Protein-coupled Receptor Stimulation Research Articles

The role of intracellular calcium and Rho kinase pathways in G protein-coupled receptor-mediated contractions of urinary bladder urothelium and lamina propria.

The influence of extracellular and intracellular calcium on smooth muscle contractile activity varies between organs. In response to G protein-coupled receptor (GPCR) stimulation, the urinary bladder detrusor muscle has shown a 70% dependence on extracellular calcium, whereas the urothelium and lamina propria (U&LP) has a 20%-50% dependence. However, as this only accounts for partial contractile activity, the contribution of intracellular calcium and calcium sensitization pathways remains unclear. This study assessed the role of intracellular signaling pathways on GPCR-mediated urinary bladder U&LP contraction. Porcine U&LP responses to activation of the Gq/11-coupled muscarinic, histamine, 5-hydroxytryptamine (serotonin), neurokinin, prostaglandin, and angiotensin II receptors were assessed with three selective inhibitors of store-released intracellular calcium, 2-aminoethyl diphenylborinate (2-APB), cyclopiazonic acid (CPA), and ruthenium red, and three Rho kinase inhibitors, fasudil, Y-27632, and GSK269962. There was no discernible impact on receptor agonist-induced contractions of the U&LP after blocking intracellular calcium pathways, suggesting that this tissue is more sensitive to alterations in the availability of extracellular calcium. However, an alternative mechanism of action for GPCR-mediated contraction was identified to be the activation of Rho kinase, such as when Y-27632 significantly reduced the GPCR-mediated contractile activity of the U&LP by approximately 50% (P < 0.05, n = 8). This suggests that contractile responses of the bladder U&LP do not involve a significant release of calcium from intracellular stores, but that Gq/11-coupled receptor activation causes calcium sensitization via Rho kinase. This study highlights a key role for Rho kinase in the urinary bladder, which may provide a novel target in the future pharmaceutical management of bladder contractile disorders.

Repeated platelet activation and the potential of previously activated platelets to contribute to thrombus formation

BackgroundEspecially in disease conditions, platelets can encounter activating agents in circulation. ObjectivesTo investigate the extent to which previously activated platelets can be reactivated and whether in-and reactivation applies to different aspects of platelet activation and thrombus formation. MethodsShort-and long-term effects of glycoprotein VI (GPVI) and G protein-coupled receptor (GPCR) stimulation on platelet activation and aggregation potential were compared via flow cytometry and plate-based aggregation. Using fluorescence and electron microscopy, we assessed platelet morphology and content, as well as thrombus formation. ResultsAfter 30 minutes of stimulation with thrombin receptor activator peptide 6 (TRAP6) or adenosine diphosphate (ADP), platelets secondarily decreased in PAC-1 binding and were less able to aggregate. The reversibility of platelets after thrombin stimulation was concentration dependent. Reactivation was possible via another receptor. In contrast, cross-linked collagen-related peptide (CRP-XL) or high thrombin stimulation evoked persistent effects in αIIbβ3 activation and platelet aggregation. However, after 60 minutes of CRP-XL or high thrombin stimulation, when αIIbβ3 activation slightly decreased, restimulation with ADP or CRP-XL, respectively, increased integrin activation again. Compatible with decreased integrin activation, platelet morphology was reversed. Interestingly, reactivation of reversed platelets again resulted in shape change and if not fully degranulated, additional secretion. Moreover, platelets that were previously activated with TRAP6 or ADP regained their potential to contribute to thrombus formation under flow. On the contrary, prior platelet triggering with CRP-XL was accompanied by prolonged platelet activity, leading to a decreased secondary platelet adhesion under flow. ConclusionThis work emphasizes that prior platelet activation can be reversed, whereafter platelets can be reactivated through a different receptor. Reversed, previously activated platelets can contribute to thrombus formation.

Jaw1/LRMP increases Ca2+ influx upon GPCR stimulation with heterogeneous effect on the activity of each ITPR subtype.

