Specific Binding of Phosphatidylinositol 4,5-Bisphosphate to Calcium-dependent Activator Protein for Secretion (CAPS), a Potential Phosphoinositide Effector Protein for Regulated Exocytosis*

Kelly M. LoyetDagger , Judith A. KowalchykDagger , Anu Chaudhary§, Jian Chen§, Glenn D. Prestwich§, and Thomas F. J. MartinDagger

From the Dagger  Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706 and the § Department of Medicinal Chemistry, University of Utah, Salt Lake City, Utah 84112

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

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.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

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.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

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).

CAPS, purified from rat brain cytosol or prepared as a baculovirus-encoded fusion protein as described previously (1, 27), was incubated with liposomes at room temperature for 1 h in 2 mM Tris, pH 8.0, 1 mM ATP, 2 mM MgCl2, 50 mM KCl, 2 mM EGTA, 1 mM dithiothreitol, either with or without Ca2+, at a mass ratio of phospholipid to CAPS of 50:1.5. Micelles and liposomes were recovered from reactions by sedimentation at 200,000 × g for 45 min. Under the centrifugation conditions used, at least 85% of the micelles and liposomes were recovered in the pellet whereas negligible amounts of CAPS was recovered in the absence of phospholipids. Pellets were washed with 1 volume of buffer, recovered by sedimentation, and resuspended in sample buffer for analysis by electrophoresis on 8% SDS gels. Supernatants were analyzed by SDS-polyacrylamide gel electrophoresis following precipitation with trichloroacetic acid. Gels were stained with Coomassie Blue or proteins were electrophoretically transferred to nitrocellulose sheets for immunoblotting with a specific CAPS rabbit polyclonal antibody generated against a CAPS peptide corresponding to amino acids 574-589. Stained gels and autoradiograms were quantitated with a Molecular Dynamics Personal Densitometer or PhosphorImager using ImageQuant software.

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).

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

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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Fig. 1.   CAPS binding to phospholipids. CAPS binding to liposomes or micelles of defined composition was measured using 50 µg of each phospholipid as described under "Experimental Procedures." The amount of CAPS bound to was determined by gel electrophoresis of the lipid pellets recovered by sedimentation followed by immunoblotting with the CAPS (574-589) antibody. CAPS binding is presented in arbitrary units representing densitometric scans of autoradiograms. The data in panels A and B are from independent experiments representative of two to three experiments. Abbreviations used are: PIP2, PtdIns(4,5)P2; PIP, PtdIns(4)P; PI, PtdIns; PE, PtdEt; PS, PtdSer; PC, PtdChol; PIP3, PtdIns(3,4,5)P3.

Equilibrium binding studies with a recombinant protein demonstrated that CAPS is a Ca2+-binding protein (27). In the present studies, Ca2+ was found to markedly inhibit CAPS binding to PtdIns(4,5)P2-containing liposomes (Fig. 2A). In contrast, the binding of CAPS to PtdChol liposomes that lacked or contained the acidic phospholipids PtdEt, PtdSer, or PtdIns was increased by the inclusion of Ca2+ in the binding mixtures. The Ca2+-dependent inhibition of binding to PtdChol/PtdIns(4,5)P2 liposomes and the stimulation of binding to PtdChol/PtdEt liposomes occurred over a similar range of Ca2+ concentrations (Fig. 2B) exhibiting half-maxima at ~10-100 µM free ionic Ca2+. This is within the range of the estimated KD (~170 µM) for the higher affinity Ca2+-binding site on CAPS that was detected in different buffer and pH conditions (27). The phospholipid composition dependence of the effects of Ca2+ on CAPS binding were particularly evident in studies where the liposome content of PtdIns(4,5)P2 was varied (Fig. 2C).


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Fig. 2.   Calcium differentially affects CAPS interactions with phospholipids. A, liposomes of the indicated composition were incubated at 0.5 mg/ml with 0.5 µM CAPS either with EGTA (cross-hatched) or with EGTA/Ca2+ (solid) to attain a free Ca2+ concentration of 100 µM. Liposomes were recovered by sedimentation and analyzed for CAPS by gel electrophoresis and Coomassie Blue staining (inset). B, similar incubations were conducted with PtdChol/PtdEt 2:1 or PtdChol/PtdIns(4,5)P2 2:1 liposomes over the range of indicated Ca2+ concentrations. 100% corresponds to maximal binding for each liposome type. C, similar incubations were conducted in the absence or presence of Ca2+ with liposomes containing PtdChol and the indicated mole fraction of PtdIns(4,5)P2. Abbreviations are defined in the legend to Fig. 1. Data shown are representative of two to three similar experiments.

