Endogenous expression and protein kinase A-dependent phosphorylation of the guanine nucleotide exchange factor Ras-GRF1 in human embryonic kidney 293 cells

We have previously reported the Ras-dependent activation of the mitogen-activated protein kinases p44 and p42, also termed extracellular signal-regulated kinases (ERK)1 and 2 (ERK1/2), mediated through Gs-coupled serotonin receptors transiently expressed in human embryonic kidney (HEK) 293 cells. Whereas Gi- and Gq-coupled receptors have been shown to activate Ras through the guanine nucleotide exchange factor (GEF) called Ras-GRF1 (CDC25Mm) by binding of Ca2+/calmodulin to its N-terminal IQ domain, the mechanism of Ras activation through Gs-coupled receptors is not fully understood. We report the endogenous expression of Ras-GRF1 in HEK293 cells. Serotonin stimulation of HEK293 cells transiently expressing Gs-coupled 5-HT7 receptors induced protein kinase A-dependent phosphorylation of the endogenous human Ras-GRF1 on Ser927 and of transfected mouse Ras-GRF1 on Ser916. Ras-GRF1 overexpression increased basal and serotonin-stimulated ERK1/2 phosphorylation. Mutations of Ser916 inhibiting (Ser916Ala) or mimicking (Ser916Asp/Glu) phosphorylation did not alter these effects. However, the deletion of amino acids 1-225, including the Ca2+/calmodulin-binding IQ domain, from Ras-GRF1 reduced both basal and serotonin-stimulated ERK1/2 phosphorylation. Furthermore, serotonin treatment of HEK293 cells stably expressing 5-HT7 receptors increased [Ca2+]i, and the serotonin-induced ERK1/2 phosphorylation was Ca2+-dependent. Therefore, both cAMP and Ca2+ may contribute to the Ras-dependent ERK1/2 activation after 5-HT7 receptor stimulation, through activation of a guanine nucleotide exchange factor with activity towards Ras.     

α1-肾上腺素受体亚型介导细胞内游离Ca2+增高的信号转导途径

本文探讨了α１ａ,α１ｂ,α１ｄ三种亚型肾上腺素受体激动时细胞内Ｃａ６２＋浓度升高的信号转导途径。在稳定表达三亚型α１－ＡＲ的ＨＥＫ２９３细胞２系中,用ｆｕｒａ－２方法细胞内Ｃａ＾２＋信号强弱的变化。结果显示,百日咳毒素对去甲肾上腺素激动三亚型α１－ＡＲ而引起的「Ｃａ＾２＋」ｉ升高无影响,Ｕ－７３１２２和ＰＭＡ明显抑制「Ｃａ＾２＋」ｉ升高．     

Characterization of P2X3, P2Y1 and P2Y4 receptors in cultured HEK293-hP2X3 cells and their inhibition by ethanol and trichloroethanol

Membrane currents and changes in the intracellular Ca2+ concentration ([Ca2+]i) were measured in HEK293 cells transfected with the human P2X3 receptor (HEK293-hP2X3). RT-PCR and immunocytochemistry indicated the additional presence of endogenous P2Y1 and to some extent P2Y4 receptors. P2 receptor agonists induced inward currents in HEK293-hP2X3 cells with the rank order of potency alpha,beta-meATP approximately ATP > ADP-beta-S > UTP. A comparable rise in [Ca2+]i was observed after the slow superfusion of ATP, ADP-beta-S and UTP; alpha,beta-meATP was ineffective. These data, in conjunction with results obtained by using the P2 receptor antagonists TNP-ATP, PPADS and MRS2179 indicate that the current response to alpha,beta-meATP is due to P2X3 receptor activation, while the ATP-induced rise in [Ca2+]i is evoked by P2Y1 and P2Y4 receptor activation. TCE depressed the alpha,beta-meATP current in a manner compatible with a non-competitive antagonism. The ATP-induced increase of [Ca2+]i was much less sensitive to the inhibitory effect of TCE than the current response to alpha,beta-meATP. The present study indicates that in HEK293-hP2X3 cells, TCE, but not ethanol, potently inhibits ligand-gated P2X3 receptors and, in addition, moderately interferes with G protein-coupled P2Y1 and P2Y4 receptors. Such an effect may be relevant for the interruption of pain transmission in dorsal root ganglion neurons following ingestion of chloral hydrate or trichloroethylene.     