Ca2+ influx upon G protein-coupled receptor (GPCR) stimulation is observed as a cytosolic Ca2+ concentration oscillation crucial to initiating downstream responses including cell proliferation, differentiation, and cell–cell communication. Although Jaw1 is known to interact with inositol 1,4,5-triphosphate receptor (ITPRs), Ca2+ channels on the endoplasmic reticulum, the function of Jaw1 in the Ca2+ dynamics with physiological stimulation remains unclear. In this study, using inducible Jaw1-expressing HEK293 cells, we showed that Jaw1 increases Ca2+ influx by GPCR stimulation via changing the Ca2+ influx oscillation pattern. Furthermore, we showed that Jaw1 increases the Ca2+ release activity of all ITPR subtypes in a subtly different manner. It is well known that the Ca2+ influx oscillation pattern varies from cell type to cell type, therefore these findings provide an insight into the relationship between the heterogeneous Ca2+ dynamics and the specific ITPR and Jaw1 expression patterns.

Differential Effects of Gq Protein-Coupled Uridine Receptor Stimulation on IL-8 Production in 1321N1 Human Astrocytoma Cells.

G-protein-coupled receptors (GPCRs) trigger various physiological functions. GPCR-mediated effects largely depend on the receptor-associated G-protein subtypes. However, compelling evidence suggests that single receptor proteins activate multiple G-protein subtypes to induce diverse physiological responses. This study compared responses mediated by three different Gq-binding uridine nucleotide receptors, P2Y2, P2Y4, and P2Y6, by measuring Ca2+ signaling and interleukin (IL)-8 production. In 1321N1 human astrocytoma cells stably expressing these receptors, agonist stimulation evoked concentration-dependent intracellular Ca2+ elevation to a similar extent. In contrast, agonist-induced IL-8 production was prominent in P2Y6-expressing cells, but not in P2Y2- and P2Y4-expressing cells. In addition to inhibition of Gq signaling, G12 signal blockade attenuated uridine 5'-diphosphate (UDP)-induced IL-8 production, suggesting the involvement of a small G-protein pathway. The Rac inhibitor EHop-16 prevented UDP-induced IL-8 release. The P2Y6-triggered IL-8 production was also inhibited by extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and protein kinase B (Akt) inhibitors. These results suggest that P2Y6 receptor-induced IL-8 production requires Gq-mediated Ca2+ signaling as well as G12-mediated activation of Rac. The results also suggest the importance of considering the involvement of multiple G proteins in understanding GPCR-mediated functions.

Role of Src homology/collagen adaptor protein p66Shc in Pyk2 translocation into mitochondria under Gq protein–coupled receptor stimulation

IntroductionMitochondrial Ca2+ (mtCa2+) uptake via a main mtCa2+ channel, mtCa2+ uniporter (MCU), mediates cell survival and death via its contribution of mitochondrial ATP synthesis and apoptotic signaling activation, respectively. Our group previously showed that Gq protein–coupled receptor (GqPCR) stimulation promotes activation of a tyrosine kinase named proline‐rich tyrosine kinase 2 (Pyk2), and its translocation from cytosol to the mitochondrial matrix, which leads to the tyrosine phosphorylation of MCU, and mtCa2+ overload, and activation of apoptotic signaling in the cardiomyocytes. However, it is still unclear how Pyk2 access from cytosol to mitochondrial matrix under GqPCR stimulation. The Src homology/collagen (Shc) adaptor protein, p66Shc, is also known to be capable to translocate to the mitochondria where it promotes increased ROS production and apoptosis. Importantly, p66Shc can bind to another tyrosine kinase Src, but its association to Pyk2 has not been well investigated.Hypothesisp66Shc serves as an important chaperon for Pyk2 translocation into the mitochondrial matrix.Aimto test whether p66Shc can associate with Pyk2.MethodsMammalian expression plasmids containing p66Shc or Pyk2 were transformed in to bacteria and purified. Overexpression of p66Shc and/or Pyk2 in HEK293T cells were performed by transient plasmid transfection. Whole cell lysates were collected and used for biochemical assay including Western blotting and co‐immunoprecipitation assay.ResultsWe confirmed that transient transfections of the plasmids containing His‐tagged p66Shc and GFP‐tagged Pyk2 were successfully yield overexpression of these proteins in HEK293Tcells. Co‐immunoprecipitation assay using GFP antibody showed that p66Shc and Pyk2 forms complex in the whole cell lysates from cells co‐transfected with two plasmids.Conclusionp66Shc can bind Pyk2 and may contribute to the mechanism for Pyk2 translocation to mitochondria. Pur next plan is to test whether the removal (overexpression) of p66Shc blocks (Pyk2 transportation and prevent excessive calcium uptake by the MCU under GqPCR stimulation both in heterologous expression system and primary cardiomyocytes.Support or Funding InformationApart of this research was supported by the Lillehei Heart Institute Summer Research Fellowship (to G.D.), American Heart Association (AHA) 18CDA34110091(to B.S.J), NIH/NHLBI R01HL136757 (to J.O.‐U.), AHA 16SDG27260248 (to J.O.‐U.), and American Physiological Society (APS) 2017 Shih‐Chun Wang Young Investigator Award.

