From the Department of Biochemistry, University of
Wisconsin, Madison, Wisconsin 53706 and the § Department of
Medicinal Chemistry, University of Utah,
Salt Lake City, Utah 84112
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ABSTRACT |
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The calcium-dependent activator protein for secretion (CAPS) is a novel neural/endocrine-specific cytosolic and peripheral membrane protein required for the Ca2+-regulated exocytosis of secretory vesicles. CAPS acts at a stage in exocytosis that follows ATP-dependent priming, which involves the essential synthesis of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2). In the present studies, CAPS is shown to bind liposomes that contain acidic phospholipids and binding was markedly enhanced by inclusion of PtdIns(4,5)P2 but not other phosphoinositides in the absence of Ca2+. PtdIns(4,5)P2, but not other phosphoinositides including PtdIns(3,4)P2 and PtdIns(3,4,5)P3, altered the susceptibility of CAPS to proteolysis by trypsin and proteinase K, suggesting that phosphoinositide binding promoted a conformational change. Photoaffinity labeling studies with a photoactivatable benzoylcinnimidyl acyl chain derivative of PtdIns(4,5)P2 confirmed the phosphoinositide-binding properties of CAPS and suggested a hydrophobic aspect of the interaction. CAPS, as one of very few characterized proteins with a binding specificity for 4-,5-phosphorylated inositides over 3-phosphorylated inositides, may function in regulated exocytosis as an effector of PtdIns(4,5)P2.
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INTRODUCTION |
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The regulated secretion of neurotransmitters and peptide hormones from neural and endocrine cells is mediated by the fusion of secretory vesicles with the plasma membrane, but the molecular mechanisms that underlie the Ca2+-dependent merger of phospholipid bilayers have not been fully elucidated. The exocytosis of large dense-core vesicles (LDCVs)1 in neuroendocrine cells can be reconstituted in broken cell (1, 2) or purified membrane preparations (3) where ATP hydrolysis is required for priming reactions that precede Ca2+-dependent membrane fusion reactions (2). LDCV fusion exhibits a dependence upon cytosolic as well as membrane-bound protein constituents (4-7), and components that operate at either the ATP-dependent or Ca2+-triggered stages of exocytosis have been characterized (1, 8-13).
At least two roles have been identified for ATP during the priming stage of exocytosis. One involves N-ethylmaleimide-sensitive factor and the ATP-dependent disassembly of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein complexes (11, 14). The SNARE proteins have been suggested to mediate specific vesicle-plasma membrane docking reactions (5, 7, 13) and are required at a late step in exocytosis for Ca2+-activated membrane fusion (12, 15). A second role for ATP is as a substrate for lipid kinases that act sequentially to catalyze phospholipid phosphorylation (10, 16-18). Phosphatidylinositol transfer protein and phosphatidylinositol-4-phosphate 5-kinase are essential cytosolic components for the reconstitution of ATP-dependent priming (9, 10) and LDCVs contain a phosphatidylinositol 4-kinase required for priming (16-18). Synthesis of PtdIns(4,5)P2 occurs during the priming step (10, 19) but the precise role of this phospholipid in Ca2+-dependent membrane fusion is unknown. The high negative charge density, the high degree of head group hydration, and the positive curvature of PtdIns(4,5)P2-containing membranes would likely increase rather than decrease the barrier to bilayer fusion (20). Hence, fusion may require segregation of these lipids by PtdIns(4,5)P2-binding proteins. In general, polyphosphoinositides may serve as spatially-localized membrane signals that recruit specific binding proteins required for signal transduction, cytoskeletal regulation, and aspects of membrane trafficking (21-23). PtdIns(4,5)P2-binding proteins that mediate the essential role of this phospholipid in regulated exocytosis remain to be identified.
