Role of Phosphoinositide Signaling in the Control of Insulin Exocytosis
Laurent Waselle,
Roy R. L. Gerona,
Nicolas Vitale,
Thomas F. J. Martin,
Marie-France Bader and
Romano Regazzi
Department of Cell Biology and Morphology, University of Lausanne (L.W., R.R.), Lausanne 1005, Switzerland; Department of Biochemistry, University of Wisconsin (R.R.L.G., T.F.J.M.), Madison, Wisconsin 53706; and Neurotransmission and Neuroendocrine Secretion, Centre National de la Recherche Scientifique, Unit 2356 (N.V., M.-F.B.), Strasbourg 67084, France
Address all correspondence and requests for reprints to: Dr. Romano Regazzi, Department of Cell Biology and Morphology, rue du Bugnon 9, 1005 Lausanne, Switzerland. E-mail: romano.regazzi{at}unil.ch.
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ABSTRACT
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Phosphoinositides (PI) are important signaling molecules involved in the regulation of vesicular trafficking. We found that phosphatidylinositol 4-phosphate (PI4P) and phosphatidylinositol 4,5-biphosphate [PI(4,5)P2] increase the secretory response triggered by 10 µM Ca2+ in streptolysin-O-permeabilized insulin-secreting INS-1E cells. In addition, nutrient-induced exocytosis was diminished in intact cells expressing constructs that sequester PI(4,5)P2 and in cells transfected with constructs that reduce by RNA interference the level of two enzymes involved in PI(4,5)P2 production, type III PI4-kinase ß and type I phosphatidylinositol 4-bisphosphate 5-kinase-
. To clarify the mechanism of action of PI, we investigated the involvement in the regulation of insulin exocytosis of three potential PI targets, phospholipase D1, the Ca2+-dependent activator protein for secretion 1, and Munc18-interacting protein 1. Transfection of insulin-secreting cells with plasmids that direct the synthesis of small interfering RNAs capable of reducing the endogenous levels of these proteins inhibited hormone release elicited by glucose- and cAMP-elevating agents without affecting basal release. Our data indicate that the production of PI(4,5)P2 is necessary for proper control of ß-cell secretion and suggest that at least part of the effect of PI on insulin exocytosis could be exerted through the activation of phospholipase D1, Ca2+-dependent activator protein for secretion 1, and Munc18-interacting protein 1.
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INTRODUCTION
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THE RELEASE OF insulin by pancreatic ß-cells plays a key role in the control of blood glucose levels. Indeed, insufficient supply of this hormone leads to hyperglycemia, glucose intolerance, and, eventually, to diabetes mellitus. Insulin exocytosis is mainly determined by plasma glucose levels and the concentrations of amino acids and fatty acids. However, a variety of neural and hormonal factors modulate the amplitude of the secretory response to nutrients (1, 2, 3). Early manifestations of ß-cell dysfunction include delayed and blunted responses to glucose and loss of tight stimulus-secretion coupling (4). To define the causes of ß-cell failure, a basic understanding of the physiology of normal insulin secretion is essential. Despite considerable progress in identification of the protein machinery required for insulin secretion (3, 5), the contribution of lipids in the regulation of ß-cell exocytosis is still largely unknown. The fusion of secretory granules with the plasma membrane necessitates the rearrangement of lipid bilayers. This rearrangement is facilitated by the presence at the site of fusion of lipids with noncylindrical shape, such as fatty acids and phosphatidic acid (6). The lipid compositions of the secretory granule membrane and the plasma membrane are also crucial for the binding of proteins involved in the control of exocytosis. In fact, accumulation of negatively charged phospholipids at the cytoplasmic leaflet of the membranes allows interaction with positively charged protein surfaces and selective recruitment of regulatory components of the exocytotic machinery. Phosphoinositides (PI) constitute an important class of negatively charged phospholipids that serve as signaling molecules in several intracellular vesicular trafficking steps, including exocytosis and endocytosis (7, 8, 9, 10). Generation of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] at defined locations in the cellular membranes permits selective recruitment of proteins endowed with specific PI(4,5)P2 binding modules, such as the pleckstrin homology (PH) domain (7, 8, 9, 10).
The major pathway for the generation of PI(4,5)P2 involves two sequential phosphorylations, at positions 4 and 5 of the inositol ring of phosphatidylinositol, by PI4- and phosphatidylinositol 4-bisphosphate (PIP) (PIP4) 5-kinases, respectively (7, 8, 9, 10). In pancreatic ß-cells, appropriate levels of PI(4,5)P2 at the plasma membrane are required to sustain insulin secretion, and a type III PI4-kinase (PI4K) has been proposed to serve as a metabolic sensor, coupling glucose metabolism to hormone release (11).
In this study we attempted to identify the PI4K and PI5K involved in the control of insulin exocytosis and to determine the roles played in this process by proteins interacting with PI(4,5)P2: phospholipase D-1 (PLD1), the Ca2+-dependent activator protein for secretion 1 (CAPS1), and Mint-1 (Munc18-interacting protein).
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RESULTS
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To evaluate the role of PIs in the regulation of ß-cell exocytosis, INS-1E cells, permeabilized with the pore-forming toxin streptolysin-O, were incubated with 10 µM PI(4)P, PI(4,5)P2, or PI(3,4,5)P3. None of the inositol-containing phospholipids affected hormone secretion occurring under resting conditions (0.1 µM free Ca2+; not shown), but the secretory response elicited by 10 µM free Ca2+ was enhanced in cells incubated with PI(4)P or PI(4,5)P2 (Fig. 1
). PI(3,4,5)P3 had no significant effect on Ca2+-induced exocytosis. To assess the involvement of PIs in nutrient-induced secretion, INS-1E cells were transiently transfected with green fluorescence protein (GFP)-tagged constructs, leading to expression of the PH domain of phospholipase C
(PLC
). The PH domain of PLC
specifically binds and sequesters PI(4,5)P2 (12). To monitor the effects of the PH domains on exocytosis, the cells were cotransfected with a second plasmid encoding human GH (hGH). Because hGH is targeted to ß-cell secretory granules and is coreleased with insulin, the secretion of this hormone serves as a reporter for exocytosis in transiently transfected cells. As shown in Fig. 2
, overexpression of the PI(4,5)P2 binding domain led to a strong reduction in stimulus-induced secretion. Basal secretion was not affected (not shown). Overexpression of a mutant of PH-PLC
that is unable to bind PI(4,5)P2 (12) displayed no effect on exocytosis (Fig. 2
). These data indicate that the presence of appropriate levels of PI(4,5)P2 is needed to sustain insulin exocytosis in INS-1E cells.