Gαq protein signaling in the bed nucleus of the stria terminalis regulate the lipopolysaccharide-induced despair-like behavior in mice.

Major depressive disorder (MDD) is highly comorbid with anxiety disorders. It has been reported that the bed nucleus of the stria terminalis (BNST) is important for the induction of anxiety and MDD. Recently, the Gαq protein signaling within the BNST is involved in the induction of anxiety through Gαq protein signaling-mediated RNA-editing of GluR2 subunit, which produces the calcium (Ca2+)-impermeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor. On the other hand, the role of Gαq protein signaling within the BNST on the induction of MDD has never been reported yet. Therefore, we investigated whether Gαq protein signaling-producing the Ca2+-impermeable AMPA receptors in the BNST is involved in the lipopolysaccharide (LPS)-induced depressive-like behavior, particularly, despair-like behavior. When mice were systemically challenged with a single dose of LPS (1.2 mg/kg, i.p.), the immobility time during tail suspension test (TST) was increased 24 h after LPS injection. However, pretreatment with bilateral intra-BNST injection of neomycin (6.5 mM, 0.125 µL/side), an inhibitor of phospholipase C that is activated by Gαq protein-coupled receptor stimulation, extended the LPS-induced increase in the immobility time of TST. Furthermore, the co-pretreatment with bilateral intra-BNST injection of neomycin with 1-naphthylacetyl spermine (3 mM, 0.125 µL/side), an antagonist of Ca2+-permeable AMPA receptor, to mimic one of the final forms of Gαq protein activation, abolished the aggravated effect of neomycin and significantly shortened the immobility time compared with the control mice with an intra-BNST injection of artificial cerebrospinal fluid before LPS injection. However, pretreatment with bilateral intra-BNST injection of MDL-12,330A (10 µM, 0.125 µL/side), an inhibitor of adenylyl cyclase that is activated by Gαs protein-coupled receptor stimulation, did not affect the LPS-induced increase in the immobility time of TST. These results indicate that the Gαq protein signaling-mediated RNA-editing of GluR2, which produces the Ca2+-impermeable AMPA receptors within the BNST, regulates the LPS-induced despair-like behavior.

G protein-coupled oestrogen receptor stimulation ameliorates iron- and ovariectomy-induced memory impairments through the cAMP/PKA/CREB signalling pathway.

Iron accumulation in the brain has been associated with neurodegenerative disorders, and imaging studies in humans indicate that iron content in brain regions correlates with poor performance in cognitive tasks. In rats, iron overload impairs memoryretention in a variety of memorytasks. Although the effects of iron on cognition in rodents are extensively reported, no previous study has been conducted in female rats. The incidence of certain dementias, such as Alzheimer's disease, is higher in women after menopause compared to aged-matched men. The role of oestrogen depletion in memory deficits in menopausal women is still a matter of debate. The present study aimed to characterise the effects of iron overload on memory in female rats by investigating the effects of ovariectomy (OVX, an experimental model of oestrogen depletion) in rats submitted to iron overload, as well as examining the effects of G protein-coupled oestrogen receptor (GPER) agonism on memory impairments induced by iron and OVX. Female rats received iron (30mgkg-1 , orally) or vehicle at postnatal days 12-14 and were submitted to OVX in adulthood. Results showed that either iron or OVX impaired memory for object placement and inhibitory avoidance. The selective GPER agonist G1, administered immediately after training, reversed both iron- and OVX-induced memory impairments. G1 effects were abolished by protein kinase A (PKA) inhibition, suggesting the involvement of the cAMP/PKA/CREB signalling pathway. The search for novel oestrogen agonists with positive effects on cognition may be promising for the development of treatments for memory disorders.

Acid Sensing Ion Channel 1 Contributes to Endothelium‐Derived Hyperpolarizing Factor Induced Vasodilation in Small Mesenteric Arteries.