Phospholipase Cbeta2 binds to and inhibits phospholipase Cdelta1

Phospholipase Cbeta (PLCbeta) isoforms, which are under the control of Galphaq and Gbetagamma subunits, generate Ca2+ signals induced by a broad array of extracellular agonists, whereas PLCdelta isoforms depend on a rise in cytosolic Ca2+ for their activation. Here we find that PLCbeta2 binds strongly to PLCdelta1 and inhibits its catalytic activity in vitro and in living cells. In vitro, this PLC complex can be disrupted by increasing concentrations of free Gbetagamma subunits. Such competition has consequences for signaling, because in HEK293 cells PLCbeta2 suppresses elevated basal [Ca2+] and inositol phosphates levels and the sustained agonist-induced elevation of Ca2+ levels caused by PLCdelta1. Also, expression of both PLCs results in a synergistic release of [Ca2+] upon stimulation in A10 cells. These results support a model in which PLCbeta2 suppresses the basal catalytic activity of PLCdelta1, which is relieved by binding of Gbetagamma subunits to PLCbeta2 allowing for amplified calcium signals.     

Signaling pathways underlying muscarinic receptor-induced [Ca2+]i oscillations in HEK293 cells

We have investigated the signaling pathways underlying muscarinic receptor-induced calcium oscillations in human embryonic kidney (HEK293) cells. Activation of muscarinic receptors with a maximal concentration of carbachol (100 microm) induced a biphasic rise in cytoplasmic calcium ([Ca2+]i) comprised of release of Ca2+ from intracellular stores and influx of Ca2+ from the extracellular space. A lower concentration of carbachol (5 microm) induced repetitive [Ca2+]i spikes or oscillations, the continuation of which was dependent on extracellular Ca2+. The entry of Ca2+ with 100 microm carbachol and with the sarcoplasmic-endoplasmic reticulum calcium ATPase inhibitor, thapsigargin, was completely blocked by 1 microm Gd3+, as well as 30-100 microm concentrations of the membrane-permeant inositol 1,4,5-trisphosphate receptor inhibitor, 2-aminoethyoxydiphenyl borane (2-APB). Sensitivity to these inhibitors is indicative of capacitative calcium entry. Arachidonic acid, a candidate signal for Ca2+ entry associated with [Ca2+]i oscillations in HEK293 cells, induced entry that was inhibited only by much higher concentrations of Gd3+ and was unaffected by 100 microm 2-APB. Like arachidonic acid-induced entry, the entry associated with [Ca2)]i oscillations was insensitive to inhibition by Gd3+ but was completely blocked by 100 microm 2-APB. These findings indicate that the signaling pathway responsible for the Ca2+) entry driving [Ca2+]i oscillations in HEK293 cells is more complex than originally thought, and may involve neither capacitative calcium entry nor a role for PLA2 and arachidonic acid.     

Opposing effects of beta-arrestin1 and beta-arrestin2 on activation and degradation of Src induced by protease-activated receptor 1

Protease-activated receptor 1 (PAR1), a G protein-coupled receptor for thrombin, is irreversibly proteolytically activated. beta-Arrestin1 and beta-arrestin2 have been reported to have different effects on signal desensitization and transduction of PAR1. In this study, we investigated whether beta-arrestin1 and beta-arrestin2 regulate Src-dependent activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) induced by PAR1 in HEK 293 cells. Our results show that PAR1-mediated activation of Src and ERK1/2 in HEK 293 cells was increased with overexpression of beta-arrestin1 or depletion of beta-arrestin2. PAR1-mediated activation of Src and ERK1/2 in HEK 293 cells was decreased or eliminated with depletion of beta-arrestin1 or overexpression of beta-arrestin2. Furthermore, depletion of beta-arrestin2 blocked PAR1-induced degradation of Src. Thus, beta-arrestin1 and beta-arrestin2 have opposing roles in regulating the activation of Src induced by PAR1. beta-Arrestin2 also appears to promote PAR1-induced degradation of Src. This degradation of Src provides a possible mechanism for terminating PAR1 signaling.     

