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• W2034896852 abstract "Synaptotagmin (Syt) II, a synaptic vesicle protein containing two copies of highly conserved protein kinase C homology regions known as the C2A and C2B domains, acts as a Ca2+ sensor and provides both phospholipid and inositol polyphosphate (IPn) recognition domains important in endo- and exocytosis. Four photoaffinity analogues of IP3, IP4, and IP6 containing a P-1- or P-2-linked 4-benzoyldihydrocinnamidyl (BZDC) photophore were used to label glutathione S-transferase (GST) fusion constructs of the Syt II-C2A and C2B domains. The P-2-linked [3H]BZDC-IP6 showed efficient, IP6-displaceable labeling of the GST-Syt II-C2B. The rank order of photocovalent modification paralleled the order of competitive displacement: IP6 (P-2-linked) > IP4 > IP3. The P-1-linked [3H]BZDC-IP6 failed to label the C2B domains. The GST-Syt III-C2B domain, which lacks IP6 binding affinity, also failed to undergo labeling by P-2-linked [3H]BZDC-IP6. When mixtures of the 32-amino acid basic peptide corresponding to the essential IPn binding region of the Syt II-C2B domain and GST-Syt II-C2B were labeled by a stoichiometric amount of P-2-linked [3H]BZDC-IP6, the two polypeptides showed equivalent affinity for the photolabel. Although the CD spectrum of this 32-mer at two pH values showed a random coil, the photoaffinity analogue of IP6 appeared to induce a binding-compatible structure in the short peptide. Synaptotagmin (Syt) II, a synaptic vesicle protein containing two copies of highly conserved protein kinase C homology regions known as the C2A and C2B domains, acts as a Ca2+ sensor and provides both phospholipid and inositol polyphosphate (IPn) recognition domains important in endo- and exocytosis. Four photoaffinity analogues of IP3, IP4, and IP6 containing a P-1- or P-2-linked 4-benzoyldihydrocinnamidyl (BZDC) photophore were used to label glutathione S-transferase (GST) fusion constructs of the Syt II-C2A and C2B domains. The P-2-linked [3H]BZDC-IP6 showed efficient, IP6-displaceable labeling of the GST-Syt II-C2B. The rank order of photocovalent modification paralleled the order of competitive displacement: IP6 (P-2-linked) > IP4 > IP3. The P-1-linked [3H]BZDC-IP6 failed to label the C2B domains. The GST-Syt III-C2B domain, which lacks IP6 binding affinity, also failed to undergo labeling by P-2-linked [3H]BZDC-IP6. When mixtures of the 32-amino acid basic peptide corresponding to the essential IPn binding region of the Syt II-C2B domain and GST-Syt II-C2B were labeled by a stoichiometric amount of P-2-linked [3H]BZDC-IP6, the two polypeptides showed equivalent affinity for the photolabel. Although the CD spectrum of this 32-mer at two pH values showed a random coil, the photoaffinity analogue of IP6 appeared to induce a binding-compatible structure in the short peptide. INTRODUCTIONThe synaptotagmins (Syts) 1The abbreviations used are: SytsynaptotagminAP-2assembly protein-2BZDC4-benzoyldihydrocinnamoylGSTglutathione S-transferaseInsinositolIP3D-myo-Ins(1,4,5)P3IP4D-myo-Ins(1,3,4,5)P4IP6Ins(1,2,3,4,5,6)P6IPninositol polyphosphatePIP2phosphatidylinositol (4,5)-bisphosphatePIP3phosphatidylinositol (3,4,5)-trisphosphatePIPnphosphoinositide polyphosphateHPLChigh pressure liquid chromatography. are synaptic vesicle proteins that play essential roles in nucleating the clathrin coat during endocytosis and in acting as Ca2+ sensors (1Geppert M. Goda Y. Hammer R.E. Li C. Rosahl T.W. Stevens C.F. SÜdhof T.C. Cell. 1994; 79: 717-727Abstract Full Text PDF PubMed Scopus (1203) Google Scholar, 2Li C. Ullrich B. Zhang J.Z. Anderson R.G.W. Brose N. SÜdhof T.C. Nature. 1995; 375: 594-599Crossref PubMed Scopus (541) Google Scholar, 3Mikoshiba K. Fukuda M. Moreira J.E. Lewis F.M.T. Sugimori M. Niinobe M. Llinas R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10703-10707Crossref PubMed Scopus (115) Google Scholar) and phosphoinositide sensors (4De Camilli P. Emr S.D. McPherson P.S. Novick P. Science. 1996; 271: 1533-1538Crossref PubMed Scopus (659) Google Scholar) during exocytosis. They are a critical part of a complex machinery of intracellular protein transport (5Rothman J.E. Protein Sci. 1996; 5: 185-194Crossref PubMed Scopus (145) Google Scholar, 6Rothman J.E. Nature. 1994; 372: 55-63Crossref PubMed Scopus (1995) Google Scholar) and the synaptic vesicle cycle (7SÜdhof T.C. Nature. 