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Fig. 1. PI(4)P and PI(4,5)P2 Potentiate Ca2+-Induced Exocytosis in Permeabilized INS-1E Cells
Cells of the insulin-secreting ß-cell line INS-1E were permeabilized with streptolysin-O and preincubated for 7 min. The cells were then incubated for another 7-min period in either a buffer containing 0.1 µM free Ca2+ (basal condition) or a buffer containing 10 µM free Ca2+ (stimulatory condition). The indicated PIs were added at a concentration of 10 µM during the preincubation and incubation periods. At the end of the incubation, the supernatants were collected and analyzed by ELISA to assess exocytosis. Ca2+-stimulated exocytosis was defined by the ratio between secretion at 10 and 0.1 µM Ca2+. The results are the mean ± SEM of five to seven independent experiments performed in triplicate. In control cells, incubation at 10 µM free Ca2+ increased hormone release by 2.7 ± 0.3-fold. **, Significantly different from control (P < 0.01).
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Fig. 2. Exocytosis of INS-1E Cells Is Inhibited by the Introduction of Protein Domains that Sequester PI(4,5)P2
INS-1E cells were transiently transfected with an empty vector (control), a construct expressing the PH domain of PLC (PLC-PH), or a construct expressing a mutant of the PH domain of PLC unable to bind PI(4,5)P2 (PLC-PH mut). Equal expression of the two GFP-tagged PH constructs was verified by analyzing the fluorescence of transfected cells. To monitor the effect on exocytosis, the cells were cotransfected with a plasmid encoding hGH. Three days later, the cells were incubated for 45 min in the presence or absence of 20 mM glucose, forskolin (1 µM), 3-isobutyl-1-methylxanthine (1 mM), and depolarizing concentrations of K+. hGH release was measured by ELISA. Stimulated exocytosis was defined by the ratio of hGH release measured in the presence and absence of secretagogues. The results are the mean ± SEM of four independent experiments performed in triplicate. In control cells, the secretagogues increased hGH release 11.9 ± 2.5-fold. **, Significantly different from control (P < 0.01).
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Next, we searched for the enzymes involved in the synthesis of the pool of PI(4,5)P2 required for ß-cell exocytosis. PI(4,5)P2 is produced by the concerted action of two kinases, PI4K and PIP5K (7, 8). Several PI4Ks with different properties and functions have been described (13). Incubation of mouse islets with phenylarsine oxide, a pharmacological inhibitor of type III PI4Ks, has been reported to inhibit insulin secretion (11). In agreement with these findings, preincubation of INS-1E cells for 30 min in the presence of phenylarsine oxide (30 µM) severely hampered the secretory response triggered by glucose- and cAMP-elevating agents (18 ± 6%; n =3) compared with control cells. PI4Kß is a type III kinase that is involved in different vesicular trafficking steps (14, 15, 16, 17). To investigate the potential role of this protein in the regulation of insulin secretion, we attempted to diminish its expression level using the RNA interference process (18). To this end we generated plasmids that allow the synthesis of short, double-stranded RNA molecules, small interfering RNA (siRNAs), directed against sequences of PI4Kß. The sequences chosen are conserved among humans, rats, and mice. To verify the efficacy of the constructs, each plasmid was transiently cotransfected in INS-1E cells with hemagglutinin (HA)-tagged PI4Kß. As shown in Fig. 3A
(top panel), coexpression of siPIK4ß-a resulted in a very strong inhibition of HA-tagged PI4Kß expression. In contrast, a second plasmid leading to the synthesis of a different siRNA (siPIK4ß-i) was inactive. The expression of other unrelated proteins, such as GFP, was not affected by the silencers (not shown). We then verified the effects of the silencers on the expression of endogenous PI4Kß. Under our experimental conditions, the transfection efficiency varied between 3050%. Despite this relatively low transfection efficacy, we were able to detect a decrease in the endogenous level of PI4Kß in homogenates of cells transfected with siPIK4ß-a, but not with siPIK4ß-i (Fig. 3A
, bottom panel). The expression of other proteins participating in the control of insulin secretion, such as granuphilin and syntaxin-1, was not affected by the silencers (not shown). This observation demonstrates that our active construct can efficiently diminish the level of PI4Kß in INS-1E cells. To determine whether silencing of PI4Kß affects the synthesis of PI(4,5)P2, INS-1E cells were transiently cotransfected with siPIK4ß-a or siPIK4ß-i and with GFP-tagged PH-PLC
. After 3 d in culture, the cells were incubated for 45 min under basal conditions or in the presence of stimulatory concentrations of glucose- and cAMP-elevating agents. They were then fixed, and the distribution of GFP-tagged PH-PLC
was determined by confocal microscopy. Under basal conditions, the ratio between the GFP fluorescence located at the plasma membrane and in the cytosol was about 3 (Fig. 3B
). This ratio was not significantly different in cells transfected with siPIK4ß-a and siPIK4ß-i. Under stimulatory conditions, the plasma membrane to cytosol ratio in cells transfected with the inactive silencer was increased, whereas in cells in which PI4Kß was silenced, the ratio tended to decrease (Fig. 3B
). These data indicate that the activity of PI4Kß is necessary to sustain PI(4,5)P2 levels in the presence of insulin secretagogues. We then tested whether the impairment in PI(4,5)P2 production is associated with a defect in secretion. Cotransfection of INS-1E cells with hGH and siPIK4ß-a or siPIK4ß-i revealed that silencing of PI4Kß results in a defect in stimulus-induced secretion (Fig. 4A
). As expected, transfection of siPIK4ß-i had no significant effect on exocytosis. Very similar data were obtained when the same experiment was performed in the well-differentiated mouse insulin-secreting cell line MIN6 B1 (19) (Fig. 4B
). These findings suggest that the activity of PI4Kß is required for maintaining an appropriate pool of PI(4)P during insulin secretion.

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Fig. 3. Silencing of PI4Kß by RNA Interference Leads to a Decrease in Plasma Membrane-Associated PI(4,5)P2
A, Upper panel, INS-1E cells were transiently cotransfected with HA-tagged PI4Kß and with an empty vector (control) or pSUPER constructs that led to the synthesis of siRNAs directed against PI4Kß (siPIK4ß-a and siPIK4ß-i). Three days later, the cells were homogenized, and the expression of HA-tagged PI4Kß was analyzed by Western blotting using an anti-HA antibody. Lower panel, INS-1E cells were transiently transfected with an empty vector (control), siPIK4ß-a, or siPIK4ß-i. Three days later, the cells were homogenized, and the expression of endogenous PIK4ß was analyzed by Western blotting using an anti-PIK4ß antibody. B, INS-1E cells were transiently transfected with a construct expressing the PH domain of PLC (PLC-PH) and with siPIK4ß-a or siPIK4ß-i. Three days later, the cells were analyzed by confocal microscopy, and the ratio between the fluorescence intensity at the plasma membrane and that in the cytosol was determined. Under stimulatory conditions, the plasma membrane/cytosol ratio of the cells transfected with siPIK4ß-a was significantly different (P < 0.01) from that of the cells transfected with the inactive silencer (siPIK4ß-i).
