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W1991937669 abstract "Synaptotagmins I and II are essential for Ca2+-regulated exocytosis of synaptic vesicles from neurons, probably serving as Ca2+ sensors. This Ca2+-sensing function is thought to be disrupted by binding of an inositol 1,3,4,5-tetrakisphosphate (IP4) to the C2B domain of synaptotagmin I or II (Fukuda, M., Moreira, J. E., Lewis, F. M. T., Sugimori, M., Niinobe, M., Mikoshiba, K., and Llinás, R. (1995) Proc. Natl. Acad. Sci. U. S. A.92, 10708–10712). Recently, several synaptotagmin isoforms, expressed outside the nervous system, have been identified in rats and proposed to be involved in constitutive vesicle traffic. To test whether the inositol high polyphosphates also regulate constitutive vesicle traffic by binding to the non-neuronal synaptotagmins, we examined the IP4 binding properties of the recombinant C2 domains of both neuronal (III, V, X, and XI) and non-neuronal (VI–VIII and IX) synaptotagmins. The C2B domains of synaptotagmins VII–IX and XI had strong IP4 binding activity, but the C2B domain of synaptotagmin VI showed very weak IP4 binding activity. In contrast, there was no significant IP4 binding activity of the C2B domains of synaptotagmins III, V, and X or any of the C2A domains. A phylogenetic tree of the C2 domains of 11 isoforms revealed that synaptotagmins III, V, VI, and X (IP4-insensitive or very weak IP4-binding isoforms) belong to the same branch. Based on the sequence comparison between the IP4-sensitive and -insensitive isoforms, we performed site-directed mutagenesis of synaptotagmin III and identified several amino acid substitutions that abolish IP4 binding activity. Our data suggest that the inositol high polyphosphates might also regulate constitutive vesicle traffic via binding to the IP4-sensitive non-neuronal synaptotagmins. Synaptotagmins I and II are essential for Ca2+-regulated exocytosis of synaptic vesicles from neurons, probably serving as Ca2+ sensors. This Ca2+-sensing function is thought to be disrupted by binding of an inositol 1,3,4,5-tetrakisphosphate (IP4) to the C2B domain of synaptotagmin I or II (Fukuda, M., Moreira, J. E., Lewis, F. M. T., Sugimori, M., Niinobe, M., Mikoshiba, K., and Llinás, R. (1995) Proc. Natl. Acad. Sci. U. S. A.92, 10708–10712). Recently, several synaptotagmin isoforms, expressed outside the nervous system, have been identified in rats and proposed to be involved in constitutive vesicle traffic. To test whether the inositol high polyphosphates also regulate constitutive vesicle traffic by binding to the non-neuronal synaptotagmins, we examined the IP4 binding properties of the recombinant C2 domains of both neuronal (III, V, X, and XI) and non-neuronal (VI–VIII and IX) synaptotagmins. The C2B domains of synaptotagmins VII–IX and XI had strong IP4 binding activity, but the C2B domain of synaptotagmin VI showed very weak IP4 binding activity. In contrast, there was no significant IP4 binding activity of the C2B domains of synaptotagmins III, V, and X or any of the C2A domains. A phylogenetic tree of the C2 domains of 11 isoforms revealed that synaptotagmins III, V, VI, and X (IP4-insensitive or very weak IP4-binding isoforms) belong to the same branch. Based on the sequence comparison between the IP4-sensitive and -insensitive isoforms, we performed site-directed mutagenesis of synaptotagmin III and identified several amino acid substitutions that abolish IP4 binding activity. Our data suggest that the inositol high polyphosphates might also regulate constitutive vesicle traffic via binding to the IP4-sensitive non-neuronal synaptotagmins. Synaptotagmins are a family of vesicle membrane proteins characterized by a short intravesicular amino terminus, a single transmembrane region, and two copies of highly conserved repeats homologous to the C2 regulatory region of protein kinase C (named the C2A and C2B domains) in the cytoplasmic domain (reviewed in Ref. 1Südhof T.C. Rizo J. Neuron. 