Doc2 alpha and Munc13-4 regulate Ca(2+) -dependent secretory lysosome exocytosis in mast cells

The Doc2 family comprises the brain-specific Doc2alpha and the ubiquitous Doc2beta and Doc2gamma. With the exception of Doc2gamma, these proteins exhibit Ca(2+)-dependent phospholipid-binding activity in their Ca(2+)-binding C2A domain and are thought to be important for Ca(2+)-dependent regulated exocytosis. In excitatory neurons, Doc2alpha interacts with Munc13-1, a member of the Munc13 family, through its N-terminal Munc13-1-interacting domain and the Doc2alpha-Munc13-1 system is implicated in Ca(2+)-dependent synaptic vesicle exocytosis. The Munc13 family comprises the brain-specific Munc13-1, Munc13-2, and Munc13-3, and the non-neuronal Munc13-4. We previously showed that Munc13-4 is involved in Ca(2+)-dependent secretory lysosome exocytosis in mast cells, but the involvement of Doc2 in this process is not determined. In the present study, we found that Doc2alpha but not Doc2beta was endogenously expressed in the RBL-2H3 mast cell line. Doc2alpha colocalized with Munc13-4 on secretory lysosomes, and interacted with Munc13-4 through its two regions, the N terminus containing the Munc13-1-interacting domain and the C terminus containing the Ca(2+)-binding C2B domain. In RBL-2H3 cells, Ca(2+)-dependent secretory lysosome exocytosis was inhibited by expression of the Doc2alpha mutant lacking either of the Munc13-4-binding regions and the inhibition was suppressed by coexpression of Munc13-4. Knockdown of endogenous Doc2alpha also reduced Ca(2+)-dependent secretory lysosome exocytosis, which was rescued by re-expression of human Doc2alpha but not by its mutant that could not bind to Munc13-4. Moreover, Ca(2+)-dependent secretory lysosome exocytosis was severely reduced in bone marrow-derived mast cells from Doc2alpha knockout mice. These results suggest that the Doc2alpha-Muunc13-4 system regulates Ca(2+)-dependent secretory lysosome exocytosis in mast cells.     

Phosphatidylinositol 4-OH kinase is a downstream target of neuronal calcium sensor-1 in enhancing exocytosis in neuroendocrine cells

Neuronal calcium sensor-1 (NCS-1), the mammalian orthologue of frequenin, belongs to a family of EF-hand-containing Ca(2+) sensors. NCS-1/frequenin has been shown to enhance synaptic transmission in PC12 cells and Drosophila and Xenopus, respectively. However, the precise molecular mechanism for the enhancement of exocytosis is largely unknown. In PC12 cells, NCS-1 potentiated exocytosis evoked by ATP, an agonist to phospholipase C-linked receptors, but had no effect on depolarization-evoked release. NCS-1 also enhanced exocytosis triggered by ionomycin, a Ca(2+) ionophore that bypasses K(+) and Ca(2+) channels. Overexpression of NCS-1 caused a shift in the dose-response curve of inhibition of ATP-evoked secretion using phenylarsine oxide, an inhibitor of phosphatidylinositol 4-OH kinase (PI4K). Plasma membrane phosphatidylinositol 4,5-bisphosphate pools were increased upon NCS-1 transfection as visualized using a phospholipase C-delta pleckstrin homology domain-green fluorescent protein construct. NCS-1-transfected cell extracts displayed increased phosphatidylinositol-4-phosphate biosynthesis, indicating an increase in PI4K activity. Mutations in NCS-1 equivalent to those that abolish the interaction of recoverin, another EF-hand-containing Ca(2+) sensor, with its downstream target rhodopsin kinase, lost their ability to enhance exocytosis. Taken together, the present data indicate that NCS-1 modulates the activity of PI4K, leading to increased levels of phosphoinositides and concomitant enhancement of exocytosis.     