The binding of PtdIns(4,5)P2 to CAPS was dose-dependent and saturable. Although CAPS bound to PtdIns(4,5)P2 with high specificity, it was with relatively low affinity. A KD for PtdIns(4,5)P2 binding was estimated in the sedimentation assay using a range of concentrations of PtdIns(4,5)P2 presented as micelles or as PtdChol-containing liposomes. An apparent KD of 50-150 µM was observed with either recombinant or native CAPS (data not shown).

The ability of CAPS to bind phospholipids was confirmed in an independent assay using immobilized CAPS incubated with 3H-liposomes of varied phospholipid composition. A glutathione S-transferase-CAPS fusion protein produced in E. coli was tethered to glutathione-Sepharose beads and incubated with [3H]PtdChol liposomes containing the acidic phospholipids PtdSer, PtdIns, PtdIns(4)P, or PtdIns(4,5)P2. Whereas glutathione-Sepharose beads with or without bound glutathione S-transferase retained only background levels of liposomes, the beads with immobilized CAPS specifically retained liposomes that contained acidic phospholipids (Fig. 3). Consistent with the sedimentation assay results, immobilized CAPS preferentially retained liposomes containing PtdIns(4,5)P2 and to a lesser extent PtdIns(4)P.


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Fig. 3.   Liposome binding study of recombinant CAPS. A glutathione S-transferase-CAPS fusion protein produced in E. coli was tethered to glutathione-Sepharose beads and compared with glutathione-Sepharose or glutathione S-transferase-Sepharose for retention of liposomes. [3H]PtdChol-containing liposomes were made to contain PtdChol/PtdSer (2:1) or PtdChol/PtdSer/phosphoinositide (2:1:1) where phosphoinositides were PtdIns, PtdIns(4)P, or PtdIns(4,5)P2. Following a 30-min incubation, beads were washed and solubilized for analysis by liquid scintillation counting. Data for the mean of duplicate incubations (except for glutathione S-transferase (GST)-Sepharose beads) are shown with bars representing the range. Abbreviations used are defined in the legend to Fig. 1.

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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Fig. 4.   PtdIns(4,5)P2 binding induces a conformational change in CAPS. CAPS was subjected to a limiting digest with trypsin or proteinase K in the presence or absence of micelles or liposomes of the indicated composition and analyzed by gel electrophoresis and Coomassie Blue staining. A, CAPS proteolysis by trypsin. Gel lanes represent recombinant CAPS (lane 1), trypsin-digested CAPS in the absence of phospholipid (lane 2), and trypsin-digested CAPS in the presence of equimolar PtdIns(4,5)P2 micelles (lane 3), PtdChol/PtdEt (2:1) liposomes (lane 4), PtdChol/PtdSer (2:1) liposomes (lane 5), or PtdChol liposomes (lane 6). Asterisks indicate full-length 163-kDa CAPS (upper) and ~60-kDa proteolytic CAPS fragments (lower) of lane 1. The top arrowhead indicates trypsin-resistant CAPS residues 371-665, while the middle and lower arrowheads show CAPS fragments that were degraded in the presence of PtdIns(4,5)P2. B-E, CAPS proteolysis by proteinase K. The four arrowheads indicate CAPS fragments identified by sequencing corresponding to residues 859-1075 (top), residues 662-860 (upper middle), residues 469-650 (lower middle), and residues 94-258 (lower). B, gel lanes represent partial digest with proteinase K in the absence of phospholipid (lane 1), in the presence of PtdIns(4,5)P2 micelles (lane 2), in the presence of liposomes containing PtdChol/PtdSer plus the indicated mol % of PtdIns(4,5)P2 (lanes 3-6), or in the presence of liposomes composed of PtdChol/PtdSer (2:1) (lane 7), PtdChol (lane 8), or PtdSer (lane 9). C, proteinase K digestions of CAPS with delayed addition of PtdIns(4,5)P2. CAPS was incubated for 30 min with proteinase K in the absence (lanes 1, 3, and 4) or presence (lanes 2 and 5) of PtdIns(4,5)P2 micelles. Additional 30-min incubations were conducted (lanes 3, 4, and 5) following addition of PtdIns(4,5)P2 to some of the incubations (lanes 4 and 5). The 25-kDa fragment (first arrowhead) generated during the first incubation (lane 1) was degraded during the second incubation when PtdIns(4,5)P2 was added (lane 4) but not in its absence (lane 3). This contrasts with the 19-kDa fragment (last arrowhead), which was preserved during the second incubation with PtdIns(4,5)P2 (lane 4). D, CAPS proteolysis by proteinase K in the presence of selected phosphoinositides. CAPS digestion was conducted in the absence of phospholipids (lane 1) or the presence of liposomes consisting of PtdChol/PtdSer (2:1) (lane 2), or PtdChol/PtdSer/phosphoinositides (2:1:1) (lanes 3-7). E, the effects of Ca2+ on CAPS proteolysis by proteinase K in the presence of phospholipids. Digestions were conducted in the absence (-) or presence (+) of 30 µM Ca2+ either in the absence (lanes 1 and 2) or in the presence (lanes 3-14) of liposomes of the indicated concentrations.

