Insulin stimulates the release of a subset of GPI-anchored proteins in a G-protein independent manner

The glycosyl-phosphatidylinositol anchored protein, membrane dipeptidase (EC 3.4.13.19) is released from the surface of 3T3-L1 adipocytes in response to insulin treatment through the action of a phospholipase C. The present study investigates the role of guanine-nucleotide binding proteins (G-proteins) in this process. Treatment of permeabilized 3T3-L1 adipocytes with GTPgammaS did not cause release of membrane dipeptidase into the medium, while GDPbetaS did not inhibit the insulin-stimulated release of membrane dipeptidase. Other activators of G-proteins, including the tetradecapeptide mastoparan, pertussis toxin and AlF3, also caused no significant release of membrane dipeptidase from the surface of the 3T3-L1 adipocytes. From these observations it is concluded that G-proteins are not involved in the insulin-stimulated release of membrane dipeptidase. Although X-Pro aminopeptidase (EC 3.4.11.9) is GPI-anchored in 3T3-L1 adipocytes as shown by digestion with bacterial phosphatidylinositol-specific phospholipase C, it was not released upon insulin treatment of the cells, indicating that only a subset of the GPI-anchored proteins are susceptible to insulin-stimulated release.     

Two isoforms of eukaryotic phospholipase C in Paramecium affecting transport and release of GPI-anchored proteins in vivo

Surface proteins anchored by a glycosylphosphatidylinositol (GPI) residue in the cell membrane are widely distributed among eukaryotic cells. The GPI anchor is cleavable by a phospholipase C (PLC) leading to the release of such surface proteins, and this process is postulated to be essential in several systems. For higher eukaryotes, the responsible enzymes have not been characterized in any detail as yet. Here we characterize six PLCs in the ciliated protozoan, Paramecium, which, in terms of catalytic domains and architecture, all show characteristics of PLCs involved in signal transduction in higher eukaryotes. We show that some of these endogenous PLCs can release GPI-anchored surface proteins in vitro: using RNA_i to reduce PLC expression results in the same effects as the application of PLC inhibitors. With two enzymes, PLC2 and PLC6, RNA_i phenotypes show strong defects in release of GPI-anchored surface proteins in vivo. Moreover, these RNA_i lines also show abnormal surface protein distribution, suggesting that GPI cleavage may influence trafficking of anchored proteins. As we find GFP fusion proteins in the cytosol and in the surface protein extracts, these PLCs obviously show unconventional translocation mechanisms. This is the first molecular data on endogenous Paramecium PLCs with the described properties affecting GPI anchors in vitro and in vivo.     

Insulin stimulates GDP release from G proteins in the rat and human liver plasma membranes

Plasma membranes (1–2 mg protein) prepared from the livers of adult male rats and human organ donors were incubated with 0.6 μM [α-³²P] guanosine triphosphate (GTP) in an adenosine triphosphate (ATP)-regenerating buffer at 37°C for 1 h; during this incubation, the [³²P]GTP is hydrolyzed and the nucleotide that is predominantly bound to the membranes is [³²P] guanosine diphosphate (GDP). [³²P]GDP release from the liver membranes was proportional to the protein concentration and increased as a function of time. At 5 mM, Ca²⁺, Mg²⁺, Mn²⁺, and Zn²⁺ maximally inhibited GDP release by 80–90%, whereas, 5 mM Cu²⁺ maximally stimulated the reaction by 100%. Therefore, cations were not included in the buffer used in the GDP release step. One μM Gpp(NH)p (5′-guanylylimidodiphosphate), a nonhydrolyzable analog of GTP, maximally stimulated [³²P]GDP release in the liver membranes by up to 30%. Although 10 nM Gpp(NH)p had no effect on GDP release, it appeared to stabilize the hormonal effect by blocking further GDP/GTP exchange. In the rat membranes, 1–100 nM glucagon (used as a positive control) stimulated [³²P]GDP release by about 17% (P < .05); similarly, 0.1–100 nM insulin stimulated [³²P]GDP release by 10–13% (P < .05). In the human membranes, 10 pM to 100 nM insulin stimulated [³²P]GDP release by 7–10%. In the rat membranes, 10 nM insulin stimulated [³²P]GDP release by 17 and 24% at 2 and 4 min, respectively (P < .05); in the human membranes, 10 nM insulin stimulated [³²P]GDP release by about 9% at 2 and 4 min. Normal rabbit IgG (used as a control for insulin receptor antibody) by itself stimulated the GDP release by rat and human membranes. However, the stimulation of the GDP release by insulin receptor antibody was consistently higher than that observed with normal rabbit IgG. Four to 15 μg of insulin receptor antibody stimulated [³²P]GDP release by 12–22% (P < .05) and 7–14% in rat and human membranes, respectively. These results indicate that ligand binding to the insulin receptor results in a functional interaction of the receptor with a guanine nucleotide-binding transducer protein (G protein) and activation of GTP/GDP exchange.     