Neuronal calcium sensor-1 and phosphatidylinositol 4-kinase beta regulate IgE receptor-triggered exocytosis in cultured mast cells

We examined the possible occurrence and function of neuronal Ca(2+) sensor 1 (NCS-1/frequenin) in the mast cell line rat basophilic leukemia, RBL-2H3. This protein has been implicated in the control of neurosecretion from dense core granules in neuronal cells as well as in the control of constitutive secretory pathways in both yeast and mammalian cells. We show that RBL-2H3 cells, secretory cells of the immune system, endogenously express the 22-kDa NCS-1 protein as well as an immune-related 50-kDa protein. Both proteins associate in vivo with phosphatidylinositol 4-kinase beta (PI4Kbeta) and colocalize with the enzyme in the Golgi region. We show further that overexpression of NCS-1 in RBL-2H3 cells stimulates the catalytic activity of PI4Kbeta, increases IgE receptor (FcepsilonRI)-triggered hydrolysis of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)), and stimulates FcepsilonRI-triggered, but not Ca(2+) ionophore-triggered, exocytosis. Conversely, expression of a kinase-dead mutant of PI4Kbeta reduces PI4Kbeta activity, decreases FcepsilonRI-stimulated phosphatidylinositol 4,5-bisphosphate hydrolysis, and blocks FcepsilonRI-triggered, but not Ca(2+) ionophore-triggered, exocytosis. Our results indicate that PI(4)P, produced by the Golgi-localized PI4Kbeta, is the rate-limiting factor in the synthesis of the pool of PI(4,5)P(2) that serves as substrate for the generation of lipid-derived second messengers in FcepsilonRI-triggered cells. We conclude that NCS-1 is involved in the control of regulated exocytosis in nonneural cells, where it contributes to stimulus-secretion coupling by interacting with PI4Kbeta and positive regulation of its activity.     

Hydrolysis of phosphatidylinositol 4,5-bisphosphate mediates calcium-induced inactivation of TRPV6 channels

TRPV6 is a member of the transient receptor potential superfamily of ion channels that facilitates Ca(2+) absorption in the intestines. These channels display high selectivity for Ca(2+), but in the absence of divalent cations they also conduct monovalent ions. TRPV6 channels have been shown to be inactivated by increased cytoplasmic Ca(2+) concentrations. Here we studied the mechanism of this Ca(2+)-induced inactivation. Monovalent currents through TRPV6 substantially decreased after a 40-s application of Ca(2+), but not Ba(2+). We also show that Ca(2+), but not Ba(2+), influx via TRPV6 induces depletion of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2) or PIP(2)) and the formation of inositol 1,4,5-trisphosphate. Dialysis of DiC(8) PI(4,5)P(2) through the patch pipette inhibited Ca(2+)-dependent inactivation of TRPV6 currents in whole-cell patch clamp experiments. PI(4,5)P(2) also activated TRPV6 currents in excised patches. PI(4)P, the precursor of PI(4,5)P(2), neither activated TRPV6 in excised patches nor had any effect on Ca(2+)-induced inactivation in whole-cell experiments. Conversion of PI(4,5)P(2) to PI(4)P by a rapamycin-inducible PI(4,5)P(2) 5-phosphatase inhibited TRPV6 currents in whole-cell experiments. Inhibiting phosphatidylinositol 4 kinases with wortmannin decreased TRPV6 currents and Ca(2+) entry into TRPV6-expressing cells. We propose that Ca(2+) influx through TRPV6 activates phospholipase C and the resulting depletion of PI(4,5)P(2) contributes to the inactivation of TRPV6.     