Several Ca2+-binding proteins are required at the Ca2+ triggering stage of exocytosis. Synaptotagmin, a possible Ca2+ sensor for regulated synaptic vesicle exocytosis (6), binds PtdIns(3,4,5)P3 in the absence of Ca2+ and PtdIns(3,4)P2 or PtdIns(4,5)P2 in the presence of Ca2+ (24). ATP-dependent priming of LDCVs is insensitive to the PtdIns 3-kinase inhibitors wortmannin and LY294002, suggesting that 3-phosphorylated inositides are not essential for exocytosis (25). A role in exocytosis for the Ca2+-dependent binding of synaptotagmin to PtdIns(4,5)P2 may be indicated by recent studies in which inositol polyphosphate inhibitory effects on evoked neurotransmitter release were reversed by preincubation with the synaptotagmin C2B antibody (26). Another Ca2+-binding protein CAPS (calcium-dependent activator protein for secretion) is essential for neurosecretion and reconstitutes LDCV exocytosis at a late post-docking step in exocytosis beyond the point of ATP-dependent priming (1, 2, 27). Here we report that CAPS is a specific PtdIns(4,5)P2-binding protein and suggest that it may serve as an effector that mediates the essential role of PtdIns(4,5)P2 in Ca2+-regulated fusion.
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EXPERIMENTAL PROCEDURES |
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Liposome Binding Assay by Sedimentation--
Phospholipids were
resuspended to 10 mg/ml in chloroform/methanol (2:1) and aliquots were
dried to a thin film under argon in glass tubes. The dried
phospholipids were resuspended in buffer by vortexing until a cloudy
suspension was formed and bath sonicated for 20 min at 25-30 °C to
form small unilamellar liposomes or micelles, which were stored at
70 °C. The bovine phospholipids PtdChol, PtdSer, PtdEt, PtdIns,
and PtdIns(4)P, and synthetic phospholipid dipalmitoyl
PtdIns(3,4)P2 were purchased from Sigma or Matreya
(Pleasant Gap, PA). Dipalmitoyl
phosphatidyl[N-methyl-3H]choline was purchased
from Amersham. Bovine PtdIns(4,5)P2 was either obtained
commercially (Sigma), was provided as a gift by Dr. R. A. Anderson
(University of Wisconsin), or was purified from bovine brain as
described (28-30). Dipalmitoyl PtdIns(3,4,5)P3, was a
generous gift from Drs. K. Fukami and T. Takenawa (Tokyo University).
Liposome Binding Assay with Immobilized CAPS-- A recombinant glutathione S-transferase-CAPS fusion protein was produced in Escherichia coli by subcloning the coding region of CAPS cDNA (10) in-frame into pGEX expression vectors (Pharmacia Biotech AB, Uppsala, Sweden) using standard methods (31). E. coli CAPS was kindly cloned and expressed by Dr. B. Porter. Glutathione-Sepharose 4B beads (Pharmacia Biotech) without bound protein or with glutathione S-transferase or glutathione S-transferase-CAPS were incubated with liposomes at room temperature for 60 min in 20 mM Hepes, pH 7.2, 100 mM KCl, 2 mM EGTA with a molar ratio of protein to phospholipid of 2.56:1. Liposomes, prepared as described above, were made at a ratio of 2:1:1 for PtdChol:PtdSer:phosphoinositide (PtdIns, PtdIns(4)P, or PtdIns(4,5)P2) or 2:1 for PtdChol:PtdSer with [3H]PtdChol, and clarified by sedimentation at 500 × g for 15 min. Incubations of liposomes with glutathione-Sepharose 4B beads were terminated by sedimentation for 5 min at 500 × g. Beads were washed with a 10-fold volume of buffer, collected at the same speed, and extracted with 10% SDS (10-fold volume). The first supernatant (A), wash supernatant (B), and bead SDS extract (C) were analyzed by liquid scintillation counting, and liposome binding was calculated as (disintegrations/min (3H)PtdChol in C)/(dpm (3H) A + B + C) × 100%.
