BRET-based assay to monitor EGFR transactivation by the AT1R reveals Gq/11 protein-independent activation and AT1R-EGFR complexes.

The type 1 angiotensin II (AngII) receptor (AT1R) transactivates the epidermal growth factor receptor (EGFR), which leads to pathological remodeling of heart, blood vessels and kidney. End-point assays are used as surrogates of EGFR activation, however these downstream readouts are not applicable to live cells, in real-time. Herein, we report the use of a bioluminescence resonance energy transfer (BRET)-based assay to assess recruitment of the EGFR adaptor protein, growth factor receptor-bound protein 2 (Grb2), to the EGFR. In a variety of cell lines, both epidermal growth factor (EGF) and AngII stimulated Grb2 recruitment to EGFR. The BRET assay was used to screen a panel of 9 G protein-coupled receptors (GPCRs) and further developed for other EGFR family members (HER2 and HER3); the AT1R was able to transactivate HER2, but not HER3. Mechanistically, AT1R-mediated ERK1/2 activation was dependent on Gq/11 and EGFR tyrosine kinase activity, whereas the recruitment of Grb2 to the EGFR was independent of Gq/11 and only partially dependent on EGFR tyrosine kinase activity. This Gq/11 independence of EGFR transactivation was confirmed using AT1R mutants and in CRISPR cell lines lacking Gq/11. EGFR transactivation was also apparently independent of ß-arrestins. Finally, we used additional BRET-based assays and confocal microscopy to provide evidence that both AngII- and EGF-stimulation promoted AT1R-EGFR heteromerization. In summary, we report an alternative approach to monitoring AT1R-EGFR transactivation in live cells, which provides a more direct and proximal view of this process, including the potential for complexes between the AT1R and EGFR.

Subcellular compartmentalization of proximal Gαq-receptor signaling produces unique hypertrophic phenotypes in adult cardiac myocytes.

G protein-coupled receptors that signal through Gαq (Gq receptors), such as α1-adrenergic receptors (α1-ARs) or angiotensin receptors, share a common proximal signaling pathway that activates phospholipase Cß1 (PLCß1), which cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) to produce inositol 1,4,5-trisphosphate (IP3) and diacylglycerol. Despite these common proximal signaling mechanisms, Gq receptors produce distinct physiological responses, yet the mechanistic basis for this remains unclear. In the heart, Gq receptors are thought to induce myocyte hypertrophy through a mechanism termed excitation-transcription coupling, which provides a mechanistic basis for compartmentalization of calcium required for contraction versus IP3-dependent intranuclear calcium required for hypertrophy. Here, we identified subcellular compartmentalization of Gq-receptor signaling as a mechanistic basis for unique Gq receptor-induced hypertrophic phenotypes in cardiac myocytes. We show that α1-ARs co-localize with PLCß1 and PIP2 at the nuclear membrane. Further, nuclear α1-ARs induced intranuclear PLCß1 activity, leading to histone deacetylase 5 (HDAC5) export and a robust transcriptional response (i.e. significant up- or down-regulation of 806 genes). Conversely, we found that angiotensin receptors localize to the sarcolemma and induce sarcolemmal PLCß1 activity, but fail to promote HDAC5 nuclear export, while producing a transcriptional response that is mostly a subset of α1-AR-induced transcription. In summary, these results link Gq-receptor compartmentalization in cardiac myocytes to unique hypertrophic transcription. They suggest a new model of excitation-transcription coupling in adult cardiac myocytes that accounts for differential Gq-receptor localization and better explains distinct physiological functions of Gq receptors.

Gaq proteins: molecular pharmacology and therapeutic potential.

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.

Decreased Gαq expression in T cells correlates with enhanced cytokine production and disease activity in systemic lupus erythematosus.

