Level Of Gq Research Articles

The TRPM7 kinase limits receptor-induced calcium release by regulating heterotrimeric G-proteins.

The melastatin-related transient receptor potential member 7 (TRPM7) is a unique fusion protein with both ion channel function and enzymatic α-kinase activity. TRPM7 is essential for cellular systemic magnesium homeostasis and early embryogenesis; it promotes calcium transport during global brain ischemia and emerges as a key player in cancer growth. TRPM7 channels are negatively regulated through G-protein-coupled receptor-stimulation, either by reducing cellular cyclic adenosine monophosphate (cAMP) or depleting phosphatidylinositol bisphosphate (PIP2) levels in the plasma membrane. We here identify that heterologous overexpression of human TRPM7-K1648R mutant will lead to disruption of protease or purinergic receptor-induced calcium release. The disruption occurs at the level of Gq, which requires intact TRPM7 kinase phosphorylation activity for orderly downstream signal transduction to activate phospholipase (PLC)β and cause calcium release. We propose that this mechanism may support limiting GPCR-mediated calcium signaling in times of insufficient cellular ATP supply.

Receptor Species-Dependent Signaling of Gqpcrs Controls IKs Activity

The membrane lipid phosphatidylinositol-4,5-bisphosphate (PIP2) interacts with signaling molecules at the plasma membrane and positively modulates KCNQ1/KCNE1 (IKs) ion channels. Membrane PIP2 is reduced by activity of phospholipase C (PLC), which in turn is controlled by Gq protein-coupled receptors (GqPCRs). Our study compared the muscarinic M1 receptor (M1-R) and the α1B adrenergic receptor (α1B-AR) in their ability to modulate IKs. We analyzed temporal aspects of receptor signaling in single HEK 293 cells and CHO cells stably expressing KCNQ1/KCNE1 using FRET-based biosensors for the Gq cycle and PLC activation. IKs was measured using voltage-clamp. Both receptors activate PLC and cause PIP2 depletion. Activation of M1-R caused robust inhibition of IKs with a kinetics that resembled the time course of PIP2-depletion and recovery of IKs upon removal of the agonist. In contrast, the α1B-AR-induced inhibition of IKs was transient and turned into activation after agonist withdrawal. This delayed response exceeded basal activation levels and occurred at a point when the PIP2 pool was completely restored. Furthermore, the α1B-AR, but not the M1-R, displayed acute desensitization in the presence of agonist that was evident on the levels of Gq and PLC activation. This indicates a feedback modulation by kinases. Indeed, global inhibition of all protein kinase C (PKC) isoforms with staurosporine removed receptor desensitization and abolished the delayed facilitation of IKs. A more specific inhibition of Ca2+-sensitive PKCs with Go6976 reduced α1B-AR desensitization but not facilitation of IKs, which was rather mediated by diacylglycerol-activated kinases. Notably, activation of M1-R and its influence on IKs were not dependent on PKC activation. We conclude from these data that fine-tuning of IKs activity via Gq is controlled by recruitment of kinases downstream of PIP2 depletion in a receptor species-specific fashion.

Altered cAMP signaling induced by lysophosphatidic acid in senescent human diploid fibroblasts

Lysophosphatidic acid (LPA) is a lipid mitogen that acts through G-protein-coupled receptors. LPA responsiveness has been reported to be dependent on the senescent state of the cells. To solve the mechanism underlying, we observed LPA-dependent cAMP status and found its age-dependent contrasting profile such as high level of cAMP in the senescent cells vs its low level in the young cells. In order to clarify the molecular mechanism of the ageing effect, we examined various molecular species involved in the cAMP signaling pathway by semi-quantitative RT-PCR. EDG-1 and EDG-4 were unchanged, but EDG-2 and EDG-7 were reduced with age. Senescent cells showed a partial reduction of Gi1, Gi2, and Gi3, but no change in the level of Gq. Decreased Gis and Gi-coupled LPA receptors may reduce the inhibitory effect of Giα on adenylyl cyclases (ACs), resulting in cAMP accumulation via activation of adenylyl cyclase in senescent fibroblasts. We also observed an age-dependent increase in some of AC isoforms: II, IV, and VI. In conclusion, multiple changes in the cAMP signaling pathway of the senescent cells might explain the altered responsiveness to the mitogenic stimuli.

