Translocation of Activator of G-protein Signaling 3 to the Golgi Apparatus in Response to Receptor Activation and Its Effect on the trans-Golgi Network

Group II activators of G-protein signaling play diverse functional roles through their interaction with Gα_i, Gα_t, and Gα_o via a G-protein regulatory (GPR) motif that serves as a docking site for Gα-GDP. We recently reported the regulation of the AGS3-Gα_i signaling module by a cell surface, seven-transmembrane receptor. Upon receptor activation, AGS3 reversibly dissociates from the cell cortex, suggesting that it may function as a signal transducer with downstream signaling implications, and this question is addressed in the current report. In HEK-293 and COS-7 cells expressing the α_2A/D-AR and Gα_i3, receptor activation resulted in the translocation of endogenous AGS3 and AGS3-GFP from the cell cortex to a juxtanuclear region, where it co-localized with markers of the Golgi apparatus (GA). The agonist-induced translocation of AGS3 was reversed by the α₂-AR antagonist rauwolscine. The TPR domain of AGS3 was required for agonist-induced translocation of AGS3 from the cell cortex to the GA, and the translocation was blocked by pertussis toxin pretreatment or by the phospholipase Cβ inhibitor {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122. Agonist-induced translocation of AGS3 to the GA altered the functional organization and protein sorting at the trans-Golgi network. The regulated movement of AGS3 between the cell cortex and the GA offers unexpected mechanisms for modulating protein secretion and/or endosome recycling events at the trans-Golgi network.     

M3 Muscarinic Receptor Interaction with Phospholipase C β3 Determines Its Signaling Efficiency

Phospholipase Cβ (PLCβ) enzymes are activated by G protein-coupled receptors through receptor-catalyzed guanine nucleotide exchange on Gαβγ heterotrimers containing G_q family G proteins. Here we report evidence for a direct interaction between M3 muscarinic receptor (M3R) and PLCβ3. Both expressed and endogenous M3R interacted with PLCβ in coimmunoprecipitation experiments. Stimulation of M3R with carbachol significantly increased this association. Expression of M3R in CHO cells promoted plasma membrane localization of YFP-PLCβ3. Deletion of the PLCβ3 C terminus or deletion of the PLCβ3 PDZ ligand inhibited coimmunoprecipitation with M3R and M3R-dependent PLCβ3 plasma membrane localization. Purified PLCβ3 bound directly to glutathione S-transferase (GST)-fused M3R intracellular loops 2 and 3 (M3Ri2 and M3Ri3) as well as M3R C terminus (M3R/H8-CT). PLCβ3 binding to M3Ri3 was inhibited when the PDZ ligand was removed. In assays using reconstituted purified components in vitro, M3Ri2, M3Ri3, and M3R/H8-CT potentiated Gα_q-dependent but not Gβγ-dependent PLCβ3 activation. Disruption of key residues in M3Ri3N and of the PDZ ligand in PLCβ3 inhibited M3Ri3-mediated potentiation. We propose that the M3 muscarinic receptor maximizes the efficiency of PLCβ3 signaling beyond its canonical role as a guanine nucleotide exchange factor for Gα.     

A naturally occurring membrane-anchored Gαs variant,XLαs,activates phospholipase Cβ4

Extra-large stimulatory Gα (XLα_s) is a large variant of G protein α_s subunit (Gα_s) that uses an alternative promoter and thus differs from Gα_s at the first exon. XLα_s activation by G protein–coupled receptors mediates cAMP generation, similarly to Gα_s; however, Gα_s and XLα_s have been shown to have distinct cellular and physiological functions. For example, previous work suggests that XLα_s can stimulate inositol phosphate production in renal proximal tubules and thereby regulate serum phosphate levels. In this study, we show that XLα_s directly and specifically stimulates a specific isoform of phospholipase Cβ (PLCβ), PLCβ4, both in transfected cells and with purified protein components. We demonstrate that neither the ability of XLα_s to activate cAMP generation nor the canonical G protein switch II regions are required for PLCβ stimulation. Furthermore, this activation is nucleotide independent but is inhibited by Gβγ, suggesting a mechanism of activation that relies on Gβγ subunit dissociation. Surprisingly, our results indicate that enhanced membrane targeting of XLα_s relative to Gα_s confers the ability to activate PLCβ4. We also show that PLCβ4 is required for isoproterenol-induced inositol phosphate accumulation in osteocyte-like Ocy454 cells. Taken together, we demonstrate a novel mechanism for activation of phosphoinositide turnover downstream of G_s-coupled receptors that may have a critical role in endocrine physiology.     

