PLCβ Signaling Research Articles

The Human Cytomegalovirus vGPCR UL33 is Essential for Efficient Lytic Replication in Epithelial Cells.

Human cytomegalovirus (HCMV) is a β-herpesvirus which is ubiquitous in the human population. HCMV has the largest genome of all known human herpesviruses, and thus encodes a large array of proteins that affect pathogenesis in different cell types. Given the large genome and the ability of HCMV to replicate in a range of cells, investigators have begun to identify viral proteins required for cell type-specific replication. There are four proteins encoded in the HCMV genome that are homologous to human G protein-coupled receptors (GPCRs); these viral-encoded GPCRs (vGPCRs) are UL33, UL78, US27, and US28. In the current study, we find that deletion of all four vGPCR genes from a clinical isolate of HCMV severely attenuates lytic replication in both primary human salivary gland epithelial cells, as well as ARPE-19 retinal epithelial cells as evidenced by significant decreases in immediate early gene expression and virus production. Deletion of UL33 from the HCMV genome also results in a failure to efficiently replicate in epithelial cells, and this defect is manifested by decreased levels of immediate early, early, and late gene expression, as well as reduced viral production. We find that similar to US28, UL33 constitutively activates Gαq-dependent PLC-β signaling to high levels in these epithelial cells. We also find that UL33 transcription is more complicated than originally believed, and there is the potential for the virus to utilize various 5' UTRs to create novel UL33 proteins that are all capable of constitutive Gαq signaling. Taken together, these studies suggest that UL33 driven signaling is important for lytic HCMV replication in cells of epithelial origin.

A new light on PLCβ!

In this issue of Cell Chemical Biology, Kim etal.1 present a novel optogenetic tool, opto-PLCβ, to control PLCβ signaling optically. In addition to eliciting PIP2 hydrolysis and downstream signaling in cells, opto-PLCβ also enabled probing the impact of PLCβ signaling on amygdala synaptic plasticity and fear learning in mice.

Spatiotemporal Optical Control of Gαq-PLCβ Interactions.

Cells experience time-varying and spatially heterogeneous chemokine signals in vivo, activating cell surface proteins including G protein-coupled receptors (GPCRs). The Gαq pathway activation by GPCRs is a major signaling axis with broad physiological and pathological significance. Compared with other Gα members, GαqGTP activates many crucial effectors, including PLCβ (Phospholipase Cβ) and Rho GEFs (Rho guanine nucleotide exchange factors). PLCβ regulates many key processes, such as hematopoiesis, synaptogenesis, and cell cycle, and is therefore implicated in terminal-debilitating diseases, including cancer, epilepsy, Huntington's Disease, and Alzheimer's Disease. However, due to a lack of genetic and pharmacological tools, examining how the dynamic regulation of PLCβ signaling controls cellular physiology has been difficult. Since activated PLCβ induces several abrupt cellular changes, including cell morphology, examining how the other pathways downstream of Gq-GPCRs contribute to the overall signaling has also been difficult. Here we show the engineering, validation, and application of a highly selective and efficient optogenetic inhibitor (Opto-dHTH) to completely disrupt GαqGTP-PLCβ interactions reversibly in user-defined cellular-subcellular regions on optical command. Using this newly gained PLCβ signaling control, our data indicate that the molecular competition between RhoGEFs and PLCβ for GαqGTP determines the potency of Gq-GPCR-governed directional cell migration.

Dissociation of the G protein βγ from the Gq–PLCβ complex partially attenuates PIP2 hydrolysis

