Activation Of Phospholipase Cbeta Research Articles

Endothelin signaling promotes melanoma tumorigenesis driven by constitutively active GNAQ.

The G-protein-coupled receptor, endothelin receptor B (EDNRB), is an important regulator of melanocyte survival and proliferation. It acts by stimulating downstream heterotrimeric G proteins, such as Gαq and Gα1 . Constitutively active, oncogenic versions of Gαq and Gα11 drive melanomagenesis, but the role of Ednrb in the context of these mutant G proteins has not been previously examined. In this paper, we used a knock-in mouse allele at the Rosa26 locus to force oncogenic GNAQQ209L expression in melanocytes in combination with Ednrb gene knockout. The resulting pathological analysis revealed that every aspect of melanomagenesis driven by GNAQQ209L was inhibited. We conclude that even in the presence of oncogenic Gαq , the Ednrb receptor activates normal Gαq and Gα11 proteins. This likely promotes tumorigenesis by activating phospholipase C-beta, the immediate effector of Gαq/11 . These findings suggest that it might be possible to target upstream receptors to offset the effects of hyperactive G proteins, recognized as the cause of a growing number of human disorders.

Intermolecular cross‐talk between the prostaglandin E2 receptor (EP)3 subtype and thromboxane A2 receptor signalling in human erythroleukaemic cells

In previous studies investigating cross-talk of signalling between prostaglandin (PG)E(2) receptor (EP) and the TPalpha and TPbeta isoforms of the human thromboxane (TX)A(2) receptor (TP), 17-phenyl trinor PGE(2)-induced desensitization of TP receptor signalling through activation of the AH6809 and SC19220-sensitive EP(1) subtype of the EP receptor family, in a cell-specific manner. Here, we sought to further investigate that cross-talk in human erythroleukaemic (HEL) 92.1.7 cells. Specificity of 17-phenyl trinor PGE(2) signalling and its possible cross-talk with signalling by TPalpha/TPbeta receptors endogenously expressed in HEL cells was examined through assessment of agonist-induced inositol 1,4,5-trisphosphate (IP)(3) generation and intracellular calcium ([Ca(2+)](i)) mobilization. While 17-Phenyl trinor PGE(2) led to activation of phospholipase (PL)Cbeta to yield increases in IP(3) generation and [Ca(2+)](i), it did not desensitize but rather augmented that signalling in response to subsequent stimulation with the TXA(2) mimetic U46619. Furthermore, the augmentation was reciprocal. Signalling by 17-phenyl trinor PGE(2) was found to occur through AH6809- and SC19920-insensitive, Pertussis toxin-sensitive, G(i)/G(betagamma)-dependent activation of PLCbeta. Further pharmacological investigation using selective EP receptor subtype agonists and antagonists confirmed that 17-phenyl trinor PGE(2)-mediated signalling and reciprocal cross-talk with the TP receptors occurred through the EP(3), rather than the EP(1), EP(2) or EP(4) receptor subtype in HEL cells. The EP(1) and EP(3) subtypes of the EP receptor family mediated intermolecular cross-talk to differentially regulate TP receptor-mediated signalling whereby activation of EP(1) receptors impaired or desensitized, while that of EP(3) receptors augmented signalling through TPalpha/TPbeta receptors, in a cell type-specific manner.

Enzymatic measurement of phosphatidic acid in cultured cells

In this work, we developed a novel enzymatic method for measuring phosphatidic acid (PA) in cultured cells. The enzymatic reaction sequence of the method involves hydrolysis of PA to produce glycerol-3-phosphate (G3P), which is then oxidized by G3P oxidase to generate hydrogen peroxide. In the presence of peroxidase, hydrogen peroxide reacted with Amplex Red to produce highly fluorescent resorufin. We found that lipase from Pseudomonas sp. can completely hydrolyze PA to G3P and FAs. The calibration curve for PA measurement was linear between 20 and 250 microM, and the detection limit was 5 microM (50 pmol in the reaction mixture). We also modified the method for the enzymatic measurement of lysophosphatidic acid. By this new method, we determined the PA content in the lipid extract from HEK293 cells. The cellular content of PA was decreased with increasing cell density but not correlated with the proliferation rate. The diacylglycerol kinase inhibitor R59949 markedly reduced the cellular PA content, suggesting the diacylglycerol kinase activity was involved in a large part of the PA production in HEK293 cells. This novel method for PA quantification is simple, rapid, specific, sensitive, and high-throughput and will help to study the biological functions of PA and its related enzymes.