Novel mechanisms for feedback regulation of phospholipase C-beta activity.

The receptor-regulated phospholipase C-beta (PLC-beta) signaling pathway is an important component in a network of signaling cascades that regulate cell function. PLC-beta signaling has been implicated in the regulation of cardiovascular function and neuronal plasticity. The Gq family of G proteins mediate receptor stimulation of PLC-beta activity at the plasma membrane. Mitogens stimulate the activity of a nuclear pool of PLC-beta. Stimulation of PLC-beta activity results in the rapid hydrolysis of phosphatidylinositol-4,5-bisphosphate, with production of inositol-1,4,5-trisphosphate and diacylglycerol, intracellular mediators that increase intracellular Ca2+ levels and activate protein kinase C activity, respectively. Diacylglycerol kinase converts diacylglycerol to phosphatidic acid, a newly emerging intracellular mediator of hormone action that targets a number of signaling proteins. Activation of the Gq linked PLC-beta signaling pathway can also generate additional signaling lipids, including phosphatidylinositol-3-phosphate and phosphatidylinositol-3,4,5-trisphosphate, which regulate the activity and/or localization of a number of proteins. Novel feedback mechanisms, directed at the level of Gq and PLC-beta, have been identified. PLC-beta and regulators of G protein signaling (RGS) function as GTPase-activating proteins on Gq to control the amplitude and duration of stimulation. Protein kinases phosphorylate and regulate the activation of specific PLC-beta isoforms. Phosphatidic acid regulates PLC-beta1 activity and stimulation of PLC-beta1 activity by G proteins. These feedback mechanisms coordinate receptor signaling and cell activation. Feedback mechanisms constitute possible targets for pharmacological intervention in the treatment of disease.

Modulation of phospholipase C pathway and level of Gq alpha/G11 alpha in rat myometrium during gestation.

The regulation of the receptor-G protein-phospholipase C (PLC) cascade was investigated in rat myometrium at midgestation (day 12) and at term (day 21) comparatively to the estrogen-treated tissue (day 0). Carbachol-mediated generation of [3H]inositol phosphates was insensitive to pertussis toxin and was enhanced at days 12 and 21 two- and threefold, respectively, with no alteration of muscarinic sites (M3 subtype). A similar increase could be detected in the production of inositol 1,4,5-trisphosphate, indicating the stimulation of a PLC degrading phosphatidylinositol 4,5-bisphosphate. AlF4- also enhanced PLC activation during gestation, suggesting pregnancy-related regulations that bypass receptor activation. Immunoreactive G proteins, Gq alpha and G11 alpha, and PLC-beta 3 were detected in all myometrial preparations. The amount of PLC-beta 3 was similar in day 0 and day 21 myometrium, although decreasing by 75% at midgestation. Of significance was the increased amount of Gq alpha in day 12 and day 21 myometrium (3- and 2-fold, respectively) which coincided with the enhanced phosphoinositide breakdown. The upregulation of Gq alpha may contribute to the enhanced PLC activity during pregnancy and at term.

Reduction in Gh protein expression is associated with cytodifferentiation of vascular smooth muscle cells.

Gh, a high molecular weight GTP-binding protein that couples alpha 1-adrenoceptors in heart and liver to phosphatidylinositol (PtdIns)-specific phospholipase C (PLC), has recently been shown to be a tissue transglutaminase type II. Transglutaminases have been suggested to play a role in the maintenance of blood vessel structure, and therefore it is possible that changes in their expression may accompany pathological states which involve phenotypic modulation of smooth muscle. Hence, we investigated the expression of Gh during differentiation of rat aortic smooth muscle cells in culture. Gh content was reduced markedly in cultured smooth muscle cells compared to freshly isolated cells as determined by Western blotting using a Gh-specific monoclonal antibody. In contrast, the level of Gq, a heterotrimeric G-protein that couples alpha 1-adrenoceptors to PLC, was maintained throughout the culture period. These findings indicate that changes in Gh expression accompany phenotypic modulation of vascular smooth muscle cells. These changes in Gh protein expression may be important in the altered responsiveness of vessels in pathological disease states.