Proteolysis of CAPS-- Proteolytic digestions of CAPS were conducted at room temperature for 60 min in either 2 mM Tris, pH 8.0, 1 mM ATP, 50 mM KCl, 2 mM EGTA, 100 mM dithiothreitol (with or without Ca2+) or in 20 mM HEPES, pH 7.2, 100 mM KCl, 2 mM EGTA, 1 mM dithiothreitol, with mass ratios of proteinase K to CAPS of 0.01:1 and trypsin to CAPS of 0.1:2. Liposomes were made as described previously and were usually added 10 min prior to protease addition. Proteinase K digestion studies employed liposomes at a mass ratio to CAPS of 6:16 for liposomes lacking phosphoinositides or at 1-1.5:16 for phosphoinositides, and trypsin digestion studies used liposomes at a mass ratio to CAPS of 1:1. Digestions were terminated by 20 mM phenylmethylsulfonyl fluoride plus 1 mM aminoethylbenzenesulfonyl fluoride (final concentrations), and the samples were analyzed by electrophoresis in 13.5% polyacrylamide SDS gels followed by Coomassie staining. Proteolytic fragments of CAPS were identified by N-terminal sequence analysis by Edman degradation chemistry using an Applied Biosystems sequencer and analyzer. Sequence analyses of some of the CAPS fragments were kindly provided by M. Jennings (Monsanto, Chesterfield, MO).
Photoaffinity Labeling with [3H]BZDC-PtdIns(4,5)P2 Analogue-- The synthesis and applications of [3H]BZDC-acyl-PtdIns(4,5)P2 have been previously described (32-34). Photolabeling studies with baculovirus-encoded recombinant CAPS were conducted on ice in 28 mM HEPES, pH 7.5, 30 mM KCl, 1 mM EGTA for 45 min at 2 cm from a 360-nm light source (Southern N.E., Ultraviolet Co., Bradford, CT) after a 10-min preincubation on ice with a 1:1 molar ratio of CAPS to [3H]BZDC-acyl-PtdIns(4,5)P2 with a specific activity of 42.5 Ci/mmol. Competition studies involved coincubation with a 1000-fold molar excess of Ins(1,4,5)P3 or PtdIns(4,5)P2 during irradiation. Incorporation of the [3H]BZDC-acyl-PtdIns(4,5)P2 probe was determined by SDS-gel electrophoresis and fluorography using EN3HANCE (NEN Life Science Products, Boston, MA) as described previously (35).
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RESULTS |
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Stereoselective Binding of Polyphosphoinositides to CAPS-- CAPS is a novel neural/endocrine-specific dimer of 145-kDa subunits that was identified by its activity in reconstituting Ca2+-dependent secretion in permeable neuroendocrine cells (1, 27). Although CAPS was initially purified as a soluble protein from rat brain cytosol, recent biochemical studies2 revealed that a substantial portion of the protein in brain homogenates localized as a peripherally-bound membrane protein. Saturable, high affinity binding of CAPS to protease-treated membranes suggested that CAPS was a phospholipid-binding protein.2 This was confirmed in direct binding studies conducted by sedimentation of liposomes of defined phospholipid composition (Fig. 1A). CAPS binding to PtdChol liposomes was negligible whereas binding to liposomes containing the acidic or neutral phospholipids PtdIns and PtdSer/PtdEt was significant. Binding was much more substantial to micelles of PtdIns(4,5)P2 and to a lesser extent PtdIns(4)P (Fig. 1A). The association of CAPS with PtdIns(4,5)P2 micelles was not attributable to the high negative charge density of this phospholipid since CAPS binding to micelles containing PtdIns(3,4,5)P3, a more highly acidic phospholipid, was similar to that of PtdIns or PtdSer/PtdEt (Fig. 1B). Similar results were obtained with phosphoinositides incorporated into liposomes with other phospholipids (see below). The results indicated that CAPS interactions with PtdIns(4,5)P2 were stereoselective for the phosphates on the inositol head group.