WDR26 Functions as a Scaffolding Protein to Promote Gβγ-mediated Phospholipase C β2 (PLCβ2) Activation in Leukocytes

We have recently identified WDR26 as a novel WD40 repeat protein that binds Gβγ and promotes Gβγ signaling during leukocyte migration. Here, we have determined the mechanism by which WDR26 enhances Gβγ-mediated phospholipase C β2 (PLCβ2) activation in leukocytes. We show that WDR26 not only directly bound Gβγ but also PLCβ2. The binding sites of WDR26 and PLCβ2 on Gβ₁γ₂ were overlapping but not identical. WDR26 used the same domains for binding Gβγ and PLCβ but still formed a signaling complex with Gβγ and PLCβ2 probably due to the fact that WDR26 formed a higher order oligomer through its Lis homology and C-terminal to LisH (LisH-CTLH) and WD40 domains. Additional studies indicated that the formation of higher order oligomers was required for WDR26 to promote PLCβ2 interaction with and activation by Gβγ. Moreover, WDR26 was required for PLCβ2 translocation from the cytosol to the membrane in polarized leukocytes, and the translocation of PLCβ2 was sufficient to cause partial activation of PLCβ2. Collectively, our data indicate that WDR26 functions as a scaffolding protein to promote PLCβ2 membrane translocation and interaction with Gβγ, thereby enhancing PLCβ2 activation in leukocytes. These findings have identified a novel mechanism of regulating Gβγ signaling through a scaffolding protein.     

Gq-coupled Purinergic Receptors Inhibit Insulin-like Growth Factor-I/Phosphoinositide 3-Kinase Pathway-dependent Keratinocyte Migration

Insulin-like growth factor-I (IGF-I) activation of phosphoinositol 3-kinase (PI3K) is an essential pathway for keratinocyte migration that is required for epidermis wound healing. We have previously reported that activation of Gα_(q/11)-coupled-P2Y₂ purinergic receptors by extracellular nucleotides delays keratinocyte wound closure. Here, we report that activation of P2Y₂ receptors by extracellular UTP inhibits the IGF-I–induced p110α-PI3K activation. Using siRNA and pharmacological inhibitors, we demonstrate that the UTP antagonistic effects on PI3K pathway are mediated by Gα_(q/11)—and not G_(i/o)—independently of phospholipase Cβ. Purinergic signaling does not affect the formation of the IGF-I receptor/insulin receptor substrate-I/p85 complex, but blocks the activity of a membrane-targeted active p110α mutant, indicating that UTP acts downstream of PI3K membrane recruitment. UTP was also found to efficiently attenuate, within few minutes, the IGF-I–induced PI3K-controlled translocation of the actin-nucleating protein cortactin to the plasma membrane. This supports the UTP ability to alter later migratory events. Indeed, UTP inhibits keratinocyte spreading and migration promoted by either IGF-I or a membrane-targeted active p110α mutant, in a Gα(q/11)-dependent manner both. These findings provide new insight into the signaling cross-talk between receptor tyrosine kinase and Gα_(q/11)-coupled receptors, which mediate opposite effects on p110α-PI3K activity and keratinocyte migration.     