The PtdIns(4,5)P2-induced increase in proteolysis by proteinase K was largely restricted to 25- and 19-kDa fragments of CAPS (Fig. 4B, lane 2, top and bottom arrowheads, respectively). Sequencing these fragments indicated that they correspond to C- and N-terminal fragments (residues 859-1075 and 94-258, respectively) of the 1289-residue CAPS protein. Identification of the proteinase K-resistant 20- and 22-kDa fragments (Fig. 4B, middle two arrowheads) by sequencing indicated that they correspond to central regions of CAPS (residues 469-650 and 662-860, respectively). Similarly, the trypsin-resistant 38-kDa fragment (Fig. 4A, top arrowhead, residues 371-665) was derived from a central region of CAPS. These results indicate that the altered proteolytic susceptibility induced in CAPS by phosphoinositide binding principally involves the N and C termini of the protein.

By conducting brief incubations, it was possible to generate the core proteolytic fragments prior to the addition of PtdIns(4,5)P2 (Fig. 4C, lane 1). Delayed addition of PtdIns(4,5)P2 was found to accelerate the proteolysis of the 25-kDa C-terminal fragment but not the 19-kDa N-terminal fragment (Fig. 4C, lane 4 versus 3). This result indicates that the 25-kDa C-terminal fragment retained PtdIns(4,5)P2-binding activity that was coupled to a conformational change. In contrast, the CAPS protein domain corresponding to the 19-kDa N-terminal fragment appears to undergo a conformational change that is indirect and secondary to phosphoinositide binding to the intact protein.

The enhanced susceptibility of CAPS to proteinase K digestion exhibited a high degree of selectivity for phosphoinositides phosphorylated at the D-4 and D-5 position of the inositol ring (Fig. 4D). PtdIns(4,5)P2 maximally increased digestion of the 19- and 25-kDa fragments whereas PtdIns(4)P enhanced cleavage to a more limited extent. PtdIns(3,4)P2 promoted a partial enhancement of proteolysis resembling that observed with PtdIns(4)P, which was preferential for the 25-kDa fragment. In contrast, PtdIns(3,4,5)P3 exerted virtually no effect on CAPS proteolysis (Fig. 4, D and E). The results indicate that CAPS exhibits a stereoselective interaction with phosphoinositides that stringently requires inositol D-4 and D-5 phosphates and the absence of a D-3 phosphate. Stereoselective phosphoinositide binding to the C-terminal domain and possibly other sites (see below) may be coupled to more global conformational changes in the CAPS protein that secondarily affect the proteolytic susceptibility of N-terminal domains.

In sedimentation assays, Ca2+ was shown to reduce phosphoinositide interactions with CAPS. Similarly, inclusion of Ca2+ in the protease digestions reversed the enhanced proteolytic susceptibility of the 19- and 25-kDa fragments promoted by PtdIns(4,5)P2 (Fig. 4E). Although Ca2+ stimulated the interactions of CAPS with PtdChol-, PtdEt-, PtdIns-, and PtdSer-containing liposomes in sedimentation assays, only subtle effects of this divalent cation on CAPS proteolysis in the presence of PtdChol/PtdSer or PtdChol/PtdSer/PtdIns liposomes were detected. These were found among the higher molecular mass fragments and corresponded to the previously reported effects of Ca2+ on CAPS proteolysis observed in the absence of lipids (27) (not shown).

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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Fig. 5.   Photoaffinity labeling with [3H]BZDC-acyl-PtdIns(4,5)P2. Photoaffinity labeling of CAPS and two CAPS proteolytic fragments with [3H]BZDC-acyl-PtdIns(4,5)P2 (lane 1, arrowheads). The BZDC-acyl-PtdIns(4,5)P2 probe and CAPS were present in equimolar amounts. Lanes 2 and 3 show parallel photoaffinity labeling reactions that contained a 1000-fold molar excess of Ins(1,4,5)P3 or PtdIns(4,5)P2, respectively.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

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 alpha -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 Cdelta (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 Cdelta (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.

    FOOTNOTES

* 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.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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