating mutations in the Gαq signaling pathway at the level of GNAQ, GNA11, or rarely CYSLTR2 or PLCβ4 are considered alterations driving proliferation in UM and several other neoplastic disorders. Here, we systematically examined the oncogenic signaling output of various mutations recurrently identified in human tumors. We demonstrate that CYSLTR2 → GNAQ/11 → PLCβ act in a linear signaling cascade that, via protein kinase C (PKC), activates in parallel the MAP-kinase and FAK/Yes-associated protein pathways. Using genetic ablation and pharmacological inhibition, we show that the PKC/RasGRP3/MAPK signaling branch is the essential component that drives the proliferation of UM. Only inhibition of the MAPK branch but not the FAK branch synergizes with inhibition of the proximal cascade, providing a blueprint for combination therapy. All oncogenic signaling could be extinguished by the novel GNAQ/11 inhibitor YM-254890, in all UM cells with driver mutation in the Gαq subunit or the upstream receptor. Our findings highlight the GNAQ/11 → PLCβ → PKC → MAPK pathway as the central signaling axis to be suppressed pharmacologically to treat for neoplastic disorders with Gαq pathway mutations.
... The heterotrimeric G protein Gq is a conserved regulator of neurotransmission in 50 metazoans. Gq is highly expressed in neurons in mammals and in the nematode C. 51 elegans (Wilkie et al. 1991;Lackner et al. 1999). In its canonical signaling pathway, Gq 52 activates phospholipase Cβ (PLCβ) to cleave the membrane lipid phosphatidylinositol 53 4,5-bisphosphate (PIP2) into inositol trisphosphate (IP3) and diacylglycerol (DAG) 54 (Rhee 2001). ...
The heterotrimeric G protein Gq regulates neuronal activity through distinct downstream effector pathways. In addition to the canonical Gq effector phospholipase Cβ, the small GTPase Rho was recently identified as a conserved effector of Gq. To identify additional molecules important for Gq signaling in neurons, we performed a forward genetic screen in the nematode Caenorhabditis elegans for suppressors of the hyperactivity and exaggerated waveform of an activated Gq mutant. We isolated two mutations affecting the MAP kinase scaffold protein KSR-1 and found that KSR-1 modulates locomotion downstream of or in parallel to the Gq-Rho pathway. Through epistasis experiments, we found that the core ERK MAPK cascade is required for Gq-Rho regulation of locomotion, but that the canonical ERK activator LET-60/Ras may not be required. Through neuron-specific rescue experiments, we found that the ERK pathway functions in head acetylcholine neurons to control Gq-dependent locomotion. Additionally, expression of activated LIN-45/Raf in head acetylcholine neurons is sufficient to cause an exaggerated waveform phenotype and hypersensitivity to the acetylcholinesterase inhibitor aldicarb, similar to an activated Gq mutant. Taken together, our results suggest that the ERK MAPK pathway modulates the output of Gq-Rho signaling to control locomotion behavior in C. elegans .
... Gaq, Gq protein a subunit, encoded by the GNAQ gene, belongs to the Gq/11 subfamily of heterotrimeric G proteins, and it is ubiquitously expressed in mammalian cells (13,14). GNAQ (Gaq) initially attracted attention for its physiological significance in the cardiovascular system (15). ...
Although guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) constitute the largest cell surface membrane receptor family and transduce thousands of extracellular signals into the cytoplasm, only four kinds of G protein α subunits (Gαs, Gαi/o, Gαq/11, and Gα12/13) are coupled to regulate cAMP or phosphatidylinositol signals. Growing evidence suggests that viruses tend to hijack GPCRs and harness their activated intracellular signaling pathways. Thus, understanding the roles of G protein signaling will further uncover the GPCR signaling pathways that are exploited by viruses. In this study, we demonstrate that the expression of GNAQ (Gq α subunit) was downregulated during viral infection and that small interfering RNA-mediated GNAQ knockdown protected host cells from both vesicular stomatitis virus (VSV) and HSV type 1 infection. Meanwhile, VSV and HSV type 1 replication was reduced significantly in Gnaq-deficient macrophages. Accordingly, the VSV distribution in the liver, spleen, and lung was reduced in Gnaq-deficient mice during VSV infection, and Gnaq-deficient mice were much more resistant to VSV infection than wild-type mice. Mechanistically, GNAQ limits type I IFN production through the canonical PLC-β/Ca2+/CALNA signaling pathway, which has been demonstrated to dephosphorylate virus-activated TANK-binding kinase 1 (TBK1). Thus, our data demonstrate that GNAQ negatively regulates the antiviral innate immune responses in a calcineurin-dependent manner. These findings also provide insights into the function and cross-talk of the classic GPCR signaling pathway with antiviral innate immune responses and suggest a potential therapeutic role for GNAQ in controlling viral diseases.
... elegans (Wilkie et al. 1991;Lackner et al. 1999). In its canonical signaling pathway, Gq activates phospholipase Cβ (PLCβ) to cleave the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) into inositol trisphosphate (IP3) and diacylglycerol (DAG) (Rhee 2001). ...
The heterotrimeric G protein Gq regulates neuronal activity through distinct downstream effector pathways. In addition to the canonical Gq effector phospholipase Cb, the small GTPase Rho was recently identified as a conserved effector of Gq. To identify additional molecules important for Gq signaling in neurons, we performed a forward genetic screen in the nematodeCaenorhabditis elegansfor suppressors of the hyperactivity and exaggerated waveform of an activated Gq mutant. We isolated two mutations affecting the MAP kinase scaffold protein KSR-1 and found that KSR-1 modulates locomotion downstream of or in parallel to the Gq-Rho pathway. Through epistasis experiments, we found that the core ERK MAPK cascade is required for Gq-Rho regulation of locomotion, but that the canonical ERK activator LET-60/Ras may not be required. Through neuron-specific rescue experiments, we found that the ERK pathway functions in head acetylcholine neurons to control Gq-dependent locomotion. Additionally, expression of activated LIN-45/Raf in head acetylcholine neurons is sufficient to cause an exaggerated waveform phenotype and hypersensitivity to the acetylcholinesterase inhibitor aldicarb, similar to an activated Gq mutant. Taken together, our results suggest that the ERK MAPK pathway modulates the output of Gq-Rho signaling to control locomotion behavior inC. elegans.
... Molecular inhibition of gustducin significantly abrogated sucralose-mediated protection of LPS-induced permeability (Fig. 5Cii). The G protein G␣q, which is highly expressed in the lung, has also been identified to play a role in sweet taste sensing (75,80). Molecular inhibition of G␣q was performed in LMVECs using siRNA (Fig. 5Di). ...
A hallmark of acute respiratory distress syndrome (ARDS) is pulmonary vascular permeability. In these settings, loss of barrier integrity is mediated by cell-contact disassembly and actin-remodelling. Studies into molecular mechanisms responsible for improving microvascular barrier function are therefore vital in the development of therapeutic targets for reducing vascular permeability in ARDS. The sweet taste receptor, T1R3 is a GPCR, activated following exposure to sweet molecules, to trigger a gustducin-dependent signal cascade. In recent years, extraoral locations for T1R3 have been identified however, no studies have focused on T1R3 within the vasculature. We hypothesise that activation of T1R3, in the pulmonary vasculature, plays a role in regulating endothelial barrier function in settings of ARDS. Our study demonstrated expression of T1R3 within the pulmonary vasculature, with a drop in expression levels following exposure to barrier disruptive agents. Exposure of lung microvascular endothelial cells to the intensely sweet molecule, sucralose, attenuated LPS- and thrombin-induced endothelial barrier dysfunction. Likewise, sucralose exposure attenuated bacteria-induced lung edema formation in vivo. Inhibition of sweet taste signalling, through zinc sulfate, T1R3 or G-protein siRNA, blunted the protective effects of sucralose on the endothelium. Sucralose significantly reduced LPS-induced increased expression or phosphorylation of key signalling molecules, Src, PAK, MLC2, HSP27 and p110αPI3K. Activation of T1R3, by sucralose, protects the pulmonary endothelium from edemagenic-agent-induced barrier disruption, potentially through abrogation of Src/PAK/p110αPI3K-mediated cell-contact disassembly and Src/MLC2/HSP27-mediated actin-remodelling. Identification of sweet taste sensing in the pulmonary vasculature may represent a novel therapeutic target to protect the endothelium in settings of ARDS.
... The Gα proteins Gαq and Gα11 are distinct gene products from the same chromosome (Wilkie et al. 1991) that have the same number of amino acids and essentially identical structures and functions (Kamato et al. 2015). Both stimulate phospholipase C-β (PLCβ) isoforms with similar efficiency (Ku et al. 1995;Strakova and Soloff 1997), thus leading to the hydrolysis of phosphatidylinositol 4,5-biphosphate (PIP 2 ), generating inositol 1,4,5-triphosphate (IP3) and 1,2-dicyaglycerol (DAG). ...
Oxytocin (OT), a hypothalamic neuropeptide involved in regulating the social behaviour of all vertebrates, has been proposed as a treatment for a number of neuropsychiatric disorders characterised by deficits in the social domain. Over the last few decades, advances focused on understanding the social effects of OT and its role in physiological conditions and brain diseases, but much less has been done to clarify the molecular cascade of events involved in mediating such effects and in particular the cellular and molecular pharmacology of OT and its target receptor (OTR) in neuronal and glial cells.
... G q is one of the four types of heterotrimeric G protein a subunits in animals (Wilkie et al. 1992). G q is widely expressed in the mammalian brain (Wilkie et al. 1991) where it acts to stimulate neuronal activity (Krause et al. 2002;Gamper et al. 2004;Coulon et al. 2010). Unlike mammals, which have four members of the G q family, Caenorhabditis elegans has only a single G q a gene egl-30 (Brundage et al. 1996). ...
The heterotrimeric G protein Gq positively regulates neuronal activity and synaptic transmission. Previously, the Rho guanine nucleotide exchange factor Trio was identified as a direct effector of Gq that acts in parallel to the canonical Gq effector phospholipase C. Here we examine how Trio and Rho act to stimulate neuronal activity downstream of Gq in the nematode Caenorhabditis elegans Through two forward genetic screens, we identify the cation channels NCA-1 and NCA-2, orthologs of mammalian NALCN, as downstream targets of the Gq/Rho pathway. By performing genetic epistasis analysis using dominant activating mutations and recessive loss-of-function mutations in the members of this pathway, we show that NCA-1 and NCA-2 act downstream of Gq in a linear pathway. Through cell-specific rescue experiments, we show that function of these channels in head acetylcholine neurons is sufficient for normal locomotion in C. elegans Our results suggest that NCA-1 and NCA-2 are physiologically relevant targets of neuronal Gq-Rho signaling in C. elegans.
... The G s and G i families regulate adenylyl cyclase activity, while G q activates phospholipase C (PLC) b and G 12/13 can activate small GTPase families [16]. The G q family consists of four members-G q , G 11 , G 14 and G 15/16 [17,18] and their respective a subunits are thus Ga q , Ga 11 , Ga 14 and Ga 15/16 . Two of these isoforms, Ga q and Ga q/11 , share 88% similarity at the amino acid sequence, and are the most prominent and ubiquitously expressed proteins in this family and are the topic of this review [19]. ...