Rac1 is essential for phospholipase C-γ2 activation in platelets

Platelet activation at sites of vascular injury is triggered through different signaling pathways leading to activation of phospholipase (PL) Cbeta or PLCgamma2. Active PLCs trigger Ca(2+) mobilization and entry, which is a prerequisite for adhesion, secretion, and thrombus formation. PLCbeta isoenzymes are activated downstream of G protein-coupled receptors (GPCRs), whereas PLCgamma2 is activated downstream of immunoreceptor tyrosine-based activation motif (ITAM)-coupled receptors, such as the major platelet collagen receptor glycoprotein (GP) VI or CLEC-2. The mechanisms underlying PLC regulation are not fully understood. An involvement of small GTPases of the Rho family (Rho, Rac, Cdc42) in PLC activation has been proposed but this has not been investigated in platelets. We here show that murine platelets lacking Rac1 display severely impaired GPVI- or CLEC-2-dependent activation and aggregation. This defect was associated with impaired production of inositol 1,4,5-trisphosphate (IP(3)) and intracellular calcium mobilization suggesting inappropriate activation of PLCgamma2 despite normal tyrosine phosphorylation of the enzyme. Rac1 ( -/- ) platelets displayed defective thrombus formation on collagen under flow conditions which could be fully restored by co-infusion of ADP and the TxA(2) analog U46619, indicating that impaired GPVI-, but not G-protein signaling, was responsible for the observed defect. In line with this, Rac1 ( -/- ) mice were protected in two collagen-dependent arterial thrombosis models. Together, these results demonstrate that Rac1 is essential for ITAM-dependent PLCgamma2 activation in platelets and that this is critical for thrombus formation in vivo.

Essential role of Pyk2 and Src kinase activation in neuropeptide-induced proliferation of small cell lung cancer cells

Neuropeptide hormones like bombesin/gastrin-releasing peptide, galanin or bradykinin, acting via auto and paracrine growth loops, represent the principal mitogens of small cell lung cancer (SCLC). These mitogenic neuropeptides activate G(q/11)-coupled receptors which stimulate phospholipase Cbeta activity, followed by rises of the intracellular calcium concentration ([Ca2+](i)) and activation of protein kinase C (PKC). We report here that proline-rich tyrosine kinase Pyk2 is highly expressed in SCLC cells and provides a functional link between neuropeptide-induced increases in [Ca2+](i) and tumor cell proliferation. Activation of Pyk2 and its association with Src kinases critically depends on the elevation of [Ca2+](i), but is independent of PKC. Src kinase activities are crucial for neuropeptide-mediated GTP-loading of Ras and activation of extracellular signal-regulated kinases in SCLC cells. Pyk2 and Src kinases essentially contribute to anchorage-independent proliferation of SCLC cells. Inhibition of either Pyk2 or Src kinases by lentiviral RNAi or pharmacological inhibition with PP2, respectively, attenuated basal and neuropeptide-elicited survival and proliferation of SCLC cells in liquid culture and in soft agar. Thus, neuropeptides stimulate anchorage-independent survival and proliferation of SCLC cells via pathways involving Pyk2 and Src kinases. Therefore, Ca2+-induced Pyk2/Src complex formation may be a rewarding molecular target for novel therapeutic strategies in SCLC cells.

Modulation of Ca2+ signals by phosphatidylinositol‐linked novel D1 dopamine receptor in hippocampal neurons

