Recurrent GNAQ mutation encoding T96S in natural killer/T cell lymphoma.

Natural killer/T cell lymphoma (NKTCL) is a rare and aggressive malignancy with a higher prevalence in Asia and South America. However, the molecular genetic mechanisms underlying NKTCL remain unclear. Here, we identify somatic mutations of GNAQ (encoding the T96S alteration of Gαq protein) in 8.7% (11/127) of NKTCL patients, through whole-exome/targeted deep sequencing. Using conditional knockout mice (Ncr1-Cre-Gnaqfl/fl), we demonstrate that Gαq deficiency leads to enhanced NK cell survival. We also find that Gαq suppresses tumor growth of NKTCL via inhibition of the AKT and MAPK signaling pathways. Moreover, the Gαq T96S mutant may act in a dominant negative manner to promote tumor growth in NKTCL. Clinically, patients with GNAQ T96S mutations have inferior survival. Taken together, we identify recurrent somatic GNAQ T96S mutations that may contribute to the pathogenesis of NKTCL. Our work thus has implications for refining our understanding of the genetic mechanisms of NKTCL and for the development of therapies.

GNAQ Negatively Regulates Antiviral Innate Immune Responses in a Calcineurin-Dependent Manner.

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-ß/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.

Role of Galphaq in pathogenesis of psoriasis, a new mechanism about the immune regulation in psoriasis.

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.

Piezo1 and Gq/G11 promote endothelial inflammation depending on flow pattern and integrin activation.

The vascular endothelium is constantly exposed to mechanical forces, including fluid shear stress exerted by the flowing blood. Endothelial cells can sense different flow patterns and convert the mechanical signal of laminar flow into atheroprotective signals, including eNOS activation, whereas disturbed flow in atheroprone areas induces inflammatory signaling, including NF-κB activation. How endothelial cells distinguish different flow patterns is poorly understood. Here we show that both laminar and disturbed flow activate the same initial pathway involving the mechanosensitive cation channel Piezo1, the purinergic P2Y2 receptor, and Gq/G11-mediated signaling. However, only disturbed flow leads to Piezo1- and Gq/G11-mediated integrin activation resulting in focal adhesion kinase-dependent NF-κB activation. Mice with induced endothelium-specific deficiency of Piezo1 or Gαq/Gα11 show reduced integrin activation, inflammatory signaling, and progression of atherosclerosis in atheroprone areas. Our data identify critical steps in endothelial mechanotransduction, which distinguish flow pattern-dependent activation of atheroprotective and atherogenic endothelial signaling and suggest novel therapeutic strategies to treat inflammatory vascular disorders such as atherosclerosis.

Loss of Gαq impairs regulatory B-cell function.

BACKGROUND: Recent studies have shown a crucial role of Gαq in immune regulation, but how Gαq modulates regulatory B-cell (Breg) function is still unclear. We address this here. METHODS: CD19+IL-10+ Bregs of wild-type (WT) and Gnaq-/- mice were analyzed by flow cytometry after stimulation by lipopolysaccharide. The WT and Gnaq-/- Bregs were isolated and cocultured with WT CD4+CD25- T cells in the presence of T-activator, and the proliferation of T cells and differentiation of regulatory T cells (Tregs) were analyzed by flow cytometry. We used inhibitors of PI3 kinase (PI3K), extracellular regulated protein kinases 1/2 (Erk1/2), and p38 mitogen-activated protein kinase (p38 MAPK) to detect the pathways involved in the regulation of Gαq on Breg differentiation, which were confirmed by western blot analysis. Furthermore, the expression level of Gαq was assessed by quantitative real-time PCR in peripheral blood mononuclear cells (PBMCs) from healthy controls and rheumatoid arthritis patients. The frequency of CD19+CD24hiCD38hi B cells in PBMCs was detected by flow cytometry, and the association of the Gαq mRNA expression level and the frequency of CD19+CD24hiCD38hi B cells was analyzed by Spearman test. RESULTS: The differentiation of CD19+IL-10+ Bregs was inhibited in the Gnaq-/- mice. In addition, Gαq depletion showed an impaired suppressive function of Bregs on T-cell proliferation, which might be due to the decreased Treg expansion. Mechanically, our data demonstrated that the PI3K, Erk1/2, and p38 MAPK signaling pathways were required for regulation of Gαq on Bregs, and blockage of these signaling pathways impaired Breg differentiation. Consistent with our previous studies, we also found a decreased frequency of CD19+CD24hiCD38hi Bregs in rheumatoid arthritis patients. As expected, a significantly positive correlation was investigated between CD19+CD24hiCD38hi Bregs with Gαq mRNA expression. CONCLUSIONS: Our results indicate that Gαq plays a critical role in the differentiation and immunosuppression of Bregs, and it may provide a new therapeutic target for autoimmune diseases.