Seven transmembrane G protein-coupled receptors (GPCRs) have gained much interest in recent years as it is the largest class among cell surface receptors. G proteins lie in the heart of GPCRs signalling and therefore can be therapeutically targeted to overcome complexities in GPCR responses and signalling. G proteins are classified into four families (Gi, Gs, G12/13 and Gq); Gq is further subdivided into four classes. Among them Gαq and Gαq/11 isoforms are most crucial and ubiquitously expressed; these isoforms are almost 88% similar at their amino acid sequence but may exhibit functional divergences. However, uncertainties often arise about Gαq and Gαq/11 inhibitors, these G proteins might also have suitability to the invention of novel-specific inhibitors for each isoforms. YM-254890 and UBO-QIC are discovered as potent inhibitors of Gαq functions and also investigated in thrombin protease-activated receptor (PAR)-1 inhibitors and platelet aggregation inhibition. The most likely G protein involved in PAR-1 stimulates responses is one of the Gαq family isoforms. In this review, we highlight the molecular structures and pharmacological responses of Gαq family which may reflect the biochemical and molecular role of Gαq and Gαq/11. The advanced understanding of Gαq and Gαq/11 role in GPCR signalling may shed light on our understanding on cell biology, cellular physiology and pathophysiology and also lead to the development of novel therapeutic agents for a number of diseases.
... 2B,C), which is clearly distinct from cells in the myeloid lineages and bone marrow (i to iv and vii, Fig. 2B,C). Ingenuity IPA analysis revealed many enriched alternatively spliced genes involved in various canonical pathways, including those in PI3K signaling in B lymphocytes and the regulation of IL-2 expression in naïve and activated T lymphocytes, in the HIPPO pathway known to play a critical role in hematopoietic stem cells (Jansson and Larsson 2012), and in Gα signaling, which is fundamental to hematopoietic cell differentiation and function ( Fig. 2D; Supplemental Table S5B; Wilkie et al. 1991). These observations suggest that regulated splicing of these genes contributes to hematopoietic lineage commitment and proliferation. ...
Myelodysplastic syndromes (MDS) are heterogeneous myeloid disorders with prevalent mutations in several splicing factors, but the splicing programs linked to specific mutations or MDS in general remain to be systematically defined. We applied RASL-seq, a sensitive and cost-effective platform, to interrogate 5502 annotated splicing events in 169 samples from MDS patients or healthy individuals. We found that splicing signatures associated with normal hematopoietic lineages are largely related to cell signaling and differentiation programs, whereas MDS-linked signatures are primarily involved in cell cycle control and DNA damage responses. Despite the shared roles of affected splicing factors in the 3' splice site definition, mutations in U2AF1, SRSF2, and SF3B1 affect divergent splicing programs, and interestingly, the affected genes fall into converging cancer-related pathways. A risk score derived from 11 splicing events appears to be independently associated with an MDS prognosis and AML transformation, suggesting potential clinical relevance of altered splicing patterns in MDS.
... Thus, we interpret the Ga 15/16 signal as further support of Ga s coupling, because we did not observe GPR37L1 signaling through either Ga i/o or Ga q/11 family members (and to our knowledge, there are no examples of GPCRs that couple exclusively to Ga 15/16 ). Furthermore, GPR37L1 abundance does not correlate with that of Ga 15/16 ; the former is abundant in the CNS (8), and the latter is abundant in stem cells and thymic epithelial cells (31) and has a low abundance in the brain (38). ...
Little is known about the pharmacology or physiology of GPR37L1, a G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor that is abundant in the cerebellum. Mice deficient in this receptor exhibit precocious cerebellar development and hypertension. We showed that GPR37L1 coupled to the G protein Gas when heterologously expressed in cultured cells in the absence of any added ligand, whereas a mutant receptor that lacked the amino terminus was inactive. Conversely, inhibition of ADAMs (a disintegrin and metalloproteases) enhanced receptor activity, indicating that the presence of the amino terminus is necessary for GPR37L1 signaling. Metalloprotease-dependent processing of GPR37L1 was evident in rodent cerebellum, where we detected predominantly the cleaved, inactive form. However, comparison of the accumulation of cAMP (adenosine 3′, 5′-monophosphate) in response to phosphodiesterase inhibition in cerebellar slice preparations from wild-type and GPR37L1-null mice showed that some constitutive signaling remained in the wild-type mice. In reporter assays of Gαs or Gαi signaling, the synthetic, prosaposin-derived peptide prosaptide (TX14A) did not increase GPR37L1 activity. Our data indicate that GPR37L1 may be a constitutively active receptor, or perhaps its ligand is present under the conditions that we used for analysis, and that the activity of this receptor is instead controlled by signals that regulate metalloprotease activity in the tissue.
... G protein α-subunit family members were first identified using affinity purification (Pang and Sternweis, 1989; and molecular cloning strategies (Strathmann and Simon, 1990;. GNAQ expression has been observed in the spleen, lung, kidney, pancreas, liver, testis, and bone-marrow adherent stromal cells (Nakamura et al., 1991;Wilkie et al., 1991;Zigman et al., 1994). The results of our study showed that GNAQ mRNA was expressed in caput, corpus, and cauda epididymis and testes, but abundantly expressed in caput and cauda epididymis. ...
The reproductive function of GNΑQ, a member of the G protein alpha subunit family, has been extensively studied in humans and rats. However, no data is available on its status in ruminants. The objectives of this study were to evaluate the expression pattern of the G-protein subunit Galphaq (GNΑQ) in the testis and epididymis of sheep by polymerase chain reaction (PCR). The mRNA expression levels were detected by real-time fluorescent quantitative PCR, and cellular localization of GNΑQ in the testis and epididymis was examined by immunohistochemistry. Additionally, GNΑQ protein was qualitatively evaluated via western blot, with the results indicating that similarities between GNΑQ mRNA levels from sheep was highly conserved with those observed in Bos taurus and Sus scrofa. Our results also indicated that GNΑQ exists in the caput and cauda epididymis of sheep, while GNΑQ in the testis and epididymis was localized to Leydig cells, spermatogonial stem cells, spermatocytes, Sertoli cells, spermatid, principal cells, and epididymis interstitial cells. The concentrations of GNΑQ mRNA and protein in the caput and cauda epididymis were significantly greater than those observed in the corpus epididymis (p < 0.01) and testis (p < 0.05). Our results indicated that GNΑQ exists at high concentrations in the caput and cauda epididymis of sheep, suggesting that GNΑQ may play an important role in gonad development and sperm maturation.
Decidualization, denoting the transformation of endometrial stromal cells into specialized decidual cells, is a prerequisite for normal embryo implantation and a successful pregnancy in human. Here, we demonstrated that knockout of Gαq lead to an aberrantly enhanced inflammatory state during decidualization. Furthermore, we showed that deficiency of Gαq resulted in over-activation of nuclear factor (NF)-κB signaling, due to the decreased expression of NFκBIA, which encode the IκB protein and is the negative regulator for NF-κB. Mechanistically, Gαq deficiency decreased the Protein kinase D (PKD, also called PKCμ) phosphorylation levels, leading to attenuated HDAC5 phosphorylation and thus its nuclear export. Aberrantly high level of nuclear HDAC5 retarded histone acetylation to inhibit the induced NFκBIA transcription during decidualization. Consistently, pharmacological activation of the PKD/PKCμ or inhibition of the HDAC5 restored the inflammatory state and proper decidual response. Finally, we disclosed that over-active inflammatory state in Gαq-deficient decidua deferred the blastocyst hatching and adhesion in vitro, and the decidual expression of Gαq was significantly lower in women with recurrent pregnancy loss compared with normal pregnancy. In brief, we showed here that Gαq as a key regulator of the inflammatory cytokine’s expression and decidual homeostasis in response to differentiation cues, which is required for successful implantation and early pregnancy.
Decidualization, denoting the transformation of endometrial stromal cells into specialized decidual cells, is a prerequisite for normal embryo implantation and a successful pregnancy in human. Here, we demonstrated that knockout of Gαq lead to an aberrantly enhanced inflammatory state during decidualization. Furthermore, we showed that deficiency of Gαq resulted in over-activation of nuclear factor (NF)-κB signaling, due to the decreased expression of NFκBIA, which encode the IκB protein and is the negative regulator for NF-κB. Mechanistically, Gαq deficiency decreased the Protein kinase D (PKD, also called PKCμ) phosphorylation levels, leading to attenuated HDAC5 phosphorylation and thus its nuclear export. Aberrantly high level of nuclear HDAC5 retarded histone acetylation to inhibit the induced NFκBIA transcription during decidualization. Consistently, pharmacological activation of the PKD/PKCμ or inhibition of the HDAC5 restored the inflammatory state and proper decidual response. Finally, we disclosed that over-active inflammatory state in Gαq-deficient decidua deferred the blastocyst hatching and adhesion in vitro, and the decidual expression of Gαq was significantly lower in women with recurrent pregnancy loss compared with normal pregnancy. In brief, we showed here that Gαq as a key regulator of the inflammatory cytokine’s expression and decidual homeostasis in response to differentiation cues, which is required for successful implantation and early pregnancy.
Decidualization, denoting the transformation of endometrial stromal cells into specialized decidual cells, is a prerequisite for normal embryo implantation and a successful pregnancy in human. Here, we demonstrated that knockout of Gαq lead to an aberrantly enhanced inflammatory state during decidualization. Furthermore, we showed that deficiency of Gαq resulted in over-activation of nuclear factor (NF)-κB signaling, due to the decreased expression of NFκBIA, which encode the IκB protein and is the negative regulator for NF-κB. Mechanistically, Gαq deficiency decreased the Protein kinase D (PKD, also called PKCμ) phosphorylation levels, leading to attenuated HDAC5 phosphorylation and thus its nuclear export. Aberrantly high level of nuclear HDAC5 retarded histone acetylation to inhibit the induced NFκBIA transcription during decidualization. Consistently, pharmacological activation of the PKD/PKCμ or inhibition of the HDAC5 restored the inflammatory state and proper decidual response. Finally, we disclosed that over-active inflammatory state in Gαq-deficient decidua deferred the blastocyst hatching and adhesion in vitro, and the decidual expression of Gαq was significantly lower in women with recurrent pregnancy loss compared with normal pregnancy. In brief, we showed here that Gαq as a key regulator of the inflammatory cytokine’s expression and decidual homeostasis in response to differentiation cues, which is required for successful implantation and early pregnancy.
Mammalian type Opsin 5 (Opn5m), a UV-sensitive GPCR opsin highly conserved in vertebrates, would provide a common basis for UV sensing from lamprey to humans. However, G protein coupled with Opn5m remains controversial due to variations in assay conditions and the origin of Opn5m across different reports. Here, we examined Opn5m from diverse species using an aequorin luminescence assay and Gα-knockout cell line. Beyond the commonly studied major Gα classes, Gαq, Gα11, Gα14, and Gα15 in the Gq class were individually investigated in this study, as they can drive distinct signaling pathways in addition to a canonical calcium response. UV light triggered a calcium response via all the tested Opn5m proteins in 293T cells, which was abolished by Gq-type Gα deletion and rescued by co-transfection with mouse and medaka Gq-type Gα proteins. Opn5m preferentially activated Gα14 and close relatives. Mutational analysis implicated specific regions, including α3-β5 and αG-α4 loops, αG and α4 helices, and the extreme C-terminus, in the preferential activation of Gα14 by Opn5m. Fluorescent in situ hybridization revealed co-expression of genes encoding Opn5m and Gα14 in the scleral cartilage of medaka and chicken eyes, supporting their physiological coupling. This suggests that the preferential activation of Gα14 by Opn5m is relevant for UV sensing in specific cell types.