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PtdIns(4,5)P2 Binding Induces an Apparent Conformational Change in CAPS-- The binding of CAPS to PtdIns(4,5)P2-containing liposomes, but not to liposomes containing other acidic phospholipids, was accompanied by an apparent conformational change in CAPS as indicated by an altered susceptibility to limited proteolysis by either trypsin or proteinase K. The major polypeptides in the purified baculovirus-encoded recombinant CAPS consisted of the full-length 163-kDa protein and ~60 kDa CAPS protein proteolytic fragments (Fig. 4A, lane 1). Limited proteolysis of this preparation with trypsin generated ~6 fragments in the 22-70-kDa range (Fig. 4A, lane 2), which were also generated in the presence of PtdChol/PtdEt, PtdChol/PtdSer, or PtdChol liposomes (Fig. 4A, lanes 4-6). In contrast, the digestion pattern was markedly altered in the presence of PtdIns(4,5)P2 micelles (Fig. 4A, lane 3, second and third arrowheads). Similar results were obtained in digestions with proteinase K, which were limited to generate several core fragments in the 18-25-kDa range that represent ~60% of the CAPS sequence (Figs. 4, B-E, arrowheads). Presentation of PtdIns(4,5)P2 in both micellar and liposome form resulted in an altered pattern of proteolysis (Fig. 4B, lanes 2-6). The molar % of PtdIns(4,5)P2 in PtdChol/PtdSer liposomes that promoted changes in proteolysis similar to those with micellar PtdIns(4,5)P2 corresponded to between 10 and 25% (Fig. 4B). As was true for trypsin, the altered proteolysis of CAPS by proteinase K was only observed with liposomes containing PtdIns(4,5)P2 but not other acidic phospholipids such as PtdSer or PtdIns (Fig. 4, B and D). Because PtdIns(4,5)P2 did not affect the proteolytic activity of trypsin or proteinase K toward other proteins tested (data not shown), the altered susceptibility of CAPS to proteolysis is inferred to represent a conformational change in the protein induced by PtdIns(4,5)P2 binding.
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Photoaffinity Labeling of CAPS with [3H]BZDC-acyl-PtdIns(4,5)P2-- Recently described (32-34) benzophenone photoaffinity derivatives of PtdIns(4,5)P2 were used to further characterize CAPS interactions with polyphosphoinositides. These derivatives probe different environments of phosphoinositide-binding sites with the [3H]BZDC-acyl-PtdIns(4,5)P2 sampling the lipid bilayer environment and the [3H]BZDC-phosphotriester-PtdIns(4,5)P2 sampling the water-head group interface (33). In preliminary experiments, [3H]BZDC-phosphotriester-PtdIns(4,5)P2, [3H]BZDC-Ins(1,4,5)P3, and [3H]BZDC-Ins(1,3,4,5)P4 probes (36) all failed to generate covalent adducts of CAPS (data not shown). In contrast, the [3H]BZDC-acyl-PtdIns(4,5)P2 photoprobe was successfully incorporated into the full-length CAPS protein as well as into two ~60-kDa fragments of CAPS (Fig. 5). CAPS derivatization with this probe was competitively inhibited by PtdIns(4,5)P2 and to a lesser extent by Ins(1,4,5)P3 (Fig. 5). Preliminary data suggest that proteinase K digestion of the [3H]BZDC-acyl-PtdIns(4,5)P2 adduct of CAPS generates several 3H-labeled fragments, one of which corresponds to the 25-kDa C-terminal fragment of CAPS (not shown).
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DISCUSSION |
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The present results indicate that CAPS is a phospholipid-binding protein that interacts generally with acidic phospholipids and specifically with PtdIns(4,5)P2. Interactions with acidic phospholipids such as PtdSer and PtdIns abundant in the cytoplasmic leaflet of membranes may be the basis for the membrane association of CAPS as a peripherally-bound protein in brain homogenates localized to plasma membrane and LDCVs.2 Interactions of CAPS with PtdIns(4,5)P2, in contrast to its interactions with other acidic phospholipids, are not based on negative charge density of the phospholipid since CAPS exquisitely discriminates PtdIns(4,5)P2 in preference to PtdIns(3,4)P2 and PtdIns(3,4,5)P3. This property distinguishes CAPS from proteins that bind acidic phospholipids nonspecifically via charge interactions (37) and implies that phosphoinositide binding involves stereoselective interactions with inositol phosphate groups. The preferential binding of PtdIns(4,5)P2 compared with PtdIns(3,4)P2 or PtdIns(3,4,5)P3 distinguishes CAPS from many other polyphosphoinositide-binding proteins that exhibit a different specificity.