Acid sensing ion channel 1 (ASIC1) belongs to the amiloride‐sensitive degenerin/epithelial sodium channel superfamily, and is permeable to both Na+ and Ca2+. ASIC channels have been found to regulate blood pressure through mechanotransduction in arterial baroreceptors and are also pH sensors in carotid body chemoreceptors. ASIC1 is additionally expressed in vascular smooth muscle and endothelial cells in a variety of vascular beds; yet little is known regarding the role of ASIC1 in the regulation of local vascular resistance. Although extracellular H+ is the recognized activator of ASIC1, we have previously shown this channel can be activated following G protein‐coupled receptor stimulation. In small mesenteric arteries, we found that ASIC1 contributes to acetylcholine (ACh)‐induced endothelial cell Ca2+ entry and vasodilation. However, the mechanisms involved in activating ASIC1 and its downstream effects on vasodilation are unknown. We hypothesized that ACh activates ASIC1‐mediated endothelial cell Ca2+ entry to mediate endothelium‐derived hyperpolarizing factor (EDHF)‐type vasodilation through activation of small (SK) and intermittent (IK) conductance Ca2+‐activated K+ channels. To test this hypothesis, we used pressure myography to study the effects of ASIC1 on ACh‐induced vasodilation (10−9 – 10−5 M) of freshly isolated mesenteric arteries (100–200 μm inner diameter), in the presence of Nω‐nitro‐L‐arginine (nitric oxide synthase inhibitor) and indomethacin (cyclooxygenase inhibitor). ACh‐induced vasodilation was abolished by specific inhibitors of SK and IK channels (apamin and TRAM‐34, respectively), confirming that the ACh‐induced dilation is EDHF‐dependent. The specific ASIC1 antagonist, psalmotoxin 1 (PcTX1; 20 nM) similarly blocks the EDHF‐mediated vasodilatory response, suggesting ASIC1 contributes to ACh‐induced vasodilation through an EDHF‐mediated pathway. Future studies will examine the upstream mechanisms involved in activation of endothelial ASIC1 and subsequent downstream effects to mediate EDHF‐dependent vasodilation. These studies suggest a role for ASIC1 as a novel contributor to agonist‐induced mesenteric vasodilation and cardiovascular homeostasis.Support or Funding InformationSupported by National Heart, Lung, and Blood Institute Grants R01 HL‐111084 (to N.L. Jernigan), T32 HL007736 (to T.C. Resta), and R01 HL132883 (to T.C. Resta).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Protein kinase D activation induces mitochondrial fragmentation and dysfunction in cardiomyocytes.

Abnormal mitochondrial morphology and function in cardiomyocytes are frequently observed under persistent Gq protein-coupled receptor (Gq PCR) stimulation. Cardiac signalling mechanisms for regulating mitochondrial morphology and function under pathophysiological conditions in the heart are still poorly understood. We demonstrate that a downstream kinase of Gq PCR, protein kinase D (PKD) induces mitochondrial fragmentation via phosphorylation of dynamin-like protein 1 (DLP1), a mitochondrial fission protein. The fragmented mitochondria enhance reactive oxygen species generation and permeability transition pore opening in mitochondria, which initiate apoptotic signalling activation. This study identifies a novel PKD-specific substrate in cardiac mitochondria and uncovers the role of PKD on cardiac mitochondria, with special emphasis on the molecular mechanism(s) underlying mitochondrial injury with abnormal mitochondrial morphology under persistent Gq PCR stimulation. These findings provide new insights into the molecular basis of cardiac mitochondrial physiology and pathophysiology, linking Gq PCR signalling with the regulation of mitochondrial morphology and function. Regulation of mitochondrial morphology is crucial for the maintenance of physiological functions in many cell types including cardiomyocytes. Small and fragmented mitochondria are frequently observed in pathological conditions, but it is still unclear which cardiac signalling pathway is responsible for regulating the abnormal mitochondrial morphology in cardiomyocytes. Here we demonstrate that a downstream kinase of Gq protein-coupled receptor (Gq PCR) signalling, protein kinase D (PKD), mediates pathophysiological modifications in mitochondrial morphology and function, which consequently contribute to the activation of apoptotic signalling. We show that Gq PCR stimulation induced by α1 -adrenergic stimulation mediates mitochondrial fragmentation in a fission- and PKD-dependent manner in H9c2 cardiac myoblasts and rat neonatal cardiomyocytes. Upon Gq PCR stimulation, PKD translocates from the cytoplasm to the outer mitochondrial membrane (OMM) and phosphorylates a mitochondrial fission protein, dynamin-like protein 1 (DLP1), at S637. PKD-dependent phosphorylation of DLP1 initiates DLP1 association with the OMM, which then enhances mitochondrial fragmentation, mitochondrial superoxide generation, mitochondrial permeability transition pore opening and apoptotic signalling. Finally, we demonstrate that DLP1 phosphorylation at S637 by PKD occurs in vivo using ventricular tissues from transgenic mice with cardiac-specific overexpression of constitutively active Gαq protein. In conclusion, Gq PCR-PKD signalling induces mitochondrial fragmentation and dysfunction via PKD-dependent DLP1 phosphorylation in cardiomyocytes. This study is the first to identify a novel PKD-specific substrate, DLP1 in mitochondria, as well as the functional role of PKD in cardiac mitochondria. Elucidation of these molecular mechanisms by which PKD-dependent enhanced fission mediates cardiac mitochondrial injury will provide novel insight into the relationship among mitochondrial form, function and Gq PCR signalling.