The kidney is a multipurpose organ that not only acts as the major filter to remove bodily wastes but also in the long‐term control of blood pressure maintenance, production of red blood cells, and synthesis of endocrine hormones. In the last quarter century, there is accumulating evidence that heterotrimeric G‐proteins play a central role in most, if not all, of these kidney functions through unexpected modes of action. One family of accessory proteins, known as activators of G‐protein signaling (AGS), has been shown to expressed in the kidney and exert various roles in affecting the magnitude, duration, and in some cases, spatial location of G‐protein subunits in the cell. In this chapter, each of the AGS protein family members will be described with their known expression profile, distribution and function in the kidney during normal and diseased conditions.
The discovery of mutated, GTPase-deficient alpha subunits of Gs or Gi2 in certain human endocrine tumors has suggested that heterotrimeric G proteins play a role in the oncogenic process. Expression of these altered forms of G alpha s or G alpha i2 proteins in rodent fibroblasts activates or inhibits endogenous adenylyl cyclase, respectively, and causes certain alterations in cell growth. However, it is not clear whether growth abnormalities result from altered cyclic AMP synthesis. In the present study, we asked whether a recently discovered family of G proteins, Gq, which does not affect adenylyl cyclase activity, but instead mediates the activation of phosphatidylinositol-specific phospholipase C harbors transforming potential. We mutated the cDNA for the alpha subunit of murine Gq in codons corresponding to a region involved in binding and hydrolysis of GTP. Similar mutations unmask the transforming potential of p21ras or activate the alpha subunits of Gs or Gi2. Our results show that when expressed in NIH 3T3 cells, activating mutations convert G alpha q into a dominant acting oncogene.
The cerebellum is essential for the control, coordination, and learning of movements, and for certain aspects of cognitive function. Purkinje cells are the sole output neurons in the cerebellar cortex and therefore play crucial roles in the diverse functions of the cerebellum. The type 1 metabotropic glutamate receptor (mGluR1) is prominently enriched in Purkinje cells and triggers downstream signaling pathways that are required for functional and structural plasticity, and for synaptic responses. To understand how mGluR1 contributes to cerebellar functions, it is important to consider not only the operational properties of this receptor, but also its spatial organization and the molecular interactions that enable its proper functioning. In this review, we highlight how mGluR1 and its related signaling molecules are organized into tightly coupled microdomains to fulfill physiological functions. We also describe emerging evidence that altered mGluR1 signaling in Purkinje cells underlies cerebellar dysfunction in ataxias of human patients and mouse models.
This article is part of the Neuropharmacology Special Issue on ‘Glutamate Receptors – mGluRs’.
The mechanisms of action of bacterial toxins have provided novel approaches to elucidating cellular and molecular regulatory mechanisms. Recently Pasteurella muHocida toxin (PMT) was shown to be an extremely effective mitogen for Swiss 3T3 cells and stimulates a phospholipase C-mediated increase in inositol phosphates in these cells. Hence, rPMT provided a novel tool for studying this signalling pathway. Here we analysed the effect of recombinant PMT (rPMT) on the G protein and tyrosine kinase-mediated pathways for the activation of phospholipase 0. The results in this thesis demonstrate that rPMT selectively potentiates neuropeptide-mediated inositol phosphate production. Treatment of Swiss 3T3 cells with a subsaturating concentration of rPMT markedly potentiated the production inositol phosphates induced by bombesin, vasopressin and endothelin in both a time- and dose-dependent manner. Under similar conditions rPMT had no potentiating effect on the production of inositol phosphates induced by PDGF (AA and BB homodimers). rPMT treatment also markedly enhanced bombesin-induced enhancement of inositol(1,4,5)trisphosphate, the direct product of phosphatidylinositol 4,5-bisphosphate hydrolysis. In addition, rPMT pretreatment greatly reduces the Ca2+-mobilising action of bombesin, consistent with Ca2+ mobilisation from a common intracellular pool. In contrast, treatment of cells with rPMT had no effect on the tyrosine phosphorylation of phospholipase Cy. Depletion of protein kinase C markedly increased the accumulation of inositol phosphates induced by rPMT in a manner similar to that observed for bombesin but not PDGF. The action of rPMT on phosphatidylinositol 4,5-bisphosphate hydrolysis also persisted in permeabilized cells. The addition of guanosine 5'-O-(β-thiodiphosphate) to permeabilized cells markedly reduced rPMT induced accumulation of inositol phosphates in a time and dose dependent manner. rPMT also increased the sensitivity of phospholipase C for free calcium. These results strongly suggest that the action of rPMT is to facilitate the coupling of G protein to phospholipase C. Recently, the pertussis toxin-insensitive Gq subfamily of G proteins have been found to couple neuropeptide receptors (including vasopressin) to the β isoform of phospholipase C. Overexpression of the alpha subunits of this G-protein family (αq and α11) by transfection in COS-1 cells resulted in an increased production of inositol phosphates induced by rPMT. These results demonstrate that Gq may be involved in the production of inositol phosphates stimulated by PMT. Serum and other growth factors are known to transiently induce the expression of the early protooncogenes c-fos and c-myc. The induction of these genes is subject to strong feedback inhibition. At least part of this autoregulatory mechanism could be the result of receptor desensitization. At the beginning of this work nothing was known about the ability of rPMT to stimulate gene expression. Since PMT by-passes receptor-mediated signal generation we have analysed the effect of rPMT on the induction of these early protooncogenes. The results in this thesis demonstrate that rPMT also stimulates the induction of c-fos and c-myc in Swiss 3T3 cells. The induction of these genes by rPMT occurs after a lag period of 3-4 h and levels of c-fos and in particular c-myc message can be detected for prolonged time periods. Maximal levels of c-fos induced by rPMT were approximately 50% of the maximum levels induced by bombesin. In contrast, maximum levels of c-myc induced by rPMT were slightly higher than maximum levels induced by bombesin. In PKC-down-regulated cells the levels of c-fos and c-myc mRNA induced by rPMT were severely attenuated but not abolished. Similar results are observed for bombesin-stimulation of c-fos and c-myc in PKC-depleted cells. These results demonstrate that rPMT stimulates early gene expression through both PKC-dependent and independent pathways. At least in the case of c-myc, the striking and prolonged expression of this protooncogene may be attributed to the persistent activation of early signalling pathways by rPMT.
Neuropeptides including bombesin and its mammalian homologue gastrin releasing peptide, stimulate proliferation and are implicated as growth factors in a variety of biological processes, including autocrine and paracrine growth of small cell lung cancer (SCLC). These peptides bind to seven transmembrane domain receptors which lack intrinsic tyrosine kinase activity and activate second messenger pathways via heterotrimeric G proteins. This thesis covers two main areas of investigation: (1) NEUROPEPTIDES AND TYROSINE PHOSPHORYLATION Recently, neuropeptides including bombesin have been shown to induce tyrosine phosphorylation in Swiss 3T3 cells, a useful model system for elucidating signal transduction pathways. The mechanism by which tyrosine phosphorylation is induced by G protein coupled receptors is unknown. This thesis shows that the non-hydrolyzable GTP analogue, GTP?S, induced tyrosine phosphorylation of multiple proteins including the focal adhesion associated proteins p125 focal adhesion kinase and paxillin in permeabilized Swiss 3T3 cells. The pattern of tyrosine phosphorylation closely resembled that induced by neuropeptides in intact cells. Our results suggest that the small GTP binding protein rhop21 rather than heterotrimeric GTP binding proteins mediates GTP?S induced tyrosine phosphorylation. The role of tyrosine phosphorylation in bombesin mediated mitogenesis is unknown. Here, it is shown that addition of the protein tyrosine kinase inhibitor [(3,4,5,-trihydroxyphenyl)-methylene]-propanedinitrile (tyrphostin) to intact Swiss 3T3 cells reduces bombesin induced tyrosine phosphorylation and inhibits bombesin mediated c-fos expression and DNA synthesis. The results demonstrate that inhibitors of tyrosine kinase activity prevent neuropeptide mediated mitogenesis and may be of therapeutic value for SCLC. (2) NOVEL SCLC THERAPIES Substance P (SP) analogues [DArg1, DPhe5, DTrp7,9, Leu11] SP and [Arg6, DTrp7,9, MePhe8] SP (6-11) inhibit the action of many different calcium mobilizing neuropeptides including vasopressin and bombesin in Swiss 3T3 cells and SCLC cell lines and block the growth of SCLC xenografts in nude mice. However, their mechanism of action is not understood. This thesis shows that these SP analogues reversibly inhibit neuropeptide- but not GTP?S-stimulated inositol phosphate production in permeabilized Swiss 3T3 cells and competitively inhibit ligand binding in intact cells or membrane preparations. The results suggest that these SP analogues block the action of vasopressin and bombesin at the receptor level. Identification of factors which stimulate SCLC cell growth is relevant to the development of novel SCLC therapies. Results presented here demonstrate that although serum mobilizes calcium and stimulates growth of SCLC cells, Ca2+ mobilization is not required for serum induced growth of these cells. The polypeptide growth factors hepatocyte growth factor and stem cell growth factor were found to stimulate colony growth of SCLC cells through a Ca2+ independent pathway. Since tyrphostin is known to inhibit polypeptide receptor tyrosine kinases and inhibits neuropeptide stimulated mitogenesis, the effect of tyrphostin with or without SP analogues was tested on SCLC growth in vitro. It is shown that tyrphostin and SP analogues synergistically inhibit SCLC cell growth.
Significance
IP 3 receptors are well-documented regulators of intracellular calcium concentration, but their role in axons of central neurons remains unexplored. Here we show that IP 3 receptors are present throughout the axon of cerebellar Purkinje cells where their activation leads to calcium release from intracellular stores. We reveal distinct, sharply delimited signaling cascades leading to IP 3 production in the axon initial segment (AIS), in the more distal part of the axon, and in synaptic terminals. In the AIS, IP 3 -induced calcium rises inhibit action potential generation, whereas similar signals in axonal terminals increase transmitter release. Metabotropic purinergic receptors appear to control the IP 3 -driven cascade. Altogether, the results reveal potent actions of IP 3 in central axons, which are both diverse and spatially constrained.
