PLC Beta Research Articles

Inhibition in Proper Ser and In Plcγ2 Synthesis Are the Main for Causing Osteoarthritis, Diabetes, and C-Lymphocytic Leukemia Diseases

Proper S6K /BTK and PLCγ2 synthesis (which regulated by Ser phosphorylation pathway) are main regulations for thromboxane-A “TXA2” synthesis, and necessary for B-cell maturations and T-cells modulations and functions. The main reasons for causing Osteoarthritis “OA” and diabetes diseases (that are linked together) are the deficiency of Ser amino acids and decreasing of Ser phosphorylation signalling pathway which necessary for proper S6K productions, where normally the Ser phosphorylation signalling pathway is the basis of Ser /Thr phosphorylation signalling and is necessary for proper Akt, S6K1 synthesis and necessary for RORs and IFNs synthesis and also necessary for running proper BTK for PLCγ2 productions , where S6K is main regulator for ATPase and for proper PLCγ1 and for PLCγ2 synthesis which necessary for bone growth and for modulating immune efficiency. Osteoarthritis “OA” is characterized by a sharp expression in Gamma-Phospholipase C-1 “PLCγ1”, with decreasing “or inhibition” in PLCγ2 “PLC beta” productions due to inhibition or mutation in S6K and then in BTK. The increasing in PLCγ1 with Deficiency in Ser amino acids will lead to deficiency in Ser phosphorylation signalling, and decreasing in synthase activity that will reflect down regulations in BTK pathways and lead to inhibition in PLCγ2 productions which will reflect diabetes (inhibition in Estrogen with the production of Androgen instead of estrogen) and can reflect Osteoarthritis “OA” prognosis depend on the percentage of Deficiency or inhibition in basic amino acids and its basic necessary signaling pathways.

Osteoarthritis Linked to Diabetes Characterized Sharp Decreasing in Ser / Proline /PLC?2 with Increasing PLC?1, Where Inhibiting S6K/BTK / PLC?2 Affect TXA2 Synthesis Cause C-Lymphocytic Leukemia

Osteoarthritis Linked to Diabetes Characterized Sharp Decreasing in Ser / Proline /PLC?2 with Increasing PLC?1, Where Inhibiting S6K/BTK / PLC?2 Affect TXA2 Synthesis Cause C-Lymphocytic Leukemia

Bitter Taste Receptors Are Expressed on Acute Myeloid Leukemia Cells and Their Activation Results in Quiescence Induction and Prevention of Migration

Acute Myeloid Leukemia (AML) is a clonal disease sprouting from a rare population of leukemic stem cells (LSCs). Over the past years, increasing interest is gaining the contribution that cell-extrinsic factors have in AML generation and maintenance. In this context, AML cell ability to sense the changes in the microenvironment is important for different cell functions, including the responsiveness to antineoplastic agents. Bitter taste receptors (T2Rs) are typical G-protein coupled receptors and are normally found on the surface of the tongue. Recent studies showed that T2Rs are widely expressed in various parts of human anatomy and have been shown to be involved in physiology of respiratory system, gastrointestinal tract and endocrine system thus suggesting a wider function in “sensing microenvironment”. So far, very few data about T2Rs expression in cancer cells are available. In particular, their expression and function in AML cells has not been investigated. Our aim was to investigate T2Rs expression and role in regulation of leukemic cell functionality.In the present work, for the first time, we showed that leukemia cell lines OCI-AML3, THP-1, KG1 and AML primary cells expressed several T2R subtypes (Fig.1A). The expression of TAS2R was associated with GPR-related downstream targets, including the beta subunit of gustducin and the PLC beta 2 (Fig.2B). Stimulation of leukemia cell lines with denatonium (DEN), a T2Rs agonist, induced intracellular Ca2+ concentration increase, thus demonstrating T2Rs functionality (Fig. 1C). To investigate whether bitter compound could modulate AML cell biological properties, we performed GEP analysis of DEN treated cells. GEP analysis identified a number of genes significantly modulated by denatonium treatment. Enrichment analysis identify significantly affected biological processes, among which, cell cycle/proliferation, apoptosis, migration/adhesion and DNA repair (Fig.1D). Specifically, leukemic cells stimulated with T2R agonist underwent down-regulation of genes involved in positive regulation of cell proliferation, migration, and cell-cycle. Whereas genes involved in cell adhesion and DNA repair were up-regulated. Functional assays confirmed molecular data. Indeed, depending on the extent of stimulation, TAS2R activation inhibited leukemia cell proliferation (Fig.2A) inducing cell cycle arrest in G0/G1 phase (Fig. 2B) or reduced cell viability inducing apoptosis (Fig. 2C), as demonstrated by caspase cascade activation and mitochondrial stress induction (Fig. 2D-E). Of note, a pronounced inhibitory effect of denatonium on leukemia cells motility, both spontaneous and in response to CXCL-12, was observed (Fig. 2F).Overall, our results indicate that in AML cells the activation of fully functional T2Rs is associated with quiescence induction and prevention of migration. These data may suggest a role for microenvironment “bitter” molecules in regulating leukemia cell functions, including lethal sensitivity to antineoplastic agents. [Display omitted] DisclosuresMartinelli:Pfizer: Consultancy; Celgene: Consultancy; Ariad/Incyte: Consultancy; Amgen: Consultancy; Johnson&Johnson: Consultancy; Roche: Consultancy.