1996; 17: 379-388Abstract Full Text Full Text PDF PubMed Scopus (396) Google Scholar). To date, at least 11 isoforms (synaptotagmins I–XI) have been described in rats or mice (2Perin M.S. Fried V.A. Mignery G.A. Jahn R. Südhof T.C. Nature. 1990; 345: 260-263Crossref PubMed Scopus (650) Google Scholar, 3Geppert M. Archer III, B.T. Südhof T.C. J. Biol. Chem. 1991; 266: 13548-13552Abstract Full Text PDF PubMed Google Scholar, 4Mizuta M. Inagaki N. Nemoto Y. Matsukura S. Takahashi M. Seino S. J. Biol. Chem. 1994; 269: 11675-11678Abstract Full Text PDF PubMed Google Scholar, 5Hilbush B.S. Morgan J.I. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8195-8199Crossref PubMed Scopus (78) Google Scholar, 6Craxton M. Goedert M. FEBS Lett. 1995; 361: 196-200Crossref PubMed Scopus (67) Google Scholar, 7Hudson A.W. Birnbaum M.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5895-5899Crossref PubMed Scopus (78) Google Scholar, 8Li C. Ullrich B. Zhang J.Z. Anderson R.G. Brose N. Südhof T.C. Nature. 1995; 375: 594-599Crossref PubMed Scopus (541) Google Scholar, 9Babity J.M. Armstrong J.N. Plumier J.C. Currie R.W. Robertson H.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2638-2641Crossref PubMed Scopus (73) Google Scholar, 10von Poser C. Ichtchenko K. Shao X. Rizo J. Südhof T.C. J. Biol. Chem. 1997; 272: 14314-14319Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). Based on their expression patterns in tissues, synaptotagmins I–V, X, and XI are classified as neuronal types (expressed abundantly in neurons), and others (synaptotagmins VI–IX) are expressed in a wide variety of tissues other than brain (so-called ubiquitous types). Most of the proteins involved in Ca2+-regulated exocytosis in neurons (e.g.synaptobrevin or syntaxin) have been reported to have homologues involved in constitutive membrane trafficking; and therefore, it has been suggested that the same protein family governs both constitutive and regulated vesicle traffic (11Bennett M.K. Scheller R.H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2559-2563Crossref PubMed Scopus (547) Google Scholar, 12Ferro-Novick S. Jahn R. Nature. 1994; 370: 191-193Crossref PubMed Scopus (559) Google Scholar, 13Söllner T. Rothman J.E. Trends Neurosci. 1994; 17: 344-348Abstract Full Text PDF PubMed Scopus (157) Google Scholar). Based on this idea, the ubiquitous isoforms of synaptotagmin are also thought to be involved in constitutive vesicle trafficking because synaptotagmin I (the best characterized neuronal type) is essential for Ca2+-regulated exocytosis in neurons and some endocrine cells (probably functioning as a Ca2+ sensor) (reviewed in Ref. 1Südhof T.C. Rizo J. Neuron. 1996; 17: 379-388Abstract Full Text Full Text PDF PubMed Scopus (396) Google Scholar). However, the exact localization and functions of the ubiquitous synaptotagmins remain unknown. Recently, we demonstrated the distinct roles of two C2 domains of synaptotagmin I (or II) in Ca2+-regulated exocytosis in the squid giant presynapse (14Mikoshiba K. Fukuda M. Moreira J.E. Lewis F.M.T. Sugimori M. Niinobe M. Llinás R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10703-10707Crossref PubMed Scopus (115) Google Scholar, 15Fukuda M. Moreira J.E. Lewis F.M.T. Sugimori M. Niinobe M. Mikoshiba K. Llinás R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10708-10712Crossref PubMed Scopus (136) Google Scholar), superior cervical ganglion cells (16Mochida S. Fukuda M. Niinobe M. Kobayashi H. Mikoshiba K. Neuroscience. 