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Fig. 4. Silencing of PI4Kß by RNA Interference Leads to a Decrease in Stimulus-Induced Exocytosis in INS-1E and MIN6 Cells
INS-1E (upper panel) or MIN6 (lower panel) were transiently cotransfected with the plasmid encoding hGH and with an empty vector (control), siPIK4ß-a or siPIK4ß-i. Three days later, the cells were incubated in the presence or absence of 20 mM glucose, 1 µM forskolin, 1 mM 3-isobutyl-1-methylxanthine, and depolarizing concentrations of K+. Exocytosis was monitored by ELISA. Stimulated exocytosis was defined as the ratio between the amount of hGH released in the presence and absence of secretagogues. The results are the mean ± SEM of three or four experiments. In control INS-1E cells, the secretagogues increased hGH release 8.2 ± 1.0-fold. In control MIN6 cells, the secretagogues increased hGH release 15.3 ± 0.6-fold. **, Significantly different from controls (P < 0.01).
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Type I PIP5K
is enriched in brain and pancreatic ß-cells (20, 21) and has very recently been involved in synaptic transmission and dense core vesicle fusion (22, 23). To evaluate the role of this enzyme in insulin exocytosis, we generated different plasmids driving the synthesis of siRNAs specifically directed against PIP5K
. As shown in Fig. 5A
, one of the plasmids (siPIP5K
-a) lowered the expression of epitope-tagged PIP5K
(upper panel) as well as that of the endogenous enzyme (lower panel). A second plasmid (siPIP5K
-i), in contrast, was without effect. PIP5K
activity was found to be required to sustain PI(4,5)P2 levels under stimulatory conditions. In fact, in the presence of insulin secretagogues, the plasma membrane to cytosol ratio of GFP-tagged PH-PLC was significantly lower in cells transfected with siPIP5K
-a compared with cells transfected with the inactive PIP5K
silencer (Fig. 5B
). Analysis of the secretory capacity of INS-1E and MIN6 cells transfected with these plasmids revealed that reduced levels of PIP5K
are associated with impairment in stimulus-induced secretion (Fig. 6
). Basal secretion, in contrast, was not affected (not shown).

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Fig. 5. Silencing of PIP5K by RNA Interference Causes a Reduction in Plasma Membrane-Associated PI(4,5)P2
A, Upper panel, INS-1E cells were transiently cotransfected with HA-tagged PIP5K and with an empty vector (control) or siRNA constructs directed against PIP5K (siPIP5K -i and siPIP5K -a). After 3 d, the expression of HA-tagged PIP5K was determined by Western blotting using an anti-HA antibody. Lower panel, INS-1E cells transiently transfected with an empty vector (control), siPIP5K -i, or siPIPK5 -a were homogenized and processed by Western blotting to assess the expression of endogenous anti-PIP5K using a specific antibody. B, INS-1E cells were transiently transfected with the PLC-PH construct and with siPIP5K -i and siPIP5K -a. Three days later, the cells were analyzed by confocal microscopy, and the ratio between the fluorescence intensity at the plasma membrane and that in the cytosol was determined. Under stimulatory conditions, the plasma membrane/cytosol ratio of the cells transfected with siPIP5K -a was significantly lower (P < 0.01) than that of the cells transfected with siPIP5K -i.
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Fig. 6. Silencing of PIP5K by RNA Interference Causes a Reduction of Stimulus-Induced Exocytosis
INS-1E (upper panel) or MIN6 (lower panel) was transiently transfected with the hGH plasmid together with either an empty vector (control) or the siRNA-encoding constructs. After 3 d in culture, the cells were incubated in the presence or absence of 20 mM glucose, 1 µM forskolin, 1 mM 3-isobutyl-1-methylxanthine, and depolarizing concentrations of K+. Stimulated exocytosis was defined as the ratio between the amount of hGH released in the presence and absence of secretagogues. The results are the mean ± SEM of three independent experiments. In control INS-1E cells, the secretagogues increased hGH release 9.2 ± 0.7-fold. In control MIN6 cells, the secretagogues increased hGH release 16.1 ± 1.7-fold. Asterisks indicate a significant difference from controls (*, P < 0.05; **, P < 0.01).
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Having demonstrated the importance of PI(4,5)P2 production for insulin secretion, we attempted to assess the contributions of known PI-binding proteins to the regulation of ß-cell exocytosis. PLD1 is the main source of phosphatidic acid, a lipid that favors vesicle fusion (24). Several studies have demonstrated that the activity of PLD1 is enhanced by PI(4,5)P2 (25, 26). We constructed two plasmids producing siRNAs specifically directed against PLD1. Transfection of siPLD1-a potently reduced the expression of exogenous GFP-tagged PLD1 (Fig. 7A
, upper panel) and endogenous PLD1 (Fig. 7A
, lower panel). In contrast, siPLD1-i was without effect. Taking into account that Western blots in Fig. 7A
were performed with samples in which only part of the cells received siPLD1-a, silencing of endogenous PLD1 in INS-1E cells is probably nearly complete. To evaluate the role of PLD1 in insulin exocytosis, the PLD1 silencers were transiently cotransfected with the plasmid encoding hGH. As shown in Fig. 7B
, stimulus-induced secretion was inhibited by siPLD1-a, but not by siPLD1-i. Overexpression of PLD1 enhanced hormone release, confirming the involvement of this enzyme in insulin exocytosis. Overexpression of PLD2, another PLD isoform with a different subcellular localization and function, was without effect (not shown).

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Fig. 7. Involvement of PLD1 in Insulin Exocytosis
A, Upper panel, INS-1E cells were transiently transfected with GFP-tagged PLD1 together with an empty pSUPER vector or with plasmids directing the synthesis of PLD1-specific siRNAs (siPLD1-a and siPLD1-i). Control cells were transfected with an empty pSUPER vector alone. The expression of GFP-tagged PLD1 was analyzed 3 d later by Western blotting using an anti-GFP antibody. Lower panel, Homogenates of INS-1E cells transiently transfected with an empty vector (control) with siPLD1-a or siPLD1-i were analyzed by Western blotting using a PLD1-specific antibody. B, INS-1E cells (left panel) and MIN6 (right panel) were cotransfected with the plasmid encoding hGH and with an empty vector (control) or vectors directing the synthesis of GFP-tagged PLD1, siPLD1-a, and siPLD1-i. After 3 d in culture, the cells were incubated in the presence or absence of secretagogues. Stimulated exocytosis was determined by calculating the ratio between the amount of hGH released in the presence and absence of stimuli. The results are the mean ± SEM of three independent experiments. In control INS-1E cells, the secretagogues increased hGH release 11.2 ± 0.2-fold. In control MIN6 cells, the secretagogues increased hGH release 15.6 ± 2.0-fold. **, Significantly different from controls (P < 0.01).
