PLCβ Activity Research Articles

Analysis of Lipid Signaling in Drosophila Photoreceptors using Mass Spectrometry.

The activation of phospholipase Cβ (PLCβ) is an essential step during sensory transduction in Drosophila photoreceptors. PLCβ activity results in the hydrolysis of the membrane lipid phosphatidylinositol 4,5 bisphosphate [PI(4,5)P2] leading ultimately to the activation of transient receptor potential (TRP) and TRP like (TRPL) channels. The activity of PLCβ also leads subsequently to the generation of many lipid species several of which have been proposed to play a role in TRP and TRPL activation. In addition, several classes of lipids have been proposed to play key roles in organizing the cell biology of photoreceptors to optimize signaling reactions for optimal sensory transduction. Historically, these discoveries have been driven by the ability to isolate Drosophila mutants for enzymes that control the levels of specific lipids and perform analysis of photoreceptor physiology in these mutants. More recently, powerful mass spectrometry methods for isolation and quantitative analysis of lipids with high sensitivity and specificity have been developed. These are particularly suited for use in Drosophila where lipid analysis is now possible from photoreceptors without the need for radionuclide labeling. In this article, the conceptual and practical considerations in the use of lipid mass spectrometry for the robust, sensitive, and accurate quantitative assessment of various signaling lipids in Drosophila photoreceptors are covered. Along with existing methods in molecular genetics and physiological analysis such lipid is likely to enhance the power of photoreceptors as a model system for discoveries in biology.

Regulation of longevity by depolarization-induced activation of PLC-β–IP3R signaling in neurons

Mitochondrial ATP production is a well-known regulator of neuronal excitability. The reciprocal influence of plasma-membrane potential on ATP production, however, remains poorly understood. Here, we describe a mechanism by which depolarized neurons elevate the somatic ATP/ADP ratio in Drosophila glutamatergic neurons. We show that depolarization increased phospholipase-Cβ (PLC-β) activity by promoting the association of the enzyme with its phosphoinositide substrate. Augmented PLC-β activity led to greater release of endoplasmic reticulum Ca2+ via the inositol trisphosphate receptor (IP3R), increased mitochondrial Ca2+ uptake, and promoted ATP synthesis. Perturbations that decoupled membrane potential from this mode of ATP synthesis led to untrammeled PLC-β-IP3R activation and a dramatic shortening of Drosophila lifespan. Upon investigating the underlying mechanisms, we found that increased sequestration of Ca2+ into endolysosomes was an intermediary in the regulation of lifespan by IP3Rs. Manipulations that either lowered PLC-β/IP3R abundance or attenuated endolysosomal Ca2+ overload restored animal longevity. Collectively, our findings demonstrate that depolarization-dependent regulation of PLC-β-IP3R signaling is required for modulation of the ATP/ADP ratio in healthy glutamatergic neurons, whereas hyperactivation of this axis in chronically depolarized glutamatergic neurons shortens animal lifespan by promoting endolysosomal Ca2+ overload.

Extended synaptotagmin regulates membrane contact site structure and lipid transfer function invivo.

Inter-organelle communication between closely apposed membranes is proposed at membrane contact sites (MCS). However, the regulation of MCS structure and their functional relevance invivo remain debated. The extended synaptotagmins (Esyt) are evolutionarily conserved proteins proposed to function at MCS. However, loss of all three Esyts in yeast or mammals shows minimal phenotypes questioning the functional importance of Esyt. We report that in Drosophila photoreceptors, MCS number is regulated by PLCβ activity. Photoreceptors of a null allele of Drosophila extended synaptotagmin (dEsyt) show loss of ER-PM MCS. Loss of dEsyt results in mislocalization of RDGB, an MCS localized lipid transfer protein, required for photoreceptor structure and function, ultimately leading to retinal degeneration. dEsyt depletion enhanced the retinal degeneration, reduced light responses and slower rates of plasma membrane PIP2 resynthesis seen in rdgB mutants. Thus, dEsyt function and PLCβ signaling regulate ER-PM MCS structure and lipid transfer in Drosophila photoreceptors.