Extracellular calcium-sensing receptor transactivates the epidermal growth factor receptor by a triple-membrane-spanning signaling mechanism

Activation of the extracellular calcium-sensing receptor (CaR) stimulates mitogen-activated protein kinases to upregulate the synthesis and secretion of parathyroid hormone related peptide (PTHrP) from cells expressing the CaR heterologously or endogenously. The current experiments demonstrate that this occurs because CaR activation "transactivates" the EGF receptor (EGFR). Time dependent increases in tyrosine phosphorylation of the EGFR after addition of extracellular calcium ([Ca2+]o, 3 mM) occurred in stably CaR-transfected HEK293 cells but not in non-transfected HEK293 cells. AG1478, an EGFR kinase inhibitor, prevented the CaR-mediated increases of pERK and PTHrP release, while AG1296, a PDGFR kinase inhibitor, had no effect. Inhibitors of matrix metalloproteinase and heparin bound-EGF prevented the CaR-mediated increases of pERK and PTHrP, consistent with a "triple-membrane-spanning signaling" requirement for transactivation of the EGFR by the CaR. Proximal and distal signal transduction cascades activated by the CaR may reflect transactivation of the EGFR by the extracellular calcium-sensing receptor.     

Activation of muscarinic receptors reduces store-operated Ca2+ entry in HEK293 cells

In many cell types membrane receptors for hormones or neurotransmitters activate a signal transduction pathway which releases Ca2+ from intracellular Ca2+ stores by the second messenger inositol 1,4,5-trisphosphate. As a consequence store-operated Ca2+ entry (SOCE) becomes activated. In the present study we addressed the question if receptor/agonist binding can modulate Ca2+ entry by mechanisms different from the store-operated one. Therefore SOCE was examined in HEK293 cells microscopically with the fura-2 technique and with patch clamp. We found that maximally preactivated SOCE could, concentration dependently, be reduced up to 80% by the muscarinic agonist acetylcholine when the cytoplasmic Ca2+ concentration was used as a measure. Muscarinic receptors seem to mediate this decrease since atropine blocked the effect completely and cell types without muscarinic receptors (BHK21, CHO) did not show acetylcholine-induced decrease of Ca2+ entry. Moreover expression of muscarinic receptor subtypes M1 and M3 in BHK21 cells established the muscarinic decrease of SOCE. Electrical measurements revealed that the membrane potential of HEK293 cells did not show any response to ACh, excluding that changes of driving forces are responsible for the block of Ca2+ entry. In contrast the electrical current which is responsible for SOCE in HEK293 cells (Ca2+ release-activated Ca2+ current (I(CRAC)) was inhibited (maximally 55%) by 10 microM ACh. From these data we conclude that in HEK293 cells a muscarinic signal transduction pathway exists which decreases the cytoplasmic Ca2+ concentration by an inhibition of I(CRAC). This mechanism may serve as a modulator of Ca2+ entry preventing a Ca2+ overload of the cytoplasm after Ca2+ store depletion.     

Phosphatidylinositol 4,5-bisphosphate hydrolysis mediates histamine-induced KCNQ/M current inhibition

The M-type potassium channel, of which its molecular basis is constituted by KCNQ2-5 homo- or heteromultimers, plays a key role in regulating neuronal excitability and is modulated by many G protein-coupled receptors. In this study, we demonstrate that histamine inhibits KCNQ2/Q3 currents in human embryonic kidney (HEK)293 cells via phosphatidylinositol 4,5-bisphosphate (PIP(2)) hydrolysis mediated by stimulation of H(1) receptor and phospholipase C (PLC). Histamine inhibited KCNQ2/Q3 currents in HEK293 cells coexpressing H(1) receptor, and this effect was totally abolished by H(1) receptor antagonist mepyramine but not altered by H(2) receptor antagonist cimetidine. The inhibition of KCNQ currents was significantly attenuated by a PLC inhibitor U-73122 but not affected by depletion of internal Ca(2+) stores or intracellular Ca(2+) concentration ([Ca(2+)](i)) buffering via pipette dialyzing BAPTA. Moreover, histamine also concentration dependently inhibited M current in rat superior cervical ganglion (SCG) neurons by a similar mechanism. The inhibitory effect of histamine on KCNQ2/Q3 currents was entirely reversible but became irreversible when the resynthesis of PIP(2) was impaired with phosphatidylinsitol-4-kinase inhibitors. Histamine was capable of producing a reversible translocation of the PIP(2) fluorescence probe PLC(delta1)-PH-GFP from membrane to cytosol in HEK293 cells by activation of H(1) receptor and PLC. We concluded that the inhibition of KCNQ/M currents by histamine in HEK293 cells and SCG neurons is due to the consumption of membrane PIP(2) by PLC.     