Représentant environ 45% de tous les gliomes, le GBM est la tumeur cérébrale la plusagressive chez l’adulte. Comme nous l’avons décrit dans l’introduction de ce manuscrit, lecaractère très hétérogène du GBM associé aux signatures moléculaires et expressions géniques,mais également aux conditions microenvironnementales hypoxique et inflammatoire,contribuent à la récidive quasi-systématique après exérèse complète-radio/chimiothérapie, etexpliquent les nombreux échecs thérapeutiques. Malgré l’arsenal thérapeutique potentiellementdisponible, appliqué parfois de manière multimodale, la survie des patients atteints de GBMn’est pas significativement améliorée, les défis à relever pour améliorer cette survie et la qualitéde vie des patients restent énormes. Ainsi, l'identification de facteurs exprimés de manièredifférentielle qui pourraient mieux définir le comportement agressif des cellules de GBMfournirait une base pour le développement de thérapies innovantes et peut-être plus efficaces.Une des caractéristiques des GBMs est leur capacité très migratoire et invasive, relayéesprincipalement par des facteurs chimiotactiques dans un microenvironnement tumoralhypoxique et inflammatoire. Les récepteurs couplés aux protéines G (RCPGs) et leurs ligands,particulièrement les RCPGs de chimiokines, surexprimés dans les GBMs et stimulant lamigration chimiotactique, l’invasion et l’angiogenèse jouent un rôle majeur dans ledéveloppement des GBMs et l'acquisition d'un phénotype agressif. Dans ce contexte, notreéquipe avait démontré que le récepteur UT de l’urotensine II (UII), une chimiokine peptidiquepro-angiogénique et pro-inflammatoire, ainsi que le système chimiokine bien connu SDF-1a/CXCR4 semblent systématiquement co-exprimés dans les GBMs, plus spécifiquement dansles zones vasculaires et périnécrotiques, montrant une corrélation avec le grade des gliomes. Invitro, nous avions aussi établi que l’UII/UT stimule la migration chimiotactique des cellules deGBM via les couplages de type Gαi/PI3K et Gα13/Rho/ROCK, des couplages précédemmentmis en évidence pour le système SDF-1α/CXCR4 et d’autres RCPGs chimiotactiques. De plus,une récente analyse de la base de données TCGA (The Cancer Genome Atlas) en composanteprincipale réalisée par Alexandre Mutel, étudiant en thèse dans l’équipe, a permis d’identifierla signature d’expression des RCPGs exprimés dans les gliomes et particulièrement dans lesGBMs, qui révèle un nombre très important de RCPGs chimiotactiques. Dans l’ensemble, leurexpression et activité signalisante redondantes fréquemment associées à la tumorigenèse, enparticulier dans les GBMs, soulignent l’intérêt d’étudier les noeuds de signalisation communs àl’ensemble de ces RCPGs chimiokines. Ces noeuds sont principalement représentés par lesprotéines G hétérotrimériques composées des sous-unité α, β et γ, qui couplent ces RCPGs etrelayent les effecteurs secondaires intracellulaires, probablement essentiels à la régulation del’agressivité des GBMs.Ainsi, l’objectif de mon travail de thèse était d’identifier les principales protéines Ga, b et gparmi les 31 protéines G exprimées chez l’Homme dans les gliomes et celles plusspécifiquement associées au degré de malignité, et à l’agressivité des GBMs puis à déterminerle rôle d’une de ces protéines G dans les mécanismes de prolifération et d’invasion de cellulesde GBM.Dans un premier temps, nous avons analysé l’expression des 31 sous-unités (15α, 5β et 11γ)de protéines G sur la base de données transcriptomiques du The Cancer Genome Atlas (TCGA),et démontré que les niveaux d'ARNm codant pour les sous-unités Gαz, Gαi1, Gβ4, Gβ5 et Gγ3sont relativement faibles dans les GBMs tandis que les sous-unités Gα12, Gα13, Gα15, Gαi2,Gαi3, Gβ2, Gγ5, Gγ11 et Gγ12 sont particulièrement surexprimées dans les GBMs et sontassociées à un mauvais pronostic en termes de récidive et de survie du patient. Nous avonsensuite confirmé par qPCR que les protéines G identifiées sont bien exprimées dans desprélèvements de GBMs de patients obtenus en collaboration avec le serviced’anatomopathologie du CHU de Rouen (Collaboration Pr A. Laquerrère) ainsi que dans 6lignées cellulaires de gliome. Les analyses des données du TCGA associées aux informationscliniques des patients indiquent que ces sous-unités sont toutes associées à un mauvais pronosticde survie chez les patients atteints de gliome. Aussi, l’inhibition de l’expression des sous-unitésGα15, Gαi3 ou Gβ2 dans la lignée de GBM U87 entraîne une diminution de laprolifération/survie et des adhésions associées à la migration cellulaire. Ainsi, nous proposonsque les protéines identifiées (Gα15, Gαi3, Gβ2, Gγ5, Gγ10, Gγ11 et Gγ12) représentent desmarqueurs de malignité des GBMs ; et contribuent au phénotype agressif d’invasivité desGBMs. Parmi ces protéines Gα, β ou γ identifiées, nous nous sommes focalisés plusparticulièrement sur Gα15, une sous-unité atypique non ubiquiste longtemps décrite commespécifique aux cellules souches hématopoïétiques. Cette dernière semble également êtreexprimée de novo dans les gliomes de haut-grade, et plus particulièrement dans les GBMsneuraux et mésenchymateux. Ainsi, nous avons recherché si l’expression de cette sous-unitéGα15 dans des cellules de GBM, contrôle les mécanismes de prolifération et d’invasivité etconstitue une potentielle cible thérapeutique.Pour mieux analyser la fonction de Gα15, nous avons utilisé la technologie siRNA, shRNAou CRISPR-Cas9 pour inhiber l’expression ou inactiver de manière stable le gène GNA15(Gα15-KO) dans les lignées cellulaires de GBM U87 et 8MG. A partir de ces cellules, nousavons démontré que la sous-expression de Gα15 ou son inactivation réduit significativement lenombre de cellules (comptage au Nucleocounter) ainsi que la viabilité des cellules de GBM(mesure de l'activité métabolique des cellules déterminée par le clivage WST-1). Ces résultatsdémontrent que Gα15 est une protéine G impliquée dans la survie des cellules de GBM, maisne modifie pas le cycle cellulaire, et ce en l’absence d’activation d’un RCPG.Le GBM se caractérise aussi par son comportement fortement invasif. Ce comportement estl'une des caractéristiques qui contribuent à la récurrence et au mauvais pronostic des GBMs. Lamigration et l'invasion des cellules tumorales est un processus coordonné en plusieurs étapescomprenant la réorganisation dynamique du cytosquelette d'actine, des protrusionsmembranaires comme les lamellipodes, la formation de complexes d’adhésion, la contractiondu corps cellulaire et le détachement de la queue cellulaire pour la migration mésenchymateuse.Dans ce contexte nous avons démontré que la répression de l’expression de Gα15 diminuesignificativement la migration des cellules de GBM dans les tests de migration en transwell etde wound healing, alors que sa surexpression favorise la migration. En évaluant la motilitécellulaire par vidéomicroscopie, nous observons que les cellules contrôles U87 émettent deslamellipodes orientées pour migrer, alors que les cellules clonales Gα15-KO ne sont paspolarisées et montrent une diminution significative de la longueur des trajectoires parcouruesassociée à une vitesse moyenne de déplacement réduite par rapport aux cellules U87. Les étudesd’immunocytochimie révèlent que l’inactivation de Gα15 entraîne une réduction significativedu nombre de complexes d’adhésion focal (FA) riches en Phospho-paxillin et en vinculine(deux protéines majeures des FA), et altère le remodelage du cytosquelette d’actine(polymérisation des fibres de stress d’actine) associé à une diminution significative del’émission des lamellipodes. Au contraire, les cellules sur-exprimant Gα15 présentent uneexacerbation de la concentration des protéines formant le complexe FA et du nombre delamellipodes. Ces observations montrent que Gα15 joue un rôle majeur dans la régulation de lamigration/invasion et la survie associées à la croissance des GBMs.Afin d’identifier les principales voies de signalisation régulées par Gα15 pour stimuler cesprocessus, nous avons analysé par Western blot les voies de signalisation oncogénique etidentifié que l’inactivation de Gα15 inhibe significativement la voie ERK1/2, réduitconsidérablement l’expression de STAT3, de Akt et de la β-caténine. De plus, l'analyse duniveau d'expression de l'ARNm codant trois effecteurs principaux de Gα15 (PLCβ1, PLCβ2 etTPR1) a révélé une diminution significative de l'expression de PLCβ2 dans les cellules U87Gα15-KO, suggérant un mécanisme commun entre PLCβ2 et Gα15 dans les GBMs. Cesdonnées permettent de proposer que Gα15 (1) stimule au moins l’activation de ERK1/2 etSTAT3 via la voie PLCβ2/PKC favorisant une haute survie/prolifération des cellules de GBM,(2) favorise la voie PI3K/Akt aboutissant à l’expression et/ou l’activation de NF-κB et de la β-caténine pour réguler l'expression de nombreux gènes pro-tumorigène, et (3) peut réguler lapolymérisation d’actine dépendante de PLCβ2 et stimuler les voies de signalisationintracellulaires (ERK, Akt,…) associées aux protéines d’adhésions paxilline et vinculine.Pour mieux comprendre si l'agressivité des gliomes favorisée par Gα15 est associée à larégulation de la transition mésenchymateuse, nos études de qPCR ou immunocytochimiquesmontrent que l’inactivation de Gα15 dans les cellules de GBM est suffisante pour diminuer demanière significative l’expression (1) des ARNm des facteurs de transcription (TFs)mésenchymateux incluant C/EBPβ, RUNX1, FOSL2, BHLHB2, ZEB1, et la translocationmembranaire de N-cadhérine, (2) de marqueurs pro-angiogéniques (Angiogénine et VEGFA)et (3) de la SERPINA1 pro-inflammatoire et du TF KLF4 marqueur de cellules souches. Enrevanche, l'expression des ARNm de répresseurs tumoraux (DKK1, ZNF238) ainsi que dumarqueur de cellules souches SOX2 activateurs de la voie DKK1, est significativement inhibéedans les cellules Gα15-KO. Ensemble, ces résultats mettent en évidence le rôle essentiel deGα15 dans l’expression marqueurs oncogénique ainsi que dans l’acquisition du phénotypemésenchymateux des gliomes, permettant la mise en place d’un microenvironnement tumoralimmunosuppresseur et angiogénique soutenant l'agressivité des GBMs.Enfin, pour évaluer l’importance de Gα15 dans la croissance et l’invasivité des GBMs invivo, des xénogreffes orthotopiques des U87 contrôles, de deux clones U87 Gα15-KO ainsi quedes U87 sur-exprimant Gα15 ont été réalisés chez la souris Nude, dans le striatum. Nos résultatsmontrent une survie significativement améliorée des souris lorsque Gα15 est inactivée (surviemédiane de 60 et 84 jours pour les clones KO 26-1 (p=0,0436) et KO 26-6 (p=0,0016)respectivement contre 44 jours pour les U87, et une survie réduite des souris Nude xénogrefféespar les cellules sur-exprimant Gα15 (50 jours contre 61 jours pour le contrôle, p=0,0326). Cesrésultats démontrent que la sous-unité Gα15 est une protéine impliquée dans l’agressivité desGBMs in vivo.En conclusion, nos résultats montrent que l’expression de Gα15 est suffisante pour maintenirla survie et entraîner la migration des cellules de GBM, et favorise l’expression ou l’activationde facteurs impliqués dans la transition mésenchymateuse, l’angiogenèse, le statut souche etl’inflammation. Bien que des expériences complémentaires s’avèrent nécessaires pourconfirmer les voies et mécanismes associés à Gα15, ces travaux originaux ouvrent des pistes etperspectives sur le potentiel diagnostique de Gα15 pour les GBM mésenchymateux etinflammatoire, et thérapeutique pour cibler les GBMs les plus invasifs et résistants.