Recent evidence indicates the existence of a putative novel phosphatidylinositol-linked D1 dopamine receptor in brain that mediates phosphatidylinositol hydrolysis via activation of phospholipase Cbeta. The present work was designed to characterize the Ca(2+) signals regulated by this phosphatidylinositol-linked D(1) dopamine receptor in primary cultures of hippocampal neurons. The results indicated that stimulation of phosphatidylinositol-linked D1 dopamine receptor by its newly identified selective agonist SKF83959 induced a long-lasting increase in basal [Ca(2+)](i) in a time- and dose-dependent manner. Stimulation was observable at 0.1 microm and reached the maximal effect at 30 microm. The [Ca(2+)](i) increase induced by 1 microm SKF83959 reached a plateau in 5 +/- 2.13 min, an average 96 +/- 5.6% increase over control. The sustained elevation of [Ca(2+)](i) was due to both intracellular calcium release and calcium influx. The initial component of Ca(2+) increase through release from intracellular stores was necessary for triggering the late component of Ca(2+) rise through influx. We further demonstrated that activation of phospholipase Cbeta/inositol triphosphate was responsible for SKF83959-induced Ca(2+) release from intracellular stores. Moreover, inhibition of voltage-operated calcium channel or NMDA receptor-gated calcium channel strongly attenuated SKF83959-induced Ca(2+) influx, indicating that both voltage-operated calcium channel and NMDA receptor contribute to phosphatidylinositol-linked D(1) receptor regulation of [Ca(2+)](i).

Constitutively active α subunits of Gq/11 and G12/13 families inhibit activation of the pro‐survival Akt signaling cascade

Accumulating evidence indicates that G protein signaling plays an active role in the regulation of cell survival. Our previous study demonstrated the regulatory effects of G(i/o) proteins in nerve growth factor-induced activation of pro-survival Akt kinase. In the present study we explored the role of various members of the G(s), G(q/11) and G(12/13) subfamilies in the regulation of Akt in cultured mammalian cells. In human embryonic kidney 293 cells transiently expressing constitutively active mutants of G alpha11, G alpha14, G alpha16, G alpha12, or G alpha13 (G alpha11QL, G alpha14QL, G alpha16QL, G alpha12QL and G alpha13QL, respectively), basal phosphorylation of Akt was attenuated, as revealed by western blotting analysis using a phosphospecific anti-Akt immunoglobulin. In contrast, basal Akt phosphorylation was unaffected by the overexpression of a constitutively active G alpha(s) mutant (G alpha(s)QL). Additional experiments showed that G alpha11QL, G alpha14QL, G alpha16QL, G alpha12QL and G alpha13QL, but not G alpha(s)QL, attenuated phosphorylation of the Akt-regulated translation regulator tuberin. Moreover, they were able to inhibit the epidermal growth factor-induced Akt activation and tuberin phosphorylation. The inhibitory mechanism of Gq family members was independent of phospholipase Cbeta activation and calcium signaling because G alpha11QL, G alpha14QL and G alpha16QL remained capable of inhibiting epidermal growth factor-induced Akt activation in cells pretreated with U73122 and the intracellular calcium chelator, BAPTA/AM. Finally, overexpression of the dominant negative mutant of RhoA blocked G alpha12QL- and G alpha13QL-mediated inhibition, suggesting that activated G alpha12 and G alpha13 inhibit Akt signaling via RhoA. Collectively, this study demonstrated the inhibitory effect of activated G alpha11, G alpha14, G alpha16, G alpha12 and G alpha13 on pro-survival Akt signaling.

Transmembrane signaling through phospholipase C‐β in the developing human prefrontal cortex

To investigate changes in muscarinic receptor-stimulated phospholipase C-beta (PLC-beta) activity during brain development, we examined the functional coupling of each of the three major protein components of the phosphoinositide system (M1, M3, and M5 muscarinic receptor subtypes; Gq/11 proteins; PLC-beta1-4 isoforms) in membrane preparations from post-mortem human prefrontal cerebral cortex collected at several stages of prenatal and postnatal development. In human prenatal brain membranes, PLC was found to be present and could be activated by calcium, but the ability of guanosine-5'-o-3 thiotriphosphate (GTPgammaS) or carbachol (in the presence of GTPgammaS) to modulate prenatal PLC-beta was significantly weaker than that associated with postnatal PLC-beta. Western blot analysis revealed that the levels of Galphaq/11 did not change significantly during development. In contrast, dramatically higher levels of expression of PLC-beta1-4 isoforms and of M1, M3, and M5 muscarinic receptors were detected in the child vs. the fetal brain, a finding that might underlie the observed increased activity of PLC. Thus, inositol phosphate production may be more efficiently regulated by altering the amount of effectors (PLC-beta1-4) and receptors (M1,3,5 subtypes) than by altering the level of Galphaq/11 subunits. These results demonstrate that different PLC isoforms are expressed in the prefrontal cortex of the developing human brain in an age-specific manner, suggesting specific roles not only in synaptic transmission but also in the differentiation and maturation of neurons in the developing brain.