Inhibition of polymorphonuclear leukocyte oxidative metabolism by exogenous phospholipase C

We studied the effects of exogenous, purified phospholipase C (PLC) on neutrophil oxidative metabolism, lysosomal enzyme release and aggregation. We found that PLC inhibited O2- and H2O2 generation and oxygen consumption, but did not alter glucose oxidation via the hexose monophosphate shunt. In contrast, we found a striking stimulation of aggregation and release of the lysosomal enzymes lysozyme and beta-glucuronidase. In experiments designed to further characterize the mechanism of the PLC effect on membrane activation we studied the effect of PLC on intracellular calcium concentration [Ca2+]i and found that PLC did not interfere with the fMLP-mediated rise in [Ca2+]i, suggesting that its inhibitory effect on the respiratory burst does not involve inhibition of early signal transduction events. In addition, we found that PLC alone results in mobilization of intracellular Ca2+ stores, consistent with its stimulatory effect on aggregation and lysosomal enzyme release.     

Involvement of protein kinase C-alpha and -epsilon in extracellular Ca(2+) signalling mediated by the calcium sensing receptor

The sensing of extracellular Ca(2+) concentration ([Ca(2+)](o)) and modulation of cellular processes associated with acute or sustained changes in [Ca(2+)](o) are cell-type specific and mediated by the calcium sensing receptor (CaR). [Ca(2+)](o) signalling requires protein kinase C (PKC), but the identity and role of PKC isoforms in CaR-mediated responses remain unclear. Here we show that high [Ca(2+)](o) activated PKC-alpha and PKC- in parathyroid cells and in human embryonic kidney (HEK293) cells overexpressing the CaR (HEK-CaR) and that this response correlated with the CaR-dependent activation of mitogen-activated protein kinases ERK1/2. Activation of ERK1/2 by acute high [Ca(2+)](o) required influx of Ca(2+)through Ni(2+)-sensitive Ca(2+)channels and phosphatidylinositol-dependent phospholipase C-beta activity. Inhibition of PKC by co-expression of dominant-negative (DN) mutants of PKC-alpha or - with the CaR attenuated sustained ERK1/2 activation. Overexpression of a PKC phosphorylation site (T888A) mutant CaR in HEK293 cells showed that this site was important for ERK1/2 activation at high [Ca(2+)](o). Activation of ERK1/2 by high [Ca(2+)](o) was not necessary for the [Ca(2+)](o)-regulated secretion of parathyroid hormone (PTH) in dispersed bovine parathyroid cells. These data suggest that the CaR-mediated [Ca(2+)](o) signal leading to regulated PTH secretion that requires diacylglycerol-responsive PKC isoforms is not mediated via the ERK pathway.     

Direct interaction and functional coupling between metabotropic glutamate receptor subtype 1 and voltage-sensitive Cav2.1 Ca2+ channel

Intracellular Ca2+ concentrations ([Ca2+]i) are regulated in a spatiotemporal manner via both entry of extracellular Ca2+ and mobilization of Ca2+ from intracellular stores. Metabotropic glutamate receptor subtype 1 (mGluR1) is a G protein-coupled receptor that stimulates the inositol 1,4,5-trisphosphate-Ca2+ signaling cascade, whereas Cav2.1 is a pore-forming channel protein of P/Q-type voltage-sensitive Ca2+ channels. In this investigation, we showed that mGluR1 and Cav2.1 are colocalized at dendrites of cerebellar Purkinje neurons and form the heteromeric assembly in both the brain and heterologously expressing COS-7 cells. This assembly occurs through the direct interaction between their carboxyl-terminal intracellular domains. Calcium imaging and whole-cell recording showed that mGluR1 inhibits Cav2.1-mediated [Ca2+]i increases and Ba2+ currents in HEK 293 cells expressing Cav2.1 with auxiliary alpha2/delta and beta1 subunits, respectively. This inhibition occurred in a ligand-independent manner and was enhanced by pre-activation of mGluR1 in a ligand-dependent manner. In contrast, simultaneous stimulation of mGluR1 and Cav2.1 induced large [Ca2+]i increases. Furthermore, the temporally regulated inhibition and stimulation of [Ca2+]i increases by mGluR1 and Cav2.1 were observed at dendrites but not soma of cultured Purkinje neurons. These data suggest that the assembly of mGluR1 and Cav2.1 provides the mechanism that ensures spatiotemporal regulation of [Ca2+]i in glutamatergic neurotransmission.     