Phospholipase C β (PLCβ), which is activated by the Gq family of heterotrimeric G proteins, hydrolyzes the inner membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2), generating diacylglycerol and inositol 1,4,5-triphosphate (IP3). Because Gq and PLCβ regulate many crucial cellular processes and have been identified as major disease drivers, activation and termination of PLCβ signaling by the Gαq subunit have been extensively studied. Gq-coupled receptor activation induces intense and transient PIP2 hydrolysis, which subsequently recovers to a low-intensity steady-state equilibrium. However, the molecular underpinnings of this equilibrium remain unclear. Here, we explored the influence of signaling crosstalk between Gq and Gi/o pathways on PIP2 metabolism in living cells using single-cell and optogenetic approaches to spatially and temporally constrain signaling. Our data suggest that the Gβγ complex is a component of the highly efficient lipase GαqGTP–PLCβ–Gβγ. We found that over time, Gβγ dissociates from this lipase complex, leaving the less-efficient GαqGTP–PLCβ lipase complex and allowing the significant partial recovery of PIP2 levels. Our findings also indicate that the subtype of the Gγ subunit in Gβγ fine-tunes the lipase activity of Gq–PLCβ, in which cells expressing Gγ with higher plasma membrane interaction show lower PIP2 recovery. Given that Gγ shows cell- and tissue-specific subtype expression, our findings suggest the existence of tissue-specific distinct Gq–PLCβ signaling paradigms. Furthermore, these results also outline a molecular process that likely safeguards cells from excessive Gq signaling.

Abstract A07: Activating mutations in uveal melanoma convey sensitivity to g-alpha q inhibition

Abstract Uveal melanoma (UVM) is the most common intraocular malignancy, which represents about 5% of all melanoma cases per year in the United States. Half of all patients eventually develop metastatic disease, resulting in an average survival rate of six months and a five-year survival rate of ~15%. Recent studies have revealed the underlying genetic landscape of uveal melanoma but have not resulted in any therapeutic options to effectively treat UVM. UVMs harbor mutually exclusive activating mutations in GPCR signaling: ~90% in G-protein alpha q (Gq) subunits GNAQ/GNA11, ~5% in the GPCR CYSLTR2, and ~4% in a downstream effector. These mutations aberrantly activate canonical PLCβ signaling: cleavage of PIP2 into DAG and IP3, both of which lead to PKC activation. In addition to uveal melanoma, recent studies have shown this exact pathway to be activated in a subset of cutaneous and mucosal melanomas as well as other diseases like blue nevi, leptomeningeal melanocytic neoplasms (LMNs), and Sturge-Weber syndrome. My project focuses on understanding how Gq signals in UVM tumorigenesis and if Gq inhibition has therapeutic potential in UVM by addressing the following aims: 1) Using a highly specific Gq inhibitor, I will ask how Gq inhibition effects different activating mutations in UVM using representative in vitro models. I have generated melanocytes dependent on different mutations in the PLCβ pathway to study pathway inhibition and therapeutic efficacy. Based on my preliminary data I will also interrogate the mechanistic basis for Gq inhibition sensitivity in GNAQQ209L driven melanocytes. 2) Determining clinical potential of this drug by testing its pharmacologic properties in vivo. Using the melanocytes I have generated along with UVM cell lines and our GEMM, we will be able to thoroughly test the drug’s efficacy against a variety of xenografts. 3) Taking advantage of the ability to inhibit active Gq, I will aim to identify other pathways activated downstream of Gq using proteomic and transcriptomic approaches. This may provide alternative therapeutic strategies to target this recalcitrant disease. The outcome of this research will indicate the effectiveness of Gq inhibition in UVM as well as elucidate currently unknown signaling pathways essential for UVM tumorigenesis. Citation Format: Tyler D. Hitchman, Emilie Ceraudo, Amanda R. Moore, Alexander N. Shoushtari, Jasmine H. Francis, Youxin Guan, Juliet Chen, Matthew T. Chang, Barry S. Taylor, Thomas P. Sakmar, Thomas Huber, Ping Chi, Yu Chen. Activating mutations in uveal melanoma convey sensitivity to g-alpha q inhibition [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr A07.

A subset of broadly responsive Type III taste cells contribute to the detection of bitter, sweet and umami stimuli.