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CAPS1 is a PI-binding protein associated with insulin-containing granules that participates in the control of exocytosis in endocrine and neuroendocrine cells (27, 28, 29, 30, 31, 32). The contribution of this protein to the regulation of the exocytotic process of ß-cells was investigated using an approach similar to that used for PLD1. Overexpression of wild-type CAPS1 did not significantly modify hormone secretion of INS-1E cells (Fig. 8B
). However, transfection of insulin-secreting cells with a plasmid (siCAPS-a) directing the synthesis of siRNAs capable of reducing the level of exogenous HA-tagged and of endogenous CAPS1 (Fig. 8A
) led to a decrease in exocytosis elicited by glucose- and cAMP-elevating agents (Fig. 8B
). This indicates that the presence of a critical level of CAPS1 is required to sustain insulin exocytosis.

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Fig. 8. CAPS1 Is Required for Insulin Exocytosis
A, Upper panel, INS-1E cells were transiently transfected with HA-tagged CAPS1 and an empty vector (control) or pSHAG1 vectors enabling the expression of CAPS1-specific siRNAs (siCAPS-a and siCAPS-i). The expression of HA-tagged CAPS1 was determined by Western blotting using an anti-HA antibody. Lower panel, Homogenates of INS-1E cells transiently transfected with an empty vector (control) with siCAPS-a or siCAPS-i were analyzed by Western blotting using a CAPS1-specific antibody. B, INS-1E cells (left panel) or MIN6 (right panel) were cotransfected with the plasmid encoding hGH and with an empty plasmid (control) or plasmids controlling the expression of CAPS1, siCAPS-a, or siCAPS-i. Three days later, the cells were exposed to basal or stimulatory conditions, and hGH release was measured by ELISA. Stimulated exocytosis was defined as the ratio between the amount of hGH secreted in the presence and absence of stimuli. The results are expressed as the mean ± SEM of three to five independent experiments performed in triplicate. In control INS-1E cells, the secretagogues elicited an increase of 11.6 ± 1.6-fold in hGH release. In control MIN6 cells, the secretagogues elicited an increase of 15.6 ± 1.9-fold in hGH release. Asterisks indicate a significant difference from controls (*, P < 0.05; **, P < 0.01).
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Mint1 is a PI-binding protein involved in synaptic transmission (33, 34). This adaptor protein is thought to recruit, at specific sites at the plasma membrane, Munc-18, a key component required for vesicle fusion (35). Mint1 is mainly expressed in neurons, but has recently also been found in pancreatic ß-cells (36). To assess the role of this protein in insulin exocytosis, we generated three plasmids directing the synthesis of siRNAs against Mint1. Transfection of siMint-a damped the expression of both exogenous and endogenous Mint1 (Fig. 9A
) and significantly reduced hormone release triggered by secretagogues (Fig. 9B
). In contrast, the other two plasmids (siMint-i.1 and siMint-i.2), which are unable to affect Mint1 expression, did not alter the secretory response of the cells. The rat sequence targeted by siMint-a is not conserved in mice. For this reason, the effect of this construct was not tested in mouse MIN6 cells.

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Fig. 9. Silencing of Mint1 Results in a Decrease in INS-1E Exocytosis
A, Upper panel, INS-1E cells were transiently transfected with Mint1 and an empty pSUPER (control) or with constructs leading to the production of Mint1-specific siRNAs (siMint-a, siMint-i.1, and siMint-i.2). The expression of Mint1 was determined by Western blotting. Lower panel, The expression of Mint1 in homogenates of INS-1E cells transiently transfected with pSUPER (control), siMint-a, siMint-i.1, or siMint-i.2 was examined by Western blotting using a Mint1 antibody. B, INS-1E cells were cotransfected with the plasmid expressing hGH and with pSUPER (control), siMint-a, siMint-i.1, or siMint-i.2. hGH release under basal or stimulatory conditions was assessed 3 d later by ELISA. The results are the mean ± SEM of three independent experiments performed in triplicate. In this series of experiments, control cells displayed an increase in hGH release of 7.1 ± 0.8-fold in the presence of glucose and cAMP-elevating agents. *, Significantly different from control (P < 0.05).
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DISCUSSION
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Insulin exocytosis is a central event in blood glucose homeostasis. The molecular mechanisms underlying this process are beginning to be elucidated, but the contribution of lipids to the regulation of secretory granule fusion is still poorly understood. PI(4,5)P2 is a negatively charged phospholipid, nonuniformly distributed on cellular membranes, that controls spatially confined membrane events, including vesicle exo- and endocytosis (7, 8, 9, 10). Exposure of pancreatic ß-cells to secretagogues is associated with increased PI turnover, and maintenance of an appropriate PI(4,5)P2 pool is a prerequisite for regulated secretion (37). Our data are consistent with a positive role for PI(4,5)P2 in the control of insulin release. In fact, in INS-1E cells exocytosis triggered by secretagogues is enhanced by introduction of PI(4,5)P2 and is impaired if the pool of this lipid is reduced by either sequestering the molecule or preventing its synthesis. The enzymes responsible for the replenishment of the pool of PI(4,5)P2 in secretagogue-stimulated ß-cells have been unknown to date. In this study we identify two PI kinases, PI4Kß and PIP5K
, that are necessary to sustain insulin exocytosis.
PI4Kß is a type III kinase involved in different vesicular trafficking events (14, 15, 16, 17). The activity of PI4Kß is positively regulated by neuronal Ca2+ sensor 1 and is a rate-limiting factor in the synthesis of the PI(4,5)P2 pool for stimulus-induced secretion in neuroendocrine and mast cells (15, 16, 17). Glucose, the main physiological stimulus for insulin release, promotes exocytosis by generating metabolic signals that lead to membrane depolarization and activation of the secretory machinery (1, 2, 3). In ß-cells, a type III PI4K has been proposed to function as a metabolic sensor, coupling glucose metabolism to insulin release (11). Moreover, in a very recent study published during the revision of this manuscript, neuronal Ca2+ sensor 1 was reported to activate PI4Kß in pancreatic ß-cells and to increase insulin release (38). Our data are fully consistent with these observations and support the idea of an important role for PI4Kß in the control of ß-cell exocytosis.