Mash1-expressing cells could differentiate to type III cells in adult mouse taste buds.

The gustatory cells in taste buds have been identified as paraneuronal; they possess characteristics of both neuronal and epithelial cells. Like neurons, they form synapses, store and release transmitters, and are capable of generating an action potential. Like epithelial cells, taste cells have a limited life span and are regularly replaced throughout life. However, little is known about the molecular mechanisms that regulate taste cell genesis and differentiation. In the present study, to begin to understand these mechanisms, we investigated the role of Mash1-positive cells in regulating adult taste bud cell differentiation through the loss of Mash1-positive cells using the Cre-loxP system. We found that the cells expressing type III cell markers-aromatic L-amino acid decarboxylase (AADC), carbonic anhydrase 4 (CA4), glutamate decarboxylase 67 (GAD67), neural cell adhesion molecule (NCAM), and synaptosomal-associated protein 25 (SNAP25)-were significantly reduced in the circumvallate taste buds after the administration of tamoxifen. However, gustducin and phospholipase C beta2 (PLC beta2)-markers of type II taste bud cells-were not significantly changed in the circumvallate taste buds after the administration of tamoxifen. These results suggest that Mash1-positive cells could be differentiated to type III cells, not type II cells in the taste buds.

Amylase expression in taste receptor cells of rat circumvallate papillae

The chemical composition of the luminal content is now accepted to have a profound influence on the performance of chemosensory receptors. Gustatory and intestinal chemoreceptors have in common their expression of molecules involved in taste sensing and signal transduction pathways. The recent finding that enterocytes of the duodenal epithelium are capable of expressing luminal pancreatic amylase suggests that taste cells of the gustatory epithelium might, in the same way, express salivary amylase in the oral cavity. Therefore, we investigated amylase expression in rat circumvallate papillae by using analyses involving immunohistochemistry, Western blot, and reverse transcription with the polymerase chain reaction. In addition, we used double-labeling confocal laser microscopy to compare amylase immunolabeling with that of the following markers: protein gene product 9.5 (PGP 9.5) and chromogranin A (CgA) for endocrine cells, alpha-gustducin and phospholipase C beta 2 (PLC beta 2) as taste-signaling molecules, and cystic fibrosis transmembrane regulator (CFTR) and Clara-cell-specific secretory protein of 10-kDa (CC10) as secretory markers. The results showed that amylase was present in some taste bud cells; its immunoreactivity was observed in subsets of cells that expressed CgA, alpha-gustducin, PLC beta 2, CFTR, or CC10. PGP 9.5 immunoreactivity was never colocalized with amylase. The data suggest that amylase-positive cells constitute an additional subset of taste receptor cells also associated with chemoreceptorial and/or secretory molecules, confirming the occurrence of various pathways in taste buds.

Nuclear PLC Beta 1 is required for 3T3-L1 adipocyte differentiation and regulates expression of the cyclin D3–cdk4 complex

A phosphoinositide signalling cycle is present in the nucleus, independent of that which occurs at the plasma membrane. The key enzyme involved in this cycle is phospholipase (PLC) β1. This nuclear cycle has been shown to be involved in both cell proliferation and differentiation. Here, we report that nuclear PLCβ1 activity is upregulated during differentiation of 3T3-L1 adipocytes. During differentiation there are two phases of PLCβ1 activity; the first occurs within 5 min of treatment with differentiation media, does not require new PLCβ1 to enter the nucleus and is regulated by pERK and PKC α while the second phase occurs from day 2 of differentiation, requires new PLCβ1 protein to enter the nucleus and is independent of regulation by pERK and PKC α. Over-expression with the PLC mutants, Δmk (which lacks the ERK phosphorylation site) and M2B (which lacks the nuclear localisation sequence), revealed that both phases of PLCβ1 activity are required for terminal differentiation to occur. Inhibition of PLCβ1 activity prevents the upregulation of cyclinD3 and cdk4 protein, suggesting that PLCβ1 plays a role in the control of the cell cycle during differentiation. These results indicate nuclear PLCβ1 as a key regulator of adipocyte differentiation.