Taste receptor cells use multiple signaling pathways to detect chemicals in potential food items. These cells are functionally grouped into different types: Type I cells act as support cells and have glial-like properties; Type II cells detect bitter, sweet, and umami taste stimuli; and Type III cells detect sour and salty stimuli. We have identified a new population of taste cells that are broadly tuned to multiple taste stimuli including bitter, sweet, sour, and umami. The goal of this study was to characterize these broadly responsive (BR) taste cells. We used an IP3R3-KO mouse (does not release calcium (Ca2+) from internal stores in Type II cells when stimulated with bitter, sweet, or umami stimuli) to characterize the BR cells without any potentially confounding input from Type II cells. Using live cell Ca2+ imaging in isolated taste cells from the IP3R3-KO mouse, we found that BR cells are a subset of Type III cells that respond to sour stimuli but also use a PLCβ signaling pathway to respond to bitter, sweet, and umami stimuli. Unlike Type II cells, individual BR cells are broadly tuned and respond to multiple stimuli across different taste modalities. Live cell imaging in a PLCβ3-KO mouse confirmed that BR cells use this signaling pathway to respond to bitter, sweet, and umami stimuli. Short term behavioral assays revealed that BR cells make significant contributions to taste driven behaviors and found that loss of either PLCβ3 in BR cells or IP3R3 in Type II cells caused similar behavioral deficits to bitter, sweet, and umami stimuli. Analysis of c-Fos activity in the nucleus of the solitary tract (NTS) also demonstrated that functional Type II and BR cells are required for normal stimulus induced expression.

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.

An unexpected INAD PDZ tandem-mediated plcβ binding in Drosophila photo receptors.

INAD assembles key enzymes of the Drosophila compound eye photo-transduction pathway into a supramolecular complex, supporting efficient and fast light signaling. However, the molecular mechanism that governs the interaction between INAD and NORPA (phospholipase Cβ, PLCβ), a key step for the fast kinetics of the light signaling, is not known. Here, we show that the NORPA C-terminal coiled-coil domain and PDZ-binding motif (CC-PBM) synergistically bind to INAD PDZ45 tandem with an unexpected mode and unprecedented high affinity. Guided by the structure of the INAD-NORPA complex, we discover that INADL is probably a mammalian counterpart of INAD. The INADL PDZ89 tandem specifically binds to PLCβ4 with a mode that is strikingly similar to that of the INAD-NORPA complex, as revealed by the structure of the INADL PDZ89-PLCβ4 CC-PBM complex. Therefore, our study suggests that the highly specific PDZ tandem - PLCβ interactions are an evolutionarily conserved mechanism in PLCβ signaling in the animal kingdom.

The N-terminal homology (ENTH) domain of Epsin 1 is a sensitive reporter of physiological PI(4,5)P2 dynamics

Phospholipase Cβ (PLCβ)-induced depletion of phosphatidylinositol-(4,5)-bisphosphate (PI(4,5)P2) transduces a plethora of signals into cellular responses. Importance and diversity of PI(4,5)P2-dependent processes led to strong need for biosensors of physiological PI(4,5)P2 dynamics applicable in live-cell experiments. Membrane PI(4,5)P2 can be monitored with fluorescently-labelled phosphoinositide (PI) binding domains that associate to the membrane depending on PI(4,5)P2 levels. The pleckstrin homology domain of PLCδ1 (PLCδ1-PH) and the C-terminus of tubby protein (tubbyCT) are two such sensors widely used to study PI(4,5)P2 signaling. However, certain limitations apply to both: PLCδ1-PH binds cytoplasmic inositol-1,4,5-trisphosphate (IP3) produced from PI(4,5)P2 through PLCβ, and tubbyCT responses do not faithfully report on PLCβ-dependent PI(4,5)P2 dynamics. In searching for an improved biosensor, we fused N-terminal homology domain of Epsin1 (ENTH) to GFP and examined use of this construct as genetically-encoded biosensor for PI(4,5)P2 dynamics in living cells. We utilized recombinant tools to manipulate PI or Gq protein-coupled receptors (GqPCR) to stimulate PLCβ signaling and characterized PI binding properties of ENTH-GFP with total internal reflection (TIRF) and confocal microscopy. ENTH-GFP specifically recognized membrane PI(4,5)P2 without interacting with IP3, as demonstrated by dialysis of cells with the messenger through a patch pipette. Utilizing Ci-VSP to titrate PI(4,5)P2 levels, we found that ENTH-GFP had low PI(4,5)P2 affinity. Accordingly, ENTH-GFP was highly sensitive to PLCβ-dependent PI(4,5)P2 depletion, and in contrast to PLCδ1-PH, overexpression of ENTH-GFP did not attenuate GqPCR signaling. Taken together, ENTH-GFP detects minute changes of PI(4,5)P2 levels and provides an important complementation of experimentally useful reporters of PI(4,5)P2 dynamics in physiological pathways.