The Small G Protein Rac1 Activates Phospholipase Cδ1 through Phospholipase Cβ2

Rac1, which is associated with cytoskeletal pathways, can activate phospholipase Cβ2 (PLCβ2) to increase intracellular Ca²⁺ levels. This increased Ca²⁺ can in turn activate the very robust PLCδ1 to synergize Ca²⁺ signals. We have previously found that PLCβ2 will bind to and inhibit PLCδ1 in solution by an unknown mechanism and that PLCβ2·PLCδ1 complexes can be disrupted by Gβγ subunits. However, because the major populations of PLCβ2 and PLCδ1 are cytosolic, their regulation by Gβγ subunits is not clear. Here, we have found that the pleckstrin homology (PH) domains of PLCβ2 and PLCβ3 are the regions that result in PLCδ1 binding and inhibition. In cells, PLCβ2·PLCδ1 form complexes as seen by Förster resonance energy transfer and co-immunoprecipitation, and microinjection of PHβ2 dissociates the complex. Using PHβ2 as a tool to assess the contribution of PLCβ inhibition of PLCδ1 to Ca²⁺ release, we found that, although PHβ2 only results in a 25% inhibition of PLCδ1 in solution, in cells the presence of PHβ2 appears to eliminates Ca²⁺ release suggesting a large threshold effect. We found that the small plasma membrane population of PLCβ2·PLCδ1 is disrupted by activation of heterotrimeric G proteins, and that the major cytosolic population of the complexes are disrupted by Rac1 activation. Thus, the activity of PLCδ1 is controlled by the amount of bound PLCβ2 that changes with displacement of the enzyme by heterotrimeric or small G proteins. Through PLCβ2, PLCδ1 activation is linked to surface receptors as well as signals that mediate cytoskeletal pathways.     

Opposite Effects of Neuropeptide FF on Central Antinociception Induced by Endomorphin-1 and Endomorphin-2 in Mice

Neuropeptide FF (NPFF) is known to be an endogenous opioid-modulating peptide. Nevertheless, very few researches focused on the interaction between NPFF and endogenous opioid peptides. In the present study, we have investigated the effects of NPFF system on the supraspinal antinociceptive effects induced by the endogenous µ-opioid receptor agonists, endomorphin-1 (EM-1) and endomorphin-2 (EM-2). In the mouse tail-flick assay, intracerebroventricular injection of EM-1 induced antinociception via µ-opioid receptor while the antinociception of intracerebroventricular injected EM-2 was mediated by both µ- and κ-opioid receptors. In addition, central administration of NPFF significantly reduced EM-1-induced central antinociception, but enhanced EM-2-induced central antinociception. The results using the selective NPFF₁ and NPFF₂ receptor agonists indicated that the EM-1-modulating action of NPFF was mainly mediated by NPFF₂ receptor, while NPFF potentiated EM-2-induecd antinociception via both NPFF₁ and NPFF₂ receptors. To further investigate the roles of µ- and κ-opioid systems in the opposite effects of NPFF on central antinociception of endomprphins, the µ- and κ-opioid receptors selective agonists DAMGO and {"type":"entrez-nucleotide","attrs":{"text":"U69593","term_id":"4205069","term_text":"U69593"}}U69593, respectively, were used. Our results showed that NPFF could reduce the central antinociception of DAMGO via NPFF₂ receptor and enhance the central antinociception of {"type":"entrez-nucleotide","attrs":{"text":"U69593","term_id":"4205069","term_text":"U69593"}}U69593 via both NPFF₁ and NPFF₂ receptors. Taken together, our data demonstrate that NPFF exerts opposite effects on central antinociception of endomorphins and provide the first evidence that NPFF potentiate antinociception of EM-2, which might result from the interaction between NPFF and κ-opioid systems.     

Two Nucleotide Transport Proteins in Chlamydia trachomatis, One for Net Nucleoside Triphosphate Uptake and the Other for Transport of Energy

The genome of Chlamydia trachomatis, one of the most prominent human pathogens, contains two structural genes coding for proteins, herein called Npt1_Ct and Npt2_Ct (nucleoside phosphate transporters 1 and 2 of C. trachomatis), exhibiting 68 and 61% similarity, respectively, to the ATP/ADP transporter from the intracellular bacterium Rickettsia prowazekii at the deduced amino acid level. Hydropathy analysis and sequence alignments suggested that both proteins have 12 transmembrane domains. The putative transporters were expressed as histidine-tagged proteins in Escherichia coli to study their biochemical properties. His₁₀-Npt1_Ct catalyzed ATP and ADP transport in an exchange mode. The apparent K_m values were 48 (ATP) and 39 (ADP) μM. ATP and ADP transport was specific since AMP, GTP, CTP, UTP, dATP, dCTP, dGTP, and dTTP did not inhibit uptake. In contrast, His₁₀-Npt2_Ct transported all four ribonucleoside triphosphates with apparent K_m values of 31 μM (GTP), 302 μM (UTP), 528 μM (CTP), and 1,158 μM (ATP). Ribonucleoside di- and monophosphates and deoxyribonucleotides were not substrates. The protonophore m-chlorocarbonylcyanide phenylhydrazone abolished uptake of all nucleoside triphosphates by Npt2_Ct. This observation indicated that His₁₀-Npt2_Ct acts as a nucleosidetriphosphate/H⁺ symporter energized by the proton motive force across the Escherichia coli cytoplasmic membrane. We conclude that Npt1_Ct provides chlamydiae with energy whereas Npt2_Ct catalyzes the net uptake of ribonucleoside triphosphates required for anabolic reactions.     