Identification of a cAMP-response element in the regulator of G-protein signaling-2 (RGS2) promoter as a key cis-regulatory element for RGS2 transcriptional regulation by angiotensin II in cultured vascular smooth muscles

Mice deficient in regulator of G-protein signaling-2 (RGS2) have severe hypertension, and RGS2 genetic variations occur in hypertensive humans. A potentially important negative feedback loop in blood pressure homeostasis is that angiotensin II (Ang II) increases vascular smooth muscle cell (VSMC) RGS2 expression. We reported that Group VIA phospholipase A(2) (iPLA(2)β) is required for this response (Xie, Z., Gong, M. C., Su, W., Turk, J., and Guo, Z. (2007) J. Biol. Chem. 282, 25278-25289), but the specific molecular causes and consequences of iPLA(2)β activation are not known. Here we demonstrate that both protein kinases C (PKC) and A (PKA) participate in Ang II-induced VSMC RGS2 mRNA up-regulation, and that actions of PKC and PKA precede and follow iPLA(2)β activation, respectively. Moreover, we identified a conserved cAMP-response element (CRE) in the murine RGS2 promoter that is critical for cAMP-response element-binding protein (CREB) binding and RGS2 promoter activation. Forskolin-stimulated RGS2 mRNA up-regulation is inhibited by CREB sequestration or specific disruption of the CREB-RGS2 promoter interaction, and Ang II-induced CREB phosphorylation and nuclear localization are blocked by iPLA(2)β pharmacologic inhibition or genetic ablation. Ang II-induced intracellular cyclic AMP accumulation precedes CREB phosphorylation and is diminished by inhibiting iPLA(2), cyclooxygenase, or lipoxygenase. Moreover, three single nucleotide polymorphisms identified in hypertensive patients are located in the human RGS2 promoter CREB binding site. Point mutations corresponding to these single nucleotide polymorphisms interfere with stimulation of human RGS2 promoter activity by forskolin. Our studies thus delineate a negative feedback loop to attenuate Ang II signaling in VSMC with potential importance in blood pressure homeostasis and the pathogenesis of human essential hypertension.     

Ceramide induces release of mitochondrial proapoptotic proteins in caspase-dependent and -independent manner in HT-29 cells

In this study, the release of mitochondrial proapoptotic intermembrane space proteins induced by exogenous C2-ceramide in human colon carcinoma (HT-29) cell line was investigated. HT-29 cells were treated with 12.5, 25 and 50 μmol/L C2-ceramide in vitro. Flow cytometer was used to detect the mitochondrial membrane potential (△Ψm). Subcellular fractions were extracted by Mitochondrial/Cytosol Fractionation Kit after C2-ceramide treatment for 24 h. SDS-PAGE was used to determine the level of cytochrome c (Cyt c), high temperature requirement A2 (HtrA2) and second mitochondrial-derived activator of caspases (Smac) released from mitochondria, the expression of X-linked inhibitor of apoptosis protein (XIAP) and caspase-3 for 24 h. The results showed that △Ψm began to decrease from 6 h after 25 and 50 μmol/L C2-ceramide treatment (P＜0.05) and cyclosporin A (CsA) could inhibit the collapse of △Ψm through regulating mitochondrial membrane permeability transition pore. There was no effect of C2-ceramide on the expression of Cyt c, HtrA2 and Smac in the total levels. 12.5, 25 and 50 μmol/L C2-ceramide could induce Cyt c, HtrA2 and Smac to release from mitochondria to cytosol and down-regulate the expression of XIAP (P＜0.05). Also there was expression of cleaved caspase-3 with C2-ceramide treatment. After the treatment with caspase inhibitor, C2-ceramide still induced the release of Cyt c and HtrA2, but Smac did not. Therefore, C2-ceramide could induce apoptosis of HT-29 cells through the mitochondria pathway. The release of Cyt c, HtrA2 and Smac from mitochondria did not occur via the same mechanism, the release of Cyt c and HtrA2 was caspase-independent and the release of Smac was caspase-dependent.     