1995; 375: 645-653Crossref PubMed Scopus (1760) Google Scholar). In addition to a short N-terminal intravesicular region and single transmembrane domain, Syts have two copies of highly conserved repeats, known as the C2A and C2B domains, which are homologous to the C2 regulatory region of protein kinase C (8Perin M.S. Fried V.A. Mignery G.A. Jahn R. SÜdhof T.C. Nature. 1990; 345: 260-261Crossref PubMed Scopus (648) Google Scholar). In particular, the C2B domain appears to play several roles. First, the C2B domain of mouse Syt II shows specific binding to high polyphosphate inositols (IPns) (9Fukuda M. Aruga J. Niinobe M. Aimoto S. Mikoshiba K. J. Biol. Chem. 1994; 269: 29206-29211Abstract Full Text PDF PubMed Google Scholar), a feature that is not shared by the highly homologous C2A domain nor with the C2 domains of other proteins such as rabphilin. Moreover, the C2B domain is necessary but not sufficient for IPn binding. Although Syt II and IV show high affinity binding of IP4 and IP6, the Syt III-C2B domain shows negligible binding, despite a high sequence identity with the Syt II-C2B domain, including the 32-residue region examined by mutational analysis (10Fukuda M. Kojima T. Aruga J. Niinobe M. Mikoshiba K. J. Biol. Chem. 1995; 270: 26523-26527Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). Inhibition of C2B function in the squid giant axon disrupts synaptic vesicle release and recycling (11Fukuda M. Moreira J.E. Lewis F.M.T. Sugimori M. Niinobe M. Mikoshiba K. Llinas R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10708-10712Crossref PubMed Scopus (135) Google Scholar). Similarly, Caenorhabditis elegans mutants lacking Syt or simply the C2B domain cannot recycle synaptic vesicles (12Jorgensen E.M. Hartweig E. Schuske K. Nonet M.L. Jin Y. Horvitz H.R. Nature. 1995; 378: 196-199Crossref PubMed Scopus (264) Google Scholar), and the presence of Syt I in cerebrospinal fluid of Alzheimer's patients (13Davidsson P. Jahn R. Bergquist J. Ekman R. Blennow K. Mol. Chem. Neuropathol. 1996; 27: 195-210Crossref PubMed Scopus (74) Google Scholar) may provide clues to the disruption of synaptic function in humans. The C2B domain acts as a high affinity receptor for clathrin assembly protein AP-2 (14Zhang J.Z. Davletov B.A. Sudhof T.C. Anderson R.G.W. Cell. 1994; 78: 751-760Abstract Full Text PDF PubMed Scopus (431) Google Scholar), and Ca2+ appears to mediate Syt dimerization via the C2B domain (15Chapman E.R. An S. Edwardson J.M. Jahn R. J. Biol. Chem. 1996; 271: 5844-5849Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar).Synaptotagmin II isolated from mouse cerebellum shows high affinity binding to Ins(1,3,4,5)P4 with a KD of 30 μM (16Niinobe M. Yamaguchi Y. Fukuda M. Mikoshiba K. Biochem. Biophys. Res. Commun. 1994; 205: 1036-1042Crossref PubMed Scopus (59) Google Scholar) and 117 nM for the GST-Syt II-C2B construct (9Fukuda M. Aruga J. Niinobe M. Aimoto S. Mikoshiba K. J. Biol. Chem. 1994; 269: 29206-29211Abstract Full Text PDF PubMed Google Scholar). Curiously, the rank order of IPns binding for native Syt II was Ins(1,3,4,5,6)-P5 > (1,3,4,5)-P4 > Ins(1,2,3,4,5,6)-P6 > Ins(1,4,5)-P3, whereas for the GST-Syt II-C2B domain the order was IP6 > IP5 > IP4 > IP3. Deletion mutants allowed mapping of the IPn binding site to the central region of the mouse Syt II-C2B domain, specifically residues 315-346 (IHLMQNGKRLKKKKTTVKKKTLNPYFNESFSF) (9Fukuda M. Aruga J. Niinobe M. Aimoto S. Mikoshiba K. J. Biol. Chem. 1994; 269: 29206-29211Abstract Full Text PDF PubMed Google Scholar). In order to obtain direct evidence for the binding site of IPns in the C2B domain, to examine the InsPn selectivity of the domains, to explore the Ca2+ dependence of binding, to determine the role of the central 315-346 peptide, and to evaluate the reason for the failure of Syt III-C2B domain to bind IPns, we undertook a series of photoaffinity labeling experiments using four tritium-labeled, benzophenone-containing derivatives of IP3, IP4, and IP6 (17Prestwich G.D. Dormán G. Elliott J.T. Marecak D.F. Chaudhary A. Photochem. Photobiol. 1996; Google Scholar).DISCUSSIONThe availability of tetherable analogues of the inositol polyphosphates and phosphoinositide polyphosphates (25Prestwich G.D. Acc. Chem. Res. 1996; 29: 503-513Crossref Scopus (107) Google Scholar), including affinity purification resins and benzophenone-containing photoaffinity labels (17Prestwich G.D. Dormán G. Elliott J.T. Marecak D.F. Chaudhary A. Photochem. Photobiol. 