Regulation of the AGS3·Gαi Signaling Complex by a Seven-transmembrane Span Receptor

G-protein signaling modulators (GPSM) play diverse functional roles through their interaction with G-protein subunits. AGS3 (GPSM1) contains four G-protein regulatory motifs (GPR) that directly bind Gα_i free of Gβγ providing an unusual scaffold for the “G-switch” and signaling complexes, but the mechanism by which signals track into this scaffold are not well understood. We report the regulation of the AGS3·Gα_i signaling module by a cell surface, seven-transmembrane receptor. AGS3 and Gα_i1 tagged with Renilla luciferase or yellow fluorescent protein expressed in mammalian cells exhibited saturable, specific bioluminescence resonance energy transfer indicating complex formation in the cell. Activation of α₂-adrenergic receptors or μ-opioid receptors reduced AGS3-RLuc·Gα_i1-YFP energy transfer by over 30%. The agonist-mediated effects were inhibited by pertussis toxin and co-expression of RGS4, but were not altered by Gβγ sequestration with the carboxyl terminus of GRK2. Gα_i-dependent and agonist-sensitive bioluminescence resonance energy transfer was also observed between AGS3 and cell-surface receptors typically coupled to Gα_i and/or Gα_o indicating that AGS3 is part of a larger signaling complex. Upon receptor activation, AGS3 reversibly dissociates from this complex at the cell cortex. Receptor coupling to both Gαβγ and GPR-Gα_i offer additional flexibility for systems to respond and adapt to challenges and orchestrate complex behaviors.     

Leucine Facilitates Insulin Signaling through a Gαi Protein-dependent Signaling Pathway in Hepatocytes

In this study, we addressed the direct effect of leucine on insulin signaling. In investigating the associated mechanisms, we found that leucine itself does not activate the classical Akt- or ERK1/2 MAP kinase-dependent signaling pathways but can facilitate the insulin-induced phosphorylations of Akt⁴⁷³ and ERK1/2 in a time- and dose-dependent manner in cultured hepatocytes. The leucine-facilitated insulin-induced phosphorylation of Akt at residue 473 was not affected by knocking down the key component of mTORC1 or -2 complexes but was blocked by inhibition of c-Src (PP2), PI3K ({"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002), Gα_i protein (pertussis toxin or siRNA against Gα_i1 gene, or β-arrestin 2 (siRNA)). Similarly, the leucine-facilitated insulin activation of ERK1/2 was also blunted by pertussis toxin. We further show that leucine facilitated the insulin-mediated suppression of glucose production and expression of key gluconeogenic genes in a Gα_i1 protein-dependent manner in cultured primary hepatocytes. Together, these results show that leucine can directly facilitate insulin signaling through a Gα_i protein-dependent intracellular signaling pathway. This is the first evidence showing that macronutrients like amino acid leucine can facilitate insulin signaling through G proteins directly.     

Distinctive G Protein-Dependent Signaling by Protease-Activated Receptor 2 (PAR2) in Smooth Muscle: Feedback Inhibition of RhoA by cAMP-Independent PKA