The Gαq/phospholipase Cβ signaling system represses tau aggregation

Alzheimer's disease is typified by calcium dysfunction and neurofibrillary tangles of tau aggregates along with mitotic proteins. Using PC12 cells as a model system, we determined whether the Gαq/PLCβ/ calcium signaling pathway impacts the manifestation of Alzheimer's disease. Down-regulating PLCβ significantly increases tau protein expression and causes a large increase in tau aggregation. Stimulating Gαq to activate PLCβ results in a modest reduction in tau aggregation while inhibiting PLCβ activity results in a modest enhancement of tau aggregation. These results suggest that PLCβ may effect tau aggregation by an additional mechanism that is independent of its ability to transduce calcium signals. To this end, we found that a cytosolic population of PLCβ binds to a mitotic protein found in neurofibrillary tangles, CDK18, which promotes tau phosphorylation and aggregation. Taken together, our studies show that the loss of PLCβ1 can promote Alzheimer's disease by a combination of its catalytic activity and its interaction with mitotic proteins thus offering an orthogonal method to control tau aggregation.

The Effect of Membrane Composition on PLCβ and Gαq‐mediated Activation

Phospholipase C β (PLCβ) enzymes are peripheral membrane proteins that are required for normal cardiovascular function and whose dysregulation results in cardiovascular disease. PLCβ hydrolyzes phosphatidylinositiol‐4,5‐bisphosphate (PIP2) to produce the potent second messengers inositol‐1,4,5‐triphosphate (IP3) and diacylglycerol (DAG), which increase intracellular Ca2+ and activate protein kinase C. PLCβ has low basal activity, but is activated downstream of G protein‐coupled receptors (GPCRs) through direct interactions with the heterotrimeric G protein subunits Gαq and Gβγ. While G proteins stimulate activity, they are insufficient for full activation. This suggests that the membrane is required for full activation. However, the molecular basis for how the membrane and its properties contribute to PLCβ adsorption, activity, and Gαq‐dependent activation are not well understood. We seek to understand how the membrane composition and its surface charge regulate adsorption of PLCβ to the membrane and promote interfacial activation, and the role of Gαq in this process. Using a model membrane system, we are applying an innovative combination of atomic force microscopy, Raman spectroscopy and biochemical assays to begin understanding how the membrane itself and Gαq regulate PLCβ activation. These studies provide the first structure‐based approach to understanding how the cell membrane regulates the activity of this essential effector enzyme.Support or Funding InformationAmerican Heart Association Scientist Development Grant (16SDG29920017) to A.M.L.; Purdue Doctoral Fellowship to B.N.H.; Purdue Center for Cancer ResearchThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Immune/Inflammatory Response and Hypocontractility of Rabbit Colonic Smooth Muscle After TNBS-Induced Colitis.

The contractility of colonic smooth muscle is dysregulated due to immune/inflammatory responses in inflammatory bowel diseases. Inflammation in vitro induces up-regulation of regulator of G-protein signaling 4 (RGS4) expression in colonic smooth muscle cells. To characterize the immune/inflammatory responses and RGS4 expression pattern in colonic smooth muscle after induction of colitis. Colitis was induced in rabbits by intrarectal instillation of 2,4,6-trinitrobenzene sulfonic acid (TNBS). Innate/adaptive immune response RT-qPCR array was performed using colonic circular muscle strips. At 1-9weeks after colonic intramuscular microinjection of lentivirus, the distal and proximal colons were collected, and muscle strips and dispersed muscle cells were prepared from circular muscle layer. Expression levels of RGS4 and NFκB signaling components were determined by Western blot analysis. The biological consequences of RGS4 knockdown were assessed by measurement of muscle contraction and phospholipase C (PLC)-β activity in response to acetylcholine (ACh). Contraction in response to ACh was significantly inhibited in the inflamed colonic circular smooth muscle cells. RGS4, IL-1, IL-6, IL-8, CCL3, CD1D, and ITGB2 were significantly up-regulated, while IL-18, CXCR4, CD86, and C3 were significantly down-regulated in the inflamed muscle strips. RGS4 protein expression in the inflamed smooth muscles was dramatically increased. RGS4 stable knockdown in vivo augmented ACh-stimulated PLC-β activity and contraction in colonic smooth muscle cells. Inflamed smooth muscle exhibits up-regulation of IL-1-related signaling components, Th1 cytokines and RGS4, and inhibition of contraction. Stable knockdown of endogenous RGS4 in colonic smooth muscle increases PLC-β activity and contractile responses.