In streptolysin-O-permeabilized INS-1E cells, addition of PI(4)P has effects on exocytosis similar to those of PI(4,5)P2. The most likely explanation for this finding is that PI(4)P is rapidly converted to PI(4,5)P2. PIP5K
, is a cytosolic enzyme that is activated by the small GTPase ADP-ribosylation factor 6 (39). Depletion of this enzyme from plasma membrane is associated with inhibition of dense core granule exocytosis in PC12 cells (39). In addition, PIP5K
knockout mice display multiple defects in the synaptic vesicle cycle, including exo- and endocytosis and alteration in vesicle fusion dynamics (22, 23). The role of PIP5K
in pancreatic ß-cell secretion has been unknown to date. In this study we show that silencing of PIP5K
in INS-1E and MIN6 cells results in a decrease in hormone release, indicating a critical role for this enzyme in insulin secretion. ADP-ribosylation factor 6 activity is required to sustain PI(4,5)P2 levels during ß-cell stimulation and is necessary for proper response to insulin secretagogues (37). Our findings suggest that at least part of the effect of the guanosine phosphatase could be achieved by activation of PIP5K
.
The signaling role of PI(4,5)P2 is mediated through interactions with proteins involved in membrane trafficking that are endowed with PI(4,5)P2-binding modules, such as the PH domain. In this study we investigated the roles of three PI(4,5)P2-binding proteins: PLD1, CAPS1, and Mint1. PLD1 is a key factor in the regulation of neuroendocrine secretion (40). PI(4,5)P2 binding is necessary for the activity of the enzyme, and a mutant of PLD1 that cannot interact with PIs is unable to control exocytosis (40). In ß-cells, PLD activity is increased in response to a variety of secretagogues, and blockade of phosphatidic acid formation inhibits secretion (24). We found that in INS-1E cells, alteration of endogenous levels of PLD1, the predominant PLD isoform in ß-cells (24), is paralleled by corresponding changes in hormone release elicited by secretagogues. PLD1 is associated with different cellular compartments (24, 25, 26). Our experiments do not determine which pool of PLD1 is involved in exocytosis. Indeed, insulin exocytosis could potentially be promoted by the generation of phosphatidic acid at the secretory granules membrane, the plasma membrane, or both.
CAPS1 was initially discovered as a cytosolic brain protein required for calcium-dependent dense core vesicle exocytosis in permeabilized PC12 cells (27). This protein is expressed in several neural and endocrine tissues, including pancreatic islets, indicating a more widespread role in regulated exocytosis (11, 27). Our data support an important role for CAPS1 in the control of insulin secretion, and complement results obtained in electrophysiology set ups in which CAPS1 antibodies were used to inactivate the protein (11). CAPS1 is required for a prefusion step preceding Ca2+-induced exocytosis (32), but its precise mode of action is not yet fully elucidated. The protein has two distinct membrane association domains permitting concomitant binding to plasma membrane and secretory granules (41). This property could favor the approach of the two membranes during the final steps of exocytosis. CAPS1 possesses at least two distinct PI(4,5)P2-binding domains capable of guiding the protein to specific membrane compartments in which complexes required for vesicle fusion assemble. In addition, PI(4,5)P2 binding to CAPS1 induces conformational changes that may somehow modulate the interaction with other components of the exocytotic apparatus during the final steps of the secretory pathway.
Mint1 is a multidomain adaptor protein involved in synaptic vesicle exocytosis (33, 34, 35). Mint1 was initially identified in yeast two-hybrid screens for proteins interacting with Munc18-1, one of the main syntaxin-1 partners and a key component of the exocytotic machinery (33). Mint1 was later demonstrated to associate with a number of other neuronal proteins, including amyloid precursor protein, presenilins, and Ca2+ channels (35). In addition, the phosphotyrosine-binding domain of Mint1 specifically binds PI(4,5)P2. The expression of Mint1 was thought to be restricted to neurons, but a recent study reported the presence of the protein in pancreatic islets (36). In this study, we not only confirm the presence of Mint1 in INS-1E cells, but, taking advantage of the RNA interference approach, we demonstrate that this adaptor protein is an important player in ß-cell exocytosis. In neurons, Mint1 was proposed to function by recruiting Munc18-1 in the vicinity of the site of exocytosis and to favor docking of synaptic vesicles at the plasma membrane. Because Munc18-1 is required for insulin secretion (42), it is tempting to speculate that a similar mechanism might operate in ß-cells. Although the functional impact of PI binding is not yet established, the capacity to interact with PI(4,5)P2 could help in restricting the distribution of Mint1 and, consequently, of Munc18-1 to specific sites at the plasma membrane.
In this study we investigated the role of the PI signaling pathway in the regulation of insulin secretion. Silencing or overexpression of the proteins involved in PI signaling affected stimulated exocytosis, but not basal secretion. This suggests that these components are dispensable to sustain a relatively low fusion rate, but become limiting when the cells are stimulated. Our data demonstrate that PI4Kß and PIP5K
are necessary to provide appropriate levels of PI(4,5)P2 to support insulin exocytosis in response to glucose- and cAMP-elevating agents. In addition, we have identified three proteins whose activity requires PI binding that are directly involved in the exocytotic process of pancreatic ß-cells: PLD1, CAPS1, and Mint1. Each of these proteins appears to be needed for a full response to secretagogues. However, almost complete knockdown of these components results in partial inhibition of insulin secretion. It is probably not possible to directly correlate a decrease in the expression of a given protein with an impact on exocytosis. Indeed, if the amount of the protein is not rate limiting, an effect on hormone release would be apparent only below a given threshold. Moreover, other isoforms of CAPS and Mint present in ß-cells may in part compensate for the loss of CAPS1 and Mint1.
In conclusion, we have demonstrated the importance of PI signaling in the control of insulin exocytosis. Our findings have certainly not exhausted the potential targets of PI(4,5)P2 and do not rule out other possible actions mediated by these signaling molecules on cytoskeletal proteins, ion channels, or other components of the exocytotic machinery that could influence the secretory process of pancreatic ß-cells.