Munc13-4 reconstitutes calcium-dependent SNARE-mediated membrane fusion

Munc13-4 is a widely expressed member of the CAPS/Munc13 protein family proposed to function in priming secretory granules for exocytosis. Munc13-4 contains N- and C-terminal C2 domains (C2A and C2B) predicted to bind Ca(2+), but Ca(2+)-dependent regulation of Munc13-4 activity has not been described. The C2 domains bracket a predicted SNARE-binding domain, but whether Munc13-4 interacts with SNARE proteins is unknown. We report that Munc13-4 bound Ca(2+) and restored Ca(2+)-dependent granule exocytosis to permeable cells (platelets, mast, and neuroendocrine cells) dependent on putative Ca(2+)-binding residues in C2A and C2B. Munc13-4 exhibited Ca(2+)-stimulated SNARE interactions dependent on C2A and Ca(2+)-dependent membrane binding dependent on C2B. In an apparent coupling of membrane and SNARE binding, Munc13-4 stimulated SNARE-dependent liposome fusion dependent on putative Ca(2+)-binding residues in both C2A and C2B domains. Munc13-4 is the first priming factor shown to promote Ca(2+)-dependent SNARE complex formation and SNARE-mediated liposome fusion. These properties of Munc13-4 suggest its function as a Ca(2+) sensor at rate-limiting priming steps in granule exocytosis.     

Phosphatidylinositol 4,5-bisphosphate regulates SNARE-dependent membrane fusion

Phosphatidylinositol 4,5-bisphosphate (PI 4,5-P(2)) on the plasma membrane is essential for vesicle exocytosis but its role in membrane fusion has not been determined. Here, we quantify the concentration of PI 4,5-P(2) as approximately 6 mol% in the cytoplasmic leaflet of plasma membrane microdomains at sites of docked vesicles. At this concentration of PI 4,5-P(2) soluble NSF attachment protein receptor (SNARE)-dependent liposome fusion is inhibited. Inhibition by PI 4,5-P(2) likely results from its intrinsic positive curvature-promoting properties that inhibit formation of high negative curvature membrane fusion intermediates. Mutation of juxtamembrane basic residues in the plasma membrane SNARE syntaxin-1 increase inhibition by PI 4,5-P(2), suggesting that syntaxin sequesters PI 4,5-P(2) to alleviate inhibition. To define an essential rather than inhibitory role for PI 4,5-P(2), we test a PI 4,5-P(2)-binding priming factor required for vesicle exocytosis. Ca(2+)-dependent activator protein for secretion promotes increased rates of SNARE-dependent fusion that are PI 4,5-P(2) dependent. These results indicate that PI 4,5-P(2) regulates fusion both as a fusion restraint that syntaxin-1 alleviates and as an essential cofactor that recruits protein priming factors to facilitate SNARE-dependent fusion.     

Protease-activated receptor-2 increases exocytosis via multiple signal transduction pathways in pancreatic duct epithelial cells

Protease-activated receptor-2 (PAR-2) is activated when trypsin cleaves its NH(2) terminus to expose a tethered ligand. We previously demonstrated that PAR-2 activates ion channels in pancreatic duct epithelial cells (PDEC). Using real-time optical fluorescent probes, cyan fluorescence protein-Epac1-yellow fluorescence protein for cAMP, PH(PLC-delta1)-enhanced green fluorescent protein for phosphatidylinositol 4,5-bisphosphate, and protein kinase Cgamma (PKCgamma)-C1-yellow fluorescence protein for diacylglycerol, we now define the signaling pathways mediating PAR-2 effect in dog PDEC. Although PAR-2 activation does not stimulate a cAMP increase, it induces phospholipase C to hydrolyze phosphatidylinositol 4,5-bisphosphate into inositol 1,4,5-trisphosphate and diacylglycerol. Intracellular Ca(2+) mobilization from inositol 1,4,5-trisphosphate-sensitive Ca(2+) stores and a subsequent Ca(2+) influx through store-operated Ca(2+) channels cause a biphasic increase in intracellular Ca(2+) concentration ([Ca(2+)](i)), measured with Indo-1 dye. Single-cell amperometry demonstrated that this increase in [Ca(2+)](i) in turn causes a biphasic increase in exocytosis. A protein kinase assay revealed that trypsin also activates PKC isozymes to stimulate additional exocytosis. Paralleling the increased exocytosis, mucin secretion from PDEC was also induced by trypsin or the PAR-2 activating peptide. Consistent with the serosal localization of PAR-2, 1 microm luminal trypsin did not induce exocytosis in polarized PDEC monolayers; on the other hand, 10 microm trypsin at 37 degrees C damaged the epithelial barrier sufficiently so that it could reach and activate the serosal PAR-2 to stimulate exocytosis. Thus, in PDEC, PAR-2 activation increases [Ca(2+)](i) and activates PKC to stimulate exocytosis and mucin secretion. These functions may mediate the reported protective role of PAR-2 in different models of pancreatitis.     