We examined expression of protease-activated receptors 2 (PAR2) and characterized their signaling pathways in rabbit gastric muscle cells. The PAR2 activating peptide SLIGRL (PAR2-AP) stimulated G_q, G₁₃, G_i1, PI hydrolysis, and Rho kinase activity, and inhibited cAMP formation. Stimulation of PI hydrolysis was partly inhibited in cells expressing PAR2 siRNA, Ga_q or Ga_i minigene and in cells treated with pertussis toxin, and augmented by expression of dominant negative regulator of G protein signaling (RGS4(N88S)). Stimulation of Rho kinase activity was abolished by PAR-2 or Ga₁₃ siRNA, and by Ga₁₃ minigene. PAR2-AP induced a biphasic contraction; initial contraction was selectively blocked by the inhibitor of PI hydrolysis ({"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075"}}U73122) or MLC kinase (ML-9), whereas sustained contraction was selectively blocked by the Rho kinase inhibitor (Y27632). PAR2-AP induced phosphorylation of MLC₂₀, MYPT1 but not CPI-17. PAR2-AP also caused a decrease in the association of NF-kB and PKA catalytic subunit: the effect of PAR2-AP was blocked by PAR2 siRNA or phosphorylation-deficient RhoA (RhoA(S188A)). PAR2-AP-induced degradation of IkBa and activation of NF-kB were abolished by the blockade of RhoA activity by Clostridium botulinum C3 exoenzyme suggesting RhoA-dependent activation of NF-kB. PAR2-AP-stimulated Rho kinase activity was significantly augmented by the inhibitors of PKA (myristoylated PKI), IKK2 (IKKIV) or NF-kB (MG132), and in cells expressing dominant negative mutants of IKK (IKK(K44A), IkBa (IkBa (S32A/S36A)) or RhoA(S188A), suggesting feedback inhibition of Rho kinase activity via PKA derived from NF-kB pathway. PAR2-AP induced phosphorylation of RhoA and the phosphorylation was attenuated in cells expressing phosphorylation-deficient RhoA(S188A). Our results identified signaling pathways activated by PAR2 to mediate smooth muscle contraction and a novel pathway for feedback inhibition of PAR2-stimulated RhoA. The pathway involves activation of the NF-kB to release catalytic subunit of PKA from its binding to IkBa and phosphorylation of RhoA at Ser¹⁸⁸.     

The Role of Calpain-Myosin 9-Rab7b Pathway in Mediating the Expression of Toll-Like Receptor 4 in Platelets: A Novel Mechanism Involved in α-Granules Trafficking

Toll-like receptors (TLRs) plays a critical role in innate immunity. In 2004, Aslam R. and Shiraki R. first determined that murine and human platelets express functional TLRs. Additionally, Andonegui G. demonstrated that platelets express TLR4, which contributes to thrombocytopenia. However, the underlying mechanisms of TLR4 expression by platelets have been rarely explored until now. The aim of this study was to identify the mechanism of TLR4 expression underlying thrombin treatment. The human washed platelets were used in this study. According to flowcytometry and western blot analysis, the surface levels of TLR4 were significantly enhanced in thrombin-activated human platelets and decreased by TMB-8, calpeptin, and {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075"}}U73122, but not Y27632 (a Rho-associated protein kinase ROCK inhibitor) indicating that thrombin-mediated TLR4 expression was modulated by PAR/PLC pathway, calcium and calpain. Co-immunoprecipitation (co-IP) assay demonstrated that the interaction between TLR4 and myosin-9 (a substrate of calpain) was regulated by calpain; cleavage of myosin-9 enhanced TLR4 expression in thrombin treated platelets. Transmission electron microscope data indicated that human platelets used α-granules to control TLR4 expression; the co-IP experiment suggested that myosin-9 did not coordinate with Rab7b to negatively regulate TLR4 trafficking in thrombin treated platelets. In summary, phospholipase Cγ-calpain-myosin 9-Rab7b axis was responsible for the mechanism underlying the regulation of TLR4 containing α-granules trafficking in thrombin-stimulated platelets, which was involved in coagulation.     

Role of contractile prostaglandins and Rho-kinase in growth factor-induced airway smooth muscle contraction

Background

In addition to their proliferative and differentiating effects, several growth factors are capable of inducing a sustained airway smooth muscle (ASM) contraction. These contractile effects were previously found to be dependent on Rho-kinase and have also been associated with the production of eicosanoids. However, the precise mechanisms underlying growth factor-induced contraction are still unknown. In this study we investigated the role of contractile prostaglandins and Rho-kinase in growth factor-induced ASM contraction.

