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Synamon, a Novel Neuronal Protein Interacting with Synapse-associated Protein 90/Postsynaptic Density-95-associated Protein

1999; Elsevier BV; Volume: 274; Issue: 39 Linguagem: Inglês

10.1074/jbc.274.39.27463

1083-351X

Ikuko Yao, Yutaka Hata, Kazuyo Hirao, Maki Deguchi, Nobuyuki Ide, Masakazu Takeuchi, Yoshimi Takai,

ATP Synthase and ATPases Research

Guanylate kinase-associated protein (GKAP)/SAP90/PSD-95-associated protein (SAPAP)/DLG-associated protein (DAP) is a protein of the postsynaptic density (PSD), and binds to the guanylate kinase domain of PSD-95/synapse-associated protein (SAP) 90 and synaptic scaffolding molecule. GKAP/SAPAP/DAP recruits PSD-95/SAP90 and its interacting protein, brain-enriched guanylate kinase-interacting protein, into the Triton X-100-insoluble fraction in transfected cells, suggesting that GKAP/SAPAP/DAP may link several PSD components to the Triton X-100-insoluble structures in the PSD. We have identified here a novel neuronal GKAP/SAPAP/DAP-binding protein and named it synamon. Synamon has seven ankyrin repeats at the NH2 terminus followed by one src homology 3 domain and one PSD-95/Dlg-A/ZO-1 domain, and several proline-rich regions at the carboxyl terminus. Synamon interacts with the COOH-terminal region of GKAP/SAPAP/DAP via the middle region containing a PSD-95/Dlg-A/ZO-1 domain. Synamon was coimmunoprecipitated with SAPAP from rat crude synaptosomes and colocalized with SAPAP in primary cultured rat hippocampal neurons. Because synamon is composed of various protein-interacting modules, it may also interact with proteins other than GKAP/SAPAP/DAP to organize the architecture of the PSD. Guanylate kinase-associated protein (GKAP)/SAP90/PSD-95-associated protein (SAPAP)/DLG-associated protein (DAP) is a protein of the postsynaptic density (PSD), and binds to the guanylate kinase domain of PSD-95/synapse-associated protein (SAP) 90 and synaptic scaffolding molecule. GKAP/SAPAP/DAP recruits PSD-95/SAP90 and its interacting protein, brain-enriched guanylate kinase-interacting protein, into the Triton X-100-insoluble fraction in transfected cells, suggesting that GKAP/SAPAP/DAP may link several PSD components to the Triton X-100-insoluble structures in the PSD. We have identified here a novel neuronal GKAP/SAPAP/DAP-binding protein and named it synamon. Synamon has seven ankyrin repeats at the NH2 terminus followed by one src homology 3 domain and one PSD-95/Dlg-A/ZO-1 domain, and several proline-rich regions at the carboxyl terminus. Synamon interacts with the COOH-terminal region of GKAP/SAPAP/DAP via the middle region containing a PSD-95/Dlg-A/ZO-1 domain. Synamon was coimmunoprecipitated with SAPAP from rat crude synaptosomes and colocalized with SAPAP in primary cultured rat hippocampal neurons. Because synamon is composed of various protein-interacting modules, it may also interact with proteins other than GKAP/SAPAP/DAP to organize the architecture of the PSD. postsynaptic density synapse-associated protein 90 guanylate kinase-associated protein SAP90/PSD-95-associated protein DLG-associated protein brain-enriched guanylate kinase-interacting protein synaptic scaffolding molecule amino acid src homology 3 N-methyl-d-aspartate glutathioneS-transferase The postsynaptic density (PSD)1 is a submembranous structure at the postsynaptic membrane mainly at the excitatory synapses (for review, see Refs. 1Kennedy M.B. Curr. Opin. Neurobiol. 1993; 3: 732-737Crossref PubMed Scopus (127) Google Scholar, 2Kennedy M.B. Trends Neurosci. 1997; 20: 264-268Abstract Full Text Full Text PDF PubMed Scopus (403) Google Scholar, 3Ziff E.B. Cell. 1997; 19: 1153-1174Google Scholar). The neurotransmitter receptors are assembled and fixed at the PSD, and several molecules implicated in the synaptic plasticity are also enriched. PSD-95/synapse-associated protein (SAP) 90 is involved in the molecular organization of these components of the PSD (Refs. 4Cho K.