Functional cooperation between the IP3 receptor and phospholipase C secures the high sensitivity to light of Drosophila photoreceptors in vivo.

Drosophila phototransduction is a model system for the ubiquitous phosphoinositide signaling. In complete darkness, spontaneous unitary current events (dark bumps) are produced by spontaneous single Gqα activation, while single-photon responses (quantum bumps) arise from synchronous activation of several Gqα molecules. We have recently shown that most of the spontaneous single Gqα activations do not produce dark bumps, because of a critical phospholipase Cβ (PLCβ) activity level required for bump generation. Surpassing the threshold of channel activation depends on both PLCβ activity and cellular [Ca(2+)], which participates in light excitation via a still unclear mechanism. We show here that in IP3 receptor (IP3R)-deficient photoreceptors, both light-activated Ca(2+) release from internal stores and light sensitivity were strongly attenuated. This was further verified by Ca(2+) store depletion, linking Ca(2+) release to light excitation. In IP3R-deficient photoreceptors, dark bumps were virtually absent and the quantum-bump rate was reduced, indicating that Ca(2+) release from internal stores is necessary to reach the critical level of PLCβ catalytic activity and the cellular [Ca(2+)] required for excitation. Combination of IP3R knockdown with reduced PLCβ catalytic activity resulted in highly suppressed light responses that were partially rescued by cellular Ca(2+) elevation, showing a functional cooperation between IP3R and PLCβ via released Ca(2+). These findings suggest that in contrast to the current dogma that Ca(2+) release via IP3R does not participate in light excitation, we show that released Ca(2+) plays a critical role in light excitation. The positive feedback between PLCβ and IP3R found here may represent a common feature of the inositol-lipid signaling.

Molecular mechanisms of phospholipase C‐β autoinhibition (843.10)

Phospholipase C‐β (PLCβ) enzymes hydrolyze the inner membrane lipid PIP2 to produce the second messengers inositol‐1,4,5‐triphosphate and diacylglycerol. PLCβ enzymes are characterized by low basal activity, due in part to a linker region within catalytic core that physically occludes the active site. We previously identified a highly conserved autoinhibitory helix in the PLCβ C‐terminal regulatory region that makes extensive interactions with the PLCβ catalytic core. Mutations within this interface lead to dramatic increases in basal activity and decreased thermal stability. Furthermore, this autoinhibitory helix docks in close proximity to the PLCβ active site and the regulatory linker. However, the molecular basis for how this inhibitory helix regulates basal activity, and whether or how it works in conjunction with the catalytic core regulatory linker remains unclear. We have used mass spectrometry, site‐directed mutagenesis, and biochemical assays to begin to elucidate the molecular basis of autoinhibition of lipid hydrolysis by these various regulatory elements. A better understanding of the mechanisms governing PLCβ activity are essential for developing novel therapeutic approaches for the treatment of cardiac hypertrophy and heart failure.Grant Funding Source: NIH grants HL086865 and HL071818 (J.J.G.T.) and AHA Fellowship 13POST1637009 (A.M.L.).

UV light activates a Gαq/11-coupled phototransduction pathway in human melanocytes

While short exposure to solar ultraviolet radiation (UVR) can elicit increased skin pigmentation, a protective response mediated by epidermal melanocytes, chronic exposure can lead to skin cancer and photoaging. However, the molecular mechanisms that allow human skin to detect and respond to UVR remain incompletely understood. UVR stimulates a retinal-dependent signaling cascade in human melanocytes that requires GTP hydrolysis and phospholipase C β (PLCβ) activity. This pathway involves the activation of transient receptor potential A1 (TRPA1) ion channels, an increase in intracellular Ca(2+), and an increase in cellular melanin content. Here, we investigated the identity of the G protein and downstream elements of the signaling cascade and found that UVR phototransduction is Gαq/11 dependent. Activation of Gαq/11/PLCβ signaling leads to hydrolysis of phosphatidylinositol (4,5)-bisphosphate (PIP2) to generate diacylglycerol (DAG) and inositol 1, 4, 5-trisphosphate (IP3). We found that PIP2 regulated TRPA1-mediated photocurrents, and IP3 stimulated intracellular Ca(2+) release. The UVR-elicited Ca(2+) response appears to involve both IP3-mediated release from intracellular stores and Ca(2+) influx through TRPA1 channels, showing the fast rising phase of the former and the slow decay of the latter. We propose that melanocytes use a UVR phototransduction mechanism that involves the activation of a Gαq/11-dependent phosphoinositide cascade, and resembles light phototransduction cascades of the eye.