Methods

Growth factor-induced contractions of guinea pig open-ring tracheal preparations were studied by isometric tension measurements. The contribution of Rho-kinase, mitogen-activated protein kinase (MAPK) and cyclooxygenase (COX) to these reponses was established, using the inhibitors Y-27632 (1 μM), U-0126 (3 μM) and indomethacin (3 μM), respectively. The Rho-kinase dependency of contractions induced by exogenously applied prostaglandin F_2α (PGF_2α) and prostaglandin E₂ (PGE₂) was also studied. In addition, the effects of the selective FP-receptor antagonist AL-8810 (10 μM) and the selective EP₁-antagonist AH-6809 (10 μM) on growth factor-induced contractions were investigated, both in intact and epithelium-denuded preparations. Growth factor-induced PGF_2α-and PGE₂-release in the absence and presence of Y-27632, U-0126 and indomethacin, was assessed by an ELISA-assay.

Results

Epidermal growth factor (EGF)-and platelet-derived growth factor (PDGF)-induced contractions of guinea pig tracheal smooth muscle preparations were dependent on Rho-kinase, MAPK and COX. Interestingly, growth factor-induced PGF_2α-and PGE₂-release from tracheal rings was significantly reduced by U-0126 and indomethacin, but not by Y-27632. Also, PGF_2α-and PGE₂-induced ASM contractions were largely dependent on Rho-kinase, in contrast to other contractile agonists like histamine. The FP-receptor antagonist AL-8810 (10 μM) significantly reduced (approximately 50 %) and the EP₁-antagonist AH-6809 (10 μM) abrogated growth factor-induced contractions, similarly in intact and epithelium-denuded preparations.

Conclusion

The results indicate that growth factors induce ASM contraction through contractile prostaglandins – not derived from the epithelium – which in turn rely on Rho-kinase for their contractile effects.     

Mutants in Drosophila TRPC Channels Reduce Olfactory Sensitivity to Carbon Dioxide

Background

Members of the canonical Transient Receptor Potential (TRPC) class of cationic channels function downstream of Gαq and PLCβ in Drosophila photoreceptors for transducing visual stimuli. Gαq has recently been implicated in olfactory sensing of carbon dioxide (CO₂) and other odorants. Here we investigated the role of PLCβ and TRPC channels for sensing CO₂ in Drosophila.

Methodology/Principal Findings

Through behavioral assays it was demonstrated that Drosophila mutants for plc21c, trp and trpl have a reduced sensitivity for CO₂. Immuno-histochemical staining for TRP, TRPL and TRPγ indicates that all three channels are expressed in Drosophila antennae including the sensory neurons that express CO₂ receptors. Electrophysiological recordings obtained from the antennae of protein null alleles of TRP (trp³⁴³) and TRPL (trpl³⁰²), showed that the sensory response to multiple concentrations of CO₂ was reduced. However, trpl³⁰²; trp³⁴³ double mutants still have a residual response to CO₂. Down-regulation of TRPC channels specifically in CO₂ sensing olfactory neurons reduced the response to CO₂ and this reduction was obtained even upon down-regulation of the TRPCs in adult olfactory sensory neurons. Thus the reduced response to CO₂ obtained from the antennae of TRPC RNAi strains is not due to a developmental defect.

Conclusion

These observations show that reduction in TRPC channel function significantly reduces the sensitivity of the olfactory response to CO₂ concentrations of 5% or less in adult Drosophila. It is possible that the CO₂ receptors Gr63a and Gr21a activate the TRPC channels through Gαq and PLC21C.     

Essential Role of the EF-hand Domain in Targeting Sperm Phospholipase Cζ to Membrane Phosphatidylinositol 4,5-Bisphosphate (PIP2)