1996; Google Scholar, 26Dormán G. Prestwich G.D. Biochemistry. 1994; 33: 5661-5673Crossref PubMed Scopus (766) Google Scholar) has enabled the identification of new PIPn- and IPn-binding proteins and the characterization of the ligand binding sites within these proteins. For example, four IP4 binding proteins were affinity-purified from rat brain (30Theibert A.B. Estevez V.A. Ferris C.D. Danoff S.K. Barrow R.K. Prestwich G.D. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3165-3169Crossref PubMed Scopus (105) Google Scholar) and photoaffinity labeled with [3H]BZDC-IP4 (22Estevez V.A. Prestwich G.D. J. Am. Chem. Soc. 1991; 113: 9885-9887Crossref Scopus (96) Google Scholar, 31Theibert A.B. Estevez V.A. Mourey R.J. Marecek J.F. Barrow R.K. Prestwich G.D. Snyder S.H. J. Biol. Chem. 1992; 267: 9071-9079Abstract Full Text PDF PubMed Google Scholar). Two have been further characterized; the α subunit of clathrin assembly protein (AP-2) (32Voglmaier S.M. Keen J.H. Murphy J. Ferris C.D. Prestwich G.D. Snyder S.H. Theibert A.B. Biochem. Biophys. Res. Commun. 1992; 187: 158-163Crossref PubMed Scopus (117) Google Scholar) had the highest affinity for IP6, whereas the novel cytoskeletal linker protein centaurin-α had the highest affinity for PIP3 (21Hammonds-Odie L.P. Jackson T.R. Profit A.A. Blader I.J. Turck C.W. Prestwich G.D. Theibert A.B. J. Biol. Chem. 1996; 271: 18859-18868Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). The IP3 binding site of the affinity-purified rat brain IP3 receptor (19Prestwich G.D. Marecek J.F. Mourey R.J. Theibert A.B. Ferris C.D. Danoff S.K. Snyder S.H. J. Am. Chem. Soc. 1991; 113: 1822-1825Crossref Scopus (60) Google Scholar) has been identified by sequencing a proteolytic fragment of the [3H]BZDC-IP3 photoaffinity-labeled protein (33Mourey R.J. Estevez V.A. Marecek J.F. Barrow R.K. Prestwich G.D. Snyder S.H. Biochemistry. 1993; 32: 1719-1726Crossref PubMed Scopus (60) Google Scholar). The availability of two regioisomeric-tethered derivatives of IP6 (23Chen J. Prestwich G.D. J. Labelled Cmpnd. Radiopharm. 1996; 38: 1113-1119Crossref Scopus (8) Google Scholar, 24Marecek J.F. Prestwich G.D. Tetrahedron Lett. 1991; 32: 1863-1866Crossref Scopus (24) Google Scholar) has facilitated the further study of kinases (34Abdullah M. Hughes P.J. Craxton A. Gigg R. Desai T. Marecek J.F. Prestwich G.D. Shears S.B. J. Biol. Chem. 1992; 267: 22340-22345Abstract Full Text PDF PubMed Google Scholar, 35Voglmaier S.M. Bembenek M.E. Kaplin A.I. Dormán G. Olszewski J.D. Prestwich G.D. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4305-4310Crossref PubMed Scopus (131) Google Scholar) and other proteins that interact with IP6. The P-2-tethered [3H]BZDC-IP6 prepared in this work was employed to establish the location of the IP6 binding site in the C2B region of the synaptotagmins and to confirm the ligand specificity of this domain by an independent method. In the process, the site specificity and ligand selectivity found fully validates the use of the photoaffinity labeling approach in the search for and active-site mapping of additional PIPn- and IPn-binding proteins.Six GST-Syt C2 domain-containing fusion proteins were examined in this study, using four photoaffinity probes corresponding to modifications of IP3, IP4, and IP6. The most important findings are summarized as follows. First, we observed extremely high selectivity and ligand specificity of labeling of the C2B-containing proteins in crude extracts. No other proteins were significantly labeled with the high specific activity [3H]BZDC-IPn derivatives even when the target proteins represented less than 2% of the total protein. The only exception was a hitherto unknown 45-kDa E. coli protein that showed high selectivity and specificity for binding of IP4. This observation may provide the basis for purification of this protein and the determination of its role in the biochemistry of this common bacterium. For all proteins and ligands, labeling followed an expected time course, with a maximum efficiency at 45 min.Second, labeling was highly dependent on the choice of ligand. Although P-2-linked [3H]BZDC-IP6 gave the strongest labeling of C2B-containing constructs, P-1-linked [3H]BZDC-IP4, [3H]BZDC-IP3, and [3H]BZDC-IP6 showed modest, weak, and no labeling, respectively. The failure of the P-1-tethered IP6 to label C2B domains was unexpected but an important “negative control.” It is important to recognize that the aminopropyl-tethered photophore adds significant “unnatural” lipophilicity to the soluble IPn recognition elements as well as masking one of the phosphates. The importance of the number and the positions of phosphates and the position of the linker were critical factors determining the photolabeling efficiency, not simply nonselective hydrophobic interactions of the photophore with the protein.Nonetheless, the observation that purified but not crude C2B domains could be weakly labeled by [3H]BZDC-IP3 emphasizes two important points. When only a single protein is present in a photoaffinity labeling experiment, even relatively low affinity sites may become covalently modified. The ligand may poorly associate with the target, but in the absence of alternative sites for occupancy, the irreversible nature of the photoaffinity labeling experiment allows labeled protein to accumulate.The photoaffinity probe may also mimic a different ligand. In BZDC-IP3, the presence of the P-1-O-(3-aminopropyl) linker bearing the hydrophobic tritiated 4-benzoyldihydrocinnamide photophore allows the photoaffinity ligand to resemble a 2-desacyl phosphoinositide 4,5-bisphosphate, PIP2. For example, profilin, a PIP2-binding cytosolic protein important in interactions of the actin cytoskeleton with membrane phospholipids (36Janmey P.A. Annu. Rev. Physiol. 1994; 56: 169-191Crossref PubMed Scopus (471) Google Scholar) has been labeled with [3H]BZDC-IP3, but displacement of labeling requires PIP2, not IP3 (17Prestwich G.D. Dormán G. Elliott J.T. Marecak D.F. Chaudhary A. Photochem. Photobiol. 1996; Google Scholar). The PH domain of the phospholipase C δ1 isoform can also be labeled efficiently3 with [ 3E. Tall, G. Dormán, P. Garcia, S. Shah, J. Chen, A. A. Profit, Q.-M. Gu, A. Chaudhary, G. D. Prestwich, and M. J. Rebecchi, submitted for publication.H]BZDC-IP3. Indeed, the C2B domain of Syt I has been recently implicated in binding of PIP2 at high calcium concentrations (37Schiavo G. Gu Q.-M. Prestwich G.D. SÖllner T.H. Rothman J.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13327-13332Crossref PubMed Scopus (260) Google Scholar), providing further explanation for the affinity of the photoaffinity probes for the Syt II-C2B domains employed in this study.Similarly the [3H]BZDC-IP4 probe effectively mimics PIP3. This observation in fact enabled the isolation and purification of centaurin-α, a novel multi-domain protein with both homology regions, suggesting its role in communication of membrane lipid changes to the cytoskeleton (21Hammonds-Odie L.P. Jackson T.R. Profit A.A. Blader I.J. Turck C.W. Prestwich G.D. Theibert A.B. J. Biol. Chem. 1996; 271: 18859-18868Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). Very recently, the N-terminal region of the α subunit of AP-2 was found to have high affinity for PIP3 (38Gaidarov I. Chen Q. Falck J.R. Reddy K.K. Keen J.H. J. Biol. Chem. 1996; 271: 20922-20929Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar), a finding foreshadowed by the highly selective and efficient photoaffinity labeling of AP-2 by [3H]BZDC-IP4 (31Theibert A.B. Estevez V.A. Mourey R.J. Marecek J.F. Barrow R.K. Prestwich G.D. Snyder S.H. J. Biol. Chem. 1992; 267: 9071-9079Abstract Full Text PDF PubMed Google Scholar, 32Voglmaier S.M. Keen J.H. Murphy J. Ferris C.D. Prestwich G.D. Snyder S.H. Theibert A.B. Biochem. Biophys. Res. Commun. 1992; 187: 158-163Crossref PubMed Scopus (117) Google Scholar). In unpublished work from our laboratories, 4A. A. Profit, J. M. Rabinovich, B. Mehrotra, G. D. Prestwich, and E. M. Lafer, unpublished observations. [3H]BZDC-IP4 showed efficient labeling of assembly protein AP-3, already documented to have high affinity for the higher IPns IP4, IP5, IP6, and IP7 (39Ye W. Ali N. Bembenek M.E. Shears S.B. Lafer E.M. J. Biol. Chem. 1995; 270: 1564-1568Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar), which inhibited assembly of the clathrin coat. Interestingly, PIP3 has now been shown to be more effective than the soluble IPns or other PIPns in inhibiting AP-3-mediated clathrin coat assembly. 