Reactivation of Gαi-coupled formyl peptide receptors is inhibited by Gαq-selective inhibitors when induced by signals generated by the platelet-activating factor receptor.

Formyl peptide receptor (FPR)-desensitized neutrophils display increased production/release of superoxide (O2-) when activated by platelet-activating factor (PAF), a priming of the response achieved through a unique receptor crosstalk mechanism. The aim of this study was to determine the effect of an inhibitor selective for small, heterotrimeric G proteins belonging to the Gαq subclass on that receptor crosstalk. We show that signals generated by FPRs and the PAF receptor (PAFR) induce activation of the neutrophil O2-, producing NADPH-oxidase, and that response was sensitive to Gαq inhibition in cells activated by PAF, but no inhibition was obtained in cells activated by FPR agonists. Signaling in naive neutrophils is terminated fairly rapidly, and the receptors become homologously desensitized. The downstream sensitivity to Gαq inhibition in desensitized cells displaying increased production/release of O2- through the PAFR receptor crosstalk mechanism also comprised the reactivation of the FPRs, and the activation signals were redirected from the PAFR to the desensitized/reactivated FPRs. The Gαq-dependent activation signals generated by the PAFRs activate the Gαi-coupled FPRs, a receptor crosstalk that represents a novel pathway by which G protein-coupled receptors can be regulated and signaling can be turned on and off.

Gq-Coupled Receptors in Autoimmunity.

Heterotrimeric G proteins can be divided into Gi, Gs, Gq/11, and G12/13 subfamilies according to their α subunits. The main function of G proteins is transducing signals from G protein coupled receptors (GPCRs), a family of seven transmembrane receptors. In recent years, studies have demonstrated that GPCRs interact with Gq, a member of the Gq/11 subfamily of G proteins. This interaction facilitates the vital role of this family of proteins in immune regulation and autoimmunity, particularly for Gαq, which is considered the functional α subunit of Gq protein. Therefore, understanding the mechanisms through which Gq-coupled receptors control autoreactive lymphocytes is critical and may provide insights into the treatment of autoimmune disorders. In this review, we summarize recent advances in studies of the role of Gq-coupled receptors in autoimmunity, with a focus on their pathologic role and downstream signaling.

B Lymphocyte-Specific Loss of Ric-8A Results in a Gα Protein Deficit and Severe Humoral Immunodeficiency.

Resistance to inhibitors of cholinesterase 8A (Ric-8A) is a highly evolutionarily conserved cytosolic protein initially identified in Caenorhabditis elegans, where it was assigned a regulatory role in asymmetric cell divisions. It functions as a guanine nucleotide exchange factor for Gαi, Gαq, and Gα12/13 and as a molecular chaperone required for the initial association of nascent Gα subunits with cellular membranes in embryonic stem cell lines. To test its role in hematopoiesis and B lymphocytes specifically, we generated ric8 (fl/fl) vav1-cre and ric8 (fl/fl) mb1-cre mice. The major hematopoietic cell lineages developed in the ric8 (fl/fl) vav1-cre mice, notwithstanding severe reduction in Gαi2/3, Gαq, and Gα13 proteins. B lymphocyte-specific loss of Ric-8A did not compromise bone marrow B lymphopoiesis, but splenic marginal zone B cell development failed, and B cells underpopulated lymphoid organs. The ric8 (fl/fl) mb1-cre B cells exhibited poor responses to chemokines, abnormal trafficking, improper in situ positioning, and loss of polarity components during B cell differentiation. The ric8 (fl/fl) mb1-cre mice had a severely disrupted lymphoid architecture and poor primary and secondary Ab responses. In B lymphocytes, Ric-8A is essential for normal Gα protein levels and is required for B cell differentiation, trafficking, and Ab responses.

The deficiency of Gαq leads to enhanced T-cell survival.