Sperm-specific phospholipase C-ζ (PLCζ) is widely considered to be the physiological stimulus that triggers intracellular Ca²⁺ oscillations and egg activation during mammalian fertilization. Although PLCζ is structurally similar to PLCδ1, it lacks a pleckstrin homology domain, and it remains unclear how PLCζ targets its phosphatidylinositol 4,5-bisphosphate (PIP₂) membrane substrate. Recently, the PLCδ1 EF-hand domain was shown to bind to anionic phospholipids through a number of cationic residues, suggesting a potential mechanism for how PLCs might interact with their target membranes. Those critical cationic EF-hand residues in PLCδ1 are notably conserved in PLCζ. We investigated the potential role of these conserved cationic residues in PLCζ by generating a series of mutants that sequentially neutralized three positively charged residues (Lys-49, Lys-53, and Arg-57) within the mouse PLCζ EF-hand domain. Microinjection of the PLCζ EF-hand mutants into mouse eggs enabled their Ca²⁺ oscillation inducing activities to be compared with wild-type PLCζ. Furthermore, the mutant proteins were purified, and the in vitro PIP₂ hydrolysis and binding properties were monitored. Our analysis suggests that PLCζ binds significantly to PIP₂, but not to phosphatidic acid or phosphatidylserine, and that sequential reduction of the net positive charge within the first EF-hand domain of PLCζ significantly alters in vivo Ca²⁺ oscillation inducing activity and in vitro interaction with PIP₂ without affecting its Ca²⁺ sensitivity. Our findings are consistent with theoretical predictions provided by a mathematical model that links oocyte Ca²⁺ frequency and the binding ability of different PLCζ mutants to PIP₂. Moreover, a PLCζ mutant with mutations in the cationic residues within the first EF-hand domain and the XY linker region dramatically reduces the binding of PLCζ to PIP₂, leading to complete abolishment of its Ca²⁺ oscillation inducing activity.     

Vitamin E isoforms differentially regulate intercellular adhesion molecule-1 activation of PKCα in human microvascular endothelial cells

Aims

ICAM-1-dependent leukocyte recruitment in vivo is inhibited by the vitamin E isoform d-α-tocopherol and elevated by d-γ-tocopherol. ICAM-1 is reported to activate endothelial cell signals including protein kinase C (PKC), but the PKC isoform and the mechanism for ICAM-1 activation of PKC are not known. It is also not known whether ICAM-1 signaling in endothelial cells is regulated by tocopherol isoforms. We hypothesized that d-α-tocopherol and d-γ-tocopherol differentially regulate ICAM-1 activation of endothelial cell PKC.

Results

ICAM-1 crosslinking activated the PKC isoform PKCα but not PKCβ in TNFα-pretreated human microvascular endothelial cells. ICAM-1 activation of PKCα was blocked by the PLC inhibitor {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122, ERK1/2 inhibitor PD98059, and xanthine oxidase inhibitor allopurinol. ERK1/2 activation was blocked by inhibition of XO and PLC but not by inhibition of PKCα, indicating that ERK1/2 is downstream of XO and upstream of PKCα during ICAM-1 signaling. During ICAM-1 activation of PKCα, the XO-generated ROS did not oxidize PKCα. Interestingly, d-α-tocopherol inhibited ICAM-1 activation of PKCα but not the upstream signal ERK1/2. The d-α-tocopherol inhibition of PKCα was ablated by the addition of d-γ-tocopherol.

Conclusions

Crosslinking ICAM-1 stimulated XO/ROS which activated ERK1/2 that then activated PKCα. ICAM-1 activation of PKCα was inhibited by d-α-tocopherol and this inhibition was ablated by the addition of d-γ-tocopherol. These tocopherols regulated ICAM-1 activation of PKCα without altering the upstream signal ERK1/2. Thus, we identified a mechanism for ICAM-1 activation of PKC and determined that d-α-tocopherol and d-γ-tocopherol have opposing regulatory functions for ICAM-1-activated PKCα in endothelial cells.     

Sprouty Proteins Inhibit Receptor-mediated Activation of Phosphatidylinositol-specific Phospholipase C

Sprouty (Spry) proteins are negative regulators of receptor tyrosine kinase signaling; however, their exact mechanism of action remains incompletely understood. We identified phosphatidylinositol-specific phospholipase C (PLC)-γ as a partner of the Spry1 and Spry2 proteins. Spry–PLCγ interaction was dependent on the Src homology 2 domain of PLCγ and a conserved N-terminal tyrosine residue in Spry1 and Spry2. Overexpression of Spry1 and Spry2 was associated with decreased PLCγ phosphorylation and decreased PLCγ activity as measured by production of inositol (1,4,5)-triphosphate (IP₃) and diacylglycerol, whereas cells deficient for Spry1 or Spry1, -2, and -4 showed increased production of IP₃ at baseline and further increased in response to growth factor signals. Overexpression of Spry 1 or Spry2 or small-interfering RNA-mediated knockdown of PLCγ1 or PLCγ2 abrogated the activity of a calcium-dependent reporter gene, suggesting that Spry inhibited calcium-mediated signaling downstream of PLCγ. Furthermore, Spry overexpression in T-cells, which are highly dependent on PLCγ activity and calcium signaling, blocked T-cell receptor-mediated calcium release. Accordingly, cultured T-cells from Spry1 gene knockout mice showed increased proliferation in response to T-cell receptor stimulation. These data highlight an important action of Spry, which may allow these proteins to influence signaling through multiple receptors.     