Role of phospholipase C-β in RNA interference

Phospholipase C-β (PLCβ) enzymes are activated by G proteins in response to agents such as hormones and neurotransmitters, and have been implicated in leukemias and neurological disorders. PLCβ activity causes an increase in intracellular calcium which ultimately leads to profound changes in the cell. PLCβ localizes to three cellular compartments: the plasma membrane, the cytosol and the nucleus. Under most cell conditions, the majority of PLCβ localizes to the plasma membrane where it interacts with G proteins. In trying to determine the factors that localize PLCβ to the cytosol and nucleus, we have recently identified the binding partner, TRAX. TRAX is a nuclease and part of the machinery involved in RNA interference. This review discusses the interaction between PLCβ and TRAX, and its repercussions in G protein signaling and RNA silencing.

Gβγ-mediated activation of protein kinase D exhibits subunit specificity and requires Gβγ-responsive phospholipase Cβ isoforms

BackgroundProtein kinase D (PKD) constitutes a novel family of serine/threonine protein kinases implicated in fundamental biological activities including cell proliferation, survival, migration, and immune responses. Activation of PKD in these cellular activities has been linked to many extracellular signals acting through antigen receptor engagement, receptor tyrosine kinases, as well as G protein-coupled receptors. In the latter case, it is generally believed that the Gα subunits of the Gq family are highly effective in mediating PKD activation, whereas little is known with regard to the ability of Gβγ dimers and other Gα subunits to stimulate PKD. It has been suggested that the interaction between Gβγ and the PH domain of PKD, or the Gβγ-induced PLCβ/PKC activity is critical for the induction of PKD activation. However, the relative contribution of these two apparently independent events to Gβγ-mediated PKD activation has yet to be addressed.ResultsIn this report, we demonstrate that among various members in the four G protein families, only the Gα subunits of the Gq family effectively activate all the three PKD isoforms (PKD1/2/3), while Gα subunits of other G protein families (Gs, Gi, and G12) are ineffective. Though the Gα subunits of Gi family are unable to stimulate PKD, receptors linked to Gi proteins are capable of triggering PKD activation in cell lines endogenously expressing (HeLa cells and Jurkat T-cells) or exogenously transfected with (HEK293 cells) Gβγ-sensitive PLCβ2/3 isoforms. This indicates that the Gi-mediated PKD activation is dependent on the released Gβγ dimers upon stimulation. Further investigation on individual Gβγ combinations (i.e. Gβ1 with Gγ1–13) revealed that, even if they can stimulate the PLCβ activity in a comparable manner, only those Gβ1γ dimers with γ2, γ3, γ4, γ5, γ7, and γ10 can serve as effective activators of PKD. We also demonstrated that Gi-mediated PKD activation is essential for the SDF-1α-induced chemotaxis on Jurkat T-cells.ConclusionsOur current report illustrates that Gβγ dimers from the Gi proteins may activate PKD in a PLCβ2/3-dependent manner, and the specific identities of Gγ components within Gβγ dimers may determine this stimulatory action.

Detailed Analysis of Phospholipase C-β Activity in Living Cells

Phospholipase C-β (PLC-β) catalyzes the hydrolysis of the plasma membrane (PM) lipid phosphatidylinositol-(4,5)-bisphosphate (PI(4,5)P2), thereby reducing the PM concentration of its precursor PI(4)P. A recent report proposed independent PI(4,5)P2 and PI(4)P PM pools with essential physiological functions, but it remains unknown whether PLC-β targets to these pools differentially.Here, we investigated the time course and specificity of PLC-β-dependent phospholipid depletion in living CHO cells using genetically-encoded phospholipid sensors, total internal reflection fluorescence microscopy and whole cell patch clamp. PLC-β mediated PI(4,5)P2 hydrolysis was detected reliably using KCNQ (Kv7)-mediated K+ currents, the PI(4,5)P2 reporters PLCδ1-PH and Epsin ENTH, but not with the C-terminus of Tubby protein. Analogous experiments showed that activation of PLC-β did not reduce the concentration of PI(4) detected with the PI(4)P-specific reporter Osbp-PH. When PLC-β3 was heterologously overexpressed, Tubby-Cterm and Osbp-PH reported on the PLC-β-induced changes of PI(4,5)P2 and PI(4)P, respectively.In summary, we present detailed real-time analysis of PLC-β activity in living cells. Our findings indicate that phospholipid sensors may detect different phospholipid pools that are accessible to PLC-β differentially. Hence, this work supports the presence of functional phospholipid pools in living cells.This work was supported by Deutsche Forschungsgemeinschaft through SFB 593 (TP A12) to D.O.