Insulin stimulates the release from liver plasma membranes of a chemical modulator of pyruvate dehydrogenase

Incubation of a rat liver particulate fraction with physiological concentrations of insulin enhances the production of a small molecular weight substance which modulates adipocyte as well as liver mitochondrial pyruvate dehydrogenase. While low concentrations of insulin enhance production of this activity, levels of greater than 10^?9M produce significantly less. Similarly, while increasing concentrations of mediator cause increased stimulation of pyruvate dehydrogenase activity, higher concentrations no longer exhibit this effect. The putative insulin mediator was partially purified on HPLC and Sephadex G-25 columns. Its molecular weight was about 1000–2000. These results indicate the presence of a chemical mediator of insulin action in liver similar to that observed in other insulin target tissues.     

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.     

Bradykinin stimulates phospholipase D in PC12 cells by a mechanism which is independent of increases in intracellular Ca2+

These experiments were designed to learn the role of bradykinin induced changes in intracellular Ca²⁺ in the activation of phospholipase D activity in PC12 cells. Ionomycin at a concentration of 0.1M caused an increase in intracellular Ca²⁺ comparable to bradykinin, but had no effect on phospholipase D activity. Carbachol, ATP, and thapsigargin also increased intracellular Ca²⁺ but had no effect on phospholipase D activity. Increases in intracellular Ca²⁺ may be a necessary but not a sufficient factor in the activation of phospholipase D. To investigate this issue, the bradykinin induced increase in intracellular Ca²⁺ was blocked by preincubating the cells in Ca²⁺-free media plus EGTA or in media containing the intracellular Ca²⁺ chelator BAPTA/AM. These preincubations completely blocked the bradykinin induced increase in intracellular Ca²⁺ but only attenuated the bradykinin mediated activation of phospholipase D. Physiological increases in intracellular Ca²⁺ apparently do not mediate the effect of bradykinin on phospholipase D.     

A conserved protein interaction interface on the type 5 G protein beta subunit controls proteolytic stability and activity of R7 family regulator of G protein signaling proteins

Regulators of G protein signaling (RGS) proteins of the R7 subfamily limit signaling by neurotransmitters in the brain and by light in the retina. They form obligate complexes with the Gβ5 protein that are subject to proteolysis to control their abundance and alter signaling. The mechanisms that regulate this proteolysis, however, remain unclear. We used genetic screens to find mutations in Gβ5 that selectively destabilize one of the R7 RGS proteins in Caenorhabditis elegans. These mutations cluster at the binding interface between Gβ5 and the N terminus of R7 RGS proteins. Equivalent mutations within mammalian Gβ5 allowed the interface to still bind the N-terminal DEP domain of R7 RGS proteins, and mutant Gβ5-R7 RGS complexes initially formed in cells but were then rapidly degraded by proteolysis. Molecular dynamics simulations suggest the mutations weaken the Gβ5-DEP interface, thus promoting dynamic opening of the complex to expose determinants of proteolysis known to exist on the DEP domain. We propose that conformational rearrangements at the Gβ5-DEP interface are key to controlling the stability of R7 RGS protein complexes.     

Protein kinase C inhibitor chelerythrine attenuates the morphine-induced excitatory amino acid release and reduction of the antinociceptive effect of morphine in rats injected intrathecally with pertussis toxin

Neuropathic pain syndromes respond poorly to opioid treatment. In our previous studies, we found that intrathecal (i.t.) injection of pertussis toxin (PTX) produces thermal hyperalgesia, which is poorly responsive to morphine and is accompanied by an increase in cerebrospinal fluid (CSF) levels of excitatory amino acids (EAAs) and protein kinase C (PKC) activation. In the present study, rats were implanted with an i.t. catheter for drug injection and a microdialysis probe for CSF dialysate collection. On the fourth day after injection of PTX (2 microg, i.t.), there was a significant reduction in the antinociceptive effect of morphine (10 microg, i.t.) which was accompanied by an increase in levels of EAAs. Pretreatment with the PKC inhibitor, chelerythrine (25 microg, i.t.) one hour before morphine injection markedly inhibited both effects. These results suggest that, in PTX-treated rats, PKC plays an important role in inhibiting the morphine-induced spinal EAA release, which might be related to the reduced antinociceptive effect of morphine.     