-O. Hunt C.A. Kennedy M.B. Neuron. 1992; 9: 929-942Abstract Full Text PDF PubMed Scopus (996) Google Scholar and 5Kistner U. Wenzel B.M. Veh R.W. Cases-Langhoff C. Garner A.M. Appeltauer U. Voss B. Gundelfinger E.D. Garner C.C. J. Biol. Chem. 1993; 268: 4580-4583Abstract Full Text PDF PubMed Google Scholar; for review, see Refs. 6Garner C. Kindler S. Trends Cell Biol. 1996; 19: 429-433Abstract Full Text PDF Scopus (63) Google Scholar, 7Gomperts S.N. Cell. 1996; 84: 69-662Abstract Full Text Full Text PDF Scopus (219) Google Scholar, 8Sheng M. Neuron. 1996; 17: 575-578Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar, 9Craven S.E. Bredt D.S. Cell. 1998; 93: 495-498Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar, 10O'Brien R.J. Lau L.F. Huganir R.L. Curr. Opin. Neurobiol. 1998; 8: 364-369Crossref PubMed Scopus (241) Google Scholar, 11Hata Y. Takai Y. Cell. Mol. Life Sci.,. 1999; (in press)PubMed Google Scholar) and essential for learning and memory (12Migaud M. Charlesworth P. Dempster M. Webster L.C. Watabe A.M. Makhisson M. He Y. Ramsay M.F. Morris R.G.M. Morrison J.H. O'Dell T.J. Grant S.G.N. Nature. 1998; 386: 433-439Crossref Scopus (948) Google Scholar). PSD-95/SAP90 is a member of membrane-associated guanylate kinases (for review, see Ref. 13Anderson J.M. Curr. Biol. 1996; 6: 382-384Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar), and binds to guanylate kinase-associated protein (GKAP)/SAP90/PSD-95-associated protein (SAPAP)/DLG-associated protein (DAP) and brain-enriched guanylate kinase-interacting protein (BEGAIN)via the guanylate kinase domain (14Kim E. Naisbitt S. Hsueh Y.-P. Rao A. Rothschild A. Craig A.M. Sheng M. J. Cell Biol. 1997; 136: 669-678Crossref PubMed Scopus (424) Google Scholar, 15Takeuchi M. Hata Y. Hirao K. Toyoda A. Irie M. Takai Y. J. Biol. Chem. 1997; 272: 11943-11951Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar, 16Satoh K. Yanai H. Senda T. Kohu K. Nakamura T. Okumura N. Matsumine A. Kobayashi S. Toyoshima K. Akiyama T. Genes Cells. 1997; 2: 415-424Crossref PubMed Scopus (112) Google Scholar, 17Deguchi M. Hata Y. Takeuchi M. Ide N. Hirao K. Yao I. Irie M. Toyoda A. Takai Y. J. Biol. Chem. 1998; 273: 26269-26272Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). In this paper, we have described GKAP/SAPAP/DAP as SAPAP just for simplicity. SAPAP is Triton X-100-insoluble in both the brain homogenate and the transfected cells. It links PSD-95/SAP90 to the Triton X-100-insoluble structures of transfected Chinese hamster ovary cells (17Deguchi M. Hata Y. Takeuchi M. Ide N. Hirao K. Yao I. Irie M. Toyoda A. Takai Y. J. Biol. Chem. 1998; 273: 26269-26272Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). BEGAIN is also recruited by SAPAP into the Triton X-100-insoluble fraction with PSD-95/SAP90 in Chinese hamster ovary cells (17Deguchi M. Hata Y. Takeuchi M. Ide N. Hirao K. Yao I. Irie M. Toyoda A. Takai Y. J. Biol. Chem. 1998; 273: 26269-26272Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). SAPAP interacts with another neuronal membrane-associated guanylate kinase, synaptic scaffolding molecule (S-SCAM), which also provides scaffolds for the components of synaptic junctions (18Hirao K. Hata Y. Ide N. Takeuchi M. Irie M. Yao I. Deguchi M. Toyoda A. Sudhof T.C. Takai Y. J. Biol. Chem. 1998; 273: 21105-21110Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). Thereby, SAPAP is a key protein in the architecture of the PSD. The detergent-insolubility of SAPAP is conferred by its NH2-terminal and middle regions. The middle region contains 5 repeats of 14 amino acids (aa) and is involved in the interaction with PSD-95/SAP90 (14Kim E. Naisbitt S. Hsueh Y.-P. Rao A. Rothschild A. Craig A.M. Sheng M. J. Cell Biol. 1997; 136: 669-678Crossref PubMed Scopus (424) Google Scholar, 15Takeuchi M. Hata Y. Hirao K. Toyoda A. Irie M. Takai Y. J. Biol. Chem. 1997; 272: 11943-11951Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar, 16Satoh K. Yanai H. Senda T. Kohu K. Nakamura T. Okumura N. Matsumine A. Kobayashi S. Toyoshima K. Akiyama T. Genes Cells. 