Feline iris melanoma, the most common feline intraocular tumour, has a reported metastatic rate of 19–63%. However, there is a lack of knowledge about its molecular biology. Previous studies have reported that feline iris melanomas do not harbour mutations comparable to common mutations found in their human counterpart. Nevertheless, there are differences in the gene expression patterns. The aim of this study was to investigate the protein expression of B-RAF oncogene serine/threonine kinase (BRAF), G protein subunit alpha q (GNAQ) and 11 (GNA11), KIT proto-oncogene receptor tyrosine kinase (KIT), and Ras association family member 1 (RASSF1) in feline iris melanomas.
Fifty-seven formalin-fixed paraffin embedded (FFPE) iris melanomas and 25 FFPE eyes without ocular abnormalities were stained with antibodies against the respective proteins using immunofluorescence. Averaged pixel intensities/μm² and percentage of stained area from total tissue area were measured and the results were compared. Compared to the control group, iris melanomas showed overexpression of BRAF, GNAQ, GNA11 and KIT. The higher expression of BRAF, GNAQ, GNA11 and KIT in feline iris melanomas suggest that these proteins may play a key role in the development of feline iris melanomas and KIT may present a possible target for future therapies in cats with feline iris melanomas.
Background
Acute myeloid leukemia (AML) is a genetically heterogeneous disease characterized by uncontrolled proliferation of precursor myeloid-lineage cells in the bone marrow. AML is also characterized with patients with poor long-term survival outcomes due to relapse. Many efforts have been made to understand the biological heterogeneity of AML and the challenges to develop new therapies are therefore enormous. G protein-coupled receptors (GPCRs) are a large attractive drug targeted family of transmembrane proteins, and aberrant GPCR expression and GPCR-mediated signaling have been implicated in leukemogenesis of AML. This review aims to identify the molecular players of GPCR signaling, focusing on the hematopoietic system, which are involved in AML to help developing novel drug targets and therapeutic strategies.
Methods
We undertook an exhaustive and structured search of bibliographic databases for research focusing in GPCR, GPCR signaling and expression in AML.
Results and Conclusion
Many scientific reports were found with compelling evidence for involvement of aberrant GPCR expression and perturbed GPCR-mediated signaling in the development of AML. The comprehensive analysis of GPCR in AML provides potential clinical biomarkers for prognostication, disease monitoring and therapeutic guidance. It will also help to provide marker panels for monitoring in AML. We conclude that GPCR-mediated signaling is contributing to leukemogenesis of AML, and postulate that mass spectrometry-based protein profiling of primary AML cells will accelerate the discovery of potential GPCR related biomarkers for AML.
Psoriasis (PSO) is a chronic inflammatory skin disease characterized with skin lesions and abnormal keratinocyte proliferation. The immune dysregulation is involved in the pathogenesis of PSO. However, the detail of immune regulation in PSO is still not very clear. Gαq, the alpha subunit of the Gq protein, played important role in the immune regulation. In this study, we aimed to investigate whether Gαq was involved in the pathogenesis of PSO. We detected the Gαq expression level and analyzed its relationship with test parameters of PSO patients. Furthermore, we used imiquimod to induce PSO mouse model in Gnaq -/- bone marrow (BM) chimeric mice. The inflammatory cytokines and its correlation with Gαq expression were analyzed both in PSO patients and mice. The results showed that the Gαq expression in PSO patients was much lower and negatively correlated with PSO Area and Severity Index (PASI), CRP, cholesterol and low-density lipoprotein. In PSO mice models, the skin lesion and keratinocyte proliferation were much more serious in Gnaq -/- BM chimeric mice. Also, the proportions of Th17 cells in Gnaq -/- BM chimeric mice were much higher than WT mice. Furthermore, the IL-17A and TNF-α in Gnaq -/- chimeric mice were also higher. Moreover, IL-17A and TNF-α in PSO patients were negatively associated with the Gαq expression. Our results indicated that Gαq was involved in the pathogenesis of PSO, and its regulation on Th17 cell differentiation and cytokine production might contribute to part of the mechanism of immune dysfunction of PSO.
G protein-coupled receptors (GPCRs) are the largest class of drug targets, largely owing to their druggability, diversity and physiological efficacy. Many drugs selectively target specific subtypes of GPCRs, but high specificity for individual GPCRs may not be desirable in complex multifactorial disease states in which multiple receptors may be involved. One approach is to target G protein subunits rather than the GPCRs directly. This approach has the potential to achieve broad efficacy by blocking pathways shared by multiple GPCRs. Additionally, because many GPCRs couple to multiple G protein signalling pathways, blocking specific G protein subunits can 'bias' GPCR signals by inhibiting only a subset of these signals. Molecules that target G protein α or βγ-subunits have been developed and show strong efficacy in multiple preclinical disease models and biased inhibition of G protein signalling. In this Review, we discuss the development and characterization of G protein α and βγ-subunit ligands and the preclinical evidence that this exciting new approach has potential for therapeutic efficacy in a number of indications, such as pain, thrombosis, asthma and heart failure.
Multicellular organisms have developed highly efficient regulatory networks to control cell proliferation. These involve cellular interactions with positive and negative diffusible modulators as well as with the extracellular matrix proteins. In fully mature organisms, the cells of many tissues and organs are maintained in a nonproliferating state, but can be stimulated to resume DNA synthesis and cell division in response to external stimuli such as hormones, antigens or growth factors. In this manner the growth of individual cells is regulated according to the requirements of the whole organism. The regulation of normal cell proliferation is, therefore, central to many physiological processes, including embryogenesis, growth and development, tissue repair and immune responses.
G proteins classically associated with the 7TM or serpentine receptors generally exist as a heterotrimeric complex consisting of α, β and γ subunits. These proteins serve as transducers of extracellular signal from plasma membrane to cellular interior. Binding of G protein-coupled receptor (GPCR) with a ligand leads to their activation, followed by that of Gα, which then dissociates from Gβγ subunits and initiate downstream effects through plasma membrane-localized effectors. This plasma membrane-restricted view of G proteins was challenged when they were found to be present in various intracellular locations such as endomembranes, cytoskeleton, mitochondria, and nucleus thereby opening up newer spatial domains for the action of these signaling molecules. Many recent studies have addressed the spatiotemporal dynamics underlying this atypical distribution and the G proteins have been shown to undergo activation-dependent as well as activation-independent relocalization. This spatially ‘directed’ targeting of G proteins provides them rapid access to intracellular communication network without relying on diffusible second messengers. Here, we present a consolidated review of the existing knowledge about the presence and physiological roles for G proteins in these atypical locations, along with the mechanistic knowhow presently known about underlying processes.
Background & Aims
Proliferation, differentiation, and morphogenesis of the intestinal epithelium are tightly regulated by a number of molecular pathways. Coordinated action of intestine is achieved by gastrointestinal hormones, most of which exert these actions through G-protein–coupled receptors. We herein investigated the role of Gαq/11-mediated signaling in intestinal homeostasis.
Methods
Intestinal tissues from control (Gnaqflox/floxGna11+/+), Int-Gq knock-out (KO) (VilCre+/-Gnaqflox/floxGna11+/+), G11 KO (Gnaqflox/floxGna11-/-), and Int-Gq/G11 double knock-out (DKO) (VilCre+/-Gnaqflox/floxGna11-/-) mice were examined by microscopy, transmission electron microscopy, and immunohistochemistry. The effect of Gαq/11-mediated signaling was studied in the cell lineage, proliferation, and apoptosis. Dextran sodium sulfate (DSS) colitis was induced to study the role of Gαq/11 in colon.
Results
Paneth cells were enlarged, increased in number, and mislocalized in Int-Gq/G11 DKO small intestine. Paneth cells also reacted with PAS and Muc2 antibody, indicating an intermediate character of Paneth and goblet cells. The nuclear β-catenin, T-cell factor 1, and Sox9 expression were reduced severely in the crypt base of Int-Gq/G11 DKO intestine. Proliferation was activated in the crypt base and apoptosis was enhanced along the crypt. Int-Gq/G11 DKO mice were susceptible to DSS colitis. Proliferation was inhibited in the crypt of unaffected and regenerative areas. Cystic crypts, periodic acid–Schiff–positive cells, and Muc2-positive cells were unusually observed in the ulcerative region.
Conclusions
The Gαq/11-mediated pathway plays a pivotal role in the preservation of intestinal homeostasis, especially in Paneth cell maturation and positioning. Wnt/β-catenin signaling was reduced significantly in the crypt base in Gαq/G11-deficient mice, resulting in the defective maturation of Paneth cells, induction of differentiation toward goblet cells, and susceptibility to DSS colitis.
Background:
Mucosal melanoma (MM) is a rare subtype of melanoma in Caucasians with extremely poor prognosis, and therapy strategy has not been clearly established for MM. We aimed to investigate the genetic aberrations possibly applicable in targeted therapy of MM. We examined the somatic mutations of GNAQ and GNA11 (GNAQ/11, encoding the guanine nucleotide-binding alpha subunits) in MM and evaluated their correlation to clinicopathologic features of MM.
Methods:
This study collected samples from primary lesions of 284 MM patients. Tissue samples were analysed for mutations in exons 4 and 5 of GNAQ/11 in genomic DNA by polymerase chain reaction amplification and Sanger sequencing. Correlations of GNAQ/11 mutations to clinicopathologic features and prognosis of MM were evaluated.
Results:
The overall mutation frequency of GNAQ/11 in MM was 9.5% (27 in 284), with a frequency of 4.6% and 4.9% for GNAQ and GNA11 mutations, respectively. The mutations in exon 5 of GNAQ/11 occurred exclusively in codon 209. GNAQ(Q209L) was the most prevalent variation (92.3% of missense GNAQ mutations). GNAQ/11 mutations were not significantly associated with age, gender, ulceration, and primary anatomic site. The median overall survival for MM patients with GNAQ mutations (16.0 versus 26.0 months, P = 0.031) or GNA11 mutations (15.0 versus 26.0 months, P = 0.039) were significantly shorter than those for patients with wild-type GNAQ and GNA11, respectively.
Conclusions:
Our study suggests that GNAQ and GNA11 mutations occur frequently in MM and may be a prognostic factor for MM. Our data implicate that GNAQ/11 may be potential targets for targeted therapy of MM.
A wide variety of stimuli send signals into target cells by stimulating the hydrolysis of a minor phospholipid component of the inner leaflet of the plasma membrane, phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]. This reaction is catalyzed by an inositol lipid-specific phospholipase C (PLC) and produces two compounds each with intracellular second messenger functions. The lipid product of this reaction is 1,2-diacylglycerol which activates a Ca2+ and phospholipid-dependent protein kinase, protein kinase C. D-Myoinositol 1,4,5,-trisphosphate [Ins(l,4,5)P3] is a water-soluble product of this reaction that releases Ca2+ ions from stores within the cell (Berridge and Irvine 1987; Kikkawa et al. 1989).