Cane Toad Skin Extract-Induced Upregulation and Increased Interaction of Serotonin 2A and D2 Receptors via Gq/11 Signaling Pathway in CLU213 Cells.

Recent evidences show that activation of serotonin 2A receptors (5-HT2A R) by agonists is significant in improving therapeutic activity of disease conditions, such as obsessive-compulsive disorder (OCD). Though the exact molecular mechanism is still not well understood, it is thought to involve agonist-driven, enhanced expression of 5-HT2A R in certain areas of brain, such as the pre-frontal cortex (PFC). Several other reports have also demonstrated association of OCD with lower dopamine receptor (D2 R) availability, primarily in the striatum of the brain along with dysfunction of 5-HT2A R-D2 R heteromer regulation. We thus hypothesized that compound(s) interacting with this molecular mechanism could be developed as drugs for long-term beneficial effects against OCD. In the present study, we have obtained experimental evidence in cultured neuronal cells (CLU213) that aqueous extract (AE, 50 μg/mL, P < 0.05) of the Australian cane toad skin significantly increased the levels of 5-HT2A R and D2 R protein and mRNA expression. AE was also found to enhance the interaction between 5-HT2A R and D2 R and formation of expression of 5-HT2A R-D2 R heteromer using co-immunoprecipitation and Western blot. Further investigation showed the involvement of classical signaling pathway (Gq/11 -PLCβ) along with c-FOS transcription factor preferentially in 5-HT2A -mediated agonist activation. These results obtained demonstrated that AE upregulates 5-HT2A R by a mechanism that appears to involve Gq/11 -PLCβ signaling pathway and c-FOS transcription factor activation. We indicate this enhanced 5-HT2A R and D2 R expression and their interaction to induce increased 5-HT2A R-D2 R heteromer formation by exposure to AE might provide a molecular mechanism to develop potential novel drug candidates to ameliorate OCD symptoms. J. Cell. Biochem. 118: 979-993, 2017. © 2016 Wiley Periodicals, Inc.

Apelin-13 enhances arcuate POMC neuron activity via inhibiting M-current.

The hypothalamus is a key element of the neural circuits that control energy homeostasis. Specific neuronal populations within the hypothalamus are sensitive to a variety of homeostatic indicators such as circulating nutrient levels and hormones that signal circulating glucose and body fat content. Central injection of apelin secreted by adipose tissues regulates feeding and glucose homeostasis. However, the precise neuronal populations and cellular mechanisms involved in these physiological processes remain unclear. Here we examine the electrophysiological impact of apelin-13 on proopiomelanocortin (POMC) neuron activity. Approximately half of POMC neurons examined respond to apelin-13. Apelin-13 causes a dose-dependent depolarization. This effect is abolished by the apelin (APJ) receptor antagonist. POMC neurons from animals pre-treated with pertussis toxin still respond to apelin, whereas the Gβγ signaling inhibitor gallein blocks apelin-mediated depolarization. In addition, the effect of apelin is inhibited by the phospholipase C and protein kinase inhibitors. Furthermore, single-cell qPCR analysis shows that POMC neurons express the APJ receptor, PLC-β isoforms, and KCNQ subunits (2, 3 and 5) which contribute to M-type current. Apelin-13 inhibits M-current that is blocked by the KCNQ channel inhibitor. Therefore, our present data indicate that apelin activates APJ receptors, and the resultant dissociation of the Gαq heterotrimer triggers a Gβγ-dependent activation of PLC-β signaling that inhibits M-current.