1997; 2: 415-424Crossref PubMed Scopus (112) Google Scholar). A proline-rich region that fits to the consensus motif for the binding of the src homology 3 (SH3) domain exists immediately after the middle region (19Sparks A.B. Rider J.E. Hoffman N.G. Fowlkes D.M. Quilliam L.A. Kay B.K. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1540-1544Crossref PubMed Scopus (329) Google Scholar). The carboxyl-terminal region is Triton X-100-soluble and well conserved among the four isoforms of SAPAP, suggesting that it may have some function. We have searched for a SAPAP-binding protein, identified a novel neuronal protein, which binds to the carboxyl-terminal region of SAPAP, and named it synamon. Rat brain yeast two-hybrid library was constructed using pVP16 vector and screened (20Hata Y. Butz S. Sudhof T.C. J. Neurosci. 1996; 16: 2488-2494Crossref PubMed Google Scholar). Rat brain cDNA libraries (Stratagene andCLONTECH) were screened with the [α-32P]dCTP-labeled random-primed probes (20Hata Y. Butz S. Sudhof T.C. J. Neurosci. 1996; 16: 2488-2494Crossref PubMed Google Scholar). Various expression vectors were constructed by conventional molecular biology techniques and polymerase chain reaction method using pBTM116, pCMV Myc, pClneo Myc, and pGex4T-1 (Amersham Pharmacia Biotech). pBTM116 SAPAP2–1 contains full-length SAPAP2. The following constructs contain the following aa residues of SAPAP1: pCMV Myc SAPAP1–1, 1–992; pCMV Myc SAPAP1–2, 1–477; pCMV Myc SAPAP1–6, 478–992; pCMV Myc SAPAP1–8, 568–992; pClneo Myc SAPAP1–8, 800–992; and pClneo Myc SAPAP1–22, 800–985. The following constructs contain the following aa residues of synamon: pGex4T-1 synamon-16, 1977–2091; pGex4T-1 synamon-18, 616–1110; and pGex4T-1 synamon-19, 863–1337. Rabbit polyclonal antibodies were raised against the product of pGex4T-1 synamon-16, which is the COOH-terminal region of synamon. The anti-SAPAP antibody was described previously (15Takeuchi M. Hata Y. Hirao K. Toyoda A. Irie M. Takai Y. J. Biol. Chem. 1997; 272: 11943-11951Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar). The mouse monoclonal anti-Myc-tag antibody was obtained from American Type Culture Collection. The rhodamine-conjugated and fluorescein isothiocyanate-conjugated second antibodies for dual labeling were purchased from Chemicon. The monoclonal anti-NMDA receptor 1 antibody was a gift of Dr. Nils Brose (Max Planck Institute, Goettingen, Germany). COS cells were cultured in Dulbecco's modified Eagle medium with 10% fetal bovine serum under 10% CO2 at 37 °C and transfected with various Myc-tagged constructs using the DEAE-dextran method (15Takeuchi M. Hata Y. Hirao K. Toyoda A. Irie M. Takai Y. J. Biol. Chem. 1997; 272: 11943-11951Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar). COS cells of two 10-cm plates were homogenized in 0.6 ml of 20 mmHepes/NaOH, pH 8.0, containing 6 m urea and 1% (w/v) Triton X-100, and centrifuged at 100,000 × g for 15 min. The supernatant was dialyzed against 2 liters of 20 mmHepes/NaOH, pH 8.0, containing 100 mm NaCl overnight, then centrifuged at 100,000 × g for 15 min to remove the debris, and used as the COS cell extracts. 0.5-ml aliquots of the extracts of COS cells expressing various Myc-tagged constructs of SAPAP1 were incubated with 800 pmol of various GST fusion proteins fixed on 20 μl of glutathione-Sepharose 4B beads. After the beads were washed with Buffer A (20 mm Hepes/NaOH, pH 8.0, containing 100 mm NaCl and 1% (w/v) Triton X-100), the proteins on the beads were detected by immunoblotting using the anti-Myc antibody. The urea/detergent extracts of rat crude synaptosomes were prepared as described (17Deguchi M. Hata Y. Takeuchi M. Ide N. Hirao K. Yao I. Irie M. Toyoda A. Takai Y. J. Biol. Chem. 1998; 273: 26269-26272Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). 