Urinary Bladder Dysfunction in Transgenic Sickle Cell Disease Mice

Background

Urological complications associated with sickle cell disease (SCD), include nocturia, enuresis, urinary infections and urinary incontinence. However, scientific evidence to ascertain the underlying cause of the lower urinary tract symptoms in SCD is lacking.

Objective

Thus, the aim of this study was to evaluate urinary function, in vivo and ex vivo, in the Berkeley SCD murine model (SS).

Methods

Urine output was measured in metabolic cage for both wild type and SS mice (25-30 g). Bladder strips and urethra rings were dissected free and mounted in organ baths. In isolated detrusor smooth muscle (DSM), relaxant response to mirabegron and isoproterenol (1nM-10μM) and contractile response to (carbachol (CCh; 1 nM-100μM), KCl (1 mM-300mM), CaCl₂ (1μM-100mM), α,β-methylene ATP (1, 3 and 10 μM) and electrical field stimulation (EFS; 1-32 Hz) were measured. Phenylephrine (Phe; 10nM-100μM) was used to evaluate the contraction mechanism in the urethra rings. Cystometry and histomorphometry were also performed in the urinary bladder.

Results

SS mice present a reduced urine output and incapacity to produce typical bladder contractions and bladder emptying (ex vivo), compared to control animals. In DSM, relaxation in response to a selective β3-adrenergic agonist (mirabegron) and to a non-selective β-adrenergic (isoproterenol) agonist were lower in SS mice. Additionally, carbachol, α, β-methylene ATP, KCl, extracellular Ca²⁺ and electrical-field stimulation promoted smaller bladder contractions in SS group. Urethra contraction induced by phenylephrine was markedly reduced in SS mice. Histological analyses of SS mice bladder revealed severe structural abnormalities, such as reductions in detrusor thickness and bladder volume, and cell infiltration.

Conclusions

Taken together, our data demonstrate, for the first time, that SS mice display features of urinary bladder dysfunction, leading to impairment in urinary continence, which may have an important role in the pathogenesis of the enuresis and infections observed the SCD patients.     

Vascular smooth muscle cells from small human omental arteries express P2X1 and P2X4 receptor subunits

Stimulation of P2X receptors by ATP in vascular smooth muscle cells (VSMCs) is proposed to mediate vascular tone. However, understanding of P2X receptor-mediated actions in human blood vessels is limited, and therefore, the current work investigates the role of P2X receptors in freshly isolated small human gastro-omental arteries (HGOAs). Expression of P2X1 and P2X4 receptor subunit messenger RNA (mRNA) and protein was identified in individual HGOA VSMCs using RT-PCR and immunofluorescent analysis and using Western blot in multi-cellular preparations. ATP of 10 μmol/l and αβ-meATP of 10 μmol/l, a selective P2X receptor agonist, evoked robust increases in [Ca²⁺]_i in fluo-3-loaded HGOA VSMCs. Pre-incubation with 1 μmol/l NF279, a selective P2X receptor antagonist, reduced the amplitude of αβ-meATP-induced increase in [Ca²⁺]_i by about 70 %. ATP of 10 μmol/l and αβ-meATP of 10 μmol/l produced similar contractile responses in segments of HGOA, and these contractions were greatly reduced by 2 μmol/l NF449, a selective P2X receptor inhibitor. These data suggest that VSMCs from HGOA express P2X1 and P2X4 receptor subunits with homomeric P2X1 receptors likely serving as the predominant target for extracellular ATP.

Electronic supplementary material

The online version of this article (doi:10.1007/s11302-014-9415-6) contains supplementary material, which is available to authorized users.