A new target for G protein signaling.

G protein-coupled receptor stimulation inhibits TRPM3 channel activity through direct binding of the Gβγ subunit to the channel.

TRPC4α and TRPC4β Similarly Affect Neonatal Cardiomyocyte Survival during Chronic GPCR Stimulation.

The Transient Receptor Potential Channel Subunit 4 (TRPC4) has been considered as a crucial Ca2+ component in cardiomyocytes promoting structural and functional remodeling in the course of pathological cardiac hypertrophy. TRPC4 assembles as homo or hetero-tetramer in the plasma membrane, allowing a non-selective Na+ and Ca2+ influx. Gαq protein-coupled receptor (GPCR) stimulation is known to increase TRPC4 channel activity and a TRPC4-mediated Ca2+ influx which has been regarded as ideal Ca2+ source for calcineurin and subsequent nuclear factor of activated T-cells (NFAT) activation. Functional properties of TRPC4 are also based on the expression of the TRPC4 splice variants TRPC4α and TRPC4β. Aim of the present study was to analyze cytosolic Ca2+ signals, signaling, hypertrophy and vitality of cardiomyocytes in dependence on the expression level of either TRPC4α or TRPC4β. The analysis of Ca2+ transients in neonatal rat cardiomyocytes (NRCs) showed that TRPC4α and TRPC4β affected Ca2+ cycling in beating cardiomyocytes with both splice variants inducing an elevation of the Ca2+ transient amplitude at baseline and TRPC4β increasing the Ca2+ peak during angiotensin II (Ang II) stimulation. NRCs infected with TRPC4β (Ad-C4β) also responded with a sustained Ca2+ influx when treated with Ang II under non-pacing conditions. Consistent with the Ca2+ data, NRCs infected with TRPC4α (Ad-C4α) showed an elevated calcineurin/NFAT activity and a baseline hypertrophic phenotype but did not further develop hypertrophy during chronic Ang II/phenylephrine stimulation. Down-regulation of endogenous TRPC4α reversed these effects, resulting in less hypertrophy of NRCs at baseline but a markedly increased hypertrophic enlargement after chronic agonist stimulation. Ad-C4β NRCs did not exhibit baseline calcineurin/NFAT activity or hypertrophy but responded with an increased calcineurin/NFAT activity after GPCR stimulation. However, this effect was not translated into an increased propensity towards hypertrophy but rather less hypertrophy during GPCR stimulation. Further analyses revealed that, although hypertrophy was preserved in Ad-C4α NRCs and even attenuated in Ad-C4β NRCs, cardiomyocytes had an increased apoptosis rate and thus were less viable after chronic GPCR stimulation. These findings suggest that TRPC4α and TRPC4β differentially affect Ca2+ signals, calcineurin/NFAT signaling and hypertrophy but similarly impair cardiomyocyte viability during GPCR stimulation.