Matrix metalloproteinase-1 activates a pertussis toxin-sensitive signaling pathway that stimulates the release of matrix metalloproteinase-9

The matrix metalloproteinases (MMPs) are a family of structurally related metalloendopeptidases so named due to their propensity to target extracellular matrix (ECM) proteins. Accumulating evidence, however, suggests that these proteases cleave numerous non-ECM substrates including enzymes and cell surface receptors. MMPs may also bind to cell surface receptors, though such binding has typically been thought to mediate internalization and degradation of the bound protease. More recently, it has been shown that MMP-1 coimmunoprecipitates with the alpha2beta1 integrin, a receptor for collagen. This association may serve to localize the enzymatic activity of MMP-1 so that collagen is cleaved and cell migration is facilitated. In other studies, however, it has been shown that integrin engagement may be linked to the activation of signaling cascades including those mediated by Gialpha containing heterotrimers. As an example, alpha2beta1 can form a complex with CD47 that may associate with Gialpha. In the present study we have therefore investigated the possibility that MMP-1 may affect intracellular changes that are linked to the activation of a Gi protein-coupled receptor. We show that treatment of neural cells with MMP-1 is followed by a rapid reduction in cytosolic levels of cAMP. Moreover, MMP-1 potentiates proteinase activated receptor-1 (PAR-1) agonist-linked increases in intracellular calcium, an effect which is often observed when an agonist of a Gi protein-coupled receptor is administered in association with an agonist of a Gq coupled receptor. In addition, MMP-1 stimulates pertussis toxin sensitive release ofMMP-9 both from cultured neural cells and monocyte/macrophages. Together, these results suggest that MMP-1 signals through a pertussis toxin-sensitive G protein-coupled receptor.     

Removal of GPI-anchored membrane proteins causes clustering of lipid microdomains in the apical head area of porcine sperm

The release of extracellular proteins is a part of the sperm capacitation process; this allows the sperm surface reorganization that enables the sperm to fertilize an oocyte. Some of the components released are ‘decapacitation factors’, an uncoordinated or early release of which may cause inappropriate surface destabilization and premature capacitation. We studied the involvement of glycosylphosphatidylinositol-anchored proteins (GPI-APs) in sperm capacitation, and reported that CD52 and CD55 exhibit bicarbonate-dependent release during in vitro sperm capacitation. Treating sperm with phosphatidylinositol-specific phospholipase C (PIPLC) resulted in the enzymatic cleavage of CD55, in both capacitating and noncapacitating conditions. Moreover, PIPLC treatment in noncapacitating conditions caused surface reorganization events that included exposure of the ganglioside GM1, aggregation of flotillin-1, and the swelling of the apical acrosome region; all of which have been reported to be associated with sperm capacitation. The acrosomal swelling was monitored using wet mount atomic force microscopy, a new imaging technique that allows nanometer-level sperm surface measurements in samples hydrated with physiological buffer rather than dried. Despite these surface changes, PIPLC treatment in identical incubation conditions did not stimulate hyperactive sperm motility or protein tyrosine phosphorylation (other hallmarks of sperm capacitation in vitro). In full capacitating conditions (i.e., the presence of bicarbonate and albumin), PIPLC treatment caused sperm deterioration. The possible role of GPI-APs removal from the sperm surface during sperm capacitation is discussed.     

Insulin release independent of a rise in cytosolic free Ca2+ by forskolin and phorbol ester

The role of cytosolic free Ca2+ in insulin release was evaluated using isolated rat pancreatic islets permeabilized with digitonin and incubated in Ca-EGTA buffers to fix free Ca2+ concentration at arbitrary levels. Ca2+ induced insulin release in a concentration-dependent manner with the threshold being between 0.1 and 1 microM. The hormone release was increased by forskolin and 12-O-tetradecanoyl phorbol-13-acetate (TPA), a potent activator of adenylate cyclase and that of protein kinase C, respectively. The findings suggest that activation of both protein kinase A and protein kinase C modulate insulin release without a concomitant increase in cytosolic free Ca2+.     

Acute and Chronic Ethanol Consumption Effects on the Immunolabeling of Gq/11α Subunit Protein and Phospholipase C Isozymes in the Rat Brain