We have previously reported that Gαq, the α subunit of the Gq protein, had important roles in dendritic cell migration, B-cell survival and autoimmunity. In this study, we showed that the deficiency of Gαq led to enhanced T-cell survival. Cultured Gnaq(-/-) T cells exhibited survival advantages both in medium alone and in the presence of anti-CD3 stimulation. Gnaq(-/-) T cells still exhibited a survival advantage when they were cultured in the presence of interleukin (IL)-2 or IL-7. Gnaq(-/-) T cells were more resistant to activation-induced cell death (AICD) in vitro. The survival advantage of Gnaq(-/-) T cells was further confirmed by transferring T cells into syngeneic hosts in vivo. Gαq deficiency might promote T-cell survival by upregulated Bcl-xL expression and downregulated Fas and FasL expressions. Furthermore, upon T-cell receptor (TCR) ligation, Akt activity was increased in Gnaq(-/-) T cells in comparison with wild-type (WT) T cells. The survival advantage of Gnaq(-/-) T cells was significantly attenuated after adding Akt inhibitor. Taken together, our data demonstrated a negative role of Gαq in regulating T-cell survival.

The DAF-16/FOXO transcription factor functions as a regulator of epidermal innate immunity.

The Caenorhabditis elegans DAF-16 transcription factor is critical for diverse biological processes, particularly longevity and stress resistance. Disruption of the DAF-2 signaling cascade promotes DAF-16 activation, and confers resistance to killing by pathogenic bacteria, such as Pseudomonas aeruginosa, Staphylococcus aureus, and Enterococcus faecalis. However, daf-16 mutants exhibit similar sensitivity to these bacteria as wild-type animals, suggesting that DAF-16 is not normally activated by these bacterial pathogens. In this report, we demonstrate that DAF-16 can be directly activated by fungal infection and wounding in wild-type animals, which is independent of the DAF-2 pathway. Fungal infection and wounding initiate the Gαq signaling cascade, leading to Ca(2+) release. Ca(2+) mediates the activation of BLI-3, a dual-oxidase, resulting in the production of reactive oxygen species (ROS). ROS then activate DAF-16 through a Ste20-like kinase-1/CST-1. Our results indicate that DAF-16 in the epidermis is required for survival after fungal infection and wounding. Thus, the EGL-30-Ca(2+)-BLI-3-CST-1-DAF-16 signaling represents a previously unknown pathway to regulate epidermal damage response.

Activation of Gq proteins coupled with 5-HT2 receptors in rat cerebral cortical membranes assessed by antibody-capture scintillation proximity assay/[S]GTPγS binding.

BACKGROUND/AIMS: Functional activation of Gq coupled with 5-HT2 receptors was investigated in rat cerebral cortical membranes. METHODS: Antibody-capture scintillation proximity assay (SPA)/[(35)S]GTPγS binding with anti-Gαq antibody was performed. RESULTS: The specific [(35)S]GTPγS binding to Gαq was increased by 5-hydroxytryptamine (5-HT) in a concentration-dependent but unsaturable manner. The increase elicited by micromolar concentrations of 5-HT was inhibited completely by ketanserin, whereas it inhibited the response by submillimolar to millimolar concentrations of 5-HT only partially. Analysis of the concentration-dependent increases by 5-HT in the absence and presence of ketanserin, methiothepin, WAY100635, and pirenzepine clearly indicates that there are two distinct components of 5-HT-stimulated [(35)S]GTPγS binding, one of which is a pharmacologically relevant increase elicited by lower concentrations (-30 µmol/l) of 5-HT mediated through 5-HT2 receptors and the other is pharmacologically undefined stimulation by higher concentrations of 5-HT. When 5-HT and carbachol were added simultaneously, there was apparently lack of additivity. CONCLUSION: It is concluded that by means of antibody-capture SPA/[(35)S]GTPγS binding it is possible to detect two distinct components of 5-HT-elicited activation of Gq shared by M1 muscarinic receptors, one of which is mediated through 5-HT2 receptors and the other is derived from unknown origin in rat cerebral cortical membranes.

CCR1-mediated STAT3 tyrosine phosphorylation and CXCL8 expression in THP-1 macrophage-like cells involve pertussis toxin-insensitive Gα(14/16) signaling and IL-6 release.