1997; 77: 937-943Crossref PubMed Scopus (56) Google Scholar), chromaffin cells (17Ohara-Imaizumi M. Fukuda M. Niinobe M. Misonou H. Ikeda K. Murakami T. Kawasaki M. Mikoshiba K. Kumakura K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 287-291Crossref PubMed Scopus (67) Google Scholar), and insulin-secreting cells (18Lang J. Fukuda M. Zhang H. Mikoshiba K. Wollheim C.B. EMBO J. 1997; 16: 5837-5846Crossref PubMed Scopus (102) Google Scholar) by using specific antibodies against each C2 domain. The C2A domain is crucial for Ca2+-regulated exocytosis and is directly involved in the fusion of synaptic vesicles with the presynaptic plasma membrane. This fusion step was strongly inhibited by binding of an inositol high polyphosphate (inositol 1,3,4,5-tetrakisphosphate (IP4), 1The abbreviations used are: IP4, inositol 1,3,4,5-tetrakisphosphate; IP5, inositol 1,3,4,5,6-pentakisphosphate; IP6, inositol hexakisphosphate; PCR, polymerase chain reaction; Syt, synaptotagmin; GST, glutathione S-transferase. inositol 1,3,4,5,6-pentakisphosphate (IP5), and inositol hexakisphosphate (IP6)) to the C2B domain of synaptotagmin I (or II) (15Fukuda M. Moreira J.E. Lewis F.M.T. Sugimori M. Niinobe M. Mikoshiba K. Llinás R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10708-10712Crossref PubMed Scopus (136) Google Scholar, 16Mochida S. Fukuda M. Niinobe M. Kobayashi H. Mikoshiba K. Neuroscience. 1997; 77: 937-943Crossref PubMed Scopus (56) Google Scholar, 17Ohara-Imaizumi M. Fukuda M. Niinobe M. Misonou H. Ikeda K. Murakami T. Kawasaki M. Mikoshiba K. Kumakura K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 287-291Crossref PubMed Scopus (67) Google Scholar, 19Niinobe M. Yamaguchi Y. Fukuda M. Mikoshiba K. Biochem. Biophys. Res. Commun. 1994; 205: 1036-1042Crossref PubMed Scopus (59) Google Scholar, 20Fukuda M. Aruga J. Niinobe M. Aimoto S. Mikoshiba K. J. Biol. Chem. 1994; 269: 29206-29211Abstract Full Text PDF PubMed Google Scholar, 21Llinás R. Sugimori M. Lang E.J. Morita M. Fukuda M. Niinobe M. Mikoshiba K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12990-12993Crossref PubMed Scopus (88) Google Scholar, 22Fukuda M. Kojima T. Aruga J. Niinobe M. Mikoshiba K. J. Biol. Chem. 1995; 270: 26523-26527Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 23Mehrotra B. Elliott J.T. Chen J. Olszewski J.D. Profit A.A. Chaudhary A. Fukuda M. Mikoshiba K. Prestwich G.D. J. Biol. Chem. 1997; 272: 4237-4244Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). In chromaffin cells particularly, IP5 is suggested to function as a fusion clamp for exocytosis because IP5 is rapidly accumulated after depolarizing stimulation (17Ohara-Imaizumi M. Fukuda M. Niinobe M. Misonou H. Ikeda K. Murakami T. Kawasaki M. Mikoshiba K. Kumakura K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 287-291Crossref PubMed Scopus (67) Google Scholar). These observations raised the possibility that inositol high polyphosphates may also regulate other types of vesicle traffic (e.g. constitutive) via binding to ubiquitous members of the synaptotagmin family (24Fukuda M. Mikoshiba K. Bioessays. 1997; 19: 593-603Crossref PubMed Scopus (89) Google Scholar). To address this question, we examined the inositol high polyphosphate binding properties of the C2 domains of all synaptotagmin isoforms identified to date (synaptotagmins I–XI) as an indicator of IP4 binding activity. In this study, we show that the C2B domains, but not the C2A domains, of non-neuronal synaptotagmin isoforms (VII–IX) also have strong IP4 binding activities. In addition, we newly identified a subclass of synaptotagmins deficient in IP4 binding activity, despite having a putative IP4-binding sequence as determined previously (20Fukuda M. Aruga J. Niinobe