Several proteins exhibit a binding preference for
PtdIns(3,4,5)P3 or PtdIns(3,4)P2 over
PtdIns(4,5)P2. These include the adapter proteins AP-2 and
AP-3 (38, 39), the brain protein -centaurin (40), the secretory
vesicle protein synaptotagmin (in the absence of Ca2+)
(24), the barbed-end capping protein gelsolin (Ref. 41, but see
Footnote 3), the actin monomer-binding
protein profilin (42), SH2 domains of several proteins (43), and the PH
domains of Bruton's tyrosine kinase, Ras-GAP1, GRP-1, Akt, and
phospholipase C
(44-47). This large group of identified D-3
phosphoinositide-binding proteins represents potential effectors for
signaling mechanisms involving the products of PtdIns 3-kinase.
In contrast, the PH domain of dynamin exhibits a specificity similar to that of CAPS for 4- and 5-phosphorylated inositides in preference to 3-phosphorylated inositides (44, 48, 49). The actin-capping CapZ-related proteins and gelsolin3 exhibit a similar specificity (50). Hence, CAPS is one of very few cellular proteins identified to date that exhibit a very strong stereoselective preference for D-4,D-5 phosphoinositides over D-3 phosphoinositides. This small group of proteins represents candidates for effectors of the signaling roles of D-4,D-5 phosphoinositides in membrane trafficking and cytoskeletal regulation.
PH domains are the best characterized motifs for stereoselective interactions with phosphoinositides where binding appears to be mediated largely if not entirely through interactions with the phosphorylated inositol ring (51, 52). This may also be the case for many other proteins such as synaptotagmin where binding of inositol polyphosphates and polyphosphoinositides exhibit identical affinities (24). In contrast, for several characterized phosphoinositide-binding proteins, interactions with the diacylglycerol moiety is an important determinant of specificity. A dual role of acyl chains and head group specificity has been demonstrated for AP-3 binding to PtdIns(3,4,5)P3, where deacylation reduced the binding affinity almost 20-fold (39). Similarly, deacylated glycerophosphorylinositols and deglycerinated inositol phosphates were completely ineffective as inhibitors of gelsolin's actin severing activity (53). Photoaffinity labeling studies suggest that CAPS interactions with PtdIns(4,5)P2 may require hydrophobic interactions with the fatty acyl chains in addition to polar interactions with inositol phosphates. This was indicated by the failure to productively cross-link CAPS with BZDC-phosphotriester-InsPx or PtdIns(4,5)P2 probes. BZDC-phosphotriester-InsPx probes, with the photoactivatable BZDC group at the water-lipid interface have been effectively cross-linked to several other phosphoinositide-binding proteins (34). In contrast, effective photoaffinity labeling of CAPS with the [3H]BZDC-acyl-PtdIns(4,5)P2 probe with the photoactivatable BZDC group on an acyl chain in a predominantly hydrophobic environment, indicates a role for hydrophobic interactions in CAPS-phosphoinositide binding. This binding also suggests that CAPS penetrates into the phospholipid bilayer because the acyl chain benzophenone embedded in the micellar interior is within photocovalent modification distance (3.1 angstroms) of CAPS protein residues. These observations indicate that CAPS interactions with phosphoinositides are quite distinct from those mediated by PH and other domains that preferentially or exclusively interact with the phosphoinositol head group. Because the CAPS protein sequence does not exhibit identifiable motifs such as those of a PH domain, structural studies of CAPS will likely reveal a novel basis for phosphoinositide interactions.
A conformational change in CAPS induced by interactions with PtdIns(4,5)P2 can be inferred from the increased susceptibility of regions of the protein to partial proteolytic digestion. In agreement with the liposome-binding studies, the altered protease susceptibility of CAPS induced by phosphoinositide binding exhibited stereoselectivity for the D-4 and D-5 position phosphates of the inositol head group. N- and C-terminal domains of CAPS were exposed to proteolysis upon PtdIns(4,5)P2 binding while the central region of the protein remained protected. Because PtdIns(4,5)P2 enhanced the proteolysis of a C-terminal but not N-terminal fragment in a CAPS proteolytic digest, it can be inferred that at least one binding site is present in the C-terminal region (residues 859-1022), which has been confirmed by preliminary photoaffinity labeling studies with [3H]BZDC-acyl-PtdIns(4,5)P2. These photocovalent modification studies indicated that other regions of CAPS also interact with PtdIns(4,5)P2,4 which could generate a global conformational change in the protein. Conformational changes promoted by phosphoinositide interactions have been reported to alter the function of several proteins. The interaction of PtdIns(4,5)P2 with vinculin affects its conformation and unmasks binding sites for talin and actin (54). Likewise, gelsolin undergoes a conformational change upon binding of PtdIns(4,5)P2 that inhibits its actin filament-severing and barbed-end capping activity (55). In addition, profilin undergoes a conformational change associated with its altered actin monomer sequestering activity (56). Conformational changes induced by PtdIns(4,5)P2 binding to CAPS may have important implications for the function of CAPS on the membrane during stages of exocytosis.