Agonists of CCR1 contribute to hypersensitivity reactions and atherosclerotic lesions, possibly via the regulation of the transcription factor STAT3. CCR1 was demonstrated to use pertussis toxin-insensitive Gα(14/16) to stimulate phospholipase Cß and NF-κB, whereas both Gα(14) and Gα(16) are also capable of activating STAT3. The coexpression of CCR1 and Gα(14/16) in human THP-1 macrophage-like cells suggests that CCR1 may use Gα(14/16) to induce STAT3 activation. In this study, we demonstrated that a CCR1 agonist, leukotactin-1 (CCL15), could indeed stimulate STAT3 Tyr(705) and Ser(727) phosphorylation via pertussis toxin-insensitive G proteins in PMA-differentiated THP-1 cells, human erythroleukemia cells, and HEK293 cells overexpressing CCR1 and Gα(14/16). The STAT3 Tyr(705) and Ser(727) phosphorylations were independent of each other and temporally distinct. Subcellular fractionation and confocal microscopy illustrated that Tyr(705)-phosphorylated STAT3 translocated to the nucleus, whereas Ser(727)-phosphorylated STAT3 was retained in the cytosol after CCR1/Gα(14) activation. CCL15 was capable of inducing IL-6 and IL-8 (CXCL8) production in both THP-1 macrophage-like cells and HEK293 cells overexpressing CCR1 and Gα(14/16). Neutralizing Ab to IL-6 inhibited CCL15-mediated STAT3 Tyr(705) phosphorylation, whereas inhibition of STAT3 activity abolished CCL15-activated CXCL8 release. The ability of CCR1 to signal through Gα(14/16) provides a linkage for CCL15 to regulate IL-6/STAT3-signaling cascades, leading to expression of CXCL8, a cytokine that is involved in inflammation and the rupture of atherosclerotic plaque.

The Gαq/11 proteins contribute to T lymphocyte migration by promoting turnover of integrin LFA-1 through recycling.

The role of Gαi proteins coupled to chemokine receptors in directed migration of immune cells is well understood. In this study we show that the separate class of Gαq/11 proteins is required for the underlying ability of T cells to migrate both randomly and in a directed chemokine-dependent manner. Interfering with Gαq or Gα11 using dominant negative cDNA constructs or siRNA for Gαq causes accumulation of LFA-1 adhesions and stalled migration. Gαq/11 has an impact on LFA-1 expression at plasma membrane level and also on its internalization. Additionally Gαq co-localizes with LFA-1- and EEA1-expressing intracellular vesicles and partially with Rap1- but not Rab11-expressing vesicles. However the influence of Gαq is not confined to the vesicles that express it, as its reduction alters intracellular trafficking of other vesicles involved in recycling. In summary vesicle-associated Gαq/11 is required for the turnover of LFA-1 adhesion that is necessary for migration. These G proteins participate directly in the initial phase of recycling and this has an impact on later stages of the endo-exocytic pathway.

Behavioral and immune responses to infection require Gαq- RhoA signaling in C. elegans.

Following pathogen infection the hosts' nervous and immune systems react with coordinated responses to the danger. A key question is how the neuronal and immune responses to pathogens are coordinated, are there common signaling pathways used by both responses? Using C. elegans we show that infection by pathogenic strains of M. nematophilum, but not exposure to avirulent strains, triggers behavioral and immune responses both of which require a conserved Gαq-RhoGEF Trio-Rho signaling pathway. Upon infection signaling by the Gαq pathway within cholinergic motorneurons is necessary and sufficient to increase release of the neurotransmitter acetylcholine and increase locomotion rates and these behavioral changes result in C. elegans leaving lawns of M. nematophilum. In the immune response to infection signaling by the Gαq pathway within rectal epithelial cells is necessary and sufficient to cause changes in cell morphology resulting in tail swelling that limits the infection. These Gαq mediated behavioral and immune responses to infection are separate, act in a cell autonomous fashion and activation of this pathway in the appropriate cells can trigger these responses in the absence of infection. Within the rectal epithelium the Gαq signaling pathway cooperates with a Ras signaling pathway to activate a Raf-ERK-MAPK pathway to trigger the cell morphology changes, whereas in motorneurons Gαq signaling triggers behavioral responses independent of Ras signaling. Thus, a conserved Gαq pathway cooperates with cell specific factors in the nervous and immune systems to produce appropriate responses to pathogen. Thus, our data suggests that ligands for Gq coupled receptors are likely to be part of the signals generated in response to M. nematophilum infection.

Expression of G protein αq Subunit is Decreased in Lymphocytes from Patients with Rheumatoid Arthritis and is Correlated with Disease Activity.