M. Aimoto S. Mikoshiba K. J. Biol. Chem. 1994; 269: 29206-29211Abstract Full Text PDF PubMed Google Scholar, 22Fukuda M. Kojima T. Aruga J. Niinobe M. Mikoshiba K. J. Biol. Chem. 1995; 270: 26523-26527Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). Interestingly, this class of synaptotagmins (III, V, VI, and X) is structurally related and distinguished from other isoforms by phylogenetic trees of the C2 domains. We further determined the conserved amino acid substitutions that abolish IP4 binding activity by site-directed mutagenesis. On the basis of these results, we discuss the functional difference between IP4-sensitive and -insensitive synaptotagmins in vesicular trafficking. cDNAs encoding two C2 domains of synaptotagmin isoforms (V–VII, X, and XI) were amplified by reverse transcriptase-polymerase chain reaction (PCR) from mouse cerebellum cDNAs using the following primers designed on the basis of rat sequences (8Li C. Ullrich B. Zhang J.Z. Anderson R.G. Brose N. Südhof T.C. Nature. 1995; 375: 594-599Crossref PubMed Scopus (541) Google Scholar, 9Babity J.M. Armstrong J.N. Plumier J.C. Currie R.W. Robertson H.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2638-2641Crossref PubMed Scopus (73) Google Scholar, 10von Poser C. Ichtchenko K. Shao X. Rizo J. Südhof T.C. J. Biol. Chem. 1997; 272: 14314-14319Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar) with the addition of appropriate restriction endonuclease sites: SytV, 5′-CGGGATCCGGGAGGAGAAGTAACAGCAA-3′ (sense) and 5′-GGAATTCTCATCGTTTCTCCAGCAGAG-3′ (antisense); SytVI, 5′-CGGGATCCAGAGGCAACATGGCGGATAA-3′ (sense) and 5′-GGAATTCTCACAACCGGGGGGTTCCCT-3′ (antisense); SytVII, 5′-CGTGATCAGAGGAGGATGAGGCCCATGA-3′ (sense) and 5′-GGAATTCCACAGGCTGCCGGGGACGAG-3′ (antisense); SytX, 5′-CGGGATCCGAGCCTGCAATAAAAATCAG-3′ (sense) and 5′-GGAATTCTCACAAGGGGTGCCAGTGTGTGA (antisense); and SytXI, 5′- GAAGATCTATGGCTGAGATCACAAATAT-3′ (sense) and 5′-CCAATTGTTAGTACTCGCTCAGACTGT-3′ (antisense). Reactions were carried out for 30 cycles, each consisting of denaturation at 94 °C for 1 min, annealing at 50 °C for 2 min, and extension at 72 °C for 2 min. After digestion with BamHI, BglII, orFbaI and EcoRI or MunI, the PCR products were purified on an agarose gel, extracted with a Geneclean II kit (BIO 101, Inc.), and then inserted into theBamHI-EcoRI site of the pGEX-2T vector (Amersham Pharmacia Biotech). Only the nucleotide sequences coding for two C2 domains were sequenced in both directions using a BcaBEST dideoxy sequencing kit (Takara Shuzo). As compared with rat sequences, several amino acid substitutions were found in mouse SytVI (Ser at position 471 was altered to Asn, S471N), SytVII (I218V), SytX (S263F and M362I), and SytXI (G188D and I357V). These changes are probably not due to PCR-induced errors because they were also found in two independent PCR products of each synaptotagmin. Using primers based on the mouse nucleotide sequences obtained above and the data base for mouse SytVIII, fragments encoding the C2A or C2B domains of synaptotagmin isoforms (SytV-, SytVII-, SytVIII-, SytX-, and SytXI-C2A or -C2B and SytVI- and SytIX-C2B) were amplified by PCR. After digestion withBamHI or BglII and EcoRI, the PCR products were inserted into the BamHI-EcoRI site of the pGEX-2T vector and verified by DNA sequencing. pGEX-2T vectors carrying mouse SytI–IV-C2A or -C2B and SytVI- and SytIX-C2A were constructed as described previously (20Fukuda M. Aruga J. Niinobe M. Aimoto S. Mikoshiba K. J. Biol. Chem. 1994; 269: 29206-29211Abstract Full Text PDF PubMed Google Scholar, 22Fukuda M. Kojima T. Aruga J. Niinobe M. Mikoshiba K. J. Biol. Chem. 1995; 270: 26523-26527Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 25Fukuda M. Kojima T. Mikoshiba