Cyclin-dependent kinase 5 associated with p39 promotes Munc18-1 phosphorylation and Ca(2+)-dependent exocytosis

Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine protein kinase that requires association with a regulatory protein, p35 or p39, to form an active enzyme. Munc18-1 plays an essential role in membrane fusion, and its function is regulated by phosphorylation. We report here that both p35 and p39 were expressed in insulin-secreting beta-cells, where they exhibited individual subcellular distributions and associated with membranous organelles of different densities. Overexpression of Cdk5, p35, or p39 showed that Cdk5 and p39 augmented Ca(2+)-induced insulin exocytosis. Suppression of p39 and Cdk5, but not of p35, by antisense oligonucleotides selectively inhibited insulin exocytosis. Transient transfection of primary beta-cells with Munc18-1 templates mutated in potential Cdk5 or PKC phosphorylation sites, in combination with Cdk5 and the different Cdk5 activators, suggested that Cdk5/p39-promoted Ca(2+)-dependent insulin secretion from primary beta-cells by phosphorylating Munc18-1 at a biochemical step immediately prior to vesicle fusion.     

Barium and Calcium Stimulate Secretion from Digitonin-Permeabilized Bovine Adrenal Chromaffin Cells by Similar Pathways

We compared the characteristics of secretion stimulated by EGTA-buffered Ba(2+)- and Ca(2+)-containing solutions in digitonin-permeabilized bovine adrenal chromaffin cells. Half-maximal secretion occurred at approximately 100 microM Ba2+ or 1 microM Ca2+. Ba(2+)-stimulated release was not due to release of sequestered intracellular Ca2+ because at a constant free Ba2+ concentration, increasing unbound EGTA did not diminish the extent of release due to Ba2+. The maximal extents of Ba(2+)- and Ca(2+)-dependent secretion in the absence of MgATP were identical. MgATP enhanced Ba(2+)-induced secretion to a lesser extent than Ca(2+)-induced secretion. Half-maximal concentrations of Ba2+ and Ca2+, when added together to cells, yielded approximately additive amounts of secretion. Maximal concentrations of Ba2+ and Ca2+ when added together to cells for 2 or 15 min were not additive. Tetanus toxin inhibited Ba(2+)- and Ca(2+)-dependent secretion to a similar extent. Ba2+, unlike Ca2+, did not activate polyphosphoinositide-specific phospholipase C. These data indicate that (1) Ba2+ directly stimulates exocytosis, (2) Ba(2+)-induced secretion is stimulated to a lesser extent than Ca(2+)-dependent secretion by MgATP, (3) Ba2+ and Ca2+ use similar pathways to trigger exocytosis, and (4) exocytosis from permeabilized cells does not require activation of polyphosphoinositide-specific phospholipase C.     

Phosphatidylinositol 4,5-bisphosphate rescues TRPM4 channels from desensitization

TRPM4 is a Ca(2+)-activated nonselective cation channel that regulates membrane potential in response to intracellular Ca(2+) signaling. In lymphocytes it plays an essential role in shaping the pattern of intracellular Ca(2+) oscillations that lead to cytokine secretion. To better understand its role in this and other physiological processes, we investigated mechanisms by which TRPM4 is regulated. TRPM4 was expressed in ChoK1 cells, and currents were measured in excised patches. Under these conditions, TRPM4 currents were activated by micromolar concentrations of cytoplasmic Ca(2+) and progressively desensitized. Here we show that desensitization can be explained by a loss of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) from the channels. Poly-l-lysine, a PI(4,5)P(2) scavenger, caused rapid desensitization, whereas MgATP, at concentrations that activate lipid kinases, promoted recovery of TRPM4 currents. Application of exogenous PI(4,5)P(2) to the intracellular surface of the patch restored the properties of TRPM4 currents. Our results suggest that PI(4,5)P(2) acts to uncouple channel opening from changes in the transmembrane potential, allowing current activation at physiological voltages. These data argue that hydrolysis of PI(4,5)P(2) underlies desensitization of TRPM4 and support the idea that PI(4,5)P(2) is a general regulator for the gating of TRPM ion channels.     