Endogenous Regulator of G-Protein Signaling Proteins Regulate the Kinetics of Gαq/11-Mediated Modulation of Ion Channels in Central Nervous System Neurons

Slow synaptic potentials are generated when metabotropic G-protein-coupled receptors activate heterotrimeric G-proteins, which in turn modulate ion channels. Many neurons generate excitatory postsynaptic potentials mediated by G-proteins of the Galphaq/11 family, which in turn activate phospholipase C-beta. Accessory GTPase-activating proteins (GAPs) are thought to be required to accelerate GTP hydrolysis and rapidly turn off G-proteins, but the involvement of GAPs in neuronal Galphaq/11 signaling has not been examined. Here, we show that regulator of G-protein signaling (RGS) proteins provide necessary GAP activity at neuronal Galphaq/11 subunits. We reconstituted inhibition of native 2-pore domain potassium channels in cerebellar granule neurons by expressing chimeric Galpha subunits that are activated by Galphai/o-coupled receptors, thus bypassing endogenous Galphaq/11 subunits. RGS-insensitive variants of these chimeras mediated inhibition of potassium channels that developed and recovered more slowly than inhibition mediated by RGS-sensitive (wild-type) chimeras or native Galphaq/11 subunits. These changes were not accompanied by a change in agonist sensitivity, as might be expected if RGS proteins acted primarily as effector antagonists. The slowed recovery from potassium channel inhibition was largely reversed by an additional mutation that mimics the RGS-bound state. These results suggest that endogenous RGS proteins regulate the kinetics of rapid Galphaq/11-mediated signals in central nervous system neurons by providing GAP activity.

Pasteurella multocida Toxin-induced Activation of RhoA Is Mediated via Two Families of Gα Proteins, Gαq and Gα12/13

Pasteurella multocida toxin (PMT) is a potent mitogen, which is known to activate phospholipase Cbeta by stimulating the alpha-subunit of the heterotrimeric G protein G(q). PMT also activates RhoA and RhoA-dependent pathways. Using YM-254890, a specific inhibitor of G(q/11), we studied whether activation of RhoA involves G proteins other than G(q/11). YM-254890 inhibited PMT or muscarinic M3-receptor-mediated stimulation of phospholipase Cbeta at similar concentrations in HEK293m3 cells. In these cells, PMT-induced RhoA activation and enhancement of RhoA-dependent luciferase activity were partially inhibited by YM-254890. In Galpha(q/11)-deficient fibroblasts, PMT induced activation of RhoA, increase in RhoA-dependent luciferase activity, and increase in ERK phosphorylation. None of these effects were influenced by YM-254890. However, RhoA activation by PMT was inhibited by RGS2, RGS16, lscRGS, and dominant negative G(13)(GA), indicating involvement of Galpha(12/13) in the PMT effect on RhoA. In Galpha(12/13) gene-deficient cells, PMT-induced stimulation of RhoA, luciferase activity, and ERK phosphorylation were blocked by YM-254890, indicating the involvement of G(q). Infection with a virus harboring the gene of Galpha(13) reconstituted the increase in RhoA-dependent luciferase activity by PMT even in the presence of YM-254890. The data show that YM-254890 is able to block PMT activation of Galpha(q) and indicate that, in addition to Galpha(q), the Galpha(12/13) G proteins are targets of PMT.

Molecular Signaling Through G-Protein-Coupled Receptors and the Control of Intracellular Calcium in Myometrium