Heterotrimeric G-proteins, which are involved in a variety of hormonal and sensory signal transductions in mammalian cells, consist of three subunits: α (39-52kDa), β (35-36 kDa), and γ (7-10 kDa). They are widely distributed among different tissues and organisms and their structures are highly conserved. This chapter briefly reviews the structure of the cDNAs and the genes for various G-protein α, β, and γ subunits from mammalian cells and the occurrence of G-protein genes in lower eukaryotes.
Signal-transducing G-proteins are heterotrimers in which molecular diversity exists for each of the subunits. The genes known thus far (16 a, 4 β, and 5 γ) and splice variants for 2 α’s allow for a calculated 400 combinations. Restriction in expression and lack of functional connotation of some of the splice variants decrease this number to a much smaller, but still considerable number of G-proteins that may be mediating receptor signals in a cell. Correlations between molecular diversity and function are being steadily worked out and an update follows.
The C-terminal portion of G(alpha) proteins plays a key role in their selective activation by cognate receptors. alpha (2A)-Adrenoceptors (alpha (2A)-ARs) can differentially inhibit or stimulate adenylyl cyclases by the activation of distinct G(i/o) and G(s) protein families. The implication of the C-terminal portion of G(alphao) and G(alphas) proteins in their activation by alpha (2A)-ARs was analyzed by constructing mutant G(alphao) proteins in which each of the last five amino acid positions were exchanged for those corresponding to a G(alphas) protein. Agonist-dependent, pertussis toxin-resistant binding of guanosine 5'-O-(3-[S-35]thio)triphosphate (S-35]GTP gammaS) revealed that the degree of positive efficacy of clonidine was highly dependent on the presence of a G(alphao) protein-derived Gly amino acid as the -3 residue at the C-terminal portion of the protein. In contrast, antagonist properties for clonidine were observed for those mutants carrying a G(alphas) protein-derived Glu residue at this position. (-)-Epinephrine yielded almost similar maximal [S-35]GTP gammaS binding responses, but its potency was decreased 22- to 150-fold at the -3 Glu containing mutant G(alphao) proteins compared with those mutants containing a Gly. A 9- to 39-fold increase in the alpha (2A)-AR agonist equilibrium dissociation constants further reflected changes in the G(alpha) protein-induced alpha (2A)-AR state mediated by the specific Gly to Glu mutation in the C-terminal portion of the G(alpha) protein. The present data emphasize the unique role of the -3 position at the G(alpha) protein C-terminal portion, independent of its surrounding peptidic environment, in constraining a structure favorable for activated receptor interaction and transmission of the mutation-induced conformational change from the G(alphao) protein to the alpha (2A)-AR.
A stromal cell line, GY30, was cloned from mouse bone marrow adherent cell layers. In culture, GY30 cells sustain the production of granulocyte-macrophage progenitor cells (GM-CFU) but fail to support the survival of pluripotential stem cells (CFU-S). GY30 cells secrete two growth factor activities distinct from interleukin-3 (IL-3), IL-2, and macrophage colony-stimulating factor (M-CSF) but functionally similar to GM-CSF and G-CSF. The production of both CSFs is increased 70- to 200-fold by treating GY30 cells with lipopolysaccharide or IL-1. RNA blot analysis reveals the presence of GM-CSF and G-CSF transcripts and demonstrates that IL-1 regulates the production of both factors at the mRNA level. Further, these studies show that the GM-CSF secreted by GY30 cells is structurally similar to the GM-CSF produced by activated T cells.
Heterotrimeric guanine nucleotide binding proteins (G proteins) transduce signals from cell-surface receptors to intracellular effector proteins. Two forms of stimulatory G protein (Gs) alpha-like subunit have been described in Drosophila melanogaster. To examine the function of these subunits we have used vaccinia virus vectors to express both proteins in cyc- cells, a murine S49 cell line deficient for Gs alpha activity. Receptor-independent activation of each Drosophila Gs alpha has demonstrated that both forms are capable of activating mammalian adenylyl cyclase and thus have the activity expected of stimulatory G proteins. However, the Drosophila Gs alpha subunits interact poorly with mammalian Gs-coupled receptors. These observations have helped to identify a region of high variability in Gs alpha proteins that may be important for receptor interactions.
Virus-transformed pre-B cells undergo ordered immunoglobulin (Ig) gene rearrangements during culture. We devised a series of highly sensitive polymerase chain reaction assays for Ig gene rearrangement and unrearranged Ig gene segment transcription to study both the possible relationship between these processes in cultured pre-B cells and the role played by heavy (H) chain (mu) protein in regulating gene rearrangement. Our analysis of pre-B cell cultures representing various stages of maturity revealed that transcription of each germline Ig locus precedes or is coincident with its rearrangement. Cell lines containing one functional rearranged H chain allele, however, continue to transcribe and to rearrange the allelic, unrearranged H chain locus. These cell lines appear to initiate but not terminate rearrangement events and therefore provide information about the requirements for activating rearrangement but not about allelic exclusion mechanisms.
Many lipids or lipid-derived products generated by phospholipases acting on phospholipids in membranes are implicated as mediators and second messengers in signal transduction. Our current understanding of the primary sequence relationships within the class of extracellular phospholipase A2's and among the numerous forms of the mammalian phosphatidylinositol-specific phospholipase C's is reviewed. New results suggesting roles for these phospholipases as well as other phospholipases such as phospholipase C and D acting on phosphatidlycholine in generating arachidonic acid for eicosanoid biosynthesis, inositol phosphates for Ca2+ mobilization, and diglyceride for protein kinase C activation through receptor-mediated processes, are discussed. In addition, the possible role of phospholipases acting on sphingolipids such as sphinglomyelinase in generating lipid mediators is considered.
Guanine nucleotide-binding protein (G protein)-mediated signal transduction constitutes a common mechanism by which cells receive and respond to a diverse set of environmental signals. Many of the signals involved in the developmental life cycle of the slime mold Dictyostelium have been postulated to be transduced by such pathways and, in some cases, these pathways have been demonstrated to be dependent on specific G proteins. Using the polymerase chain reaction, we have identified two additional Dictyostelium G alpha genes, G alpha 4 and G alpha 5, that are developmentally regulated. Transcripts from both of these genes are primarily expressed during the multicellular stages of development, suggesting possible roles in cell differentiation or morphogenesis. The entire G alpha 4 gene was sequenced and found to encode a protein consisting of 345 amino acids. The G alpha 4 subunit is homologous to other previously identified G alpha subunits, including the Dictyostelium G alpha 1 (43% identity) and G alpha 2 (41% identity) subunits. However, the G alpha 4 subunit contains some unusual sequence divergences in residues highly conserved among most eukaryotic G alpha subunits, suggesting that G alpha 4 may be a member of another class of G alpha subunits.
Cloning of a complementary DNA (cDNA) for Gz alpha, a newly appreciated member of the family of guanine nucleotide-binding regulatory proteins (G proteins), has allowed preparation of specific antisera to identify the protein in tissues and to assay it during purification from bovine brain. Additionally, expression of the cDNA in Escherichia coli has resulted in the production and purification of the recombinant protein. Purification of Gz from bovine brain is tedious, and only small quantities of protein have been obtained. The protein copurifies with the beta gamma subunit complex common to other G proteins; another 26-kDa GTP-binding protein is also present in these preparations. The purified protein could not serve as a substrate for NAD-dependent ADP-ribosylation catalyzed by either pertussis toxin or cholera toxin. Purification of recombinant Gz alpha (rGz alpha) from E. coli is simple, and quantities of homogeneous protein sufficient for biochemical analysis are obtained. Purified rGz alpha has several properties that distinguish it from other G protein alpha subunit polypeptides. These include a very slow rate of guanine nucleotide exchange (k = 0.02 min-1), which is reduced greater than 20-fold in the presence of mM concentrations of Mg2+. In addition, the rate of the intrinsic GTPase activity of Gz alpha is extremely slow. The hydrolysis rate (kcat) for rGz alpha at 30 degrees C is 0.05 min-1, or 200-fold slower than that determined for other G protein alpha subunits. rGz alpha can interact with bovine brain beta gamma but does not serve as a substrate for ADP-ribosylation catalyzed by either pertussis toxin or cholera toxin. These studies suggest that Gz may play a role in signal transduction pathways that are mechanistically distinct from those controlled by the other members of the G protein family.
We have isolated a gene coding for a G protein alpha subunit from the flowering plant Arabidopsis thaliana. This gene, named GPA1, was isolated by using a DNA probe generated by polymerase chain reaction based on protein sequences from mammalian and yeast G protein alpha subunits. The sequences of genomic and cDNA clones indicate that GPA1 has 14 exons, and the deduced amino acid sequence shows that the GPA1 gene product (GP alpha 1) has 383 amino acid residues (44,582 Da). The GP alpha 1 protein exhibits similarity to all known G protein alpha subunits--36% of its amino acids are identical and 73% are similar (identical and conservative changes) to mammalian inhibitory guanine nucleotide-binding regulatory factor alpha subunits and transducins. Furthermore, the GP alpha 1 protein has all of the consensus regions for a GTP-binding protein. The GPA1-encoded mRNA of 1.55 kilobases is most abundant in vegetative plant tissues, as determined by RNA blot analysis. Restriction fragment length polymorphism mapping experiments show that GPA1 is approximately 1.2 centimorgans from the visible marker er on chromosome 2.
Novel G protein alpha subunits were purified from rat brain by an affinity matrix containing immobilized beta gamma subunits (Pang, I.-H., and Sternweis, P. C. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 7814-7818). They were unique based on the following criteria. These alpha subunits migrated differently through polyacrylamide gels with an apparent molecular mass of 42 kDa. They did not behave similarly to the other brain G proteins by conventional chromatographic techniques. Antisera raised against a common region of known alpha subunits failed to recognize these 42-kDa polypeptides. Finally, primary sequences of tryptic fragments of these proteins contain regions homologous to, yet unique from, the other alpha subunits. Sequences are identical with one or more members of a new family of alpha subunits recently identified by molecular genetic techniques (Strathmann, M., Wilke, T. M., and Simon, M. I. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 7407-7409); most of the primary sequence identifies an alpha subunit labeled alpha q. These polypeptides were not substrates for ADP-ribosylation catalyzed by pertussis toxin. They bound GTP gamma S only with slow rates and low stoichiometry. Antisera to peptides based on primary sequence were specific for the new alpha subunits and indicate that they are widely distributed at low levels in different tissues but more concentrated in brain and lung. This procedure provides a means of preparing native G proteins that have a potential role as modulators of pertussis toxin-insensitive regulatory pathways.