Gαq, the alpha subunit of Gq, a member of the Gq/11 sub-family, was reported to inhibit phosphatidylinositol-3-Kinase (PI3K) activation and prevent the activation of Akt. Previous studies demonstrated that mice losing Gαq in their immune system could spontaneously develop inflammatory arthritis. In this study, we showed that the Gαq expressions at mRNA and protein levels in the peripheral blood lymphocytes (PBLs) from patients with rheumatoid arthritis (RA) were significantly decreased in comparison of which in healthy individuals. The expression levels of Gαq mRNA in PBLs from patients with RA were correlated with RA disease activity (DAS28), anti-cyclic citrullinated protein antibodies, C-reactive protein and rheumatoid factor. We also demonstrated that Gαq controlled the apoptosis of RA PBLs through regulating the activity of Mcl-1 and caspase-3. These data suggested that Gαq might be involved in the pathogenesis of RA by regulating PBLs apoptosis.

Muscarinic acetylcholine receptor-mediated activation of G(q) in rat brain membranes determined by guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTPγS) binding using an anti-G protein scintillation proximity assay.

In the present study, we performed antibody-capture guanosine-5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPγS) scintillation proximity assay (SPA), in which immuno-capture of Gα subunits following [(35)S]GTPγS binding was combined with SPA technology, in rat brain membranes. Preliminary experiments using a series of agonists and commercially available anti-Gα antibodies indicated the increase in specific [(35)S]GTPγS binding to Gα(q) determined with the anti-Gα antibody sc-393 and evoked by carbamylcholine chloride (CCh) was pharmacologically relevant. The experimental conditions were optimized as for the concentrations of GDP, MgCl(2), and NaCl, the dilution of the anti-Gα(q) antibody, and membrane protein contents incubated. Under the optimized conditions, CCh-stimulated specific [(35)S]GTPγS binding to Gα(q) in a concentration-dependent and saturable manner with an EC(50) of around 10 µM in all of the membranes prepared from rat hippocampus, cerebral cortex, and striatum. The maximum responses were varied according to the brain regions, with the rank order in magnitude of hippocampus > cerebral cortex > striatum. The addition of MT-7, a snake toxin with high selectivity for M(1) over the other muscarinic acetylcholine receptors (mAChRs) (M(2)-M(5)), almost completely extinguished CCh-stimulated [(35)S]GTPγS binding to Gα(q), even at a concentration as low as 1 nM. These results indicate that the functional coupling between M(1) mAChR and Gα(q) can be investigated in rat native brain membranes by means of antibody-capture SPA/[(35)S]GTPγS binding assay. The assay developed in the present study would provide a useful strategy for investigation of possible pathophysiological alterations in neuropsychiatric disorders such as Alzheimer's disease and schizophrenia as well as for drug discovery.

Enzymatic actions of Pasteurella multocida toxin detected by monoclonal antibodies recognizing the deamidated α subunit of the heterotrimeric GTPase Gq.

Pasteurella multocida toxin (PMT) is a virulence factor responsible for the pathogenesis of some Pasteurellosis. PMT exerts its toxic effects through the activation of heterotrimeric GTPase (G(q), G(12/13) and G(i))-dependent pathways, by deamidating a glutamine residue in the α subunit of these GTPases. However, the enzymatic characteristics of PMT are yet to be analyzed in detail because the deamidation has only been observed in cell-based assays. In the present study, we developed rat monoclonal antibodies, specifically recognizing the deamidated Gα(q), to detect the actions of PMT by immunological techniques such as western blotting. Using the monoclonal antibodies, we found that the toxin deamidated Gα(q) only under reducing conditions. The C-terminal region of PMT, C-PMT, was more active than the full-length PMT. The C3 domain possessing the enzyme core catalyzed the deamidation in vitro without any other domains. These results not only support previous observations on toxicity, but also provide insights into the enzymatic nature of PMT. In addition, we present several lines of evidence that Gα(11), as well as Gα(q), could be a substrate for PMT.

CCR1-mediated activation of Nuclear Factor-kappaB in THP-1 monocytic cells involves Pertussis Toxin-insensitive Galpha(14) and Galpha(16) signaling cascades.