The synthesis of PtdIns(4,5)P2 is an essential
ATP-dependent reaction that primes the exocytotic apparatus
for Ca2+-activated fusion (10, 19). A specific requirement
for 4- and 5-phosphorylated inositides was inferred by the
identification of PtdIns(4)P 5-kinase as a required priming factor
(10), by the finding that PtdIns 4-kinase activity is essential for
priming (18) and by the demonstration of inhibitory effects on priming of reagents specific for D-4,D-5 phosphoinositides such as
phospholipase C (10). Recent studies have found that high
concentrations (>10 molar %) of PtdIns(4,5)P2 are
synthesized on the cytoplasmic leaflet of the LDCV membrane during
priming in PC12 cells,5
consistent with the localization of PtdIns 4-kinase to the vesicle membrane (16, 17). In the present study, similar concentrations of
PtdIns(4,5)P2 in liposomes promoted a conformational change in CAPS. Because CAPS is required for Ca2+-activated
exocytosis at a step following ATP-dependent priming (27),
an attractive possibility is that CAPS is an effector protein that
mediates the essential role of PtdIns(4,5)P2 in
exocytosis.
The precise role of PtdIns(4,5)P2 in priming exocytosis and the detailed mechanism of CAPS action remain to be elucidated, but the characterization of CAPS as a specific binding protein for D-4,D-5 phosphoinositides suggests a plausible connection. CAPS is associated with the plasma membrane and LDCVs in brain tissue where it may be peripherally-bound to the membrane by interactions with acidic phospholipids such as PtdSer and PtdIns.2 The synthesis of PtdIns(4,5)P2 on docked LDCVs during priming may recruit CAPS to the plasma membrane-vesicle interface. Conformational changes promoted by PtdIns(4,5)P2 binding may allow penetration of CAPS into the bilayers and enable this large (290 kDa) dimeric protein to mediate increased contacts between the fusion partner membranes. Increased Ca2+, which inhibits PtdIns(4,5)P2 binding but enhances general phospholipid binding to CAPS, could trigger rearrangements that facilitate fusion acting in concert with other Ca2+-activated membrane-interacting proteins such as synaptotagmin. While speculative, this model is predictive for future studies that assess the importance of PtdIns(4,5)P2 binding for CAPS action in exocytosis.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 The abbreviations used are: LDCV, large dense-core vesicle; PtdIns, phosphatidylinositol; PtdIns(4)P, phosphatidylinositol 4-monophosphate; PtdIns(4,5)P2, phosphatidylinositol 4,5-bisphosphate; PtdIns(3,4)P2, phosphatidylinositol 3,4-bisphosphate; PtdIns(3,4,5)P3, phosphatidylinositol 3,4,5-trisphosphate; Ins(1,4,5)P3, inositol 1,4,5-trisphosphate; Ins(1,3,4,5)P4, inositol 1,3,4,5-tetrakisphosphate; PtdChol, phosphatidylcholine; PtdSer, phosphatidylserine; PtdEt, phosphatidylethanolamine; CAPS, calcium-dependent activator protein for secretion; BZDC, 1-O-[3-(4-benzoyldihydrocinnamidyl)propyl]-; PH, pleckstrin homology.
2 B. Berwin, E. Floor, and T. F. J. Martin, manuscript submitted.
3 H. Yin and J. Chen, manuscript submitted.
4 K. M. Loyet, unpublished results.
5 K. M. Loyet, K. Fukami, T. Takenawa, and T. F. J. Martin, manuscript in preparation.
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REFERENCES |
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