Activation of phosphatidylinositol-linked novel D1 dopamine receptor contributes to the calcium mobilization in cultured rat prefrontal cortical astrocytes.

Recent evidences indicate the existence of an atypical D(1) dopamine receptor other than traditional D(1) dopamine receptor in the brain that mediates PI hydrolysis via activation of phospholipase C(beta) (PLC(beta)). To further understand the basic physiological function of this receptor in brain, the effects of a selective phosphoinositide (PI)-linked D(1) dopamine receptor agonist SKF83959 on cytosolic free calcium concentration ([Ca(2+)](i)) in cultured rat prefrontal cortical astrocytes were investigated by calcium imaging. The results indicated that SKF83959 caused a transient dose-dependent increase in [Ca(2+)](i). Application of D(1) receptor, but not D(2), alpha(1) adrenergic, 5-HT receptor, or cholinergic antagonist prevented SKF83959-induced [Ca(2+)](i) rise, indicating that activation of the D(1) dopamine receptor was essential for this response. Increase in [Ca(2+)](i) was a two-step process characterized by an initial increase in [Ca(2+)](i) mediated by release from intracellular stores, supplemented by influx through voltage-gated calcium channels, receptor-operated calcium channels, and capacitative Ca(2+) entry. Furthermore, SKF83959-stimulated increase in [Ca(2+)](i) was abolished following treatment with a PLC inhibitor. Overall, these results suggested that activation of D(1) receptor by SKF83959 mediates a dose-dependent mobilization of [Ca(2+)](i) via the PLC signaling pathway in cultured rat prefrontal cortical astrocytes.

The Role of PLCβ1 in the Control of Oocyte Meiosis During Folliculogenesis

The dynamics of the subcellular distribution of PLCbeta1 was investigated during meiosis competence acquisition and meiosis resumption in relation to oocyte diameter and to nonsurrounded-nucleolus or surrounded-nucleolus chromatin configurations. Oocytes collected after both in vivo and in vitro folliculogenesis were studied. In both conditions, at the beginning of the process, most oocytes exhibited a nuclear PLCbeta 1 associated with a nonsurrounded-nucleolus chromatin configuration. Then at the final stage of the process, the factors shifted mainly toward a cytoplasmic PLCbeta1 and a surrounded-nucleolus chromatin configuration, typical of a preovulatory fertilizable oocyte. Additionally, only germinal vesicle oocytes with a diameter > 75 microm, and exhibiting cytoplasmic PLC beta1 distribution and surrounded-nucleolus chromatin configuration, resumed meiosis. Our findings demonstrate a strong correlation between oocyte diameter, chromatin configuration, and PLCbeta1 localization. Thus, PLCbeta1 localization appears to be a key factor determining the progressive acquisition of meiotic competence and final resumption of meiosis.

Phospholipase C β3 deficiency leads to macrophage hypersensitivity to apoptotic induction and reduction of atherosclerosis in mice

Atherosclerosis is an inflammatory disease that is associated with monocyte recruitment and subsequent differentiation into lipid-laden macrophages at sites of arterial lesions, leading to the development of atherosclerotic plaques. PLC is a key member of signaling pathways initiated by G protein-coupled ligands in macrophages. However, the role of this enzyme in the regulation of macrophage function is not known. Here, we studied macrophages from mice lacking PLC beta2, PLC beta3, or both PLC isoforms and found that PLC beta3 is the major functional PLC beta isoform in murine macrophages. Although PLC beta3 deficiency did not affect macrophage migration, adhesion, or phagocytosis, it resulted in macrophage hypersensitivity to multiple inducers of apoptosis. PLC beta3 appeared to regulate this sensitivity via PKC-dependent upregulation of Bcl-XL. The significance of PLC beta signaling in vivo was examined using the apoE-deficient mouse model of atherosclerosis. Mice lacking both PLC beta3 and apoE exhibited fewer total macrophages and increased macrophage apoptosis in atherosclerotic lesions, as well as reduced atherosclerotic lesion size when compared with mice lacking only apoE. These results demonstrate what we believe to be a novel role for PLC activity in promoting macrophage survival in atherosclerotic plaques and identify PLC beta3 as a potential target for treatment of atherosclerosis.