Cannabinoid receptor agonists upregulate and enhance serotonin 2A (5‐HT2A) receptor activity via ERK1/2 signaling

Recent behavioral studies suggest that nonselective agonists of cannabinoid receptors may regulate serotonin 2A (5-HT(2A)) receptor neurotransmission. Two cannabinoids receptors are found in brain, CB1 and CB2 receptors, but the molecular mechanism by which cannabinoid receptors would regulate 5-HT(2A) receptor neurotransmission remains unknown. Interestingly, we have recently found that certain cannabinoid receptor agonists can specifically upregulate 5-HT(2A) receptors. Here, we present experimental evidence that rats treated with a nonselective cannabinoid receptor agonist (CP 55,940, 50 µg/kg, 7 days) showed increases in 5-HT(2A) receptor protein levels, 5-HT(2A) receptor mRNA levels, and 5-HT(2A) receptor-mediated phospholipase C beta (PLCβ) activity in prefrontal cortex (PFCx). Similar effects were found in neuronal cultured cells treated with CP 55,940 but these effects were prevented by selective CB2, but not selective CB1, receptor antagonists. CB2 receptors couple to the extracellular kinase (ERK) signaling pathway by Gα(i/o) class of G-proteins. Noteworthy, GP 1a (selective CB2 receptor agonist) produced a strong upregulation of 5-HT(2A) receptor mRNA and protein, an effect that was prevented by selective CB2 receptor antagonists and by an ERK1/2 inhibitor, PD 198306. In summary, our results identified a strong cannabinoid-induced upregulation of 5-HT(2A) receptor signaling in rat PFCx. Our cultured cell studies suggest that selective CB2 receptor agonists upregulate 5-HT(2A) receptor signaling by activation of the ERK1/2 signaling pathway. Activity of cortical 5-HT(2A) receptors has been associated with several physiological functions and neuropsychiatric disorders such as stress response, anxiety and depression, and schizophrenia. Therefore, these results may provide a molecular mechanism by which activation of cannabinoid receptors might be relevant to the pathophysiology of some cognitive and mood disorders in humans.

Fever-like temperature modification differentially affects in vitro signaling of bradykinin B1 and B2 receptors

The bradykinin (BK) B(2) and B(1) receptors (B(2)R, B(1)R) belong to the rhodopsin-like G protein-coupled receptors (GPCRs) and are involved in (patho)physiological processes such as blood pressure regulation or inflammation. They mediate the effects of the pro-inflammatory peptides bradykinin/kallidin and desArg(9)-BK/desArg(10)-kallidin, respectively. Whereas the B(2)R is constitutively expressed and gets internalized upon activation, the B(1)R is especially induced by inflammatory mediators and responds to stimulation with increased surface receptor numbers. Stimulation of both receptors activates phospholipase Cβ (PLCβ) and mitogen activated protein kinase (MAPK) signaling. Because inflammatory processes are characterized by heat (fever), we analyzed the effect of increased temperature (41°C vs. 37°C) on B(1)R and B(2)R signaling in HEK 293 and IMR 90 cells. Our results show that signaling of both receptors is temperature-sensitive, however to a different extent and with regard to the investigated pathways. Comparing PLCβ activity and Ca(2+)-regulated signals, a temperature-dependent increase was only observed for B(1)R but not for B(2)R activation, whereas MAPK activities were doubled at 41°C for both receptors. Taken together, our findings suggest that the observed temperature sensitivity of B(1)R-induced PLCβ activation is B(1)R-specific. In contrast, the enhanced stimulation of MAPK activity under hyperthermic conditions appears to be a common phenomenon for GPCRs.