Cellular mechanisms regulating myometrial intracellular free calcium (Ca2+(i)) are addressed in this review, with emphasis on G-protein-coupled receptor pathways. An increase in myometrial Ca2+(i) results in phosphorylation of myosin light chain, an increase in myosin adenosine monophosphatase (ATPase) activity and contraction. Dephosphorylation of myosin light chain and a decline in Ca2+(i) are associated with relaxation. Increases in Ca2+(i) are controlled by multiple signaling pathways, including receptor-mediated activation of phospholipase Cbeta (PLCbeta), leading to release of Ca2+ from intracellular stores. Ca2+ also enters myometrial cells through plasma membrane Ca2+ channels. Conversely, adenosine triphosphate (ATP)-dependent Ca2+ pumps lower Ca2+(i) concentrations and potassium channels promote hyperpolarization that can decrease Ca2+ entry. Receptor-coupled pathways that promote uterine relaxation primarily involve activation of cyclic adenosine monophosphate (cAMP)- or cyclic guanosine monophosphate (cGMP)-stimulated protein kinases that phosphorylate proteins regulating Ca2+ homeostasis. cAMP has inhibitory effects on myometrial contractile activity, agonist-stimulated phosphatidylinositide turnover and increases in Ca2+(i). Some of these effects require association of protein kinase A (PKA) with a plasma membrane-associated A-kinase-anchoring-protein (AKAP). Near term in the rat, there is a decline in the plasma membrane localization of PKA associated with this anchoring protein. This correlates with changes in the regulation of signaling pathways controlling Ca2+(i). L-type voltage-operated Ca2+ entry is an important regulator of myometrial contraction. In addition, putative signal-regulated or capacitative Ca2+ channel proteins, TrpCs, are expressed in myometrium, and signal-regulated Ca2+ entry is observed in human myometrial cells. This Ca2+ entry mechanism may play a significant role in the control of myometrial Ca2+(i) dynamics and myometrial contraction. The regulation of myometrial Ca2+(i) is complex. Understanding the mechanisms involved may lead to design of tocolytics that target multiple pathways and achieve improved suppression of premature labor.

A Novel Gαq/11-selective Inhibitor

YM-254890, which was isolated from the culture broth of Chromobacterium sp., inhibits ADP-induced platelet aggregation and has antithrombotic and thrombolytic effects. YM-254890 blocks Galpha(q/11)-coupled ADP receptor P2Y1-mediated Ca(2+) mobilization. Here we report that YM-254890 is a selective Galpha(q/11) inhibitor. YM-254890 blocked Ca(2+) mobilization mediated by several Galpha(q/11)-coupled receptors but not by Galpha(i)- or Galpha(15)-coupled receptor, indicating that phospholipase Cbeta activation and subsequent signaling molecules are not the target of YM-254890. YM-254890 completely prevented the serum response factor (SRF)-mediated gene transcription induced by Galpha(q)R183C, which is constitutively active in a receptor-dependent manner because of its reduced k(cat) of GTP hydrolysis. Conversely, YM-254890 had only a modest effect on the SRF-mediated gene transcription by Galpha(q)Q209L, which is GTPase-deficient (activated) Galpha(q). These suggested that the acting point of YM-254890 is receptor-Galpha(q) interaction or the subsequent guanine nucleotide exchange step. The fact that YM-254890 (i) inhibited the SRF-mediated gene transcription by Galpha(qi5), which interacts with Galpha(i)-coupled receptor and possesses the effector function of Galpha(q), and (ii) had no effect on the K(d) value of high affinity [(3)H]2MeSADP binding to P2Y1, which reflects the agonist-receptor-Galpha ternary complex, suggested that receptor-Galpha(q/11) interaction is not the target of YM-254890. On the other hand, specific [(35)S]GTPgammaS binding to Galpha(q/11) stimulated by the M1 muscarinic acetylcholine receptor and P2Y1 were inhibited by YM-254890. These data indicate that YM-254890 blocks the exchange of GDP for GTP in Galpha(q/11) activation. This novel Galpha(q/11)-selective inhibitor is a promising and powerful tool for studying Galpha(q/11) protein activation, Galpha(q/11) -coupled receptor signaling, and Galpha(q/11)-mediated biological events.

Differential Sensitivity of Phosphatidylinositol 3-Kinase p110γ to Isoforms of G Protein βγ Dimers