The Phosphoinositide-Phospholipase C (PI-PLC) Pathway in the Mouse Oocyte

As highlighted in this review, the phosphoinositide-phospholipase C pathway is strongly implicated in the control of mouse oocyte meiosis. The pathway becomes progressively functional as oocyte growth advances, and it appears to play a role in the G2/M transition when meiosis resumes, at least in the in vitro spontaneous model. Even if the inositol 1,4,5-trisphosphate receptors are present from the beginning, they function and release Ca2+ when the follicular antrum appears. Phospholipase C beta1 (PLC beta 1) is first exclusively localized to the nucleus and then migrates to the cytoplasm when the oocyte is fully grown. During oocyte maturation PLC beta 1 is active in the cytoplasm before it migrates and becomes active in the nucleus just prior to germinal vesicle breakdown. Because a similar circuit is observed for protein kinase C alpha (PKC alpha), PKC beta 1, PKC beta 2, and active mitogen-activated protein kinase, it is tempting to envisage that a feedback loop occurs between these pathways as demonstrated in other cell types. The chronology of these molecular movements into the oocyte reveals the particular and important role of the nucleus phosphoinositide cycle during oocyte meiosis. It appears also that this chronology is crucial and that defects leading to an inappropriate intracellular localization can have dramatic consequences. Such anomalies can prevent the production of competent oocytes and lead to fertility problems.

Stable Association between Gαq and Phospholipase Cβ1 in Living Cells

Signal transduction through G alpha(q) involves stimulation of phospholipase C beta (PLC beta) that results in increased intracellular Ca2+ and activation of protein kinase C. We have measured complex formation between G alpha(q) and PLC beta1 in vitro and in living PC12 and HEK293 cells by fluorescence resonance energy transfer. In vitro measurements show that PLC beta1 will bind to G alpha(q)(guanosine 5'-3-O-(thio)triphosphate) and also to G alpha(q)(GDP), and the latter association has a different protein-protein orientation. In cells, image analysis of fluorescent-tagged proteins shows that G alpha(q) is localized almost entirely to the plasma membrane, whereas PLC beta1 has a significant cytosolic population. By using fluorescence resonance energy transfer, we found that these proteins are pre-associated in the unstimulated state in PC12 and HEK293 cells. By determining the cellular levels of the two proteins in transfected versus nontransfected cells, we found that under our conditions overexpression should not significantly promote complex formation. G alpha(q)-PLC beta1 complexes are observed in both single cell measurements and measurements of a large (i.e. 10(6)) cell suspension. The high level (approximately 40% maximum) of FRET is surprising considering that G alpha(q) is more highly expressed than PLC beta1 and that not all PLC beta1 is plasma membrane-localized. Our measurements suggest a model in which G proteins and effectors can exist in stable complexes prior to activation and that activation is achieved through changes in intermolecular interactions rather than diffusion and association. These pre-formed complexes in turn give rise to rapid, localized signals.

Chronic intake of caffeine during gestation down regulates metabotropic glutamate receptors in maternal and fetal rat heart

Caffeine is the most widely consumed substance in the world which antagonizes adenosine effects. Adenosine acting through A(1) receptors inhibits glutamate release which binds to metabotropic glutamate receptors (mGluRs). Recently, we have shown that maternal caffeine intake during gestation causes down-regulation of A(1) and metabotropic glutamate receptors in the brain of both rat mothers and fetuses. In the present work we provide evidence that caffeine also affects receptors in hearts, causing a decrease in mGluRs from both maternal and fetal hearts. A decrease in G(q/11) and PLC beta(1) proteins level was also observed in both tissues. However, phospholipase C activity was only affected in fetal heart, being significantly decreased. These results suggest an in vivo cross-talk mechanism between adenosine and glutamate receptors in peripheral tissues. Therefore, special attention should be paid to caffeine ingestion during gestation.