Phosphatidylinositol 4,5-bisphosphate mediates Ca2+-induced platelet alpha-granule secretion: evidence for type II phosphatidylinositol 5-phosphate 4-kinase function

To understand the molecular basis of granule release from platelets, we examined the role of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) in alpha-granule secretion. Streptolysin O-permeabilized platelets synthesized PtdIns(4,5)P(2) when incubated in the presence of ATP. Incubation of streptolysin O-permeabilized platelets with phosphatidylinositol-specific phospholipase C reduced PtdIns(4,5)P(2) levels and resulted in a dose- and time-dependent inhibition of Ca(2+)-induced alpha-granule secretion. Exogenously added PtdIns(4,5)P(2) inhibited alpha-granule secretion, with 80% inhibition at 50 microm PtdIns(4,5)P(2). Nanomolar concentrations of wortmannin, 33.3 microm LY294002, and antibodies directed against PtdIns 3-kinase did not inhibit Ca(2+)-induced alpha-granule secretion, suggesting that PtdIns 3-kinase is not involved in alpha-granule secretion. However, micromolar concentrations of wortmannin inhibited both PtdIns(4,5)P(2) synthesis and alpha-granule secretion by approximately 50%. Antibodies directed against type II phosphatidylinositol-phosphate kinase (phosphatidylinositol 5-phosphate 4-kinase) also inhibited both PtdIns(4,5)P(2) synthesis and Ca(2+)-induced alpha-granule secretion by approximately 50%. These antibodies inhibited alpha-granule secretion only when added prior to ATP exposure and not when added following ATP exposure, prior to Ca(2+)-mediated triggering. The inhibitory effects of micromolar wortmannin and anti-type II phosphatidylinositol-phosphate kinase antibodies were additive. These results show that PtdIns(4,5)P(2) mediates platelet alpha-granule secretion and that PtdIns(4,5)P(2) synthesis required for Ca(2+)-induced alpha-granule secretion involves the type II phosphatidylinositol 5-phosphate 4-kinase-dependent pathway.     

Non-additive potentiation of glutamate release by phorbol esters and metabotropic mGlu7 receptor in cerebrocortical nerve terminals

We recently showed that prolonged activation of metabotropic glutamate receptor 7 (mGlu7) potentiates glutamate release. This signalling involves phospholipase C activation via a pertussis toxin insensitive G protein and the subsequent hydrolysis of phosphatidylinositol (4,5)-bisphosphate. Release potentiation is independent of protein kinase C activation but it is dependent on the downstream release machinery, as reflected by the concomitant translocation of active zone Munc13-1 protein from the soluble to particulate fractions. Here we show that phorbol ester and mGlu7 receptor-dependent facilitation of neurotransmitter release is not additive, suggesting they share a common signalling mechanism. However, release potentiation is restricted to release sites that express N-type Ca(2+) channels, because phorbol ester and mGlu7 receptor-mediated release potentiation are absent in nerve terminals from mice lacking N-type Ca(2+) channels. In addition, phorbol esters but not mGlu7 receptors potentiate release at nerve terminals with P/Q-type Ca(2+) channels, although only under restricted conditions of Ca(2+) influx. The differential effect of phorbol esters at nerve terminals with either N- or P/Q-type Ca(2+) channels seems to be unrelated to the type Munc13 isoform expressed, and it is more likely dependent on other properties of the release machinery.     