PREX1 Protein Function Is Negatively Regulated Downstream of Receptor Tyrosine Kinase Activation by p21-activated Kinases (PAKs)

Downstream of receptor tyrosine kinase and G protein-coupled receptor (GPCR) stimulation, the phosphatidylinositol 3,4,5-trisphosphate (PIP3)-dependent Rac exchange factor (PREX) family of guanine nucleotide exchange factors (GEFs) activates Rho GTPases, leading to important roles for PREX proteins in numerous cellular processes and diseases, including cancer. PREX1 and PREX2 GEF activity is activated by the second messengers PIP3 and Gβγ, and further regulation of PREX GEF activity occurs by phosphorylation. Stimulation of receptor tyrosine kinases by neuregulin and insulin-like growth factor 1 (IGF1) leads to the phosphorylation of PREX1; however, the kinases that phosphorylate PREX1 downstream of these ligands are not known. We recently reported that the p21-activated kinases (PAKs), which are activated by GTP-bound Ras-related C3 botulinum toxin substrate 1 (Rac1), mediate the phosphorylation of PREX2 after insulin receptor activation. Here we show that certain phosphorylation events on PREX1 after insulin, neuregulin, and IGF1 treatment are PAK-dependent and lead to a reduction in PREX1 binding to PIP3 Like PREX2, PAK-mediated phosphorylation also negatively regulates PREX1 GEF activity. Furthermore, the onset of PREX1 phosphorylation was delayed compared with the phosphorylation of AKT, supporting a model of negative feedback downstream of PREX1 activation. We also found that the phosphorylation of PREX1 after isoproterenol and prostaglandin E2-mediated GPCR activation is partially PAK-dependent and likely also involves protein kinase A, which is known to reduce PREX1 function. Our data point to multiple mechanisms of PREX1 negative regulation by PAKs within receptor tyrosine kinase and GPCR-stimulated signaling pathways that have important roles in diseases such as diabetes and cancer.

GPCR-G Protein-β-Arrestin Super-Complex Mediates Sustained G Protein Signaling

Classically, G protein-coupled receptor (GPCR) stimulation promotes G protein signaling at the plasma membrane, followed by rapid β-arrestin-mediated desensitization and receptor internalization into endosomes. However, it has been demonstrated that someGPCRs activate G proteins from within internalized cellular compartments, resulting in sustained signaling. We have used a variety of biochemical, biophysical, and cell-based methods to demonstrate the existence, functionality, and architecture of internalized receptor complexes composed of a single GPCR, β-arrestin, and G protein. These super-complexes or "megaplexes" more readily form at receptors that interact strongly with β-arrestins via a C-terminal tail containing clusters of serine/threonine phosphorylation sites. Single-particle electron microscopy analysis of negative-stained purified megaplexes reveals that a single receptor simultaneously binds through its core region with G protein and through its phosphorylated C-terminal tail with β-arrestin. The formation of such megaplexes provides a potential physical basis for the newly appreciated sustained G protein signaling from internalized GPCRs.

TRPM7 kinase activity regulates murine mast cell degranulation.

The Mg(2+) and Ca(2+) conducting transient receptor potential melastatin 7 (TRPM7) channel-enzyme (chanzyme) has been implicated in immune cell function. Mice heterozygous for a TRPM7 kinase deletion are hyperallergic, while mice with a single point mutation at amino acid 1648, silencing kinase activity, are not. As mast cell mediators trigger allergic reactions, we here determine the function of TRPM7 in mast cell degranulation and histamine release. Our data establish that TRPM7 kinase activity regulates mast cell degranulation and release of histamine independently of TRPM7 channel function. Our findings suggest a regulatory role of TRPM7 kinase activity on intracellular Ca(2+) and extracellular Mg(2+) sensitivity of mast cell degranulation. Transient receptor potential melastatin 7 (TRPM7) is a divalent ion channel with a C-terminally located α-kinase. Mice heterozygous for a TRPM7 kinase deletion (TRPM7(+/∆K) ) are hypomagnesaemic and hyperallergic. In contrast, mice carrying a single point mutation at amino acid 1648, which silences TRPM7 kinase activity (TRPM7(KR) ), are not hyperallergic and are resistant to systemic magnesium (Mg(2+) ) deprivation. Since allergic reactions are triggered by mast cell-mediated histamine release, we investigated the function of TRPM7 on mast cell degranulation and histamine release using wild-type (TRPM7(+/+) ), TRPM7(+/∆K) and TRPM7(KR) mice. We found that degranulation and histamine release proceeded independently of TRPM7 channel function. Furthermore, extracellular Mg(2+) assured unperturbed IgE-DNP-dependent exocytosis, independently of TRPM7. However, impairment of TRPM7 kinase function suppressed IgE-DNP-dependent exocytosis, slowed the cellular degranulation rate, and diminished the sensitivity to intracellular calcium (Ca(2+) ) in G protein-induced exocytosis. In addition, G protein-coupled receptor (GPCR) stimulation revealed strong suppression of histamine release, whereas removal of extracellular Mg(2+) caused the phenotype to revert. We conclude that the TRPM7 kinase activity regulates murine mast cell degranulation by changing its sensitivity to intracellular Ca(2+) and affecting granular mobility and/or histamine contents.