Regulation of the TRP calcium channel by eye‐PKC in Drosophila

The goal of this research study is to investigate the regulation of Drosophila visual signaling by eye-protein kinase C (eye-PKC). Drosophila visual signaling involves a G-protein coupled PLC beta cascade, initiated by light activation of rhodopsin. Rhodopsin, activates a Gq heterotrimeric protein that stimulates PLC beta. PLC beta catalyzes the hydrolysis of PIP2 into IP3 and DAG, resulting in depolarization of the photoreceptor cell via two plasma membrane channels: transient receptor potential (TRP) calcium channel and TRP-like nonselective cation channel. Deactivation of visual signaling is mediated by eye-PKC, which is activated by both DAG and calcium. Eye-PKC, PLC beta and TRP are constitutively attached to a scaffold protein named INAD. The in vivo function of eye-PKC is dependent on its interaction with INAD. Previously, TRP has been shown to be phosphorylated in vitro by eye-PKC. To investigate the regulation of TRP by eye-PKC, first we will map the eye-PKC phosphorylation sites on TRP. By using a combination of pull-down and in vitro kinase assays of various substrates, we have demonstrated phosphorylation of the TRP C-terminal tail by eye-PKC. Using various substrates and site-directed mutagenesis, we determined that TRP is phosphorylated by eye-PKC at serine 982. These results were confirmed by mass spectrometry analysis of both in vitro and in vivo phosphorylated TRP. We are now analyzing the phenotype of transgenic flies expressing a modified TRP (Ser982Ala) by electroretinogram recordings. This study will give us a better understanding of how phosphorylation modulates TRP channel activity. This research was supported by NIH grant EY09743 (to B.-H.S).

Phosphatidyl‐inositide signalling proteins in a novel class of sensory cells in the mammalian olfactory epithelium

Ciliated sensory neurons, supporting cells and basal stem cells represent major cellular components of the main olfactory epithelium in mammals. Here we describe a novel class of sensory cells in the olfactory neuroepithelium. The cells express phospholipase C beta-2 (PLC beta2), transient receptor potential channels 6 (TRPC6) and inositol 3, 4, 5-trisphosphate receptors type III (InsP3R-III). Unlike ciliated olfactory neurons, they express neither olfactory marker protein nor centrin, adenylyl cyclase or cyclic nucleotide-gated cation channels. Typical components of the cytoskeleton of microvilli, ezrin and actin are found co-localized with PLC beta2 and TRPC6 in apical protrusions of the cells. In Ca2+-imaging experiments, the cells responded to odours. They express neuronal marker proteins and possess an axon-like process, but following bulbectomy the cells do not degenerate. Our results suggest a novel class of microvillous secondary chemosensory cells in the mammalian olfactory system. These cells, which utilize phosphatidyl-inositides in signal transduction, represent about 5% of all olfactory cells. Their abundance indicates that they play an important role in stimulus-dependent functions and/or the regeneration of the olfactory system.

Nuclear phospholipase C β1, regulation of the cell cycle and progression of acute myeloid leukemia

A large number of observations have hinted at the fact that location impinges on function of some of the main players of nuclear inositol lipid cycle. PLC beta1 is a well-known example, given that it has been shown that only the enzyme located in the nucleus targets the cyclin D3/cdk4 complex, playing, in turn, a key role in the control of normal progression through the G1 phase of the cell cycle. The PLC beta1 gene, which is constituted of 36 small exons and large introns, maps on the short arm of human chromosome 20 (20pl2, nearby markers D20S917 and D20S177) with the specific probe (PAC clone HS881E24) spanning from exon 19 to 32 of the gene itself. The chromosome band 20pl2 has been shown to be rearranged in human diseases such as solid tumors without a more accurate definition of the alteration, maybe because of the absence of candidate genes or specific probes. Moreover, non-specific alterations in chromosome 20 have been found in patients affected by MDS and acute myeloid leukemia AML. MDS is an adult hematological disease that evolves into AML in about 30% of the cases. The availability of a highly specific probe gave an opportunity to perform in patients affected with MDS/AML, associated with normal karyotype, painting and FISH analysis aimed to check the PLC beta1 gene, given that this signaling molecule is a key player in the control of some checkpoints of the normal progression through the cell cycle. FISH analysis disclosed in a small group of MDS/AML patients with normal karyotype the monoallelic deletion of the PLC beta1 gene. In contrast, PLC beta4, another gene coding for a signaling molecule, located on 20pl2.3 at a distance as far as less than 1 Mb from PLC beta1, is unaffected in MDS patients with the deletion of PLC beta1 gene, hinting at an interstitial deletion. The MDS patients, bearing the deletion, rapidly evolved to AML, whilst the normal karyotype MDS patients, showing non-deletion of PLC beta1 gene, are still alive at least 24 months after the diagnosis. The immunocytochemical analysis using an anti PLC beta1 monoclonal antibody showed that all the AML/MDS patients who were normal at FISH analysis also had normal staining of the nucleus, which is a preferential site for PLC beta1. In contrast, the monoallelic deletion gave rise to a dramatic decrease of the nuclear staining suggesting a decreased expression of the nuclear PLC beta1. The reported data strengthen the contention of a key role played by PLC beta1 in the nucleus, suggest a possible involvement of PLC beta1 in the progression of MDS to AML and pave the way for a larger investigation aimed at identifying a possible high risk group among MDS patients with a normal karyotype.