The ability of G protein alpha and betagamma subunits to activate the p110gamma isoform of phosphatidylinositol 3-kinase (PtdIns 3-kinase) was examined using pure, recombinant G proteins and the p101/p110gamma form of PtdIns 3-kinase reconstituted into synthetic lipid vesicles. GTP-activated Gs, Gi, Gq, or Go alpha subunits were unable to activate PtdIns 3-kinase. Dimers containing Gbeta(1-4) complexed with gamma2-stimulated PtdIns 3-kinase activity about 26-fold with EC50 values ranging from 4 to 7 nm. Gbeta5gamma2 was not able to stimulate PtdIns 3-kinase despite producing a 10-fold activation of avian phospholipase Cbeta. A series of dimers with beta subunits containing point mutations in the amino acids that undergo a conformational change upon interaction of betagamma with phosducin (beta1H311Agamma2, beta1R314Agamma2, and beta1W332Agamma2) was tested, and only beta1W332Agamma2 inhibited the ability of the dimer to stimulate PtdIns 3-kinase. Dimers containing the beta1 subunit complexed with a panel of different Ggamma subunits displayed variation in their ability to stimulate PtdIns 3-kinase. The beta1gamma2, beta1gamma10, beta1gamma12, and beta1gamma13 dimers all activated PtdIns 3-kinase about 26-fold with 4-25 nm EC50 values. The beta1gamma11 dimer, which contains the farnesyl isoprenoid group and is highly expressed in tissues containing the p101/p110gamma form of PtdIns 3-kinase, was ineffective. The role of the prenyl group on the gamma subunit in determining the activation of PtdIns 3-kinase was examined using gamma subunits with altered CAAX boxes directing the addition of farnesyl to the gamma2 subunit and geranylgeranyl to the gamma1 and gamma11 subunits. Replacement of the geranylgeranyl group of the gamma2 subunit with farnesyl inhibited the activity of beta1gamma2 on PtdIns 3-kinase. Conversely, replacement of the farnesyl group on the gamma1 and gamma11 subunit with geranylgeranyl restored almost full activity. These findings suggest that all beta subunits, with the exception of beta5, interact equally well with PtdIns 3-kinase. In contrast, the composition of the gamma subunit and its prenyl group markedly affects the ability of the betagamma dimer to stimulate PtdIns 3-kinase.

Action of Pasteurella multocida Toxin Depends on the Helical Domain of Gαq

Pasteurella multocida produces a 146-kDa protein toxin (PMT), which activates multiple cellular signal transduction pathways, resulting in the activation of phospholipase Cbeta, RhoA, Jun kinase, and extracellular signal-regulated kinase. Using Galpha(q)/Galpha(11) -deficient cells, it was shown that the PMT-induced pleiotropic effects are mediated by Galpha(q) but not by the highly related Galpha(11) protein (Zywietz, A., Gohla, A., Schmelz, M., Schultz, G., and Offermanns, S. (2001) J. Biol. Chem. 276, 3840-3845). Here we studied the molecular basis of the unique specificity of PMT to distinguish between Galpha(q) and/or Galpha(11). Infection of Galpha(q) -deficient cells with retrovirus-encoding Galpha(q) caused reconstitution of PMT-induced activation of phospholipase Cbeta, whereas Galpha(11) -encoding virus did not reconstitute PMT activity. Chimeras between Galpha(q) and/or Galpha(11) revealed that a peptide region of Galpha(q), covering amino acid residues 105-113, is essential for the action of PMT to activate phospholipase Cbeta. Exchange of glutamine 105 or asparagine 109 of Galpha(11), which are located in the all-helical domain of the Galpha subunit, with the equally positioned histidines of Galpha(q), renders Galpha(11) capable of transmission PMT-induced phospholipase Cbeta activation. The data indicate that the all-helical domain of Galpha(q) is essential for the action of PMT and suggest an essential functional role of this domain in signal transduction via G(q) proteins.

Regulation of cyclin-dependent kinase 5 and calcium/calmodulin-dependent protein kinase II by phosphatidylinositol-linked dopamine receptor in rat brain.