5W. Hao, Z. Tan, K. Prasad, K. K. Reddy, J. Chen, G. D. Prestwich, J. R. Falck, S. B. Shears, and E. M. Lafer (1997) J. Biol. Chem. 272, in press. Finally, the labeling of purified Syt II-C2B domains by [3H]BZDC-IP4 can be understood in terms of the affinity of Syt C2 domains for PIP3 at low calcium concentrations (37Schiavo G. Gu Q.-M. Prestwich G.D. SÖllner T.H. Rothman J.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13327-13332Crossref PubMed Scopus (260) Google Scholar).The relative importance of the phosphate groups in binding to the C2B domains was examined in an independent set of NMR studies (40Clark B.A. Biological Applications of NMR: Structure Determination and Relaxation Studies.Ph. D. Thesis, State University of New York at Stony Brook, NY. 1995; Google Scholar). The relative rate enhancements, which depend on increases in motional correlation time, were determined for clearly resolved phosphorus and proton signals of the photoaffinity ligand in the presence and the absence of GST-Syt II-C2B. Thus, 1H and 31P NMR of 10 mM BZDC-IP4 were first determined at 298 K for 20 mM sodium acetate buffer containing 0.1 mM EDTA, pH 5.5; next, 0.04 molar equivalent of purified GST-Syt II-C2B (0.4 mM) was added to the solution, and the chemical shifts and relaxation times were remeasured. The signals exhibiting the largest rate enhancements included P-5, H-5, and H-6, whereas the P-1 and side chain aminopropyl relaxation rates were essentially unaffected by the binding to the C2B domain. Thus, the 4,5,6 region of the inositol ring is intimately involved in the binding to C2B, whereas the aminopropyl tether retains motional freedom even in the bound state (40Clark B.A. Biological Applications of NMR: Structure Determination and Relaxation Studies.Ph. D. Thesis, State University of New York at Stony Brook, NY. 1995; Google Scholar).Third, labeling was well correlated with reported relative IPn binding affinity as determined by equilibrium methods using displacement of [3H]IP4 with unlabeled IPns (9Fukuda M. Aruga J. Niinobe M. Aimoto S. Mikoshiba K. J. Biol. Chem. 1994; 269: 29206-29211Abstract Full Text PDF PubMed Google Scholar). Thus, using [3H]BZDC-IP6 as the probe, displacement of 50% of the labeling required about a 150-fold molar excess of IP6, whereas little or no displacement was observed with IP4 or IP3 at six times this concentration of displacer. When [3H]BZDC-IP4 labeling was examined in detail (data not shown), IP6 was again the most effective competing ligand.Fourth, labeling was correlated with binding affinities of C2B domains for the functionally diverse Syt II and Syt III constructs. Consistent with the [3H]IP4 binding affinities (10Fukuda M. Kojima T. Aruga J. Niinobe M. Mikoshiba K. J. Biol. Chem. 1995; 270: 26523-26527Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar), the GST-Syt III-C2B domain was not covalently labeled upon irradiation with [3H]BZDC-IP6. Moreover, purified GST fusion proteins in which [3H]IP4 binding had been partially restored also recovered the ability to be photoaffinity labeled. Thus, the C-terminal truncated GST-Syt III-C2B(δC) showed partial unmasking of the IPn binding domain.Fifth, labeling by P-2-linked [3H]BZDC-IP6 correlated with the known affinities of the two different C2 domains, which show different calcium ion-dependent properties (41Sugita S. Hata Y. SÜdhof T.C. J. Biol. Chem. 1996; 271: 1262-1265Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). The C2A domain acts as a calcium sensor (3Mikoshiba K. Fukuda M. Moreira J.E. Lewis F.M.T. Sugimori M. Niinobe M. Llinas R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10703-10707Crossref PubMed Scopus (115) Google Scholar) during endocytosis, and three-dimensional structures have been described (42Sutton R.B. Davletov B.A. Berghuis A.M. Sudhof T.C. Sprang S.R. Cell. 1995; 80: 929-938Abstract Full Text PDF PubMed Scopus (602) Google Scholar, 43Shao X.G. Davletov B.A. Sutton R.B. Sudhof T.C. Rizo J. Science. 