4-ml aliquots of the extracts were incubated with 30 μl of the anti-SAPAP serum or the preimmune serum fixed on protein G-Sepharose Fast Flow beads. After the beads were washed three times with Buffer A, the proteins on the beads were detected by immunoblotting using the anti-synamon antibody. Subcellular fractionation of rat brain, primary cultures of rat hippocampal neurons, immunocytostaining, SDS-polyacrylamide gel electrophoresis, and protein determination were performed as described (15Takeuchi M. Hata Y. Hirao K. Toyoda A. Irie M. Takai Y. J. Biol. Chem. 1997; 272: 11943-11951Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar). Northern and Western blottings were performed using multiple tissue Northern blots (CLONTECH) and ECL reagents (Amersham Pharmacia Biotech), respectively. We performed the yeast two-hybrid screening of a rat brain cDNA library using a bait containing full-length SAPAP2 (pBTM116 1305–1). We obtained 6 positive independent clones from 8 × 105 clones; 4 clones were PSD-95/SAP90, and the remaining two clones, pPrey 3102 and 3103, were encoded novel proteins. pPrey 3102 and pPrey 3103 interacted with not only SAPAP2 but also SAPAP1, -3, and -4 in the yeasts (data not shown). We started the cDNA library screenings to obtain the full-length sequences of these two genes to find that they are alternative splicing isoforms. We obtained the presumptive full-length coding sequence of synamon through the conventional hybridization screening and polymerase chain reaction (Fig. 1). The protein, synamon, was composed of 2,158 aa. The NH2 terminus had 7 ankyrin repeats followed by one SH3 and one PDZ domain. pPrey 3103 contained the residues 404–902 of synamon with an insert of 9 aa (Ile-Leu-Ile-Asp-Gly-Ile-Asp-Ser-Gly) between the residues 644 and 645 (at the triangle in Fig. 1). Several independent clones contained the same insert at the same position. pPrey 3102 contained the residues 646–776 of synamon preceded by an additional 50 aa, which were unique for pPrey 3102. The constructs used in this study were prepared from the clone without the insert. Synamon had the homology toCaenorhabditis elegans C33B4.3, which had 6 ankyrin repeats and one PDZ domain without an SH3 domain. The software TopPred in the simple modular architecture research tool predicted the residues 1793–1813 as a transmembrane domain (21Schultz J. Milpetz F. Bork P. Ponting C.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5857-5864Crossref PubMed Scopus (2972) Google Scholar), but another software, SOSUI in GenomeNet WWW server, predicted a soluble protein. Synamon has several proline-rich regions in the COOH-terminal region (boxed in Fig. 1). We confirmed the binding of synamon to SAPAP in the crude synaptosomes. Synamon was coimmunoprecipitated with SAPAP from the rat crude synaptosomes (Fig. 2 A). Next, we determined which region of SAPAP was involved in the interaction with synamon. Both pPrey 3102 and pPrey 3103 contained the region around the PDZ domain of synamon (underlined in Fig. 1), and this region was conceivably the SAPAP-interacting region of synamon. We prepared two GST fusion proteins of synamon, GST-synamon-18 and -19, and tested the interactions with various Myc-tagged constructs of SAPAP1 (Fig. 2 B). GST-synamon-18 covered the putative SAPAP-interacting region around the PDZ domain, whereas GST-synamon-19 did not contain it. GST-Synamon-18 interacted with full-length SAPAP1 and its COOH-terminal region, but not with the NH2-terminal region (Fig. 2 C). The deletion of COOH-terminal 7 amino acids of SAPAP1 abolished the interaction with GST-synamon-18. GST-synamon-19 did not interact with any Myc-tagged constructs of SAPAP1. The GST-synamon-18 and -19 did not cover the whole sequence of synamon, and we could not exclude the possibility that some untested region of synamon binds to the NH2-terminal and/or middle regions of SAPAP1. The current result, however, indicates that the synamon-interacting region