Antisera directed against specific subunits of guanine nucleotide-binding regulatory proteins (G proteins) were used to immunoprecipitate these polypeptides from metabolically labeled cells. This technique detects, in extracts of a human astrocytoma cell line, the alpha subunits of Gs (stimulatory) (alpha 45 and alpha 52), a 41-kDa subunit of Gi (inhibitory) (alpha 41), a 40-kDa protein (alpha 40), and the 36-kDa beta subunit. No protein that comigrated with the alpha subunit of Go (unknown function) (alpha 39) was detected. In cells grown in the presence of [3H]myristic acid, alpha 41 and alpha 40 contained 3H label, while the beta subunit did not. Chemical analysis of lipids attached covalently to purified alpha 41 and alpha 39 from bovine brain also revealed myristic acid. Similar analysis of brain G protein beta and gamma subunits and of Gt (transducin) subunits (alpha, beta, and gamma) failed to reveal fatty acids. The fatty acid associated with alpha 41, alpha 40, and alpha 39 was stable to treatment with base, suggesting that the lipid is linked to the polypeptide via an amide bond. These GTP binding proteins are thus identified as members of a select group of proteins that contains myristic acid covalently attached to the peptide backbone. Myristate may play an important role in stabilizing interactions of G proteins with phospholipid or with membrane-bound proteins.
We have examined the arrangement of immunoglobulin heavy chain constant (CH) and joining (JH) region genes in murine T cell hybrid lines and in T lymphomas. CH genes derived from both parental cell types were present in all hybrids for which polymorphism in sequences flanking CH genes permitted us to distinguish parental CH genes. All T lymphomas and T cell hybrids retained the C alpha gene in germ-line configuration and all but one cell line had germ-line C mu genes. Novel DNA fragments reactive with JH probes were observed in six of nine T cell hybrids, as well as in two T lymphomas, WEHI7.1 and YAC-1, but not in the fusion parent, BW5147. No RNA homologous to C gamma 2b, C alpha, or lambda genes was detected in any of the T cell lines. T cell lines contained poly(A)+ RNA homologous to a C mu cDNA probe. More importantly, in several cell lines the C mu RNAs were associated with membrane-bound polyribosomes. These results suggest that both JH rearrangements and C mu RNA production occur in at least some mature, antigen-specific T cells. They may therefore reflect events in normal T cell development and function related to those involved in the generation of the T receptor for antigen.
The primary structure of G proteins as deduced from purified proteins and cloned subunits is presented. When known, their functions are discussed, as are recent data on direct regulation of ionic channels by G proteins. Experiments on expression of α subunits, either in bacteria or by in vitro translation of mRNA synthesized from cDNA are presented as tools for definitive assignment of function to a given G protein.The dynamics of G protein-mediated signal transduction are discussed. Key points include the existence of two superimposed regulatory cycles in which upon activation by GTP, G proteins dissociate into α and βγ and their dissociated α subunits hydrolyze GTP. The action of receptors to catalyze rather than regulate by allostery the activation of G proteins by GTP is emphasized, as is the role of subunit dissociation, without which receptors could not act as catalysts.Current views of intramembrane networking of G protein-mediated receptor-effector coupling are discussed.To facilitate the reading of this review, we have presented the various subtopics of this rapidly expanding field in sections I–IX, each of which is organized as a self-contained sub-chapter that can be read independently of the others.
A chemically induced leukemia of BDF1 mice, designated 70Z/3, was adapted to tissue culture in our laboratory. A minority of these cells displayed surface immunoglobulin (sIg) as detected by immunofluorescence with rhodamine-labeled class (IgM)-specific antibodies. Addition of the B cell mitogen, LPS, to the cultures induced sIg expression on all of these cells. The kinetics of this transition were dependent on the dose of LPS. As little as 0.1 µg/ml induced sIg on >97% of the cells within 36 hr. DxS also induced sIg whereas other mitogens (Con A, PPD) or inducing agents (DMSO, dimethyl formamide, butyric acid), tested over a wide range of concentrations, failed to induce sIg expression.
The cells bear H-2 antigens but lack IgD, IgG, IgA, Ia, and Thy 1.2. Exposure to LPS had no effect on the presence or absence of these structures. A small percentage of cells possessed receptors for complement and for the Fc portion of immunoglobulin. Cytoplasmic IgM was detectable within all of the cells, and constitutive production of small quantities of IgM was confirmed by SDS-polyacrylamide gel electrophoresis of serologic precipitates of cell lysates after pulsing with radioactive amino acids. Using similar methods, we failed to detect active secretion of immunoglobulin.
Thus, this cell line has properties similar to cytoplasmic Ig+, surface Ig- cells found in immature tissues and in bone marrow of mice and humans that are thought to be immediate precursors of sIg+ B lymphocytes. It may provide a model for studying the mechanism of LPS activation and the molecular events associated with externalization of cell surface receptors.
The alpha subunit of the guanine nucleotide-binding protein Go ("o" for other) is believed to mediate signal transduction between a variety of receptors and effectors. cDNA clones encoding two forms of Go alpha subunit were isolated from a mouse brain library. These two forms, which we call GoA alpha and GoB alpha, appear to be the products of alternative splicing. GoA alpha differs from GoB alpha over the C-terminal third of the deduced protein sequence. Both forms are predicted to be substrates for ADP-ribosylation by pertussis toxin. GoA alpha transcripts are present in a variety of tissues but are most abundant in brain. The GoB alpha transcript is expressed at highest levels in brain and testis. It is possible that GoA alpha and GoB alpha have different functions.
Many hormones, neurotransmitters and growth factors, on binding to G protein-coupled receptors or receptors possessing tyrosine kinase activity, increase intracellular levels of the second messengers inositol 1,4,5-trisphosphate and 1,2-diacylglycerol. This is due to activation of phosphoinositide-specific phospholipase(s) C (PLC), the isozymes of which are classified into groups, alpha, beta, gamma and delta. The beta, gamma and delta groups themselves contain PLC isozymes which have both common and unique structural domains. Only the gamma 1 isozyme has been implicated in a signal transduction mechanism. This involves association with, and tyrosine phosphorylation by, the ligand-bound epidermal growth factor and platelet-derived growth factor receptors, probably by means of the PLC-gamma 1-specific src homology (SH2) domain. Because EGF receptor-mediated tyrosine phosphorylation of PLC-gamma 1 stimulates catalytic activity in vitro and G proteins have been implicated in the activation of PLC, we investigated which PLC isozymes are subject to G protein regulation. We have purified an activated G protein alpha subunit that stimulates partially purified phospholipase C and now report that this G protein specifically activates the beta 1 isozyme, but not the gamma 1 and delta 1 isozymes of phospholipase C. We also show that this protein is related to the Gq class of G protein alpha subunits.
The hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) by phospholipase C yields the second messengers inositol 1,4,5-trisphosphate (InsP3) and 1,2-diacylglycerol. This activity is regulated by a variety of hormones through G protein pathways. However, the specific G protein or proteins involved has not been identified. The alpha subunit of a newly discovered pertussis toxin-insensitive G protein (Gq) has recently been isolated and is now shown to stimulate the activity of polyphosphoinositide-specific phospholipase C (PI-PLC) from bovine brain. Both the maximal activity and the affinity of PI-PLC for calcium ion were affected. These results identify Gq as a G protein that regulates PI-PLC.
GTPases are conserved molecular switches, built according to a common structural design. Rapidly accruing knowledge of individual GTPases--crystal structures, biochemical properties, or results of molecular genetic experiments--support and generate hypotheses relating structure to function in other members of the diverse family of GTPases.
The heterotrimeric guanine nucleotide-binding proteins (G proteins) act as switches that regulate information processing circuits
connecting cell surface receptors to a variety of effectors. The G proteins are present in all eukaryotic cells, and they
control metabolic, humoral, neural, and developmental functions. More than a hundred different kinds of receptors and many
different effectors have been described. The G proteins that coordinate receptor-effector activity are derived from a large
gene family. At present, the family is known to contain at least sixteen different genes that encode the alpha subunit of
the heterotrimer, four that encode beta subunits, and multiple genes encoding gamma subunits. Specific transient interactions
between these components generate the pathways that modulate cellular responses to complex chemical signals.
Signal-transduction pathways mediated by guanine nucleotide-binding regulatory proteins (G proteins) determine many of the responses of hematopoietic cells. A recently identified gene encoding a G protein alpha subunit, G alpha 16, is specifically expressed in human cells of the hematopoietic lineage. The G alpha 16 cDNA encodes a protein with predicted Mr of 43,500, which resembles the G q class of alpha subunits and does not include a pertussis toxin ADP-ribosylation site. In comparison with other G protein alpha subunits, the G alpha 16 predicted protein has distinctive amino acid sequences in the amino terminus, the region A guanine nucleotide-binding domain, and in the carboxyl-terminal third of the protein. Cell lines of myelomonocytic and T-cell phenotype express the G alpha 16 gene, but no expression is detectable in two B-cell lines or in nonhematopoietic cell lines. G alpha 16 gene expression is down-regulated in HL-60 cells induced to differentiate to neutrophils with dimethyl sulfoxide. Antisera generated from synthetic peptides that correspond to two regions of G alpha 16 specifically react with a protein of 42- to 43-kDa in bacterial strains that overexpress G alpha 16 and in HL-60 membranes. This protein is decreased in membranes from dimethyl sulfoxide-differentiated HL-60 cells and is not detectable in COS cell membranes. The restricted expression of this gene suggests that G alpha 16 regulates cell-type-specific signal-transduction pathways, which are not inhibited by pertussis toxin.
Heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins) are central to the signaling processes of multicellular organisms. We have explored the diversity of the G protein subunits in mammals and found evidence for a large family of genes that encode the alpha subunits. Amino acid sequence comparisons show that the different alpha subunits fall into at least three classes. These classes have been conserved in animals separated by considerable evolutionary distances; they are present in mammals, Drosophila, and nematodes. We have now obtained cDNA clones encoding two murine alpha subunits, G alpha 12 and G alpha 13, that define a fourth class. The translation products are predicted to have molecular masses of 44 kDa and to be insensitive to ADP-ribosylation by pertussis toxin. They share 67% amino acid sequence identity with each other and less than 45% identity with other alpha subunits. Their transcripts can be detected in every tissue examined, although the relative levels of the G alpha 13 message appear somewhat variable.
Somatic mutations in a subset of growth hormone (GH)-secreting pituitary tumors convert the gene for the alpha polypeptide chain (alpha s) of Gs into a putative oncogene, termed gsp. These mutations, which activate alpha s by inhibiting its guanosine triphosphatase (GTPase) activity, are found in codons for either of two amino acids, each of which is completely conserved in all known G protein alpha chains. The likelihood that similar mutations would activate other G proteins prompted a survey of human tumors for mutations that replace either of these two amino acids in other G protein alpha chain genes. The first gene so far tested, which encodes the alpha chain of Gi2, showed mutations that replaced arginine-179 with either cysteine or histidine in 3 of 11 tumors of the adrenal cortex and 3 of 10 endocrine tumors of the ovary. The mutant alpha i2 gene is a putative oncogene, referred to as gip2. In addition, gsp mutations were found in 18 of 42 GH-secreting pituitary tumors and in an autonomously functioning thyroid adenoma. These findings suggest that human tumors may harbor oncogenic mutations in various G protein alpha chain genes.