PLCζ causes Ca(2+) oscillations in mouse eggs by targeting intracellular and not plasma membrane PI(4,5)P(2)

Sperm-specific phospholipase C ζ (PLCζ) activates embryo development by triggering intracellular Ca(2+) oscillations in mammalian eggs indistinguishable from those at fertilization. Somatic PLC isozymes generate inositol 1,4,5-trisphophate-mediated Ca(2+) release by hydrolyzing phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) in the plasma membrane. Here we examine the subcellular source of PI(4,5)P(2) targeted by sperm PLCζ in mouse eggs. By monitoring egg plasma membrane PI(4,5)P(2) with a green fluorescent protein-tagged PH domain, we show that PLCζ effects minimal loss of PI(4,5)P(2) from the oolemma in contrast to control PLCδ1, despite the much higher potency of PLCζ in eliciting Ca(2+) oscillations. Specific depletion of this PI(4,5)P(2) pool by plasma membrane targeting of an inositol polyphosphate-5-phosphatase (Inp54p) blocked PLCδ1-mediated Ca(2+) oscillations but not those stimulated by PLCζ or sperm. Immunolocalization of PI(4,5)P(2), PLCζ, and catalytically inactive PLCζ (ciPLCζ) revealed their colocalization to distinct vesicular structures inside the egg cortex. These vesicles displayed decreased PI(4,5)P(2) after PLCζ injection. Targeted depletion of vesicular PI(4,5)P(2) by expression of ciPLCζ-fused Inp54p inhibited the Ca(2+) oscillations triggered by PLCζ or sperm but failed to affect those mediated by PLCδ1. In contrast to somatic PLCs, our data indicate that sperm PLCζ induces Ca(2+) mobilization by hydrolyzing internal PI(4,5)P(2) stores, suggesting that the mechanism of mammalian fertilization comprises a novel phosphoinositide signaling pathway.     

Munc18-1 and Munc18-2 proteins modulate beta-cell Ca2+ sensitivity and kinetics of insulin exocytosis differently

Fast neurotransmission and slower hormone release share the same core fusion machinery consisting of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins. In evoked neurotransmission, interactions between SNAREs and the Munc18-1 protein, a member of the Sec1/Munc18 (SM) protein family, are essential for exocytosis, whereas other SM proteins are dispensable. To address if the exclusivity of Munc18-1 demonstrated in neuroexocytosis also applied to fast insulin secretion, we characterized the presence and function of Munc18-1 and its closest homologue Munc18-2 in β-cell stimulus-secretion coupling. We show that pancreatic β-cells express both Munc18-1 and Munc18-2. The two Munc18 homologues exhibit different subcellular localization, and only Munc18-1 redistributes in response to glucose stimulation. However, both Munc18-1 and Munc18-2 augment glucose-stimulated hormone release. Ramp-like photorelease of caged Ca(2+) and high resolution whole-cell patch clamp recordings show that Munc18-1 and Munc18-2 overexpression shift the Ca(2+) sensitivity of the fastest phase of insulin exocytosis differently. In addition, we reveal that Ca(2+) sensitivity of exocytosis in β-cells depends on the phosphorylation status of the Munc18 proteins. Even though Munc18-1 emerges as the key SM-protein determining the Ca(2+) threshold for triggering secretory activity in a stimulated β-cell, Munc18-2 has the ability to increase Ca(2+) sensitivity and thus mediates the release of fusion-competent granules requiring a lower cytoplasmic-free Ca(2+) concentration, [Ca(2+)](i)(.) Hence, Munc18-1 and Munc18-2 display distinct subcellular compartmentalization and can coordinate the insulin exocytotic process differently as a consequence of the actual [Ca(2+)](i).     

Relationship between arachidonic acid release and Ca2(+)-dependent exocytosis in digitonin-permeabilized bovine adrenal chromaffin cells.

The relationship between Ca2(+)-dependent arachidonic acid release and exocytosis from digitonin-permeabilized bovine adrenal chromaffin cells was investigated. The phospholipase A2 inhibitors mepacrine, nordihydroguaiaretic acid and indomethacin had no effect on either arachidonic acid release or secretion. The phospholipase A2 activator melittin had no effect on secretion. The specific diacylglycerol lipase inhibitor RG80267 had no effect on secretion, but decreased basal arachidonic acid release to such an extent that the level of arachidonic acid in treated cells in response to 10 microM-Ca2+ was equivalent to that of control cells in the absence of Ca2+. Staurosporine, a protein kinase C inhibitor, was found to abolish Ca2(+)-dependent arachidonic acid release completely, but had only a slight inhibitory effect on Ca2(+)-dependent secretion. It is concluded that arachidonic acid is not essential for Ca2(+)-dependent exocytosis in adrenal chromaffin cells.     