1996; 273: 248-251Crossref PubMed Scopus (294) Google Scholar) for this critical domain. The phospholipid selectivities of several Syts have been described in detail, and the Syt IV C2A domain also appears to function as a calcium sensor (27Fukuda M. Kojima T. Mikoshiba K. J. Biol. Chem. 1996; 271: 8430-8434Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Consistent with the absence of high affinity [3H]IP4 binding to C2A (10Fukuda M. Kojima T. Aruga J. Niinobe M. Mikoshiba K. J. Biol. Chem. 1995; 270: 26523-26527Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar), the GST-Syt II-C2A domain was not labeled by [3H]BZDC-IP6. Preliminary observations suggest Ca2+-dependent labeling of this domain with [3H]BZDC-IP3, consistent with binding to other anionic phospholipids such as phosphatidylserine. In contrast, the C2B domain has been implicated in binding to IPns (9Fukuda M. Aruga J. Niinobe M. Aimoto S. Mikoshiba K. J. Biol. Chem. 1994; 269: 29206-29211Abstract Full Text PDF PubMed Google Scholar), PIPns (38Gaidarov I. Chen Q. Falck J.R. Reddy K.K. Keen J.H. J. Biol. Chem. 1996; 271: 20922-20929Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar), AP-2 (14Zhang J.Z. Davletov B.A. Sudhof T.C. Anderson R.G.W. Cell. 1994; 78: 751-760Abstract Full Text PDF PubMed Scopus (431) Google Scholar), and other elements of the synaptic vesicle fusion machinery (44Schiavo G. Gmachi M.J.S. Steinbeck G. SÖllner T.H. Rothman J.E. Nature. 1995; 378: 733-736Crossref PubMed Scopus (155) Google Scholar). The efficient labeling of the GST Syt II-C2B domain (and GST-Syt II-C2A+C2B domain) with [3H]BZDC-IP6 is consistent with the different affinities of these two protein kinase C2 homology domains.Sixth, a synthetic 32-residue peptide corresponding to the base-rich central region of the C2B domain, identified by mutational and truncation/deletion methods as crucial to high affinity [3H]IP4 binding (9Fukuda M. Aruga J. Niinobe M. Aimoto S. Mikoshiba K. J. Biol. Chem. 1994; 269: 29206-29211Abstract Full Text PDF PubMed Google Scholar, 10Fukuda M. Kojima T. Aruga J. Niinobe M. Mikoshiba K. J. Biol. Chem. 1995; 270: 26523-26527Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar), also showed high affinity for the [3H]BZDC-IP6 photoaffinity probe. Indeed, when an equimolar ratio of 32-mer to GST-Syt II-C2B was labeled with a stoichiometric amount of this photoaffinity label, the full-length C2B domain and 32-mer were equally labeled. Increasing amounts of peptide were able to completely sequester the photoprobe from the C2B domain. This observation is highly significant in that it represents the smallest minidomain known to demonstrate selective, high affinity binding to IP6 or its analogues. Surprisingly, this peptide failed to exhibit secondary structure, other than random coil, in aqueous buffers at two pH values in the presence and the absence of IP6. It is conceivable, in view of the dimerization of Syts via their C2B domains (15Chapman E.R. An S. Edwardson J.M. Jahn R. J. Biol. Chem. 1996; 271: 5844-5849Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar), that the peptide is only able to adopt an IP6-binding conformation in the presence of a preorganized IP6-C2B complex. However, the 32-mer peptide could be labeled successfully with [3H]BZDC-IP6 even in the absence of the GST Syt II-C2B protein (data not shown), and this labeling can be reduced by addition of IP6 as a competitor. This suggests that BZDC-IP6 may induce a structure in the otherwise random coil polypeptide. The labeling was maximal with [3H]BZDC-IP6 relative to the [3H]BZDC-IP3 and IP4 photoprobes, supporting the higher affinity of the 32-mer peptide for IP6.A related C2B domain has been identified in a novel IP4-binding protein from rat brain and both human and porcine platelets (45Cullen P.J. Hsuan J.J. Truong O. Letcher A.J. Jackson T.R. Dawson A.P. Irvine R.F. Nature. 1995; 376: 527-530Crossref PubMed Scopus (286) Google Scholar); this protein also possesses GTPase-activating activity not found in other IP4-binding proteins. This protein has high specificity for IP4, with 1000-fold lower affinity for IP6. The central IPn binding sequence would have missing or moved basic residues found in the IP6 binding region of C2B with a central region of EAKKTKVKKK (human) as opposed to KKKKTTVKKK (mouse). A preliminary report suggested that a 25-mer peptide that includes this recognition site could bind IP4 with high affinity via a “basket-like” structure, allowing the lysines or arginines to interact with the phosphates of the IP4 (46Irvine R. Cullen P. Curr. Biol. 1996; 6: 537-540Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). The data described herein constitute the experimental evidence in support of this hypothesis.Synaptotagmin is essential for several aspects of synaptic vesicle dynamics. In particular, strong biological evidence now supports a critical role for the C2B domain in recycling of synaptic vesicles. This evidence is based on the accumulation of synaptic vesicles in C. elegans mutants lacking the C2B domain of their Syt (12Jorgensen E.M. Hartweig E. Schuske K. Nonet M.L. Jin Y. Horvitz H.R. Nature. 