Receptor sequestration in response to β-arrestin-2 phosphorylation by ERK1/2 governs steady-state levels of GPCR cell-surface expression

MAPKs are activated in response to G protein-coupled receptor (GPCR) stimulation and play essential roles in regulating cellular processes downstream of these receptors. However, very little is known about the reciprocal effect of MAPK activation on GPCRs. To investigate possible crosstalk between the MAPK and GPCRs, we assessed the effect of ERK1/2 on the activity of several GPCR family members. We found that ERK1/2 activation leads to a reduction in the steady-state cell-surface expression of many GPCRs because of their intracellular sequestration. This subcellular redistribution resulted in a global dampening of cell responsiveness, as illustrated by reduced ligand-mediated G-protein activation and second-messenger generation as well as blunted GPCR kinases and β-arrestin recruitment. This ERK1/2-mediated regulatory process was observed for GPCRs that can interact with β-arrestins, such as type-2 vasopressin, type-1 angiotensin, and CXC type-4 chemokine receptors, but not for the prostaglandin F receptor that cannot interact with β-arrestin, implicating this scaffolding protein in the receptor's subcellular redistribution. Complementation experiments in mouse embryonic fibroblasts lacking β-arrestins combined with in vitro kinase assays revealed that β-arrestin-2 phosphorylation on Ser14 and Thr276 is essential for the ERK1/2-promoted GPCR sequestration. This previously unidentified regulatory mechanism was observed after constitutive activation as well as after receptor tyrosine kinase- or GPCR-mediated activation of ERK1/2, suggesting that it is a central node in the tonic regulation of cell responsiveness to GPCR stimulation, acting both as an effector and a negative regulator.

Structural Analysis of PTM Hotspots (SAPH-ire) – A Quantitative Informatics Method Enabling the Discovery of Novel Regulatory Elements in Protein Families

Predicting the biological function potential of post-translational modifications (PTMs) is becoming increasingly important in light of the exponential increase in available PTM data from high-throughput proteomics. We developed structural analysis of PTM hotspots (SAPH-ire)—a quantitative PTM ranking method that integrates experimental PTM observations, sequence conservation, protein structure, and interaction data to allow rank order comparisons within or between protein families. Here, we applied SAPH-ire to the study of PTMs in diverse G protein families, a conserved and ubiquitous class of proteins essential for maintenance of intracellular structure (tubulins) and signal transduction (large and small Ras-like G proteins). A total of 1728 experimentally verified PTMs from eight unique G protein families were clustered into 451 unique hotspots, 51 of which have a known and cited biological function or response. Using customized software, the hotspots were analyzed in the context of 598 unique protein structures. By comparing distributions of hotspots with known versus unknown function, we show that SAPH-ire analysis is predictive for PTM biological function. Notably, SAPH-ire revealed high-ranking hotspots for which a functional impact has not yet been determined, including phosphorylation hotspots in the N-terminal tails of G protein gamma subunits—conserved protein structures never before reported as regulators of G protein coupled receptor signaling. To validate this prediction we used the yeast model system for G protein coupled receptor signaling, revealing that gamma subunit–N-terminal tail phosphorylation is activated in response to G protein coupled receptor stimulation and regulates protein stability in vivo. These results demonstrate the utility of integrating protein structural and sequence features into PTM prioritization schemes that can improve the analysis and functional power of modification-specific proteomics data.

Gβ4γ1 as a modulator of M3 muscarinic receptor signalling and novel roles of Gβ1 subunits in the modulation of cellular signalling

Much is known about the how Gβγ subunits regulate effectors in response to G protein-coupled receptor stimulation. However, there is still a lot we don't know about how specific combinations of Gβ and Gγ are wired into different signalling pathways. Here, using an siRNA screen for different Gβ and Gγ subunits, we examined an endogenous M3 muscarinic receptor signalling pathway in HEK 293 cells. We observed that Gβ4 subunits were critical for calcium signalling and a downstream surrogate measured as ERK1/2 MAP kinase activity. A number of Gγ subunits could partner with Gβ4 but the best coupling was seen via Gβ4γ1. Intriguingly, knocking down Gβ1 actually increased signalling through the M3-mAChR most likely via an increase in Gβ4 levels. We noted that Gβ1 occupies the promoter of Gβ4 and may participate in maturation of its mRNA. This highlights a new role for Gβγ signalling beyond their canonical roles in cellular signalling.