Aberrant T cell immune responses appear central to the development of systemic lupus erythematosus (SLE). We previously reported that Gαq, the alpha subunit of Gq, regulates T and B cell immune responses, promoting autoimmunity. To address whether Gαq contributes to the pathogenesis of SLE, Gαq mRNA expression was studied using real time-PCR in PBMCs and T cells from SLE patients as well as age- and sex-matched healthy controls. Our results showed that Gαq mRNA expression was decreased in PBMCs and T cells from SLE patients compared to healthy individuals. Correlation analyses showed that Gαq expression in T cells from SLE patients was associated with disease severity (as per SLE Disease Activity Index), the presence of lupus nephritis, and expression of Th1, Th2 and Th17 cytokines. In keeping with clinical results, T-helper cell subsets (Th1, Th2 and Th17) were over-represented in Gαq knockout mice. In addition, Gαq expression in SLE T cells was negatively correlated with the expression of Bcl-2, an anti-apoptotic gene, and positively correlated with the expression of Bax, a pro-apoptotic gene. These data suggest that reduced Gαq levels in T cells may promote enhanced and prolonged T cell activation, contributing to the clinical manifestations of SLE.

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

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

GNA15 expression in small intestinal neuroendocrine neoplasia: functional and signalling pathway analyses.

Gastroenteropancreatic neuroendocrine neoplasia (GEP-NEN) comprises a heterogeneous group of tumours that exhibit widely divergent biological behaviour. The identification of new targetable GPCR-pathways involved in regulating cell function could help to identify new therapeutic strategies. We assessed the function of a haematopoietic stem cell heterotrimeric G-protein, Gα15, in gut neuroendocrine cell models and examined the clinical implications of its over expression. Functional assays were undertaken to define the role of GNA15 in the small intestinal NEN cell line KRJ-I and in clinical samples from small intestinal NENs using quantitative polymerase chain reaction, western blot, proliferation and apoptosis assays, immunoprecipitation, immunohistochemistry (IHC) and automated quantitative analysis (AQUA). GNA15 was not expressed in normal neuroendocrine cells but was overexpressed in GEP-NEN cell lines. In KRJ-I cells, decreased expression of GNA15 was associated with inhibition of proliferation, activation of apoptosis and differential effects on pro-proliferative ERK, NFκB and Akt pathway signalling. Moreover, Gα15 was demonstrated to couple to the ß1 adrenergic receptor and modulated proliferative signals through this GPCR. Transcript and protein levels of GNA15 were significantly elevated in primary and metastatic tumours compared to normal mucosa and were particularly increased in low Ki-67 expressing tumours. IHC and AQUA revealed that a higher Gα15 expression was associated with a poorer survival. GNA15 may have a pathobiological role in SI-NENs. Targeting this signalling mediator could provide an opportunity for the development of new therapeutic strategies for this tumour type.

Levobupivacaine differentially suppresses platelet aggregation by modulating calcium release in a dose-dependent manner.

OBJECTIVE: Levobupivacaine, an amide local anesthetic widely used in regional anesthesia, is reported in recent studies that it is a potent inhibitor of platelet functions. However, the concentrations of levobupivacaine were limitedly estimated in these reports. Additionally, the mechanisms by which it affects platelet function and blood coagulation is still not entirely known. The purpose of this study was to further investigate its effects on platelet function and the possible signaling mechanisms under various concentrations of levobupivacaine. METHODS: Blood samples collected from healthy volunteers were separated into whole blood, platelet-rich-plasma and washed platelets. The effect of levobupivacaine on platelet aggregation was studied using platelet function analyzer (PFA-100) and platelet aggregometer. Agonist-induced platelet adenosine triphosphate (ATP) release, cytosolic calcium mobilization, thromboxane B2 (TxB2) secretion and platelet P-selectin translocation under various concentrations of levobupivacaine were investigated. RESULTS: Our results indicated that levobupivacaine possessed negative effect on platelet aggregation. The closure times of (PFA-100) were lengthened and the agonist-induced platelet aggregation was significantly attenuated by levobupivacaine even at a low dose (50 µgml(-1)). Pretreatment with levobupivacaine produced significant changes in agonist-induced platelet P-selectin translocation, ATP release, thromboxane A2 (TxA2) production, and calcium mobilization in a dose-dependent manner. The p38 mitogen-activated protein kinases (MAPK), protein kinase C (PKC) δ subtype, cytosolic phospholipase A2 (cPLA2), and protein kinase B (PKB or Akt) were involved in collagen-induced platelet signaling, which would be responsible for antiplatelet effects of levobupivacaine. CONCLUSION: We explored possible targets of levobupivacaine on platelets aggregation signaling mechanisms. Our data revealed that p38 MAPK, PKC δ subtype, cPLA2, and Akt were pathways involved in collagen-induced platelet signaling, which might be responsible for antiplatelet effects of levobupivacaine. Our study did provide direct evidence bolstering the critical mechanisms of levobupivacaine within different contexts. Additionally, levobupivacaine imposed a negative effect on platelet aggregation through multiple signaling pathways.