Regulation and Activation of Phospholipase Cβ

Phospholipase Cβ (PLCβ), which catalyzes the hydrolysis of phosphatidylinositol‐4,5‐bisphosphate (PIP2) to the second messengers diacylglycerol (DAG) and inositol‐1,4,5‐triphosphate (IP3), is activated in response to specific extracellular stimuli through interactions with the heterotrimeric G protein Gαq. However, the structural and functional mechanisms underlying the regulation of PLCβ activity is poorly understood. In this work we present two new structures of PLCβ homologs purified from endogenous sources that include a highly conserved element from the C‐terminal regulatory domain. This conserved element forms a microdomain that interacts with a conserved surface of the catalytic core. Deletion of or mutation of residues within this microdomain in PLCβ3 results in increased PLCβ activity. Based on the recent structure of the Gαq‐PLCβ3 complex, this microdomain is likely displaced from the catalytic core upon Gαq binding. Thus, the C‐terminal microdomain appears to represent an autoinhibitory element that modulates the basal activity of PLCβ and is involved in allosteric changes mediated by Gαq.Support was provided by NIH grants HL086865 and HL071818.

Direct Activation of Human Phospholipase C by Its Well Known Inhibitor U73122

Phospholipase C (PLC) enzymes are an important family of regulatory proteins involved in numerous cellular functions, primarily through hydrolysis of the polar head group from inositol-containing membrane phospholipids. U73122 (1-(6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione), one of only a few small molecules reported to inhibit the activity of these enzymes, has been broadly applied as a pharmacological tool to implicate PLCs in diverse experimental phenotypes. The purpose of this study was to develop a better understanding of molecular interactions between U73122 and PLCs. Hence, the effects of U73122 on human PLCβ3 (hPLCβ3) were evaluated in a cell-free micellar system. Surprisingly, U73122 increased the activity of hPLCβ3 in a concentration- and time-dependent manner; up to an 8-fold increase in enzyme activity was observed with an EC50=13.6±5 μm. Activation of hPLCβ3 by U73122 required covalent modification of cysteines as evidenced by the observation that enzyme activation was attenuated by thiol-containing nucleophiles, l-cysteine and glutathione. Mass spectrometric analysis confirmed covalent reaction with U73122 at eight cysteines, although maximum activation was achieved without complete alkylation; the modified residues were identified by LC/MS/MS peptide sequencing. Interestingly, U73122 (10 μm) also activated hPLCγ1 (>10-fold) and hPLCβ2 (∼2-fold); PLCδ1 was neither activated nor inhibited. Therefore, in contrast to its reported inhibitory potential, U73122 failed to inhibit several purified PLCs. Most of these PLCs were directly activated by U73122, and a simple mechanism for the activation is proposed. These results strongly suggest a need to re-evaluate the use of U73122 as a general inhibitor of PLC isozymes.

Characterization of Melatonin Receptors and Signaling Pathways in Gastrointestinal Smooth Muscle

Melatonin, a close derivative of serotonin, is involved in physiological regulation of circadian rhythms. In the gastrointestinal (GI) system, melatonin exhibits endocrine, paracrine and autocrine actions and is implicated in the regulation of GI motility. Generally, melatonin actions oppose the stimulatory actions of serotonin on motility. However, it is not known whether melatonin can also act directly on GI smooth muscle cells.AIMTo determine the expression of melatonin receptors in smooth muscle and identify their signaling pathways.METHODSMuscle cells were isolated from rabbit stomach by enzymatic digestion. Expression of melatonin receptors, MT1 and MT2, was determined by RT‐PCR and Western blot. G protein activity was measured by melatonin‐induced increase in Gα binding to [35S]GTPγS. PLC‐β activity was measured by ion‐exchange chromatography. Cytosolic Ca2+ was measured in fura‐2 loaded cells and muscle contraction by scanning micrometry.RESULTSMT1 was detected by RT‐PCR and western blot. Melatonin activated Gαq, but not Gαs, Gαi1, Gαi2, or Gαi3. Consistent with activation of Gαq, melatonin stimulated PI hydrolysis, increased cytosolic Ca2+, and elicited muscle contraction.CONCLUSION. Gastric smooth muscle cells express receptors for melatonin (MT1) coupled to Gq. The receptors mediate stimulation of PLC‐β activity and increase in cytosolic Ca2+, and elicit muscle contraction.