Endothelin A and endothelin B receptors differ in their ability to stimulate ERK1/2 activation

Endothelin-1 (ET-1) acts on two different G protein-coupled receptors, namely the endothelin A (ET(A)) and the endothelin B (ET(B)) receptors. Both receptor subtypes show differences in their tissue expression and signal transduction. In the present study, we compared the ability of ET(A) and ET(B) receptors to stimulate extracellular signal-regulated kinase 1/2 (ERK1/2). In addition, we analyzed the role of the extracellular N terminus for ERK1/2 activation, because the ET(B) receptor undergoes an agonist-dependent N-terminal proteolysis. ET-1 stimulation of HEK293 cells stably expressing the ET(A) receptor induced a monophasic, but sustained ERK1/2 activation, whereas the ET(B) receptor showed a biphasic ERK1/2 activation. A truncated mutant ET(B) receptor, lacking the proteolytically cleaved N terminus (delta2-64 ET(B)) revealed only a monophasic and transient ERK1/2 activation. Treatment of HEK293 delta2-64 ET(B) cell clones with ET-1 and a synthetic NT27-64 peptide, corresponding to the N-terminally cleaved fragment of the ET(B) receptor and ET-1, did not restore the biphasic activation of ERK1/2. A chimeric ET(B) receptor in which the N terminus was replaced by the N terminus of the ET(A) receptor elicited biphasic ERK1/2 activation. The presented data suggest that an intact N terminus of the ET(B) receptor is necessary for the second phase of ERK1/2 activation. However, it appears that the length of the N terminus rather than a specific sequence motif is required for biphasic ERK1/2 activation.     

Apo-ghrelin receptor forms heteromers with DRD2 in hypothalamic neurons and is essential for anorexigenic effects of DRD2 agonism

We identified subsets of neurons in the brain that coexpress the dopamine receptor subtype-2 (DRD2) and the ghrelin receptor (GHSR1a). Combination of FRET confocal microscopy and Tr-FRET established the presence of GHSR1a:DRD2 heteromers in hypothalamic neurons. To interrogate function, mice were treated with the selective DRD2 agonist cabergoline, which produced anorexia in wild-type and ghrelin?/? mice; intriguingly, ghsr?/? mice were refractory illustrating dependence on GHSR1a, but not ghrelin. Elucidation of mechanism showed that formation of GHSR1a:DRD2 heteromers allosterically modifies canonical DRD2 dopamine signaling resulting in Gβγ subunit-dependent mobilization of [Ca2?](i) independent of GHSR1a basal activity. By targeting the interaction between GHSR1a and DRD2 in wild-type mice with a highly selective GHSR1a antagonist (JMV2959) cabergoline-induced anorexia was blocked. Inhibiting dopamine signaling in subsets of neurons with a GHSR1a antagonist has profound therapeutic implications by providing enhanced selectivity because neurons expressing DRD2 alone would be unaffected.     