Abstract: The goal of this investigation was to examine whether postreceptor sites [G_q/11 protein and phospholipase C (PLC) isozymes] of the phosphoinositide signal transduction system are involved in neuroadaptational mechanisms in the brain during chronic ethanol consumption. It was observed that acute ethanol treatment has no effect on the immunolabeling of PLC-β₁, -γ₁, and -δ₁ and the α subunit of G_q/11 protein in the rat cortex as determined by western blotting using specific monoclonal antibodies. On the other hand, chronic ethanol consumption (15 days) resulted in a significant decrease in the immunolabeling of PLC-β₁, whereas under identical conditions, the immunolabeling of PLC-γ₁ and -δ₁ isozymes was not significantly altered. The decreased immunolabeling of PLC-β₁ during chronic ethanol consumption was not altered by 24 h of withdrawal after 15 days of ethanol consumption. The immunolabeling of the α subunit of G_q/11 protein was significantly decreased after 15 days of ethanol consumption but had returned to normal levels after 24 h of ethanol withdrawal. Also, chronic ethanol treatment resulted in a significant decrease in phosphatidylinositol 4,5-bisphosphate-specific PLC activity, which remained the same after 24 h of ethanol withdrawal. These results suggest that decreased PLC activity during ethanol consumption and its withdrawal may be due to decreased protein levels of the G_q/11 protein-coupled PLC-β₁ isozyme but not the PLC-γ₁ or -δ₁ isozyme in the rat cortex. It is possible that changes in the protein levels of the G_q/11 protein-coupled PLC-β₁ isozyme and in PLC activity in the brain may be involved in the cellular adaptation to chronic ethanol exposure.     

Pharmacological and Genetic Targeting of the PI4KA Enzyme Reveals Its Important Role in Maintaining Plasma Membrane Phosphatidylinositol 4-Phosphate and Phosphatidylinositol 4,5-Bisphosphate Levels

Phosphatidylinositol 4-kinase type IIIα (PI4KA) is a host factor essential for hepatitis C virus replication and hence is a target for drug development. PI4KA has also been linked to endoplasmic reticulum exit sites and generation of plasma membrane phosphoinositides. Here, we developed highly specific and potent inhibitors of PI4KA and conditional knock-out mice to study the importance of this enzyme in vitro and in vivo. Our studies showed that PI4KA is essential for the maintenance of plasma membrane phosphatidylinositol 4,5-bisphosphate pools but only during strong stimulation of receptors coupled to phospholipase C activation. Pharmacological blockade of PI4KA in adult animals leads to sudden death closely correlating with the drug''s ability to induce phosphatidylinositol 4,5-bisphosphate depletion after agonist stimulation. Genetic inactivation of PI4KA also leads to death; however, the cause in this case is due to severe intestinal necrosis. These studies highlight the risks of targeting PI4KA as an anti-hepatitis C virus strategy and also point to important distinctions between genetic and pharmacological studies when selecting host factors as putative therapeutic targets.     

Insulin stimulates the activity of a protamine kinase in isolated rat hepatocytes

Treatment of isolated rat hepatocytes with 10-100 nM insulin for 5-10 min increased by about 2-fold the activity of a protamine kinase which exhibited properties similar to those of a protamine kinase from bovine kidney (Damuni, Z., Amick, G. D., and Sneed, T. R. (1989) J. Biol. Chem. 264, 6412-6416). Half-maximal increase in protamine kinase activity occurred at about 1 nM insulin. This effect of insulin was detected only when 25 mM NaF or 50 mM KPO4 were included in the homogenization buffers and was not prevented by preincubation of the hepatocytes with 10 microM cycloheximide. Insulin stimulation of protamine kinase was maintained following chromatography of extracts on protamine-agarose, DEAE-cellulose, and Sephacryl S-200 gel filtration. The apparent Mr of the protamine kinase from control and insulin-treated hepatocytes was 45,000 as estimated by gel permeation chromatography. Experiments utilizing partially purified protamine kinase from control and insulin-treated hepatocytes indicated that insulin did not affect the apparent Km for protamine, Mg2+, or ATP, but increased the Vmax for the protamine kinase reaction by 1.6-2-fold. Incubation with the catalytic subunit of protein phosphatase 2A completely inactivated the protamine kinase from control and insulin-treated cells. The results indicate that the insulin-stimulated increase in protamine kinase activity may be due to a covalent modification, possibly phosphorylation, of the protamine kinase.     

Expression and potential function of Rho family small G proteins in cells of the mammalian seminiferous epithelium

Dynamic cellular rearrangements involving the actin cytoskeleton are required of both Sertoli and germ cells during spermatogenesis. Rho family small G proteins have been implicated in the control of the actin cytoskeleton in numerous cell types. Therefore, RhoA and Rac1 were investigated in Sertoli and germ cells. RhoA and Rac1 have been detected at both the mRNA and protein levels in these cells. In addition, Sertoli cell L-selectin is shown to interact with actin binding proteins, potentially providing a link between L-selectin and Rac1 signaling. Finally, inactivation of Sertoli cell Rho family proteins yields disruption of the actin cytoskeleton.