The RhoA-specific guanine nucleotide exchange factor p63RhoGEF binds to activated Gα 16 and inhibits the canonical phospholipase Cβ pathway

Heterotrimeric G proteins regulate diverse physiological processes by modulating the activities of intracellular effectors. Members of the Gα q family link G protein-coupled receptor activation to phospholipase Cβ (PLCβ) activity and intracellular calcium signaling cascades. However, they differ markedly in biochemical properties as well as tissue distribution. Recent findings have shown that some of the cellular activities of Gα q family members are independent of PLCβ activation. A guanine nucleotide exchange factor, p63RhoGEF, has been shown to interact with Gα q proteins and thus provides linkage to RhoA activation. However, it is not known if p63RhoGEF can associate with other Gα q family members such as Gα 16. In the present study, we employed co-immunoprecipitation studies in HEK293 cells to demonstrate that p63RhoGEF can form a stable complex with the constitutively active mutant of Gα 16 (Gα 16QL). Interestingly, overexpression of p63RhoGEF inhibited Gα 16QL-induced IP 3 production in a concentration-dependent manner. The binding of PLCβ 2 to Gα 16QL could be displaced by p63RhoGEF. Similarly, p63RhoGEF inhibited the binding of tetratricopeptide repeat 1 to Gα 16QL, leading to a suppression of Gα 16QL-induced Ras activation. In the presence of p63RhoGEF, Gα 16QL-induced STAT3 phosphorylation was significantly reduced and Gα 16QL-mediated SRE transcriptional activation was attenuated. Taken together, these results suggest that p63RhoGEF binds to activated Gα 16 and inhibits its signaling pathways.

PI(3,4,5)P3 potentiates phospholipase C-β activity

Phospholipase C-β (PLC-β) isozymes are key effectors in G protein-coupled signaling pathways. Previously, we showed that PLC-β1 and PLC-β3 bound immobilized PIP3. In this study, PIP3 was found to potentiate Ca2+-stimulated PLC-β activities using an in vitro reconstitution assay. LY294002, a specific PI 3-kinase inhibitor, significantly inhibited 10 min of agonist-stimulated total IP accumulation. Both LY294002 and wortmannin inhibited 90 sec of agonist-stimulated IP3 accumulation in intact cells. Moreover, transfected p110CAAX, a constitutively activated PI 3-kinase catalytic subunit, increased 90 sec of oxytocin-stimulated IP3 accumulation. Receptor-ligand binding assays indicated that LY294002 did not affect G protein-coupled receptors directly, suggesting a physiological role for PIP3 in directly potentiating PLC-β activity. When coexpressed with p110CAAX, fluorescence-tagged PLC-β3 was increasingly localized to the plasma membrane. Additional observations suggest that the PH domain of PLC-β is not important for p110CAAX-induced membrane association.

G Protein-coupled Receptor (GPCR) Kinase 2 Regulates Agonist-independent Gq/11 Signaling from the Mouse Cytomegalovirus GPCR M33

The mouse cytomegalovirus M33 protein is highly homologous to mammalian G protein-coupled receptors (GPCRs) yet functions in an agonist-independent manner to activate a number of classical GPCR signal transduction pathways. M33 is functionally similar to the human cytomegalovirus-encoded US28 GPCR in its ability to induce inositol phosphate accumulation, activate NF-kappaB, and promote smooth muscle cell migration. This ability to promote cellular migration suggests a role for viral GPCRs like M33 in viral dissemination in vivo, and accordingly, M33 is required for efficient murine cytomegalovirus replication in the mouse. Although previous studies have identified several M33-induced signaling pathways, little is known regarding the membrane-proximal events involved in signaling and regulation of this receptor. In this study, we used recombinant retroviruses to express M33 in wild-type and Galpha(q/11)(-/-) mouse embryonic fibroblasts and show that M33 couples directly to the G(q/11) signaling pathway to induce high levels of total inositol phosphates in an agonist-independent manner. Our data also show that GRK2 is a potent regulator of M33-induced G(q/11) signaling through its ability to phosphorylate M33 and sequester Galpha(q/11) proteins. Taken together, the results from this study provide the first genetic evidence of a viral GPCR coupling to a specific G protein signaling pathway as well as identify the first viral GPCR to be regulated specifically by both the catalytic activity of the GRK2 kinase domain and the Galpha(q/11) binding activity of the GRK2 RH domain.