1995; 378: 196-199Crossref PubMed Scopus (264) Google Scholar) and from the inhibition of synaptic vesicle recycling in squid (Loglio pealei) giant synapse preterminals when injected with antibodies to the this C2B domain (11Fukuda M. Moreira J.E. Lewis F.M.T. Sugimori M. Niinobe M. Mikoshiba K. Llinas R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10708-10712Crossref PubMed Scopus (135) Google Scholar). The high inositol polyphosphates (IPns, n = 4, 5, or 6) can block synaptic neurotransmitter release and may regulate the fusion step preceding exocytosis (11Fukuda M. Moreira J.E. Lewis F.M.T. Sugimori M. Niinobe M. Mikoshiba K. Llinas R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10708-10712Crossref PubMed Scopus (135) Google Scholar). The emergence of PIP3 as a preferred ligand for inhibition of clathrin coating mediated by AP-2 (38Gaidarov I. Chen Q. Falck J.R. Reddy K.K. Keen J.H. J. Biol. Chem. 1996; 271: 20922-20929Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar) and AP-35 may suggest that the IPns (n = 4, 5, or 6) may not be the actual physiological ligands involved. An important research area would now be studies directed at determining the nature and flux of PIPn and IPn ligands actually involved in the endo- and exocytotic events in vivoConclusionFour photoaffinity analogues of IP3, IP4, and IP6 have been employed to document the selectivity and specificity of different inositol polyphosphates toward the C2 domains of two Syts. We made five key observations. First, labeling of the C2B-containing proteins in crude extracts showed extremely high selectivity and ligand specificity. Second, labeling was highly dependent on ligand, with [3H]BZDC-IP6 showing the optimal labeling of C2B domains. Third, labeling was well correlated with reported relative IPn binding affinities as determined by equilibrium methods using displacement of [3H]IP4 with unlabeled IPns. This validates photolabeling as a methodology for identification of novel proteins that bind IPns. Fourth, labeling was correlated with [3H]IP4 binding affinities of C2B domains for the functionally diverse Syt II and Syt III constructs and for the C2A versus C2B domains of Syt II. Fifth, a 32-residue base-rich peptide located in the central region of the C2B domain exhibited high affinity binding to the photoaffinity label when employed in competition with the full-length C2B domain. This observation opens the door to use of peptides in physiological experiments to modulate IPn-receptor binding in a wide variety of systems. Future studies on active site mapping and structure determination of domain-IPn complexes will also be realizable goals given these new data. INTRODUCTIONThe synaptotagmins (Syts) 1The abbreviations used are: SytsynaptotagminAP-2assembly protein-2BZDC4-benzoyldihydrocinnamoylGSTglutathione S-transferaseInsinositolIP3D-myo-Ins(1,4,5)P3IP4D-myo-Ins(1,3,4,5)P4IP6Ins(1,2,3,4,5,6)P6IPninositol polyphosphatePIP2phosphatidylinositol (4,5)-bisphosphatePIP3phosphatidylinositol (3,4,5)-trisphosphatePIPnphosphoinositide polyphosphateHPLChigh pressure liquid chromatography. are synaptic vesicle proteins that play essential roles in nucleating the clathrin coat during endocytosis and in acting as Ca2+ sensors (1Geppert M. Goda Y. Hammer R.E. Li C. Rosahl T.W. Stevens C.F. SÜdhof T.C. Cell. 1994; 79: 717-727Abstract Full Text PDF PubMed Scopus (1203) Google Scholar, 2Li C. Ullrich B. Zhang J.Z. Anderson R.G.W. Brose N. SÜdhof T.C. Nature. 1995; 375: 594-599Crossref PubMed Scopus (541) Google Scholar, 3Mikoshiba K. Fukuda M. Moreira J.E. Lewis F.M.T. Sugimori M. Niinobe M. Llinas R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10703-10707Crossref PubMed Scopus (115) Google Scholar) and phosphoinositide sensors (4De Camilli P. Emr S.D. McPherson P.S. Novick P. Science. 1996; 271: 1533-1538Crossref PubMed Scopus (659) Google Scholar) during exocytosis. They are a critical part of a complex machinery of intracellular protein transport (5Rothman J.E. Protein Sci. 1996; 5: 185-194Crossref PubMed Scopus (145) Google Scholar, 6Rothman J.E. Nature. 