A brain dopamine receptor that modulates phosphatidylinositol (PI) metabolism via the activation of phospholipase Cbeta (PLCbeta) has been described previously. The present study aims to define the downstream signaling cascade initiated by the PI-linked dopamine receptor. Incubation of rat brain frontal cortical slices with 6-chloro-7,8-dihydroxy-3-methyl-1-(3-methylphenyl)-2,3,4,5-tetrahydro-1H-3-benzazepine (SKF83959), a recently identified selective agonist of the PI-linked D1-like dopamine receptor, elicited transient time- and dose-dependent stimulations of cyclin-dependent kinase 5 (cdk5) and calcium/calmodulin-dependent protein kinase II (CaMK II) activities. The stimulation of these kinases is blocked by 20 microM R-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SCH23390) or the PLCbeta antagonist 1-[6-[[17beta-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U-73122) and is attenuated by the protein kinase inhibitor calphostin C or by the intracellular calcium chelator BAPTA, indicating that SKF83959 stimulates cdk5 and CaMK II activities via a PI-linked D1-like dopamine receptor, and PLCbeta and is dependent on protein kinase C and calcium. Although cdk5 and CaMK II are physically associated in native brain tissue, no change in this association was observed in response to SKF83959 stimulation or to the inhibition of either cdk5 by roscovitine or of CaMK by 2-[N-(2-hydroxyethyl)]-N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine) (KN93), suggesting that SKF83959-mediated stimulation of cdk5 or CaMK II is independent of the other kinase and that the association of the two kinases is not modulated by change of kinase activity. Moreover, we found that cdk5 phosphorylates dopamine and cAMP-regulated phosphoprotein at Thr75, whereas CaMK II is responsible for the activation of cAMP response element-binding protein in response to SKF83959 stimulation. The present data provide the first insight into the signaling mechanism for the PI-linked dopamine receptor. This information, in turn, may help in exploring the functional consequences of stimulation of this brain receptor.

Differential postmortem delay effect on agonist-mediated phospholipase Cbeta activity in human cortical crude and synaptosomal brain membranes.

The phosphoinositide signal transduction system, and particularly, phospholipase Cbeta isozymes, are relevant in the etiopathogeny of human neuropsychiatric pathologies such as depression. Stimulation of phospholipase Cbeta activity by muscarinic receptors and G proteins was determined in crude and synaptosomal membrane preparations from nine postmortem human frontal cortices (postmortem delay range 8 to 50 h). Thus, the phospholipase Cbeta activity was determined by measuring the hydrolysis of exogenous [3H]-phosphatidylinositol 4,5-bisphosphate. There was a postmortem delay-mediated decrease in the PIP2 hydrolysis irrespective of the membrane preparation used (P < 0.05). Moreover, there were statistically significant differences for exponential decay curve parameters (K factor and Span) of PLCbeta activity induced by agonist-mediated activation between crude and synaptosomal membrane preparations. These results show that the postsynaptic component of the PLCbeta activity is more sensible to the postmortem delay effect.

Differential chemokine activation of CC chemokine receptor 1‐regulated pathways: ligand selective activation of Gα 14‐coupled pathways

Chemokines regulate the chemotaxis, development, and differentiation of many cell types enabling the regulation of routine immunosurveillance and immunological adaptation. CC chemokine receptor 1 (CCR1) is the target of 11 chemokines. This promiscuity of receptor-ligand interactions and the potential for functional redundancy has led us to investigate the selective activation of CCR1-coupled pathways by known CCR1 agonists. Chemokines leukotactin-1, macrophage inflammatory protein (MIP)-1alpha, monocyte chemotactic peptide (MCP)-3, RANTES, and MIP-1delta all inhibited adenylyl cyclase activity in cells transiently transfected with CCR1. In contrast, only MIP-1delta was unable to signal via G14-, G16- or chimeric 16z44-coupled pathways. In a stable cell line expressing CCR1 and Galpha14, all of these five chemokines along with hemofiltrate CC chemokine (HCC)-1 and myeloid progenitor inhibitory factor (MPIF)-1 were able to stimulate G(i/o)-coupled pathways, but MIP-1delta, HCC-1 and MPIF-1 were unable to activate G14-mediated stimulation of phospholipase Cbeta activity. In addition, MIP-1delta was unable to promote the phosphorylation of extracellular signal-regulated kinase and c-Jun N-terminal kinase. This suggests that different chemokines are able to selectively activate CCR1-coupled pathways, probably because of different intrinsic ligand efficacies. CCR1 and Galpha14 or Galpha16 are co-expressed in several cell types and we hypothesize that selective activation of chemokine receptors provides a mechanism by which chemokines are able to fine-tune intracellular signaling pathways.