K. J. Biol. Chem. 1996; 271: 8430-8434Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Glutathione S-transferase (GST) fusion proteins of the C2A and C2B domains of mouse synaptotagmin isoforms (GST-STI–XI-C2A and -C2B, where STI–XI is synaptotagmins I–XI) were expressed inEscherichia coli JM109 and then purified by glutathione-Sepharose 4B (Amersham Pharmacia Biotech) chromatography according to the manufacturer's recommendations. GST-STI-C2A and -C2B encoded amino acids 138–266 and 268–392, respectively, of mouse synaptotagmin I. Similarly, the following fusion proteins were prepared: GST-STII-C2A, -C2B, and -C2BΔC, corresponding to amino acids 139–267, 267–393, and 267–346 of mouse synaptotagmin II, respectively; GST-STIII-C2A, -C2B, and -C2BΔC, corresponding to amino acids 290–421, 425–549, and 425–501 of mouse synaptotagmin III, respectively; GST-STIV-C2A and -C2B, corresponding to amino acids 151–281 and 281–408 of mouse synaptotagmin IV, respectively; GST-STV-C2A, -C2B, and -C2BΔC, corresponding to amino acids 5–135, 135–261, and 135–213 of mouse synaptotagmin V, respectively; GST-STVI-C2A and -C2B, corresponding to amino acids 227–357 and 357–483 of mouse synaptotagmin VI, respectively; GST-STVII-C2A and -C2B, corresponding to amino acids 132–261 and 261–387 of mouse synaptotagmin VII, respectively; GST-STVIII-C2A and -C2B, corresponding to amino acids 70–195 and 195–318 of mouse synaptotagmin VIII, respectively; GST-STIX-C2A and -C2B, corresponding to amino acids 105–234 and 233–360 of mouse synaptotagmin IX, respectively; GST-STX-C2A, -C2B, and -C2BΔC, corresponding to amino acids 228–358, 358–484, and 358–436 of mouse synaptotagmin X, respectively; and GST-STXI-C2A and -C2B, corresponding to amino acids 153–284 and 284–413 of mouse synaptotagmin XI, respectively. The amino acids of SytV–VIII, SytX, and SytXI were numbered according to previously described rat sequences (8Li C. Ullrich B. Zhang J.Z. Anderson R.G. Brose N. Südhof T.C. Nature. 1995; 375: 594-599Crossref PubMed Scopus (541) Google Scholar, 9Babity J.M. Armstrong J.N. Plumier J.C. Currie R.W. Robertson H.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2638-2641Crossref PubMed Scopus (73) Google Scholar, 10von Poser C. Ichtchenko K. Shao X. Rizo J. Südhof T.C. J. Biol. Chem. 1997; 272: 14314-14319Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). The protein concentrations of the purified recombinant proteins were initially determined using a Bio-Rad protein assay with bovine serum albumin used as a reference. Purified proteins were analyzed by 10% SDS-polyacrylamide gel electrophoresis followed by Coomassie Brilliant Blue R-250 staining. The purity of each recombinant protein was estimated by scanning the Coomassie Blue-stained SDS-polyacrylamide gel followed by densitometry with BioImage (Millipore Corp.), and the concentration of each GST fusion protein was then determined with bovine serum albumin as a standard. cDNAs encoding two C2 domains of SrgI and SytB/K were also amplified by reverse transcriptase-PCR from mouse cerebellum cDNAs using the following primers designed on the basis of rat sequences (26Thompson C.C. J. Neurosci. 1996; 16: 7832-7840Crossref PubMed Google Scholar, 27Kwon O.J. Gainer H. Wray S. Chin H. FEBS Lett. 1996; 378: 135-139Crossref PubMed Scopus (32) Google Scholar) with the addition of appropriate restriction endonuclease sites: SrgI, 5′-GAAGATCTATGGCCGTGGACGTGACAGA (sense) and 3′-CCAATTGTTAGTTTCGCCGGACTGGAT-3′ (antisense); and SytB/K, 5′-CGGGATCCATGGCGTACATCCAGTTGGA-3′ (sense) and 5′-GGAATTCTCAGGTCACCTCCAGCGAGG-3′ (antisense). Reaction conditions were the same as described above. After digestion with BamHI orBglII and EcoRI or MunI, the PCR products were purified on