Soluble sperm extract specifically recapitulates the initial phase of the Ca2+ response in the fertilized oocyte of P. occelata following a G-protein/ PLCβ signaling pathway.

Matured oocytes of the annelidan worm Pseudopotamilla occelata are fertilized at the first metaphase of the meiotic division. During the activation by fertilizing spermatozoa, the mature oocyte shows a two-step intracellular Ca2+ increase. Whereas the first Ca2+ increase is localized and appears to utilize the inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ stores, the second Ca2+ increase is global and involves Ca2+ influx via voltage-gated Ca2+ channels on the entire surface of the oocyte. To study how sperm trigger the Ca2+ increases during fertilization, we prepared soluble sperm extract (SE) and examined its ability to induce Ca2+ increases in the oocyte. The SE could evoke a Ca2+ increase in the oocyte when it was added to the medium, but not when it was delivered by microinjection. However, the second-step Ca2+ increase leading to the resumption of meiosis did not follow in these eggs. Local application of SE induced a non-propagating Ca2+ increase and formed a cytoplasmic protrusion that was similar to that created by the fertilizing sperm at the first stage of the Ca2+ response, important for sperm incorporation into the oocyte. Our results suggest that the fertilizing spermatozoon may trigger the first-step Ca2+ increase before it fuses with the oocyte in a pathway that involves the G-protein-coupled receptor and phospholipase C. Thus, the first phase of the Ca2+ response in the fertilized egg of this species is independent of the second phase of the Ca2+ increase for egg activation.

WNK1 Promotes PIP2 Synthesis to Coordinate Growth Factor and GPCR-Gq Signaling

PLC-β signaling is generally thought to be mediated by allosteric activation by G proteins and Ca(2+). Although availability of the phosphatidylinositol-4,5-biphosphate (PIP(2)) substrate is limiting in some cases, its production has not been shown to be independently regulated as a signaling mechanism. WNK1 protein kinase is known to regulate ion homeostasis and cause hypertension when expression is increased by gene mutations. However, its signaling functions remain largely elusive. Using diacylglycerol-stimulated TRPC6 and inositol trisphosphate-mediated Ca(2+) transients as cellular biosensors, we show that WNK1 stimulates PLC-β signaling in cells by promoting the synthesis of PIP(2) via stimulation of phosphatidylinositol 4-kinase IIIα. WNK1 kinase activity is not required. Stimulation of PLC-β by WNK1 and by Gα(q) are synergistic; WNK1 activity is essential for regulation of PLC-β signaling by G(q)-coupled receptors, and basal input from G(q) is necessary for WNK1 signaling via PLC-β. WNK1 further amplifies PLC-β signaling when it is phosphorylated by Akt kinase in response to insulin-like growth factor. WNK1 is a novel regulator of PLC-β that acts by controlling substrate availability. WNK1 thereby coordinates signaling between G protein and Akt kinase pathways. Because PIP(2) is itself a signaling molecule, regulation of PIP(2) synthesis by WNK1 also allows the cell to initiate PLC signaling while independently controlling the effects of PIP(2) on other targets. These findings describe a new signaling pathway for Akt-activating growth factors, a mechanism for G protein-growth factor crosstalk, and a means to independently control PLC signaling and PIP(2) availability.