The activation of exocytotic sites by the formation of phosphatidylinositol 4,5-bisphosphate microdomains at syntaxin clusters

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a minor component of the lipid bilayer but plays an important role in various cellular functions, including exocytosis and endocytosis. Recently, PI(4,5)P2 was shown to form microdomains in the plasma membrane. In this study, we investigated the relationship between the spatial organization of PI(4,5)P2 microdomains and exocytotic machineries in clonal rat pheochromocytoma PC12 cells. Both PI(4,5)P2 and syntaxin, a soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein essential for exocytosis, exhibited punctate clusters in isolated plasma membranes. The number of PI(4,5)P2 microdomains colocalizing with syntaxin clusters and large dense core vesicles (LDCVs) was decreased after catecholamine release. Alternatively, the expression of type I phosphatidylinositol-4-phosphate 5-kinase (PIP5KI) increased the number of PI(4,5)P2 microdomains at syntaxin clusters with docked LDCVs and enhanced exocytotic activity, possibly by increasing the number of release sites. About half of the PI(4,5)P2 microdomains were not colocalized with Thy-1, a specific marker of lipid rafts, and the colocalization of transfected PIP5KI with syntaxin clusters was observed. These results suggest that the formation of PI(4,5)P2 microdomains at syntaxin clusters with docked LDCVs is essential for Ca2+-dependent exocytosis.     

Munc13 homology domain-1 in CAPS/UNC31 mediates SNARE binding required for priming vesicle exocytosis

Neuropeptide and peptide hormone secretion from neural and endocrine cells occurs by Ca(2+)-triggered dense-core vesicle exocytosis. The membrane fusion machinery consisting of vesicle and plasma membrane SNARE proteins needs to be assembled for Ca(2+)-triggered vesicle exocytosis. The related Munc13 and CAPS/UNC31 proteins that prime vesicle exocytosis are proposed to promote SNARE complex assembly. CAPS binds SNARE proteins and stimulates SNARE complex formation on liposomes, but the relevance of SNARE binding to CAPS function in cells had not been determined. Here we identify a core SNARE-binding domain in CAPS as corresponding to Munc13 homology domain-1 (MHD1). CAPS lacking a single helix in MHD1 was unable to bind SNARE proteins or to support the Ca(2+)-triggered exocytosis of either docked or newly arrived dense-core vesicles. The results show that MHD1 is a SNARE-binding domain and that SNARE protein binding is essential for CAPS function in dense-core vesicle exocytosis.     

A pleckstrin homology domain specific for phosphatidylinositol 4, 5-bisphosphate (PtdIns-4,5-P2) and fused to green fluorescent protein identifies plasma membrane PtdIns-4,5-P2 as being important in exocytosis

Kinetically distinct steps can be distinguished in the secretory response from neuroendocrine cells with slow ATP-dependent priming steps preceding the triggering of exocytosis by Ca(2+). One of these priming steps involves the maintenance of phosphatidylinositol 4, 5-bisphosphate (PtdIns-4,5-P(2)) through lipid kinases and is responsible for at least 70% of the ATP-dependent secretion observed in digitonin-permeabilized chromaffin cells. PtdIns-4,5-P(2) is usually thought to reside on the plasma membrane. However, because phosphatidylinositol 4-kinase is an integral chromaffin granule membrane protein, PtdIns-4,5-P(2) important in exocytosis may reside on the chromaffin granule membrane. In the present study we have investigated the localization of PtdIns-4,5-P(2) that is involved in exocytosis by transiently expressing in chromaffin cells a pleckstrin homology (PH) domain that specifically binds PtdIns-4, 5-P(2) and is fused to green fluorescent protein (GFP). The PH-GFP protein predominantly associated with the plasma membrane in chromaffin cells without any detectable association with chromaffin granules. Rhodamine-neomycin, which also binds to PtdIns-4,5-P(2), showed a similar subcellular localization. The transiently expressed PH-GFP inhibited exocytosis as measured by both biochemical and electrophysiological techniques. The results indicate that the inhibition was at a step after Ca(2+) entry and suggest that plasma membrane PtdIns-4,5-P(2) is important for exocytosis. Expression of PH-GFP also reduced calcium currents, raising the possibility that PtdIns-4,5-P(2) in some manner alters calcium channel function in chromaffin cells.     