an agarose gel and extracted with a Geneclean II kit and then inserted into the BamHI-EcoRI site of the pGEX-2T vector. Only the nucleotide sequences coding for two C2 domains were sequenced in both directions using a BcaBEST dideoxy sequencing kit. As compared with rat sequences, three amino acid substitutions were found in mouse SrgI (D315E, T317S, and A393V). These changes are probably not due to PCR-induced errors because they were also found in two independent PCR products. Using primers based on the mouse nucleotide sequences obtained above, fragments encoding the C2A or C2B domains of SrgI and SytB/K were amplified by PCR. After digestion with BamHI or BglII andEcoRI, the PCR products were inserted into theBamHI-EcoRI site of the pGEX-2T vector and verified by DNA sequencing. GST-Srg-C2A, -C2B, and -C2BΔC contained amino acids 149–278, 278–404, and 278–356, respectively, of mouse SrgI; and GST-STB/K-C2A, -C2B, and -C2BΔC contained amino acids 180–315, 315–443, and 315–394, respectively, of mouse SytB/K. Site-directed mutagenesis of GST-STIII-C2Bα-(P505F,E509Q,N510K) and GST-STIII-C2Bβ7-(H525K,V531K,C532I,R533F) was carried out by means of two-step PCR as follows (22Fukuda M. Kojima T. Aruga J. Niinobe M. Mikoshiba K. J. Biol. Chem. 1995; 270: 26523-26527Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). In GST-STIII-C2Bα-(P505F,E509Q,N510K), for example, the right and left halves of the C2B domain were separately amplified with two pairs of oligonucleotides (primer A (5′-CGGGATCCGAAAAGGCAGATCTTGGGGA-3′) and mutagenic primer B (5′-GCGACGTCGAACACCAGGGCCT-3′) (left half); mutagenic primer C (5′-TCGACGTCGCTTTCGAGAGCGTGCAGAAAGTGGGTCTCAG-3′) and primer D (5′-GGAATTCATCTCTGCCCAGTGTTCTC-3′) (right half)). The two resulting PCR fragments were digested with AatII (underlined), ligated to each other, and reamplified with primers A and D. The obtained PCR fragment encoding the mutant C2B domain of synaptotagmin III (P505F,E509Q,N510K) was subcloned into theBamHI-EcoRI site of pGEX-2T and verified by DNA sequencing. Site-directed mutagenesis of GST-STIII-C2Bloopβ7–8-(E537N,A539T,D540G,G543L) was achieved by PCR using primer A and a mutagenic primer (primer E, 5′-GGAATTCATCTCTGCCCAGTGTTCTCTGAGGTGTGGGCCGGTAGCGTTTGGGCCCACG-3′). The obtained PCR fragment encoding the mutant C2B domain of synaptotagmin III (E537N,A539T,D540G,G543L) was subcloned into theBamHI-EcoRI site of the pGEX-2T vector and verified by DNA sequencing. Other plasmids encoding the mutant C2B domain of synaptotagmin were similarly constructed by means of PCR using mutagenic primers. Measurement of IP4 binding was performed as described previously (20Fukuda M. Aruga J. Niinobe M. Aimoto S. Mikoshiba K. J. Biol. Chem. 1994; 269: 29206-29211Abstract Full Text PDF PubMed Google Scholar) with slight modifications. Briefly, the buffer system was changed from 20 mm Tris-HCl (pH 8.0) to 50 mm HEPES-KOH (pH 7.2) because GST-STII-C2B showed stronger IP4 binding activity in the latter buffer. GST fusion proteins (1–2.5 μg) were incubated with 9.6 nm [3H]IP4 (specific radioactivity of 777 GBq/mmol; NEN Life Science Products) in 49 μl of 50 mm HEPES-KOH (pH 7.2) for 10 min at 4 °C. The sample was then mixed with 1 μl of 50 mg/ml γ-globulins and 50 μl of a solution containing 30% (w/v) polyethylene glycol 6000 and 50 mm HEPES-KOH (pH 7.2) and placed on ice for 5 min. The precipitate obtained by centrifugation at 10,000 × gfor 5 min was solubilized in 500 μl of Solvable (Packard Instrument Co.), and radioactivity was measured in Aquasol 2 (Packard Instrument Co.) with a liquid scintillation counter. Nonspecific binding was determined in the presence of 10 μm nonradioactive IP4 Multiple sequence alignment of the C2 domains of synaptotagmin