Dynamic evolution of the adenine nucleotide translocase interactome during chemotherapy-induced apoptosis.

The mitochondrial permeability transition pore complex (PTPC) is involved in the control of the mitochondrial membrane permeabilization during apoptosis, necrosis and autophagy. Indeed, the adenine nucleotide translocator (ANT) and the voltage-dependent anion channel (VDAC), two major components of PTPC, are the targets of a variety of proapoptotic inducers. Using co-immunoprecipitation and proteomic analysis, we identified some of the interacting partners of ANT in several normal tissues and human cancer cell lines. During chemotherapy-induced apoptosis, some of these interactions were constant (e.g. ANT-VDAC), whereas others changed strongly concomitantly with the dissipation of the mitochondrial transmembrane potential and until nuclear degradation occurred (e.g. Bax, Bcl-2, subunits of the respiratory chain, a subunit of the phosphatase PP2A, phospholipase PLC beta 4 and IP3 receptor). In addition, a glutathione-S-transferase (GST) interacts with ANT in normal tissue, in colon carcinoma cells and in vitro. This interaction is lost during apoptosis induction, suggesting that GST behaves as an endogenous repressor of PTPC and ANT pore opening. Thus, ANT is connected to mitochondrial proteins as well as to proteins from other organelles such as the endoplasmic reticulum forming a dynamic polyprotein complex. Changes within this ANT interactome coordinate the lethal response of cells to apoptosis induction.

Assay for G protein-dependent activation of phospholipase C beta using purified protein components.

The activity of mammalian phosphoinositide-specific phospholipase C beta (PLC beta) is regulated by the alpha q family of G protein alpha subunits and by beta gamma subunits thought to be released from Gi. Interactions between G protein subunits and PLC beta can be assayed by measuring the stimulation of PLC beta enzymatic activity on reconstituting the purified G protein subunits with purified PLC beta on artificial phospholipid vesicles containing the substrate, phosphatidylinositol-4,5-bisphosphate (PIP2). These vesicles are doped with [3H]-inositol PIP2 and the rate of hydrolysis is determined by quantitating the amount of [3H]-inositol triphosphate (IP3) released from the vesicle into the aqueous phase. This assay provides a relatively simple method for assessing the activity PLC activity and its ability to be regulated by beta gamma and alpha(q) subunits. It can also be used to assess the functionality of the components after modification by mutagenesis, chemical modification, or in the presence of competing molecules.

Activation of Platelet-activating Factor Receptor-coupled Gαq Leads to Stimulation of Src and Focal Adhesion Kinase via Two Separate Pathways in Human Umbilical Vein Endothelial Cells

Platelet-activating factor (PAF), a phospholipid second messenger, has diverse physiological functions, including responses in differentiated endothelial cells to external stimuli. We used human umbilical vein endothelial cells (HUVECs) as a model system. We show that PAF activated pertussis toxin-insensitive G alpha(q) protein upon binding to its seven transmembrane receptor. Elevated cAMP levels were observed via activation of adenylate cyclase, which activated protein kinase A (PKA) and was attenuated by a PAF receptor antagonist, blocking downstream activity. Phosphorylation of Src by PAF required G alpha(q) protein and adenylate cyclase activation; there was an absolute requirement of PKA for PAF-induced Src phosphorylation. Immediate (1 min) PAF-induced STAT-3 phosphorylation required the activation of G alpha(q) protein, adenylate cyclase, and PKA, and was independent of these intermediates at delayed (30 min) and prolonged (60 min) PAF exposure. PAF activated PLC beta 3 through its G alpha(q) protein-coupled receptor, whereas activation of phospholipase C gamma 1 (PLC gamma 1) by PAF was independent of G proteins but required the involvement of Src at prolonged PAF exposure (60 min). We demonstrate for the first time in vascular endothelial cells: (i) the involvement of signaling intermediates in the PAF-PAF receptor system in the induction of TIMP2 and MT1-MMP expression, resulting in the coordinated proteolytic activation of MMP2, and (ii) a receptor-mediated signal transduction cascade for the tyrosine phosphorylation of FAK by PAF. PAF exposure induced binding of p130(Cas), Src, SHC, and paxillin to FAK. Clearly, PAF-mediated signaling in differentiated endothelial cells is critical to endothelial cell functions, including cell migration and proteolytic activation of MMP2.