Bombyx adipokinetic hormone receptor activates extracellular signal-regulated kinase 1 and 2 via G protein-dependent PKA and PKC but β-arrestin-independent pathways

Neuropeptides of the adipokinetic hormone (AKH) family are among the best studied hormone peptides. They play important roles in insect hemolymph sugar homeostasis, larval lipolysis, and storage-fat mobilization. Mechanistic investigations have shown that, upon AKH stimulation, adipokinetic hormone receptor (AKHR) couples to a Gs protein and enhances adenylate cyclase activity, leading to intracellular cAMP accumulation. However, the underlying molecular mechanism by which this signaling pathway connects to extracellular signal-regulated kinase 1/2 (ERK1/2) remains to be elucidated. Using HEK293 cells stably or transiently expressing AKHR, we demonstrated that activation of AKHR elicited transient phosphorylation of ERK1/2. Our investigation indicated that AKHR-mediated activation of ERK1/2 was significantly inhibited by H-89 (protein kinase A inhibitor), Go6983, and GF109203X (protein kinase C inhibitors) but not by U73122 (PLC inhibitor) or FIPI (PLD inhibitor). Moreover, AKHR-induced ERK1/2 phosphorylation was blocked by the calcium chelators EGTA and BAPTA-AM. Furthermore, ERK1/2 activation in both transiently and stably AKHR-expressing HEK293 cells was found to be sensitive to pretreatment of pertussis toxin, whereas AKHR-mediated ERK1/2 activation was insensitive to siRNA-induced knockdown of β-arrestins and to pretreatment of inhibitors of EGFR, Src, and PI3K. On the basis of our data, we propose that activated AKHR signals to ERK1/2 primarily via PKA- and calcium-involved PKC-dependent pathways. Our current study provides the first in-depth study defining the mechanisms of AKH-mediated ERK activation through the Bombyx AKHR.     

Ca2+ signaling in prothoracicotropic hormone-stimulated prothoracic gland cells of Manduca sexta: evidence for mobilization and entry mechanisms

Prothoracicotropic hormone (PTTH) stimulates ecdysteroidogenesis in lepidopteran prothoracic glands (PGs), thus indirectly controlling molting and metamorphosis. PTTH triggers a signal transduction cascade in PGs that involves an early influx of Ca2+. Although the importance of Ca2+ has been long known, the mechanism(s) of PTTH-stimulated changes in cytoplasmic Ca2+ [Ca2+]i are not yet well understood. PGs from the fifth instar of Manduca sexta were exposed to PTTH in vitro. The resultant changes in [Ca2+]i were measured using ratiometric analysis of a fura-2 fluorescence signal in the presence and absence of inhibitors of specific cellular signaling mechanisms. The phospholipase C (PLC) inhibitor U-73122 nearly abolished the PTTH-stimulated increase in [Ca2+]i, as well as PTTH-stimulated ecdysteroidogenesis and extracellular-signal regulated kinase phosphorylation, thus establishing a role for PLC and implicating inositol trisphosphate (IP3) in PTTH signal transduction. Two antagonists of the IP3 receptor, 2-APB and TMB-8, likewise blocked the [Ca2+]i response by a mean of 92%. We describe for the first time the presence of Ca2+ oscillations in PTTH-stimulated cells in Ca2+-free medium. External Ca2+ entered PG cells via at least two routes: store-operated (capacitative) Ca2+ entry channels and L-type voltage-gated Ca2+ channels. We propose that PTTH initiates a transductory cascade typical of many G-protein coupled receptors, involving both Ca2+ mobilization and entry pathways.     

The T-cell antigen receptor regulates sustained increases in cytoplasmic free Ca2+ through extracellular Ca2+ influx and ongoing intracellular Ca2+ mobilization.

Signal transduction by the T-cell antigen receptor involves the turnover of polyphosphoinositides and an increase in the concentration of cytoplasmic free Ca2+ ([Ca2+]i). This increase in [Ca2+]i is due initially to the release of Ca2+ from intracellular stores, but is sustained by the influx of extracellular Ca2+. To examine the regulation of sustained antigen-receptor-mediated increases in [Ca2+]i, we studied the relationships between extracellular Ca2+ influx, the mobilization of Ca2+ from intracellular stores, and the contents of inositol polyphosphates after stimulation of the antigen receptor on a human T-cell line, Jurkat. We demonstrate that sustained antigen-receptor-mediated increases in [Ca2+]i are associated with ongoing depletion of intracellular Ca2+ stores. When antigen-receptor-ligand interactions are disrupted, [Ca2+]i and inositol 1,4,5-trisphosphate return to basal values over 3 min. Under these conditions, intracellular Ca2+ stores are repleted if extracellular Ca2+ is present. There is a tight temporal relationship between the fall in [Ca2+]i, the return of inositol 1,4,5-trisphosphate to basal values, and the repletion of intracellular Ca2+ stores. Reversal of the increase in [Ca2+]i preceeds any fall in inositol tetrakisphosphate by 2 min. These studies suggest that sustained antigen-receptor-induced increases in [Ca2+]i, although dependent on extracellular Ca2+ influx, are also regulated by ongoing inositol 1,4,5-trisphosphate-mediated intracellular Ca2+ mobilization. In addition, an elevated concentration of inositol tetrakisphosphate in itself is insufficient to sustain an increase in [Ca2+]i within Jurkat cells.     