of SAPAP1 is the COOH-terminal region and distinct from the PSD-95/SAP90-interacting middle region. Northern blot analysis revealed a 9.0-kilobase message only in brain (Fig. 3 A). No message was detected in heart, spleen, lung, liver, kidney, skeletal muscles, or testis. Western blot analysis showed a signal with a molecular mass of 288 kDa only in brain (Fig. 3 B). The signal of a molecular mass of 182 kDa was also detected. Because the calculated molecular weight of synamon is 225,508, the 288-kDa protein is likely to be synamon and the 182-kDa protein may be a degradation product or a short isoform. In rat hippocampal neurons, synamon was localized in the cell body and the dendrites (Fig.4 A). On the dendrites, synamon was colocalized with NMDA receptor 1 (Fig. 4 A). Because SAPAP was colocalized with NMDA receptor 1 (Fig. 4 B), synamon was considered to be colocalized with SAPAP in rat hippocampal neurons.Figure 4Subcellular localization of synamon in neurons. Panel A, synamon and NMDA receptor 1 in rat primary cultured hippocampal neurons. Rat hippocampal neurons were double-stained with the polyclonal rabbit anti-synamon and the monoclonal mouse NMDA receptor 1 antibodies. a, synamon; andb, NMDA receptor 1. Panel B, SAPAP and NMDA receptor 1 in rat primary cultured hippocampal neurons. Rat hippocampal neurons were double-stained with the polyclonal rabbit anti-SAPAP and the monoclonal mouse NMDA receptor 1 antibodies. a, SAPAP; and b, NMDA receptor 1. The bar indicates 10 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In this paper, we have identified a novel neuronal protein interacting with GKAP/SAPAP/DAP, synamon, by the yeast two-hybrid screening. Synamon was coimmunoprecipitated with GKAP/SAPAP/DAP from rat crude synaptosomes. Synamon was not remarkably enriched in the PSD fraction and distributed in the soma and neurites of rat primary cultured hippocampal neurons. However, at dendrites, synamon was colocalized with NMDA receptors and GKAP/SAPAP/DAP. These findings suggest that synamon interacts with GKAP/SAPAP/DAP in vivo. Synamon has a characteristic molecular structure composed of ankyrin repeats, an SH3 domain, a PDZ domain, and proline-rich sequences. Synamon interacts with the COOH-terminal region of GKAP/SAPAP/DAP via its middle region containing a PDZ domain. Although ligands for domains other than the PDZ domain of synamon need to be clarified, synamon may interact with unidentified molecules and link them to GKAP/SAPAP/DAP to form the architecture of the PSD. We submitted the sequence of synamon to the GenBank™ in November, 1998 and corrected a sequencing error in May, 1999. After the submission of this work to the Journal of Biological Chemistry, one group reported the same protein as a protein interacting with GKAP/SAPAP/DAP, cortactin, and Homer and named it Shank (Naisbitt, S., Kim, E., Tu, J. C., Xiao, B., Sala, C., Valtschanoff, J., Weinberg, R. J., Worley, P. F., and Sheng, M. (1999)Neuron 23,569–582 and Tu, J. C., Xiao, B., Naisbitt, S., Yuan, J. P., Petralia, R. S., Brakeman, P., and Doan, A., Aakalu, V., K., Lanahan, A. A., Sheng, M., and Worley, P. F. (1999)Neuron 23,583–592). Another group directly submitted a gene named SPANK-1 to the GenBank™ (AF159016) (Tobaben, S., Sudhof, T. C., and Stahl, B.). Shank 1a, synamon, and SPANK-1 are identical. The protein named ProSAP1/CortBP1 was identified as a synaptic cortactin-binding protein (Boeckers, T. M., Kruetz, M. R., Winter, C., Zuschratter, W., Smalla, K.-H., Sanmarti-Vila, L., Wex, H., Langnaese, K., Bockmann, J., Garner, C. C., and Gundelfinger, E. D. (1999) J. Neurosci. 19,6506–6518). The authors compared the sequences of ProSAP1/CortBP1 and synamon and discussed that synamon was an isoform of ProSAP1/CortBP1. Although the authors used the sequence of synamon that we submitted originally and that had a sequencing error, their conclusion was correct, and Shank la/synamon/SPANK-1 is an isoform of ProSAP1/CortBP1.