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MATERIALS AND METHODS
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The antibody against type I PI5K
was provided by Dr. G. Di Paolo (Yale University, New Haven, CT). Antibodies against PI4Kß, PLD1, and Mint1 were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY), BioSource International (Camarillo, CA), and BD Transduction Laboratories (San Diego, CA), respectively. The antibody against CAPS1 has been described previously (27). Secondary antibodies coupled to horseradish peroxidase were purchased from Sigma-Aldrich Corp. (St. Louis, MO). Phosphatidyl-D-myo-inositol-4-phosphate, phosphatidyl-D-myo-inositol-4,5-bisphosphate, and phosphatidylinositol-3,4,5-trisphosphate were obtained from Calbiochem (San Diego, CA). Phenylarsine oxide was purchased from Sigma-Aldrich Corp. The pSUPER plasmid (43) was provided by Dr. Agami (Netherlands Cancer Institute, Amsterdam, The Netherlands). The pSHAG1 vector was obtained from Dr. G. J. Hannon (Cold Spring Harbor Laboratory, Plainview, NY). The plasmids encoding GFP-tagged, wild-type PH-PLC
and PH-PLC
(S34T, R40L) were gifts from Dr. R. Holz (Michigan University, Ann Arbor, MI). HA-tagged PI4Kß, GFP-tagged PLD1, and Mint1 were provided by Dr. M. A. De Matteis (Consorzio Mario Negri Sud, Santa Maria Imbaro, Italy), Dr. M. A. Frohman (State University of New York, New York, NY), and Dr. T. Südhof (Texas University, Dallas, TX), respectively. Generation of HA-tagged PIP5K
and HA-tagged CAPS1 expression vectors has been described previously (39, 41).
Preparation of Silencing Vectors for PI4Kß, PI5K
, PLD1, Mint1, and CAPS1
Mammalian expression vectors directing the synthesis of siRNAs targeted against PI4Kß, PIP5K
, PLD1, or Mint1 were prepared according to the method of Brummelkamp et al. (43). Two cDNA fragments encoding a 19-nucleotide sequence derived from the target transcript and separated from its reverse 19-nucleotide complement by a short spacer were synthesized by MWG Biotech Co. (Ebersberg, Germany), annealed, and cloned in front of the H1-RNA promoter in the pSUPER vector (43). The silencers were generated using the following sequences: rat PI4Kß [accession no. NM_031083; nucleotides 875893 (active) and 18691887 (inactive)], rat PIP5K
[accession no. XM_234925; nucleotides 19081926 (active) and 10771095 (inactive)], rat PLD1 [accession no. NM_030992; nucleotides 26042622 (active) and 369387 (inactive)], and Mint1 [accession no. AF029105; nucleotides 13661384, siMint-a nucleotides 13981416, iMint-i.1; nucleotides 175317771, iMint-i.2]1 silencers were constructed by inserting short hairpin oligonucleotides including sequences 39023930 (active) and 75102 (inactive) of rat CAPS1 (accession no. NM_013219) in pSHAG1. This vector contains a U6 promoter and BseRI and BamHI cloning sites in a pENTR/D topo backbone (44). Each silencer was tested by Western blotting for its effect on the expression of its specific target and other unrelated proteins, such as GFP, granuphilin, and syntaxin-1.
Cell Culture and Transfection
The rat pancreatic ß-cell line INS-1E was cultured in RPMI 1640 medium supplemented with 50 µM ß-mercaptoethanol, 100 µM sodium pyruvate, and 5% fetal calf serum as previously described (45, 46). The mouse pancreatic ß-cell line MIN6 B1 was cultured as previously described (19). Transient transfection was performed using the Effectene reagent (Qiagen, Santa Clarita, CA) according to the manufacturers instructions. In all experiments the DNA/Effectene ratio was 1:25. Under our experimental conditions, transfection efficiency, estimated by counting the fraction of fluorescent cells after transfection with a plasmid encoding GFP, ranged from 3050%.
Secretion Assay
INS-1E and MIN6 B1 cells were transiently cotransfected with a plasmid encoding hGH and with plasmids encoding either the RNAi silencers or, in some cases, constructs leading to overexpression of the proteins under study. None of the plasmids used in this study affected the total amount of hGH produced by INS-1E or MIN6 cells. Thus, in each experiment the total cellular content of hGH was equivalent for all transfectants. Three days later, the cells were preincubated for 30 min in 20 mM HEPES (pH 7.4), 128 mM NaCl, 5 mM KCl, 1 mM MgCl2, and 2.7 mM CaCl2. The cells were then incubated for 45 min at 37 C either in the same buffer (basal) or in a buffer containing 20 mM HEPES (pH 7.4), 53 mM NaCl, 80 mM KCl, 1 mM MgCl2, 2.7 mM CaCl2, 20 mM glucose, 1 µM forskolin, and 1 mM 3-isobutyl-1-methylxanthine (stimulated). Exocytosis from transfected cells was assessed by measuring by ELISA the amount of hGH released in the medium during the incubation period (Roche, Rotkreuz, Switzerland). For the experiments with streptolysin-O, INS-1E cells were rendered permeable in potassium glutamate buffer [20 mM HEPES (pH 7.0), 140 mM potassium glutamate, 5 mM NaCl, 7 mM MgSO4, 5 mM Na2ATP, and 10.2 mM EGTA] containing 1.5 IU/ml streptolysin-O for 7 min at 37 C. After permeabilization, the cells were preincubated for 7 min in potassium glutamate buffer containing 0.1 µM free Ca2+. The medium was then replaced with a potassium glutamate buffer containing either 0.1 µM (basal) or 10 µM (stimulated) free Ca2+ for another 7-min period at 37 C. PI(4)P, PI(4,5)P2, or PI(3,4,5)P3 (final concentration, 10 µM) was added during the preincubation and incubation periods. PI(4)P and PI(4,5)P2, which are water soluble, were dissolved directly in potassium glutamate buffer. PI(3,4,5)P3 was first dissolved in dimethylsulfoxide at a concentration of 10 mM and then diluted to the final concentration in potassium glutamate buffer.
Immunoblotting
The cells were transfected and grown on 24-well plates under the same conditions used for the secretion assays. After 3 d, the cells were washed twice in ice-cold PBS and harvested. Cells from one well were solubilized in 200 µl sodium dodecyl sulfate sample buffer and boiled for 10 min. Equal amounts of protein were separated on polyacrylamide gels and transferred to nitrocellulose membranes. Immunostaining with specific antibodies and horseradish peroxidase-conjugated secondary antibodies diluted 1:2000 was carried out using the enhanced chemiluminescence technique (Amersham Biosciences, Piscataway, NJ).
Assessment of PI(4,5)P2 Levels at the Plasma Membrane
INS-1E cells were transiently cotransfected with GFP-tagged PH-PLC and the indicated silencers. After 3 d in culture, the cells were washed and incubated under the same conditions as those used for the secretion assays. After 45 min, they were fixed and analyzed by confocal microscopy (Leica model TCS NT, Lasertechnik, Heidelberg, Germany). Images acquired through the center of the transfected cells were used to determine the relative fluorescence intensity of GFP-tagged PH-PLC at the plasma membrane and in the cytosol using Scion Image software (Scion Corp., Frederick, MD).