1994; 372: 55-63Crossref PubMed Scopus (1995) Google Scholar) and the synaptic vesicle cycle (7SÜdhof T.C. Nature. 1995; 375: 645-653Crossref PubMed Scopus (1760) Google Scholar). In addition to a short N-terminal intravesicular region and single transmembrane domain, Syts have two copies of highly conserved repeats, known as the C2A and C2B domains, which are homologous to the C2 regulatory region of protein kinase C (8Perin M.S. Fried V.A. Mignery G.A. Jahn R. SÜdhof T.C. Nature. 1990; 345: 260-261Crossref PubMed Scopus (648) Google Scholar). In particular, the C2B domain appears to play several roles. First, the C2B domain of mouse Syt II shows specific binding to high polyphosphate inositols (IPns) (9Fukuda M. Aruga J. Niinobe M. Aimoto S. Mikoshiba K. J. Biol. Chem. 1994; 269: 29206-29211Abstract Full Text PDF PubMed Google Scholar), a feature that is not shared by the highly homologous C2A domain nor with the C2 domains of other proteins such as rabphilin. Moreover, the C2B domain is necessary but not sufficient for IPn binding. Although Syt II and IV show high affinity binding of IP4 and IP6, the Syt III-C2B domain shows negligible binding, despite a high sequence identity with the Syt II-C2B domain, including the 32-residue region examined by mutational analysis (10Fukuda M. Kojima T. Aruga J. Niinobe M. Mikoshiba K. J. Biol. Chem. 1995; 270: 26523-26527Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). Inhibition of C2B function in the squid giant axon disrupts synaptic vesicle release and recycling (11Fukuda M. Moreira J.E. Lewis F.M.T. Sugimori M. Niinobe M. Mikoshiba K. Llinas R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10708-10712Crossref PubMed Scopus (135) Google Scholar). Similarly, Caenorhabditis elegans mutants lacking Syt or simply the C2B domain cannot recycle synaptic vesicles (12Jorgensen E.M. Hartweig E. Schuske K. Nonet M.L. Jin Y. Horvitz H.R. Nature. 1995; 378: 196-199Crossref PubMed Scopus (264) Google Scholar), and the presence of Syt I in cerebrospinal fluid of Alzheimer's patients (13Davidsson P. Jahn R. Bergquist J. Ekman R. Blennow K. Mol. Chem. Neuropathol. 1996; 27: 195-210Crossref PubMed Scopus (74) Google Scholar) may provide clues to the disruption of synaptic function in humans. The C2B domain acts as a high affinity receptor for clathrin assembly protein AP-2 (14Zhang J.Z. Davletov B.A. Sudhof T.C. Anderson R.G.W. Cell. 1994; 78: 751-760Abstract Full Text PDF PubMed Scopus (431) Google Scholar), and Ca2+ appears to mediate Syt dimerization via the C2B domain (15Chapman E.R. An S. Edwardson J.M. Jahn R. J. Biol. Chem. 1996; 271: 5844-5849Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar).Synaptotagmin II isolated from mouse cerebellum shows high affinity binding to Ins(1,3,4,5)P4 with a KD of 30 μM (16Niinobe M. Yamaguchi Y. Fukuda M. Mikoshiba K. Biochem. Biophys. Res. Commun. 1994; 205: 1036-1042Crossref PubMed Scopus (59) Google Scholar) and 117 nM for the GST-Syt II-C2B construct (9Fukuda M. Aruga J. Niinobe M. Aimoto S. Mikoshiba K. J. Biol. Chem. 1994; 269: 29206-29211Abstract Full Text PDF PubMed Google Scholar). Curiously, the rank order of IPns binding for native Syt II was Ins(1,3,4,5,6)-P5 > (1,3,4,5)-P4 > Ins(1,2,3,4,5,6)-P6 > Ins(1,4,5)-P3, whereas for the GST-Syt II-C2B domain the order was IP6 > IP5 > IP4 > IP3. Deletion mutants allowed mapping of the IPn binding site to the central region of the mouse Syt II-C2B domain, specifically residues 315-346 (IHLMQNGKRLKKKKTTVKKKTLNPYFNESFSF) (9Fukuda M. Aruga J. Niinobe M. Aimoto S. Mikoshiba K. J. Biol. Chem. 1994; 269: 29206-29211Abstract Full Text PDF PubMed Google Scholar). In order to obtain direct evidence for the binding site of IPns in the C2B domain, to examine the InsPn selectivity of the domains, to explore the Ca2+ dependence of binding, to determine the role of the central 315-346 peptide, and to evaluate the reason for the failure of Syt III-C2B domain to bind IPns, we undertook a series of photoaffinity labeling experiments using four tritium-labeled, benzophenone-containing derivatives of IP3, IP4, and IP6 (17Prestwich G.D. Dormán G. Elliott J.T. Marecak D.F. Chaudhary A. Photochem. Photobiol. 1996; Google Scholar)." @default.
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