Phosphatidylinositol4-phosphate 5-kinase prevents the decrease in the HERG potassium current induced by Gq protein-coupled receptor stimulation

The human ether-a-go-go-related gene (HERG) potassium current (IHERG) has been shown to decrease in amplitude following stimulation with Gq protein-coupled receptors (GqRs), such as α1-adrenergic and M1-muscarinic receptors (α1R and M1R, respectively), at least partly via the reduction of membrane phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). The present study was designed to investigate the modulation of HERG channels by PI(4,5)P2 and phosphatidylinositol4-phosphate 5-kinase (PI(4)P5-K), a synthetic enzyme of PI(4,5)P2. Whole-cell patch-clamp recordings were used to examine the activity of HERG channels expressed heterologously in Chinese Hamster Ovary cells. The stimulation of α1R with phenylephrine or M1R with acetylcholine decreased the amplitude of IHERG accompanied by a significant acceleration of deactivation kinetics and the effects on IHERG were significantly attenuated in cells expressing PI(4)P5-K. The density of IHERG in cells expressing GqRs alone was significantly increased by the coexpression of PI(4)P5-K without significant differences in the voltage dependence of activation and deactivation kinetics. The kinase-deficient substitution mutant, PI(4)P5-K-K138A did not have these counteracting effects on the change in IHERG by M1R stimulation. These results suggest that the current density of IHERG is closely dependent on the membrane PI(4,5)P2 level, which is regulated by PI(4)P5-K and GqRs and that replenishing PI(4,5)P2 by PI(4)P5-K recovers IHERG.

G-Protein-gekoppelte Designerrezeptoren

The signalling via G protein-coupled receptor (GPCR) stimulation is complex, as a particular GPCR may activate more than one G protein. A ligand can also activate several GPCRs with different G protein coupling specificity. To reduce the complexity of GPCR signalling, “G protein coupled designer receptors exclusively activated by designer drugs” (DREADDs) have been developed that can be activated by the compound clozapine-N-oxide. These designer receptors are powerful chemical-genetic tools to investigate the physiological functions of G protein-coupled receptors in vivo.

Phospholipase C-β1 and β4 Contribute to Non-Genetic Cell-to-Cell Variability in Histamine-Induced Calcium Signals in HeLa Cells

A uniform extracellular stimulus triggers cell-specific patterns of Ca2+ signals, even in genetically identical cell populations. However, the underlying mechanism that generates the cell-to-cell variability remains unknown. We monitored cytosolic inositol 1,4,5-trisphosphate (IP3) concentration changes using a fluorescent IP3 sensor in single HeLa cells showing different patterns of histamine-induced Ca2+ oscillations in terms of the time constant of Ca2+ spike amplitude decay and the Ca2+ oscillation frequency. HeLa cells stimulated with histamine exhibited a considerable variation in the temporal pattern of Ca2+ signals and we found that there were cell-specific IP3 dynamics depending on the patterns of Ca2+ signals. RT-PCR and western blot analyses showed that phospholipase C (PLC)-β1, -β3, -β4, -γ1, -δ3 and -ε were expressed at relatively high levels in HeLa cells. Small interfering RNA-mediated silencing of PLC isozymes revealed that PLC-β1 and PLC-β4 were specifically involved in the histamine-induced IP3 increases in HeLa cells. Modulation of IP3 dynamics by knockdown or overexpression of the isozymes PLC-β1 and PLC-β4 resulted in specific changes in the characteristics of Ca2+ oscillations, such as the time constant of the temporal changes in the Ca2+ spike amplitude and the Ca2+ oscillation frequency, within the range of the cell-to-cell variability found in wild-type cell populations. These findings indicate that the heterogeneity in the process of IP3 production, rather than IP3-induced Ca2+ release, can cause cell-to-cell variability in the patterns of Ca2+ signals and that PLC-β1 and PLC-β4 contribute to generate cell-specific Ca2+ signals evoked by G protein-coupled receptor stimulation.