Agonists of CC chemokine receptor CCR1 contribute to the pathogenesis of autoimmune and other inflammatory diseases, possibly via the regulation of the transcription factor NF-kappaB. CCR1 and CCR2b have been demonstrated to use PTX-insensitive Galpha(14) and Galpha(16) to stimulate PLCbeta in cotransfected cells, and Galpha(14) and Galpha(16) are capable of activating NF-kappaB. The coexpression of Galpha(14), Galpha(16), and CCR1 in human monocytic THP-1 cells suggests that CCR1 may use Galpha(14) or Galpha(16) to induce NF-kappaB activation. Here, we demonstrated that a CCR1 agonist, Lkn-1, stimulated NF-kappaB phosphorylation via PTX-insensitive G proteins in THP-1 cells. Lkn-1 also mediated IKK/NF-kappaB phosphorylations in HEK293 cells overexpressing CCR1 and Galpha(14/16). Using various kinase inhibitors, Raf-1, MEK1/2, PLCbeta, PKC, CaM, CaMKII, and c-Src were found to participate in Lkn-1-stimulated IKK/NF-kappaB phosphorylations in THP-1 and transfected HEK293 cells. Although c-Jun N-terminal kinase and p38 MAPK were activated by Lkn-1, they were not required in Lkn-1-induced IKK phosphorylation. The ability of CCR1 to signal through Galpha(14/16) thus provides a linkage for chemokines to regulate NF-kappaB-dependent responses.

Interplay between the heterotrimeric G-protein subunits Galphaq and Galphai2 sets the threshold for chemotaxis and TCR activation.

BACKGROUND: TCR and CXCR4-mediated signaling appears to be reciprocally regulated pathways. TCR activation dampens the chemotactic response towards the CXCR4 ligand CXCL12, while T cells exposed to CXCL12 are less prone to subsequent TCR-activation. The heterotrimeric G proteins Galphaq and Galphai2 have been implicated in CXCR4-signaling and we have recently also reported the possible involvement of Galphaq in TCR-dependent activation of Lck (Ngai et al., Eur. J. Immunol., 2008, 38: 32083218). Here we examined the role of Galphaq in migration and TCR activation. RESULTS: Pre-treatment of T cells with CXCL12 led to significantly reduced Lck Y394 phosphorylation upon TCR triggering indicating heterologous desensitization. We show that knockdown of Galphaq significantly enhanced basal migration in T cells and reduced CXCL12-induced SHP-1 phosphorylation whereas Galphai2 knockdown inhibited CXCL12-induced migration. CONCLUSION: Our data suggest that Galphai2 confers migration signals in the presence of CXCL12 whereas Galphaq exerts a tonic inhibition on both basal and stimulated migrational responses. This is compatible with the notion that the level of Galphaq activation contributes to determining the commitment of the T cell either to migration or activation through the TCR.

Effects of progesterone on motility and prostaglandin levels in the distal guinea pig colon.

Progesterone (P4) inhibits the gastrointestinal muscle contraction by downregulating Galpha(q/11) proteins that mediate contraction, by upregulating Galpha(s) proteins that mediate relaxation, and by altering the pattern of cyclooxygenase (COX) enzymes and prostaglandins. We aimed to examine whether P4 treatment of guinea pigs in vivo affects basal colon motility [basal motility index (MI)] by altering the levels and actions of PGF(2alpha) and PGE(2). Guinea pigs were treated with intramuscular (IM) P4 for 4 days. The BASAL MI, the PGF(2alpha)-induced contraction, and PGE(2)-induced inhibition of contraction were examined in muscle strips and cells. The levels of PGF(2alpha) and PGE(2) were measured by radioimmunoassay. Treatment with P4 reduced the basal MI, the levels of PGF(2alpha), and PGF(2alpha)-induced contraction. P4 increased PGE(2) levels, and PGE(2) induced relaxation. Pretreatment with IM RU-486 (10 mg/kg per day), a P4 receptor antagonist, 1 h before P4 blocked the actions of P4. The PGF(2alpha) antagonist Al-1180 abolished basal MI and PGF(2alpha)-induced contraction. N-ethylmaleimide, which blocks unoccupied membrane receptors, blocked Ach and VIP actions but had no effect on PGF(2alpha) and PGE(2) effects. A COX-1 inhibitor decreased and a COX-2 inhibitor increased PGF(2alpha) levels; GTPgammaS increased and GDPbetaS decreased the levels of PGF(2alpha). Galpha(q/11) protein antibodies (Abs) reduced PGF(2alpha) levels, and Galpha(i3) Abs blocked its motor actions. Galphas Abs increased PGF(2alpha) but decreased PGE(2) levels. We concluded that P4 decreases basal MI by reducing PGF(2alpha) levels caused by downregulation of Galpha(q/11) and that PGF(2alpha)-induced contraction was blocked by downregulating Galpha(i3). P4 also decreased the basal MI by increasing PGE(2) levels, and PGE(2) induced relaxation by upregulating Galpha(s) proteins.