Phospholipase Cβ3 Mediates LH-Induced Granulosa Cell Differentiation

Previous studies showed that under certain conditions LH can stimulate not only adenylate cyclase (AC) but also phospholipase Cβ (PLCβ) signaling in target cells; however, the physiological involvement of PLCβ in LH-induced ovarian follicular cell differentiation has not been determined. To address this, ex vivo expression analyses and specific PLCβ targeting were performed in primary bovine granulosa cells. Expression analyses in cells from small (2.0-5.9 mm), medium (6.0-9.9 mm), and ovulatory-size (10.0-13.9 mm) follicles revealed an increase in mRNA and protein levels of heterotrimeric G protein subunits-αs, -αq, -α11, and -αi2 in ovulatory-size follicles, simultaneous with a substantial increase in LH receptor expression. Among the four known PLCβ isoforms, PLCβ3 (PLCB3) was specifically up-regulated in cells from ovulatory-size follicles, in association with a predominantly cytoplasmic location of PLCB3 in these cells and a significant inositol phosphate response to LH stimulation. Furthermore, RNA interference-mediated PLCB3 down-regulation reduced the ability of LH to induce hallmark differentiation responses of granulosa cells, namely transcriptional up-regulation of prostaglandin-endoperoxide synthase 2 and down-regulation of both aromatase expression and estradiol production. Responses to the AC agonist, forskolin, however, were not affected. In addition, PLCB3 down-regulation did not alter cAMP responses to LH in granulosa cells, ruling out a primary involvement of AC in mediating the effects of PLCB3. In summary, we provide evidence of a physiological involvement of PLCβ signaling in ovulatory-size follicles and specifically identify PLCB3 as a mediator of LH-induced differentiation responses of granulosa cells.

Gα16 interacts with tetratricopeptide repeat 1 (TPR1) through its β3 region to activate Ras independently of phospholipase Cβ signaling

BackgroundG protein-coupled receptors constitute the largest family of cell surface receptors in the mammalian genome. As the core of the G protein signal transduction machinery, the Gα subunits are required to interact with multiple partners. The GTP-bound active state of many Gα subunits can bind a multitude of effectors and regulatory proteins. Yet it remains unclear if the different proteins utilize distinct or common structural motifs on the Gα subunit for binding. Using Gα16 as a model, we asked if its recently discovered adaptor protein tetratricopeptide repeat 1 (TPR1) binds to the same region as its canonical effector, phospholipase Cβ (PLCβ).ResultsWe have examined the specificity of Gα16/TPR1 association by testing a series of chimeras between Gα16 and Gαz. TPR1 co-immunoprecipitated with Gα16 and more tightly with its constitutively active Gα16QL, but not Gαz. Progressive replacement of Gα16 sequence with the corresponding residues of Gαz eventually identified a stretch of six amino acids in the β3 region of Gα16 which are responsible for TPR1 interaction and the subsequent Ras activation. Insertion of these six residues into Gαz allowed productive TPR1-interaction. Since the β3 region only minimally contributes to interact with PLCβ, several chimeras exhibited differential abilities to stimulate PLCβ and Ras. The ability of the chimeras to activate downstream transcription factors such as signal transducer and activator of transcription 3 and nuclear factor κB appeared to be associated with PLCβ signaling.ConclusionsOur results suggest that Gα16 can signal through TPR1/Ras and PLCβ simultaneously and independently. The β3 region of Gα16 is essential for interaction with TPR1 and the subsequent activation of Ras, but has relatively minor influence on the PLCβ interaction. Gα16 may utilize different structural domains to bind TPR1 and PLCβ.

Phosphatidic acid potentiates Gα q stimulation of phospholipase C-β 1 signaling

Phosphatidic acid (PA) is interactive with Gα q-linked agonists to stimulate GPCR signaling via phospholipase C-β 1 (PLC-β 1). Phorbol 12-myristate 13-acetate (PMA) increases cellular levels of PA and phospholipase D activity (PLD). This study evaluated whether PMA can stimulate PLC-β 1 activity via PA, independent of GPCR input in transfected COS 7 cells. PMA alone had little effect on PLC activity in cells co-transfected with PLC-β 1 and Gα q. Activated Gα q, induced by co-transfecting muscarinic cholinergic receptor (m1R), was necessary for stimulation of PLC-β 1 activity by PMA. Stimulation by PMA was dependent on the PA-regulatory motif of PLC-β 1 implicating PA in this mechanism. PLD1 knockdown by antisense decreased responsiveness of PLC-β 1 to both PMA and carbachol. PA alone thus has little effect on PLC-β 1 activity, but PA and PLD1 synergize with activated Gα q to stimulate PLC-β 1 signaling. Coordinate interaction with activated Gα q may serve as an important mechanism to fine tune response to ligands while preventing spurious initiation of PLC-β signaling by PA in cells.