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ACKNOWLEDGMENTS
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We thank Drs. Di Paolo, Agami, Hannon, De Matteis, Frohman, Holz, and Südhof for supplying materials, and Dr. V. Lilla for providing the MIN6 B1 clone.
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FOOTNOTES
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This work was supported by Swiss National Science Foundation Grant 3200B0-101746 (to R.R.) and National Institutes of Health Grant DK-40428 (to T.F.J.M.).
First Published Online August 4, 2005
Abbreviations: CAPS1, Ca2+-dependent activator protein for secretion 1; GFP, green fluorescence protein; h, human; HA, hemagglutinin; Mint1, Munc18-interacting protein 1; PH, pleckstrin homology; PI, phosphoinositide; PI4K, PI4-kinase; PI4P, phosphatidylinositol 4-phosphate; PI(4,5)P2, phosphatidylinositol 4,5-biphosphate; PIP4, phosphatidylinositol 4-bisphosphate; PLC
, phospholipase C
; PLD1, phospholipase D1; siRNA, small interfering RNA.
Received for publication December 23, 2004.
Accepted for publication July 29, 2005.
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REFERENCES
|
---|
- Straub SG, Sharp GW 2002 Glucose-stimulated signaling pathways in biphasic insulin secretion. Diabetes Metab Res Rev 18:451463[CrossRef][Medline]
- Wollheim CB, Maechler P 2002 ß-Cell mitochondria and insulin secretion: messenger role of nucleotides and metabolites. Diabetes 51:S37S42
- Rorsman P, Renstrom E 2003 Insulin granule dynamics in pancreatic ß cells. Diabetologia 46:10291045[CrossRef][Medline]
- Pratley RE, Weyer C 2001 The role of impaired early insulin secretion in the pathogenesis of type II diabetes mellitus. Diabetologia 44:929945[CrossRef][Medline]
- Easom RA 2000 ß-Granule transport and exocytosis. Semin Cell Dev Biol 11:253266[CrossRef][Medline]
- Chernomordik LV, Kozlov MM 2003 Protein-lipid interplay in fusion and fission of biological membranes. Annu Rev Biochem 72:175207[CrossRef][Medline]
- Martin TF 1997 Phosphoinositides as spatial regulators of membrane traffic. Curr Opin Neurobiol 7:331338[CrossRef][Medline]
- Takenawa T, Itoh T 2001 Phosphoinositides, key molecules for regulation of actin cytoskeletal organization and membrane traffic from the plasma membrane. Biochim Biophys Acta 1533:190206[Medline]
- De Matteis MA, Godi A 2004 PI-loting membrane traffic. Nat Cell Biol 6:487492[CrossRef][Medline]
- Wenk MR, De Camilli P 2004 Protein-lipid interactions and phosphoinositide metabolism in membrane traffic: insights from vesicle recycling in nerve terminals. Proc Natl Acad Sci USA 101:82628269[Abstract/Free Full Text]
- Olsen HL, Hoy M, Zhang W, Bertorello AM, Bokvist K, Capito K, Efanov AM, Meister B, Thams P, Yang SN, Rorsman P, Berggren PO, Gromada J 2003 Phosphatidylinositol 4-kinase serves as a metabolic sensor and regulates priming of secretory granules in pancreatic ß cells. Proc Natl Acad Sci USA 100:51875192[Abstract/Free Full Text]
- Varnai P, Balla T 1998 Visualization of phosphoinositides that bind pleckstrin homology domains: calcium- and agonist-induced dynamic changes and relationship to myo-[3H]inositol-labeled phosphoinositide pools. J Cell Biol 143:501510[Abstract/Free Full Text]
- Balla T 1998 Phosphatidylinositol 4-kinases. Biochim Biophys Acta 1436:6985[Medline]
- Godi A, Pertile P, Meyers R, Marra P, Di Tullio G, Iurisci C, Luini A, Corda D, De Matteis MA 1999 ARF mediates recruitment of PtdIns-4-OH kinase-ß and stimulates synthesis of PtdIns(4,5)P2 on the Golgi complex. Nat Cell Biol 1:280287[CrossRef][Medline]
- Taverna E, Francolini M, Jeromin A, Hilfiker S, Roder J, Rosa P 2002 Neuronal calcium sensor 1 and phosphatidylinositol 4-OH kinase ß interact in neuronal cells and are translocated to membranes during nucleotide-evoked exocytosis. J Cell Sci 115:39093922[Abstract/Free Full Text]
- Rajebhosale M, Greenwood S, Vidugiriene J, Jeromin A, Hilfiker S 2003 Phosphatidylinositol 4-OH kinase is a downstream target of neuronal calcium sensor-1 in enhancing exocytosis in neuroendocrine cells. J Biol Chem 278:60756084[Abstract/Free Full Text]
- Kapp-Barnea Y, Melnikov S, Shefler I, Jeromin A, Sagi-Eisenberg R 2003 Neuronal calcium sensor-1 and phosphatidylinositol 4-kinase ß regulate IgE receptor-triggered exocytosis in cultured mast cells. J Immunol 171:53205327[Abstract/Free Full Text]
- Hannon GJ 2002 RNA interference. Nature 418:244251[CrossRef][Medline]
- Lilla V, Webb G, Rickenbach K, Maturana A, Steiner DF, Halban PA, Irminger JC 2003 Differential gene expression in well-regulated and dysregulated pancreatic ß-cell (MIN6) sublines. Endocrinology 144:13681379[Abstract/Free Full Text]
- Ishihara H, Shibasaki Y, Kizuki N, Wada T, Yazaki Y, Asano T, Oka Y 1998 Type I phosphatidylinositol-4-phosphate 5-kinases. Cloning of the third isoform and deletion/substitution analysis of members of this novel lipid kinase family. J Biol Chem 273:87418748[Abstract/Free Full Text]
- Wenk MR, Pellegrini L, Klenchin VA, Di Paolo G, Chang S, Daniell L, Arioka M, Martin TF, De Camilli P 2001 PIP kinase I
is the major PI(4,5)P(2) synthesizing enzyme at the synapse. Neuron 32:7988[CrossRef][Medline]
- Di Paolo G, Moskowitz HS, Gipson K, Wenk MR, Voronov S, Obayashi M, Flavell R, Fitzsimonds RM, Ryan TA, De Camilli P 2004 Impaired PtdIns(4,5)P2 synthesis in nerve terminals produces defects in synaptic vesicle trafficking. Nature 431:415422[CrossRef][Medline]