isoforms was performed using the PILEUP program of the GCG program (Version 8.1). Calculation of genetic distance and suitable depiction of the phylogenetic tree using the neighbor joining method were performed with the SINCA program (Fujitsu). Putative IP4-binding sites were aligned referring to the multiple alignment results. Statistical analysis and curve fitting were done using the GraphPad PRISM computer program (Version 2.0). The C2 domain, originally identified as a sequence motif of protein kinase C, is a conserved protein module of ∼120 amino acids found in many proteins (28Brose N. Hofmann K. Hata Y. Südhof T.C. J. Biol. Chem. 1995; 270: 25273-25280Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar). Among them, synaptotagmins are apparently distinguished from other proteins in that they have a single transmembrane region and tandem C2 domains (C2A and C2B domains) with a short spacer. In our previous studies, we showed that neuronal synaptotagmins I, II, and IV, but not synaptotagmin III, are IP4- or inositol high polyphosphate-binding proteins (20Fukuda M. Aruga J. Niinobe M. Aimoto S. Mikoshiba K. J. Biol. Chem. 1994; 269: 29206-29211Abstract Full Text PDF PubMed Google Scholar,22Fukuda M. Kojima T. Aruga J. Niinobe M. Mikoshiba K. J. Biol. Chem. 1995; 270: 26523-26527Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 23Mehrotra B. Elliott J.T. Chen J. Olszewski J.D. Profit A.A. Chaudhary A. Fukuda M. Mikoshiba K. Prestwich G.D. J. Biol. Chem. 1997; 272: 4237-4244Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). To further examine whether other neuronal and non-neuronal isoforms of synaptotagmins are also regulated by inositol high polyphosphates like synaptotagmin I, we prepared GST fusion proteins of C2 domains of synaptotagmins V–XI and tested for their IP4 binding activity (Fig. 1 and TableI). GST-STVII, -STVIII, -STIX, and -STXI-C2B had strong IP4 binding activity like synaptotagmin II, but GST-STVI-C2B showed weak IP4 binding activity (<20% of that of GST-STII-C2B). In contrast, GST-STV and -STX and all the GST-ST-C2A fusion proteins showed no significant IP4 binding activity under our experimental conditions.Table IComparison of IP4 binding properties of tandem C2 domains of synaptotagmins, SrgI, synaptotagmin B/K, rabphilin 3A, and Doc2NameC2AC2BRef.SytI −1-a−, no significant IP4 binding activity; +++, 75–100% of the IP4 binding activity of GST-STII-C2B (see Fig.1); ++, 50–75% of the IP4 binding activity of GST-STII-C2B; +, 5–25% of the IP4 binding activity of GST-STII-C2B.+++20Fukuda M. Aruga J. Niinobe M. Aimoto S. Mikoshiba K. J. Biol. Chem. 1994; 269: 29206-29211Abstract Full Text PDF PubMed Google ScholarSytII−+++20Fukuda M. Aruga J. Niinobe M. Aimoto S. Mikoshiba K. J. Biol. Chem. 1994; 269: 29206-29211Abstract Full Text PDF PubMed Google ScholarSytIII−−22Fukuda M. Kojima T. Aruga J. Niinobe M. Mikoshiba K. J. Biol. Chem. 1995; 270: 26523-26527Abstract Full Text Full Text PDF PubMed Scopus (162) Google ScholarSytIV−+++22Fukuda M. Kojima T. Aruga J. Niinobe M. Mikoshiba K. J. Biol. Chem. 1995; 270: 26523-26527Abstract Full Text Full Text PDF PubMed Scopus (162) Google ScholarSytV−−This studySytVI−+This studySytVII−+++This studySytVIII−++This studySytIX−+++This studySytX−−This studySytXI−+++This studySrgI−−This studySytB/K−−This studyRabphilin 3A−−20Fukuda M. Aruga J. Niinobe M. Aimoto S. Mikoshiba K. J. Biol. Chem. 1994; 269: 29206-29211Abstract Full Text PDF PubMed Google ScholarDoc2α−−30Kojima T. Fukuda M. Aruga J. Mikoshiba K. J. Biochem. (Tokyo). 1996; 120: 671-676Crossref PubMed Scopus (45) Google ScholarDoc2β−−30Kojima T. Fukuda M. Aruga J. Mikoshiba K. J. Biochem. (Tokyo). 