Calmodulin Is a Phospholipase C-β Interacting Protein

Phospholipase C-beta 3 (PLC beta 3) is an important effector enzyme in G protein-coupled signaling pathways. Activation of PLC beta 3 by G alpha and G beta gamma subunits has been fairly well characterized, but little is known about other protein interactions that may also regulate PLC beta 3 function. A yeast two-hybrid screen of a mouse brain cDNA library with the amino terminus of PLC beta 3 has yielded potential PLC beta 3 interacting proteins including calmodulin (CaM). Physical interaction between CaM and PLC beta 3 is supported by a positive secondary screen in yeast and the identification of a CaM binding site in the amino terminus of PLC beta 3. Co-precipitation of in vitro translated and transcribed amino- and carboxyl-terminal PLC beta 3 revealed CaM binding at a putative amino-terminal binding site. Direct physical interaction of PLC beta 3 and PLC beta 1 isoforms with CaM is supported by pull-down of both isoenzymes with CaM-Sepharose beads from 1321N1 cell lysates. CaM inhibitors reduced M1-muscarinic receptor stimulation of inositol phospholipid hydrolysis in 1321N1 astrocytoma cells consistent with a physiologic role for CaM in modulation of PLC beta activity. There was no effect of CaM kinase II inhibitors, KN-93 and KN-62, on M1-muscarinic receptor stimulation of inositol phosphate hydrolysis, consistent with a direct interaction between PLC beta isoforms and CaM.

Gβγ Mediates the Interplay between Tubulin Dimers and Microtubules in the Modulation of Gq Signaling

Agonist stimulation causes tubulin association with the plasma membrane and activation of PLC beta 1 through direct interaction with, and transactivation of, G alpha q. Here we demonstrate that G beta gamma interaction with tubulin down-regulates this signaling pathway. Purified G beta gamma, alone or with phosphatidylinositol 4,5-bisphosphate (PIP2), inhibited carbachol-evoked membrane recruitment of tubulin and G alpha q transactivation by tubulin. Polymerization of microtubules elicited by G beta gamma overrode tubulin translocation to the membrane in response to carbachol stimulation. G beta gamma sequestration of tubulin reduced the inhibition of PLC beta 1 observed at high tubulin concentration. G beta 1 gamma 2 interacted preferentially with tubulin-GDP, whereas G alpha q was transactivated by tubulin-GTP. Prenylation of the gamma 2 polypeptide was required for G beta gamma/tubulin interaction. Both confocal microscopy and coimmunoprecipitation studies revealed the spatiotemporal pattern of G beta gamma/tubulin interaction during carbachol stimulation of neuroblastoma SK-N-SH cells. In resting cells G beta gamma localized predominantly at the cell membrane, whereas tubulin was found in well defined microtubules in the cytosol. Within 2 min of agonist exposure, a subset of tubulin translocated to the plasma membrane and colocalized with G beta. Fifteen min post-carbachol addition, tubulin and G beta colocalized in vesicle-like structures in the cytosol. G beta/tubulin colocalization increased after pretreatment of cells with the microtubule-depolymerizing agent, colchicine, and was inhibited by taxol. Taxol also inhibited carbachol-induced PIP2 hydrolysis. It is suggested that G beta gamma/tubulin interaction mediates internalization of membrane-associated tubulin at the offset of PLC beta 1 signaling. Newly cytosolic G beta gamma/tubulin complexes might promote microtubule polymerization attenuating further tubulin association with the plasma membrane. Thus G protein-coupled receptors might evoke G alpha and G beta gamma to orchestrate regulation of phospholipase signaling by tubulin dimers and control of cell shape by microtubules.