Differential Contribution of GTPase Activation and Effector Antagonism to the Inhibitory Effect of RGS Proteins on Gq-mediated Signaling in Vivo

RGS proteins act as negative regulators of G protein signaling by serving as GTPase-activating proteins (GAP) for alpha subunits of heterotrimeric G proteins (Galpha), thereby accelerating G protein inactivation. RGS proteins can also block Galpha-mediated signal production by competing with downstream effectors for Galpha binding. Little is known about the relative contribution of GAP and effector antagonism to the inhibitory effect of RGS proteins on G protein-mediated signaling. By comparing the inhibitory effect of RGS2, RGS3, RGS5, and RGS16 on Galpha(q)-mediated phospholipase Cbeta (PLCbeta) activation under conditions where GTPase activation is possible versus nonexistent, we demonstrate that members of the R4 RGS subfamily differ significantly in their dependence on GTPase acceleration. COS-7 cells were transiently transfected with either muscarinic M3 receptors, which couple to endogenous Gq protein and mediate a stimulatory effect of carbachol on PLCbeta, or constitutively active Galphaq*, which is inert to GTP hydrolysis and activates PLCbeta independent of receptor activation. In M3-expressing cells, all of the RGS proteins significantly blunted the efficacy and potency of carbachol. In contrast, Galphaq* -induced PLCbeta activation was inhibited by RGS2 and RGS3 but not RGS5 and RGS16. The observed differential effects were not due to changes in M3, Galphaq/Galphaq*, PLCbeta, or RGS expression, as shown by receptor binding assays and Western blots. We conclude that closely related R4 RGS family members differ in their mechanism of action. RGS5 and RGS16 appear to depend on G protein inactivation, whereas GAP-independent mechanisms (such as effector antagonism) are sufficient to mediate the inhibitory effect of RGS2 and RGS3.

Requirement of Gbetagamma and c-Src in D2 dopamine receptor-mediated nuclear factor-kappaB activation.

The D2 dopamine receptor (D2R) was examined for its ability to mediate nuclear factor-kappaB (NF-kappaB) activation through G proteins. Stimulation of D2R-transfected HeLa cells with its agonist quinpirole induced the expression of a NF-kappaB luciferase reporter and formation of NF-kappaB-DNA complex. This response was blocked by pertussis toxin, and by the Gbetagamma scavengers transducin and beta-adrenergic receptor kinase 1 carboxyl-terminal fragment. Unlike Gi-coupled chemoattractant receptors, D2R activated NF-kappaB without an increase in phospholipase C-beta activity, and the response was only slightly affected by the phosphoinositide 3-kinase inhibitor 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). In contrast, treatment with genistein and 4-amino-1-tert-butyl-3-(p-methylphenyl)pyrazolo[3,4-d] pyrimidine abolished the induced NF-kappaB activation, suggesting involvement of protein tyrosine kinases. Activation of D2R led to phosphorylation of c-Src at Tyr-418, and expression of a kinase-deficient c-Src inhibited D2R-mediated NF-kappaB activation. The D2R-mediated NF-kappaB activation was not dependent on epidermal growth factor (EGF) receptor transactivation since 4-(3'-chloroanilino)-6,7-dimethoxyquinazoline (AG1478), an EGF receptor-selective tyrphostin used at 1 microM, blocked EGF-induced NF-kappaB activation but not the quinpirole-induced response. In addition, the D2R-mediated NF-kappaB activation was enhanced by over-expression of beta-arrestin 1. These results suggest that D2R-mediated NF-kappaB activation requires Gbetagamma and c-Src, and possibly involves beta-arrestin 1.

Chemokines integrate JAK/STAT and G-protein pathways during chemotaxis and calcium flux responses.

The JAK/STAT (Janus kinase / signaling transducer and activator of transcription) signaling pathway is implicated in converting stationary epithelial cells to migratory cells. In mammals, migratory responses are activated by chemoattractant proteins, including chemokines. We found that by binding to seven-transmembrane G-protein-coupled receptors, chemokines activate the JAK/STAT pathway to trigger chemotactic responses. We show that chemokine-mediated JAK/STAT activation is critical for G-protein induction and for phospholipase C-beta dependent Ca(2+) flux; in addition, pharmacological inhibition of JAK or mutation of the JAK kinase domain causes defects in both responses. Furthermore, G alpha(i) association with the receptor is dependent on JAK activation, and the chemokine-mediated Ca(2+) flux that requires phospholipase C-beta activity takes place downstream of JAK kinases. The chemokines thus employ a mechanism that links heterologous signaling pathways--G proteins and tyrosine kinases--in a network that may be essential for mediating their pleiotropic responses.