Role of sphingosine kinase in Ca(2+) signalling by epidermal growth factor receptor

Contribution of sphingosine kinase (SPK)-catalyzed production of sphingosine-1-phosphate (SPP), in comparison to phospholipase C (PLC), to Ca(2+) signalling by epidermal growth factor (EGF) was studied in two HEK-293 cell clones (HEK2 and HEK3), expressing functional EGF receptors and exhibiting release of stored Ca(2+) by intracellular SPP. In HEK3 cells, EGF increased [Ca(2+)](i) and stimulated both, SPK and PLC. [Ca(2+)](i) increase, but not PLC stimulation, was strongly reduced by SPK inhibition. In HEK2 cells, EGF similarly stimulated PLC, but did not increase [Ca(2+)](i) or stimulate SPK, suggesting that intracellular SPP production plays a major role for Ca(2+) signalling by EGF in HEK-293 cells.     

Plectin regulates the signaling and trafficking of the HIV-1 co-receptor CXCR4 and plays a role in HIV-1 infection

The CXC chemokine CXCL12 and its cognate receptor CXCR4 play an important role in inflammation, human immunodeficiency virus (HIV) infection and cancer metastasis. The signal transduction and intracellular trafficking of CXCR4 are involved in these functions, but the underlying mechanisms remain incompletely understood. In the present study, we demonstrated that the CXCR4 formed a complex with the cytolinker protein plectin in a ligand-dependent manner in HEK293 cells stably expressing CXCR4. The glutathione-S-transferase (GST)-CXCR4 C-terminal fusion proteins co-precipitated with the full-length and the N-terminal fragments of plectin isoform 1 but not with the N-terminal deletion mutants of plectin isoform 1, thereby suggesting an interaction between the N-terminus of plectin and the C-terminus of CXCR4. This interaction was confirmed by confocal microscopic reconstructions showing co-distribution of these two proteins in the internal vesicles after ligand-induced internalization of CXCR4 in HEK293 cells stably expressing CXCR4. Knockdown of plectin with RNA interference (RNAi) significantly inhibited ligand-dependent CXCR4 internalization and attenuated CXCR4-mediated intracellular calcium mobilization and activation of extracellular signal regulated kinase 1/2 (ERK1/2). CXCL12-induced chemotaxis of HEK293 cells stably expressing CXCR4 and of Jurkat T cells was inhibited by the plectin RNAi. Moreover, CXCR4 tropic HIV-1 infection in MAGI (HeLa-CD4-LTR-Gal) cells was inhibited by the RNAi of plectin. Thus, plectin appears to interact with CXCR4 and plays an important role in CXCR4 signaling and trafficking and HIV-1 infection.     

Molecular and functional characterization of adipokinetic hormone receptor and its peptide ligands in Bombyx mori

Neuropeptides of the adipokinetic hormone (AKH) family are among the best studied hormone peptides, but its signaling pathways remain to be elucidated. In this study, we molecularly characterized the signaling of Bombyx AKH receptor (AKHR) and its peptide ligands in HEK293 cells. In HEK293 cells stably expressing AKHR, AKH1 stimulation not only led to a ligand concentration dependent mobilization of intracellular Ca²⁺ and cAMP accumulation, but also elicited transient activation of extracellular signal-regulated kinase 1/2 (ERK1/2) pathway. We observed that AKH receptor was rapidly internalized after AKH1 stimulation. We further demonstrated that AKH2 exhibited high activities in cAMP accumulation and ERK1/2 activation on AKHR comparable to AKH1, whereas AKH3 was much less effective.