1996; 120: 671-676Crossref PubMed Scopus (45) Google Scholar1-a −, no significant IP4 binding activity; +++, 75–100% of the IP4 binding activity of GST-STII-C2B (see Fig.1); ++, 50–75% of the IP4 binding activity of GST-STII-C2B; +, 5–25% of the IP4 binding activity of GST-STII-C2B. Open table in a new tab To understand the relationship between the molecular evolution of the C2 domains of synaptotagmins and IP4binding capacity, a phylogenetic tree of the C2 domains of synaptotagmin isoforms was constructed using the neighbor joining method (Fig. 2). In this phylogenetic tree, the C2B domain of synaptotagmin I is expressed as the most primitive or original form of the C2B domain because this domain of invertebrate synaptotagmins has been identified inDrosophila (31Perin M.S. Johnston P.A. Özcelik T. Jahn R. Francke U. Südhof T.C. J. Biol. Chem. 1991; 266: 615-622Abstract Full Text PDF PubMed Google Scholar), Caenorhabditis elegans (32Nonet M.L. Grundahl K. Meyer B.J. Rand J.B. Cell. 1993; 73: 1291-1305Abstract Full Text PDF PubMed Scopus (458) Google Scholar),Aplysia (33Martin K.C. Hu Y. Armitage B.A. Siegelbaum S.A. Kandel E.R. Kaang B.-K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11307-11311Crossref PubMed Scopus (43) Google Scholar), and squid (Loligo pealei) (14Mikoshiba K. Fukuda M. Moreira J.E. Lewis F.M.T. Sugimori M. Niinobe M. Llinás R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10703-10707Crossref PubMed Scopus (115) Google Scholar) as having a less distant genetic relationship to mouse synaptotagmin I than the other isoforms (data not shown). According to this phylogenetic tree, the C2 domains of synaptotagmin isoforms are classified into two distinct groups, C2A and C2B, and the C2 domain from the same isoform is located at very similar positions in the two groups, suggesting that mammalian synaptotagmin isoforms were separated after the tandem C2 domains had been produced. When synaptotagmins that bind IP4 strongly or weakly are solid-boxed orbroken-boxed, respectively, it is apparent that the C2B domains of synaptotagmins III, V, VI, and X (IP4-insensitive or weak binding isoforms) form a small but distinct branch (Fig. 2). To further examine whether these synaptotagmins (III, V, VI, and X) have a common sequence responsible for the lack of IP4 binding at the amino acid level, we compared the putative IP4-binding sites of all synaptotagmin isoforms as determined previously (20Fukuda M. Aruga J. Niinobe M. Aimoto S. Mikoshiba K. J. Biol. Chem. 1994; 269: 29206-29211Abstract Full Text PDF PubMed Google Scholar, 22Fukuda M. Kojima T. Aruga J. Niinobe M. Mikoshiba K. J. Biol. Chem. 1995; 270: 26523-26527Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar) (Fig. 3). However, in this region, no apparent differences were observed between IP4-sensitive and -insensitive synaptotagmin isoforms. Within the putative IP4-binding domain, three positively charged amino acids responsible for high affinity IP4binding activity (Lys at positions 327, 328, and 332 of synaptotagmin II (22Fukuda M. Kojima T. Aruga J. Niinobe M. Mikoshiba K. J. Biol. Chem. 1995; 270: 26523-26527Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar); asterisks in Fig. 3) were highly conserved among isoforms, whereas the corresponding positions in the C2A domains of virtually none of these molecules are occupied by positively charged amino acids (data not shown) (24Fukuda M. Mikoshiba K. Bioessays. 1997; 19: 593-603Crossref PubMed Scopus (89) Google Scholar). Since SytVIII-C2B lacks one of the important Lys residues (Ser at position 252, Ser-252), its IP4 binding activity was weaker than that of synaptotagmin II (Fig. 3), which is consistent with our previous mutational analysis (22" @default.
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