Heterotrimeric guanine nucleotide-binding proteins (G proteins) are integral to the signal transduction pathways that mediate the cell's response to many hormones, neuromodulators, and a variety of other ligands. While many signaling processes are guanine nucleotide dependent, the precise coupling between a variety of receptors, G proteins, and effectors remains obscure. We found that the family of genes that encode the alpha subunits of heterotrimeric G proteins is much larger than had previously been supposed. These novel alpha subunits could account for some of the diverse activities attributed to G proteins. We have now obtained cDNA clones encoding two murine alpha subunits, G alpha q and G alpha 11, that are 88% identical. They lack the site that is ordinarily modified by pertussis toxin and their sequences vary from the canonical Gly-Ala-Gly-Glu-Ser (GAGES) amino acid sequence found in most other G protein alpha subunits. Multiple mRNAs as large as 7.5 kilobases hybridize to G alpha q specific probes and are expressed at various levels in many different tissues. G alpha 11 is encoded by a single 4.0-kilobase message which is expressed ubiquitously. Amino acid sequence comparisons suggest that G alpha q and G alpha 11 represent a third class of alpha subunits. A member of this class was found in Drosophila melanogaster. This alpha subunit, DG alpha q, is 76% identical to G alpha q. The presence of the Gq class in both vertebrates and invertebrates points to a role that is central to signal transduction in multicellular organisms. We suggest that these alpha subunits may be involved in pertussis toxin-insensitive pathways coupled to phospholipase C.
The primary structure of G proteins as deduced from purified proteins and cloned subunits is presented. When known, their functions are discussed, as are recent data on direct regulation of ionic channels by G proteins. Experiments on expression of alpha subunits, either in bacteria or by in vitro translation of mRNA synthesized from cDNA are presented as tools for definitive assignment of function to a given G protein. The dynamics of G protein-mediated signal transduction are discussed. Key points include the existence of two superimposed regulatory cycles in which upon activation by GTP, G proteins dissociate into alpha and beta gamma and their dissociated alpha subunits hydrolyze GTP. The action of receptors to catalyze rather than regulate by allostery the activation of G proteins by GTP is emphasized, as is the role of subunit dissociation, without which receptors could not act as catalysts. To facilitate the reading of this review, we have presented the various subtopics of this rapidly expanding field in sections 1-1X, each of which is organized as a self-contained sub-chapter that can be read independently of the others.
The early stages of hematopoiesis have been difficult to study due to problems in obtaining homogeneous populations of progenitor cells that retain both self-renewal and differentiative capacities. We have developed an in vitro system in which transformation of murine bone-marrow cells with the BCR/ABL oncogene, a gene associated with stem-cell leukemias, leads to the outgrowth of clonal lines that have an early lymphoid progenitor cell phenotype. The progenitor cells retain immunoglobulin heavy and light chain genes in a germ-line configuration. These cells give rise in vitro to pre-B cells that have diverse diversity-joining (D-J) region rearrangements, and on transfer to mice with severe combined immune deficiency, differentiate to surface IgM+, immunoglobulin-secreting B cells that respond to T-cell help and function in an antigen-specific fashion. Although their growth is stimulated by BCR/ABL, the progenitor cells depend for continued growth on a stromal cell-derived soluble factor distinct from the pre-B-cell growth factor, interleukin 7. These findings show that BCR/ABL can promote proliferation of an early hematopoietic progenitor cell without preventing its differentiation. This system provides a means of studying the complete B-cell developmental process from clonal progenitor cell to end-stage plasma cell.
A thermostable DNA polymerase was used in an in vitro DNA amplification procedure, the polymerase chain reaction. The enzyme,
isolated from Thermus aquaticus, greatly simplifies the procedure and, by enabling the amplification reaction to be performed
at higher temperatures, significantly improves the specificity, yield, sensitivity, and length of products that can be amplified.
Single-copy genomic sequences were amplified by a factor of more than 10 million with very high specificity, and DNA segments
up to 2000 base pairs were readily amplified. In addition, the method was used to amplify and detect a target DNA molecule
present only once in a sample of 10(5) cells.
Biochemical and electrophysiological studies suggest that odorants induce responses in olfactory sensory neurons via an adenylate
cyclase cascade mediated by a G protein. An olfactory-specific guanosine triphosphate (GTP)-binding protein alpha subunit
has now been characterized and evidence is presented suggesting that this G protein, termed Golf, mediates olfaction. Messenger
RNA that encodes Golf alpha is expressed in olfactory neuroephithelium but not in six other tissues tested. Moreover, within
the olfactory epithelium, Golf alpha appears to be expressed only by the sensory neurons. Specific antisera were used to localize
Golf alpha protein to the sensory apparatus of the receptor neurons. Golf alpha shares extensive amino acid identity (88 percent)
with the stimulatory G protein, Gs alpha. The expression of Golf alpha in S49 cyc- kin- cells, a line deficient in endogenous
stimulatory G proteins, demonstrates its capacity to stimulate adenylate cyclase in a heterologous system.
Biochemical analysis has revealed a number of guanine nucleotide-binding regulatory proteins (G proteins) that mediate signal transduction in mammalian systems. Characterization of their cDNAs uncovered a family of proteins with regions of highly conserved amino acid sequence. To examine the extent of diversity of the G protein family, we used the polymerase chain reaction to detect additional gene products in mouse brain and spermatid RNA that share these conserved regions. Sequences corresponding to six of the eight known G protein alpha subunits were obtained. In addition, we found sequences corresponding to five newly discovered alpha subunits. Our results suggest that the complexity of the G protein family is much greater than previously suspected.
The STE4 and STE18 genes are required for haploid yeast cell mating. Sequencing of the cloned genes revealed that the STE4 polypeptide shows extensive homology to the beta subunits of mammalian G proteins, while the STE18 polypeptide shows weak similarity to the gamma subunit of transducin. Null mutations in either gene can suppress the haploid-specific cell-cycle arrest caused by mutations in the SCG1 gene (previously shown to encode a protein with similarity to the alpha subunit of G proteins). We propose that the products of the STE4 and STE18 genes comprise the beta and gamma subunits of a G protein complex coupled to the mating pheromone receptors. The genetic data suggest pheromone-receptor binding leads to the dissociation of the alpha subunit from beta gamma (as shown for mammalian G proteins), and the free beta gamma element initiates the pheromone response.
A subset of growth hormone-secreting human pituitary tumours carries somatic mutations that inhibit GTPase activity of a G protein alpha chain, alpha(s). The resulting activation of adenylyl cyclase bypasses the cells' normal requirement for trophic hormone. Amino acids substituted in the putative gsp oncogene identify a domain of G protein alpha-chains required for intrinsic ability to hydrolyse GTP. This domain may serve as a built-in counter-part of the separate GTPase-activating proteins required for GTP hydrolysis by small GTP-binding proteins such as p21ras.
The production of B lymphocytes and myeloid cells occurs in the bone marrow in association with a supporting population of stromal cells. To determine whether these processes are dependent upon the same or different populations of stromal cells, stromal cell lines were generated from the adherent layer of a Dexter type long-term bone marrow culture. These cultures support myeloid cells and their precursors, a B cell precursor, and the adherent layer cells with support B cell differentiation under appropriate conditions. Two of the lines examined, S10 and S17, express class I histocompatibility antigens but not other hemopoietic cell surface determinants such as Thy-1, Lyt-1, Ig, Ia, Mac-1, or BP-1. Both lines could support myelopoiesis under Dexter conditions upon seeding with nylon wool-passed bone marrow. The nylon wool passage depletes stromal cells capable of forming adherent layers in vitro but retains hemopoietic precursors. The number of cells and colony-forming units-granulocytes/macrophages in the nonadherent cell population recovered 3 wk post-seeding had increased 19-fold and 10-fold, respectively, in the reseeded cultures of S10 and S17. After 3 wk of growth in Dexter conditions, the reseeded cultures were transferred to conditions optimal for B cell differentiation described by Whitlock and Witte. After 4 wk of growth, hemopoietic cells were consistently recovered from S17 cultures but not those of S10. A proportion of these cells from S17 cultures expressed the 14.8 antigen and were surface IgM positive. Surviving hemopoietic cells present in cultures of S10 were primarily macrophages. These findings indicate that S17 but not S10 can support both myelopoiesis and B lymphopoiesis and suggest that one stromal cell population has the capacity to form a hemopoietic microenvironment for both lineages.
By analyzing the effects of single base substitutions around the ATG initiator codon in a cloned preproinsulin gene, I have identified ACCATGG as the optimal sequence for initiation by eukaryotic ribosomes. Mutations within that sequence modulate the yield of proinsulin over a 20-fold range. A purine in position -3 (i.e., 3 nucleotides upstream from the ATG codon) has a dominant effect; when a pyrimidine replaces the purine in position -3, translation becomes more sensitive to changes in positions -1, -2, and +4. Single base substitutions around an upstream, out-of-frame ATG codon affect the efficiency with which it acts as a barrier to initiating at the downstream start site for preproinsulin. The optimal sequence for initiation defined by mutagenesis is identical to the consensus sequence that emerged previously from surveys of translational start sites in eukaryotic mRNAs. The mechanism by which nucleotides flanking the ATG codon might exert their effect is discussed.
The structure of immunoglobulin-related gene was analyzed in individual Abelson murine leukemia virus (A-MuLV)-transformed lymphoid cell lines. Essentially all of these lines, whether immunoglobulin-containing or null, had DNA rearrangements in the vicinity of the JH regions on both chromosomes as well as deletions of at least 5 kb of DNA 5' to JH. None of these lines, however, except rare light chain producers, had detectable rearrangement at either their kappa or lambda light chain loci. In contrast to A-MuLV-transformants derived from bone marrow. Those from early fetal liver frequently contained more than two and sometimes 12 or more distinct, rearranged JH-containing fragments. Cellular subclones derived from these lines had a subset, usually two, of the fragments found in the parent line. Therefore, heavy chain gene rearrangement appears to precede that of light chain gene rearrangement and is still continuing in certain cultured A-MuLV transformants.
Under simple culture conditions, in vitro culture of T cell-depleted splenic mononuclear cells reliably promotes the outgrowth of B lymphoblasts. Such cells, termed BLY1, can be propagated as long-term B cell lines but are nontumorigenic in syngeneic recipients. The phenotype of BLy1 cells is unusual. They express certain B cell markers, including surface mu-lambda, I-Ak, and C3 and Fc receptors; however, delta heavy chain and kappa light chain production is absent. Although the cells do not express Thy-1 or Lyt-2, Lyt-1 is readily demonstrable. These distinctive features of BLy1 cells thus resemble a recently described B cell subset prominent early in ontogeny, and in the B cell defect of NZB mice.
The mammalian ras proteins and the bacterial elongation factors share the ability to interact with guanine nucleotides. Comparison of the amino acid sequences has revealed the presence of multiple homologous regions common to all members of the ras and elongation factor families. Two homologous regions share sequence similarities and predicted secondary structure with areas of the elongation factors which have been implicated in GTP binding, suggesting that these regions are part of a common GTP-binding domain. Two other homologous regions contain critical amino acids for the activation of the transforming potential of the mammalian ras proteins. The predicted secondary structure containing residue 61, but not 12, is the same for the ras proteins and elongation factors. It is proposed that the aligned homologous regions surrounding position 12 constitute common functional binding sites with different specificities; by analogy to other GTP-binding proteins, a likely candidate to interact at such a site is a GTPase-subunit.
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