Does mRNA level of microsomal carnitine palmitoyltransferase predict yield of peripheral blood stem cell apheresis?

Transplantation of autologous hematopoietic stem cells is a well established therapeutic procedure. Despite advances in efficacy of the stem cell mobilization and apheresis process until now a predictive factor for the expected stem cell yield before initiation of mobilization therapy could not be identified. The main objective of our study was to evaluate alterations in enzymes involved in fatty acid metabolism on the level of gene expression in mononuclear cells, as changes in relative mRNA levels of these enzymes could represent the hematopoietic regenerative potential. Data of 23 consecutive patients with different lymphoid malignancies undergoing stem cell mobilization were analyzed. Our results show that mRNA levels of microsomal carnitine palmitoyltransferase in peripheral blood mononuclear cells quantified before application of mobilization therapy correlate positively with the amount of CD34 positive cells in peripheral blood before first apheresis, in the first apheresis product and in the total harvest outcome. The association of enzymes involved in fatty acid metabolism with hematoopoiesis was further confirmed in healthy subjects on altitude-adaptation training and in proliferating or differentiating HL60 cells. This gives evidence for a possible predictive value of such analyzes though further data of a larger sample are to be collected to confirm our observations.

Dominant portion of thyrotropin-releasing hormone receptor is excluded from lipid domains. Detergent-resistant and detergent-sensitive pools of TRH receptor and Gqalpha/G11alpha protein.

Some G protein-coupled receptors might be spacially targetted to discrete domains within the plasma membrane. Here we assessed the localization in membrane domains of the epitope-tagged, fluorescent version of thyrotropin-releasing hormone receptor (VSV-TRH-R-GFP) expressed in HEK293 cells. Our comparison of three different methods of cell fractionation (detergent extraction, alkaline treatment/sonication and mechanical homogenization) indicated that the dominant portion of plasma membrane pool of the receptor was totally solubilized by Triton X-100 and its distribution was similar to that of transmembrane plasma membrane proteins (glycosylated and non-glycosylated forms of CD147, MHCI, CD29, CD44, transmembrane form of CD58, Tapa1 and Na,K-ATPase). As expected, caveolin and GPI-bound proteins CD55, CD59 and GPI-bound form of CD58 were preferentially localized in detergent-resistant membrane domains (DRMs). Trimeric G proteins G(q)alpha/G(11)alpha, G(i)alpha1/G(i)alpha2, G(s)alphaL/G(s)alphaS and Gbeta were distributed almost equally between detergent-resistant and detergent-solubilized pools. In contrast, VSV-TRH-R-GFP, Galpha, Gbeta and caveolin were localized massively only in low-density membrane fragments of plasma membranes, which were generated by alkaline treatment/sonication or by mechanical homogenization of cells. These data indicate that VSV-TRH-R-GFP as well as other transmembrane markers of plasma membranes are excluded from TX-100-resistant, caveolin-enriched membrane domains. Trimeric G protein G(q)alpha/G(11)alpha occurs in both DRMs and in the bulk of plasma membranes, which is totally solubilized by TX-100.

Loss of Gq/11 family G proteins in the nervous system causes pituitary somatotroph hypoplasia and dwarfism in mice.

Heterotrimeric G proteins of the Gq/11 family transduce signals from a variety of neurotransmitter and hormone receptors and have therefore been implicated in various functions of the nervous system. Using the Cre/loxP system, we generated mice which lack the genes coding for the alpha subunits of the two main members of the Gq/11 family, gnaq and gna11, selectively in neuronal and glial precursor cells. Mice with defective gnaq and gna11 genes were morphologically normal, but they died shortly after birth. Mice carrying a single gna11 allele survived the early postnatal period but died within 3 to 6 weeks as anorectic dwarfs. In these mice, postnatal proliferation of pituitary somatotroph cells was strongly impaired, and plasma growth hormone (GH) levels were reduced to 15%. Hypothalamic levels of GH-releasing hormone (GHRH), an important stimulator of somatotroph proliferation, were strongly decreased, and exogenous administration of GHRH restored normal proliferation. The hypothalamic effects of ghrelin, a regulator of GHRH production and food intake, were reduced in these mice, suggesting that an impairment of ghrelin receptor signaling might contribute to GHRH deficiency and abnormal eating behavior. Taken together, our findings show that Gq/11 signaling is required for normal hypothalamic function and that impairment of this signaling pathway causes somatotroph hypoplasia, dwarfism, and anorexia.