Novel function of phosphoinositide 3-kinase in T cell Ca2+ signaling. A phosphatidylinositol 3,4,5-trisphosphate-mediated Ca2+ entry mechanism

This study presents evidence that phosphoinositide (PI) 3-kinase is involved in T cell Ca(2+) signaling via a phosphatidylinositol 3,4, 5-trisphosphate PI(3,4,5)P(3)-sensitive Ca(2+) entry pathway. First, exogenous PI(3,4,5)P(3) at concentrations close to its physiological levels induces Ca(2+) influx in T cells, whereas PI(3,4)P(2), PI(4, 5)P(2), and PI(3)P have no effect on [Ca(2+)](i). This Ca(2+) entry mechanism is cell type-specific as B cells and a number of cell lines examined do not respond to PI(3,4,5)P(3) stimulation. Second, inhibition of PI 3-kinase by wortmannin and by overexpression of the dominant negative inhibitor Deltap85 suppresses anti-CD3-induced Ca(2+) response, which could be reversed by subsequent exposure to PI(3,4,5)P(3). Third, PI(3,4,5)P(3) is capable of stimulating Ca(2+) efflux from Ca(2+)-loaded plasma membrane vesicles prepared from Jurkat T cells, suggesting that PI(3,4,5)P(3) interacts with a Ca(2+) entry system directly or via a membrane-bound protein. Fourth, although D-myo-inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4, 5)P(4)) mimics PI(3,4,5)P(3) in many aspects of biochemical functions such as membrane binding and Ca(2+) transport, we raise evidence that Ins(1,3,4,5)P(4) does not play a role in anti-CD3- or PI(3,4,5)P(3)-mediated Ca(2+) entry. This PI(3,4,5)P(3)-stimulated Ca(2+) influx connotes physiological significance, considering the pivotal role of PI 3-kinase in the regulation of T cell function. Given that PI 3-kinase and phospholipase C-gamma form multifunctional complexes downstream of many receptor signaling pathways, we hypothesize that PI(3,4,5)P(3)-induced Ca(2+) entry acts concertedly with Ins(1,4,5)P(3)-induced Ca(2+) release in initiating T cell Ca(2+) signaling. By using a biotinylated analog of PI(3,4,5)P(3) as the affinity probe, we have detected several putative PI(3,4,5)P(3)-binding proteins in T cell plasma membranes.     

Phosphatidylinositol 4-phosphate 5-kinase alpha is a downstream effector of the small G protein ARF6 in membrane ruffle formation

Synthesis of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], a signaling phospholipid, is primarily carried out by phosphatidylinositol 4-phosphate 5-kinase [PI(4)P5K], which has been reported to be regulated by RhoA and Rac1. Unexpectedly, we find that the GTPgammaS-dependent activator of PI(4)P5Kalpha is the small G protein ADP-ribosylation factor (ARF) and that the activation strictly requires phosphatidic acid, the product of phospholipase D (PLD). In vivo, ARF6, but not ARF1 or ARF5, spatially coincides with PI(4)P5Kalpha. This colocalization occurs in ruffling membranes formed upon AIF4 and EGF stimulation and is blocked by dominant-negative ARF6. PLD2 similarly translocates to the ruffles, as does the PH domain of phospholipase Cdelta1, indicating locally elevated PI(4,5)P2. Thus, PI(4)P5Kalpha is a downstream effector of ARF6 and when ARF6 is activated by agonist stimulation, it triggers recruitment of a diverse but interactive set of signaling molecules into sites of active cytoskeletal and membrane rearrangement.