- Gong LW, Di Paolo G, Diaz E, Cestra G, Diaz ME, Lindau M, De Camilli P, Toomre D 2005 Phosphatidylinositol phosphate kinase type I
regulates dynamics of large dense-core vesicle fusion. Proc Natl Acad Sci USA 102:52045209[Abstract/Free Full Text]
- Hughes WE, Elgundi Z, Huang P, Frohman MA, Biden TJ 2004 Phospholipase D1 regulates secretagogue-stimulated insulin release in pancreatic ß-cells. J Biol Chem 279:2753427541[Abstract/Free Full Text]
- Brown FD, Thompson N, Saqib KM, Clark JM, Powner D, Thompson NT, Solari R, Wakelam MJ 1998 Phospholipase D1 localises to secretory granules and lysosomes and is plasma-membrane translocated on cellular stimulation. Curr Biol 8:835838[CrossRef][Medline]
- Du G, Altshuller YM, Vitale N, Huang P, Chasserot-Golaz S, Morris AJ, Bader MF, Frohman MA 2003 Regulation of phospholipase D1 subcellular cycling through coordination of multiple membrane association motifs. J Cell Biol 162:305315[Abstract/Free Full Text]
- Walent JH, Porter BW, Martin TFJ 1992 A novel 145 kd brain cytosolic protein reconstitutes Ca2+-regulated secretion in permeable neuroendocrine cells. Cell 70:765775[CrossRef][Medline]
- Loyet KM, Kowalchyk JA, Chaudhary A, Chen J, Prestwich GD, Martin TF 1998 Specific binding of phosphatidylinositol 4,5-bisphosphate to calcium-dependent activator protein for secretion (CAPS), a potential phosphoinositide effector protein for regulated exocytosis. J Biol Chem 273:83378343[Abstract/Free Full Text]
- Tandon A, Bannykh S, Kowalchyk JA, Banerjee A, Martin TFJ, Balch WE 1998 Differential regulation of exocytosis by calcium and CAPS in semi-intact synaptosomes. Neuron 21:147154[CrossRef][Medline]
- Elhamdani A, Martin TFJ, Kowalchyk JA, Artalejo CR 1999 Ca2+-dependent activator protein for secretion is critical for the fusion of dense-core vesicles with the membrane in calf adrenal chromaffin cells. J Neurosci 19:73757383[Abstract/Free Full Text]
- Rupnik M, Kreft M, Sikdar SK, Grilc S, Romih R, Zupancic G, Martin TF, Zorec R 2000 Rapid regulated dense-core vesicle exocytosis requires the CAPS protein. Proc Natl Acad Sci USA 97:56275632[Abstract/Free Full Text]
- Grishanin RN, Kowalchyk JA, Klenchin VA, Ann K, Earles CA, Chapman ER, Gerona RR, Martin TF 2004 CAPS acts at a prefusion step in dense-core vesicle exocytosis as a PIP2 binding protein. Neuron 43:551562[CrossRef][Medline]
- Okamoto M, Sudhof TC 1997 Mints, Munc18-interacting proteins in synaptic vesicle exocytosis. J Biol Chem 272:3145931464[Abstract/Free Full Text]
- Ho A, Morishita W, Hammer RE, Malenka RC, Sudhof TC 2003 A role for Mints in transmitter release: Mint 1 knockout mice exhibit impaired GABAergic synaptic transmission. Proc Natl Acad Sci USA 100:14091414[Abstract/Free Full Text]
- Biederer T, Sudhof TC 2000 Mints as adaptors. Direct binding to neurexins and recruitment of munc18. J Biol Chem 275:3980339806[Abstract/Free Full Text]
- Zhang W, Lilja L, Bark C, Berggren PO, Meister B 2004 Mint1, a Munc-18-interacting protein, is expressed in insulin-secreting ß-cells. Biochem Biophys Res Commun 320:717721[CrossRef][Medline]
- Lawrence JT, Birnbaum MJ 2003 ADP-ribosylation factor 6 regulates insulin secretion through plasma membrane phosphatidylinositol 4,5-bisphosphate. Proc Natl Acad Sci USA 100:1332013325[Abstract/Free Full Text]
- Gromada J, Bark C, Smidt K, Efanov AM, Janson J, Mandic SA, Webb DL, Zhang W, Meister B, Jeromin A, Berggren PO 2005 Neuronal calcium sensor-1 potentiates glucose-dependent exocytosis in pancreatic ß cells through activation of phosphatidylinositol 4-kinase ß. Proc Natl Acad Sci USA 102:1030310308[Abstract/Free Full Text]
- Aikawa Y, Martin TF 2003 ARF6 regulates a plasma membrane pool of phosphatidylinositol(4,5)bisphosphate required for regulated exocytosis. J Cell Biol 162:647659[Abstract/Free Full Text]
- Vitale N, Caumont AS, Chasserot-Golaz S, Du G, Wu S, Sciorra VA, Morris AJ, Frohman MA, Bader MF 2001 Phospholipase D1: a key factor for the exocytotic machinery in neuroendocrine cells. EMBO J 20:24242434[Abstract/Free Full Text]
- Grishanin RN, Klenchin VA, Loyet KM, Kowalchyk JA, Ann K, Martin TF 2002 Membrane association domains in Ca2+-dependent activator protein for secretion mediate plasma membrane and dense-core vesicle binding required for Ca2+-dependent exocytosis. J Biol Chem 277:2202522034[Abstract/Free Full Text]
- Zhang W, Efanov A, Yang SN, Fried G, Kolare S, Brown H, Zaitsev S, Berggren PO, Meister B 2000 Munc-18 associates with syntaxin and serves as a negative regulator of exocytosis in the pancreatic ß-cell. J Biol Chem 275:4152141527[Abstract/Free Full Text]
- Brummelkamp TR, Bernards R, Agami R 2002 A system for stable expression of short interfering RNAs in mammalian cells. Science 296:550553[Abstract/Free Full Text]
- Paddison PJ, Caudy AA, Bernstein E, Hannon GJ, Conklin DS 2002 Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes Dev 16:948958[Abstract/Free Full Text]
- Asfari M, Janjic D, Meda P, Li G, Halban PA, Wollheim CB 1992 Establishment of 2-mercaptoethanol-dependent differentiated insulin-secreting cell lines. Endocrinology 130:167178[Abstract]
- Merglen A, Theander S, Rubi B, Chaffard G, Wollheim CB, Maechler P 2004 Glucose sensitivity and metabolism-secretion coupling studied during two-year continuous culture in INS-1E insulinoma cells. Endocrinology 145:667678[Abstract/Free Full Text]