Defective sarcolemmal phospholipase C signaling in diabetic cardiomyopathy.

Phospholipase C (PLC) activity is known to influence cardiac function. This study was undertaken to examine the status of PLC beta3 in the cardiac cell plasma membrane (sarcolemma, SL) in an experimental model of chronic diabetes. SL membrane was isolated from diabetic rat hearts at 8 weeks after a single i.v. injection of streptozotocin (65 mg/kg body weight). The total SL PLC was decreased in diabetes and was associated with a decrease in SL PLC beta3 activity, which immunofluorescence in frozen diabetic left ventricular tissue sections revealed to be due to a decrease in PLC beta3 protein abundance. In contrast, the SL abundance of Gqalpha was significantly increased during diabetes. These changes were associated with a loss of contractile function (+/- dP/dt). A 2-week insulin treatment of 6-week diabetic animals partially normalized all of these parameters. These findings suggest a defect in PLC beta3-mediated signaling processes may contribute to the cardiac dysfunction seen during diabetes.

Dynamic regulation of a GPCR-tetraspanin-G protein complex on intact cells: central role of CD81 in facilitating GPR56-Galpha q/11 association.

By means of a variety of intracellular scaffolding proteins, a vast number of heterotrimeric G protein-coupled receptors (GPCRs) may achieve specificity in signaling through a much smaller number of heterotrimeric G proteins. Members of the tetraspanin family organize extensive complexes of cell surface proteins and thus have the potential to act as GPCR scaffolds; however, tetraspanin-GPCR complexes had not previously been described. We now show that a GPCR, GPR56/TM7XN1, and heterotrimeric G protein subunits, Galpha(q), Galpha(11), and Gbeta, associate specifically with tetraspanins and CD81, but not with other tetraspanins. CD9 Complexes of GPR56 with CD9 and CD81 remained intact when fully solubilized and were resistant to cholesterol depletion. Hence they do not depend on detergent-insoluble, raft-like membrane microdomains for stability. A central role for CD81 in promoting or stabilizing a GPR56-CD81-Galpha(q/11) complex was revealed by CD81 immunodepletion and reexpression experiments. Finally, antibody engagement of cell surface CD81 or cell activation with phorbol ester revealed two distinct mechanisms by which GPR56-CD81-Galpha(q/11) complexes can be dynamically regulated. These data reveal a potential role for tetraspanins CD9 and CD81 as GPCR scaffolding proteins.

A functional assay for G-protein-coupled receptors using stably transformed insect tissue culture cell lines.

Insect cells are an underexplored resource for functional G-protein-coupled receptor (GPCR) assays, despite a strong record in biochemical (binding) assays. Here we describe the use of vectors capable of creating stably transformed insect cell lines to generate a cell-based functional GPCR assay. This assay employs the luminescent photoprotein aequorin and the promiscuous G-protein subunit Galpha16 and is broadly applicable to human GPCRs. We demonstrate that the assay can quantitate ligand concentration-activity relationships for seven different human GPCRs, can differentiate between partial and full agonists, and can determine rank order potencies for both agonists and antagonists that match those seen with other assay systems. Human Galpha16 improves signal strength but is not required for activity with some receptors. The coexpression of human and bovine betagamma subunits and/or phospholipase Cbeta makes no difference to agonist efficacy or potency. Two different receptors expressed in the same cell line respond to their specific agonists, and two different cell lines (Sf9 and High 5) are able to functionally detect the same expressed GPCR. Sf9 cells have the capability to produce fully functional human receptors, allied to a low background of endogenous receptors, and so are a valuable system for investigating orphan GPCRs and receptor dimerization.