A Transgenic Mouse Model of Heart Failure Using Inducible Gαq

Receptors coupled to Galpha q play a key role in the development of heart failure. Studies using genetically modified mice suggest that Galpha q mediates a hypertrophic response in cardiac myocytes. Galpha q signaling in these models is modified during early growth and development, whereas most heart failure in humans occurs after cardiac damage sustained during adulthood. To determine the phenotype of animals that express increased Galpha q signaling only as adults, we generated transgenic mice that express a silent Galpha q protein (Galpha qQ209L-hbER) in cardiac myocytes that can be activated by tamoxifen. Following drug treatment to activate Galpha q Q209L-hbER, these mice rapidly develop a dilated cardiomyopathy and heart failure. This phenotype does not appear to involve myocyte hypertrophy but is associated with dephosphorylation of phospholamban (PLB), decreased sarcoplasmic reticulum Ca2+-ATPase activity, and a decrease in L-type Ca2+ current density. Changes in Ca2+ handling and decreased cardiac contractility are apparent 1 week after Galpha qQ209L-hbER activation. In contrast, transgenic mice that express an inducible Galpha q mutant that cannot activate phospholipase Cbeta (PLCbeta) do not develop heart failure or changes in PLB phosphorylation, but do show decreased L-type Ca2+ current density. These results demonstrate that activation of Galpha q in cardiac myocytes of adult mice causes a dilated cardiomyopathy that requires the activation of PLCbeta. However, increased PLCbeta signaling is not required for all of the Galpha q-induced cardiac abnormalities.

NHERF2 specifically interacts with LPA2 receptor and defines the specificity and efficiency of receptor-mediated phospholipase C-beta3 activation.

Lysophosphatidic acid (LPA) activates a family of cognate G protein-coupled receptors and is involved in various pathophysiological processes. However, it is not clearly understood how these LPA receptors are specifically coupled to their downstream signaling molecules. This study found that LPA(2), but not the other LPA receptor isoforms, specifically interacts with Na(+)/H(+) exchanger regulatory factor2 (NHERF2). In addition, the interaction between them requires the C-terminal PDZ domain-binding motif of LPA(2) and the second PDZ domain of NHERF2. Moreover, the stable expression of NHERF2 potentiated LPA-induced phospholipase C-beta (PLC-beta) activation, which was markedly attenuated by either a mutation in the PDZ-binding motif of LPA(2) or by the gene silencing of NHERF2. Using its second PDZ domain, NHERF2 was found to indirectly link LPA(2) to PLC-beta3 to form a complex, and the other PLC-beta isozymes were not included in the protein complex. Consistently, LPA(2)-mediated PLC-beta activation was specifically inhibited by the gene silencing of PLC-beta3. In addition, NHERF2 increases LPA-induced ERK activation, which is followed by cyclooxygenase-2 induction via a PLC-dependent pathway. Overall, the results suggest that a ternary complex composed of LPA(2), NHERF2, and PLC-beta3 may play a key role in the LPA(2)-mediated PLC-beta signaling pathway.

Functional relevance of the disulfide-linked complex of the N-terminal PDZ domain of InaD with NorpA.

In Drosophila, phototransduction is mediated by G(q)-activation of phospholipase C and is a well studied model system for understanding the kinetics of signal initiation, propagation and termination controlled by G proteins. The proper intracellular targeting and spatial arrangement of most proteins involved in fly phototransduction require the multi-domain scaffolding protein InaD, composed almost entirely of five PDZ domains, which independently bind various proteins including NorpA, the relevant phospho lipase C-beta isozyme. We have determined the crystal structure of the N-terminal PDZ domain of InaD bound to a peptide corresponding to the C-terminus of NorpA to 1.8 A resolution. The structure highlights an intermolecular disulfide bond necessary for high affinity interaction as determined by both in vitro and in vivo studies. Since other proteins also possess similar, cysteine-containing consensus sequences for binding PDZ domains, this disulfide-mediated 'dock-and-lock' interaction of PDZ domains with their ligands may be a relatively ubiquitous mode of coordinating signaling pathways.