Synaptic localization of SAPAP1, a synaptic membraneassociated protein Blackwell Oxford, Genes GTC © 1356-9597 February 82O ISynaptic Yao riginal Blackwell ettoal. UK Article localization Cells 2002 Science, Publishing Ltdof SAPAP Ltd
Ikuko Yao, Junko Iida, Wataru Nishimura and Yutaka Hata* Department of Medical Biochemistry, Graduate School of Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan
Abstract Background: SAPAP1 was originally identified as a protein interacting with the guanylate kinase domain of PSD-95. SAPAP1 also interacts with various proteins, including neurofilaments, synaptic scaffolding molecule (S-SCAM), nArgBP2, dynein light chain and Shank through different regions.
PSD-95 or S-SCAM. SAPAP1 was not involved in the synaptic localization of PSD-95 or S-SCAM, but affected that of Shank. The synaptic targeting of SAPAP1 was not suppressed by blocking NMDA or AMPA receptors. Fluorescent recovery after a photobleaching study revealed that SAPAP1 was immobile at synapses.
Results: We expressed various regions of SAPAP1 in hippocampal neurones. The synaptic targeting of SAPAP1 was mediated by the N-terminal region and did not depend on the interaction with
Conclusion: SAPAP1 is a component of the static core of PSD, and its dynamics are different from those of the other PSD components, PSD-95, S-SCAM and BEGAIN.
Introduction
PSD-95 and requires the activity of NMDA receptors (Deguchi et al. 1998; Yao et al. 2002). Here, we have studied SAPAP1, which also interacts with the GK domain of PSD-95 (Kim et al. 1996; Takeuchi et al. 1997; Satoh et al. 1997). There are four isoforms of SAPAP, SAPAP1 to -4, which are encoded by different genes (Takeuchi et al. 1997). The short alternative splicing product of SAPAP1 is called GK-associated protein (GKAP) (Kim et al. 1996). Human SAPAP1 and -2 correspond to DLG-associated protein (DAP) 1 and -2 (Satoh et al. 1997). All SAPAPs have five repeats of 14 amino acids in the middle region followed by the proline-rich sequence and the C-terminal PDZ-binding motif. The middle region binds the GK domains of PSD-95 and S-SCAM (Kim et al. 1996; Hirao et al. 1998).The proline-rich sequence and the PDZ-binding motif interact with the SH3 domain of nArgBP2 and the PDZ domain of Shank, respectively (Kawabe et al. 1999; Boeckers et al. 1999; Naisbitt et al. 1999; Yao et al. 1999b). SAPAP1 also binds neurofilaments and dynein light chain with distinct regions (Hirao et al. 2000; Haraguchi et al. 2000; Naisbitt et al. 2000). In this study we have examined which region of SAPAP1 is involved in its synaptic targeting, how SAPAP1 affects the localization of other PSD components, and whether NMDA
Excitatory synapses have a postsynaptic density (PSD), which is identified as an electron-dense and detergentresistant structure (Kennedy 1997; O’Brien et al. 1998; Scannevin & Huganir 2000). PSD contains receptors, signalling molecules and cytoskeletal proteins, and is organized through the sequential protein–protein interactions of these components. Because PSD is regarded as a structure underlying synaptic plasticity, it is important to study how PSD components are assembled. The prototypic synaptic membrane-associated protein, PSD-95, plays a central role in the assembly of PSD components (Cho et al. 1992; Kistner et al. 1993; Hata & Takai 1999; Sheng 2001). PSD-95 has three PSD-95/Dlg-A/ZO-1 (PDZ) domains, one src homology (SH) 3 domain, and one guanylate kinase (GK) domain. PSD-95 interacts with N-methyl-D-aspartate (NMDA) receptors by PDZ domains and induces the clustering of receptors (Kornau et al. 1995). In previous studies we showed that the synaptic targeting of brain-enriched GK domain-associated protein (BEGAIN) depends on the interaction with Communicated by : Kohei Miyazono *Correspondence: E-mail:
[email protected] © Blackwell Publishing Limited
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receptor activity is required for the synaptic localization of SAPAP1.We have also examined fluorescent recovery after photobleaching (FRAP) using green fluorescent protein (GFP)-tagged SAPAP1.
Results GFP-tagged and Myc-tagged SAPAP1 are targeted to synapses like endogenous SAPAP
To clarify the molecular determinant for the synaptic localization of SAPAP1, we made use of GFP-tagged and Myc-tagged SAPAP1 proteins containing various regions. First, we examined whether GFP-tagged and Myc-tagged SAPAP1 were targeted to synapses like endogenous SAPAP. Endogenous SAPAP was co-localized with synaptophysin (Fig. 1A). We expressed GFP-tagged SAPAP1 in neurones using Sindbis virus. After 24 h, GFP-SAPAP1 was found in clusters within dendritic spines and dendritic shafts (Fig. 1B). Clusters of GFP-SAPAP1 at dendritic spines were primarily synaptic, found by labelling for synaptophysin. We also transfected neurones with the plasmid of Myc-tagged SAPAP1-I with a calcium phosphate precipitation method on day 4. Neurones were fixed on day 14 and immunostained with the anti-Myc and the anti-synaptophysin antibodies. Myc-SAPAP1-I also formed clusters that were co-localized with synaptophysin (Fig. 1C). These results support the idea that GFP-SAPAP1 and Myc-SAPAP1-I are targeted to synapses like the endogenous SAPAP in neurones. The N-terminal region of SAPAP1 mediates the synaptic targeting
Subsequent to this, we expressed Myc-tagged proteins containing various regions of SAPAP1 in neurones by
transfection (Fig. 2A). Myc-SAPAP1-VI containing the C-terminal region and Myc-SAPAP1-IX containing the middle GK domain-binding region were diffusely distributed (Fig. 2Ba,b). Myc-SAPAP1-X, which covered only the N-terminal region, formed clusters, which were apposed to those of synaptophysin (Fig. 2Bc). The results of quantitative analyses are summarized in Fig. 2C. These data indicate that the N-terminal region of SAPAP1 is necessary and sufficient to mediate the synaptic targeting. The synaptic targeting of SAPAP is independent of PSD-95 or S-SCAM
As we have previously reported, BEGAIN, which interacts with the GK domain of PSD-95, is tethered to synapses by PSD-95 (Yao et al. 2002). The result above, however, implies that the synaptic targeting of SAPAP1 is independent of the interaction with PSD-95 or SSCAM. To confirm that PSD-95 or S-SCAM did not affect the localization of endogenous SAPAP, we overexpressed the GK domain of PSD-95 or S-SCAM by transfection. Myc-PSD-95–4, which contained the GK domain of PSD-95 and inhibited the synaptic targeting of BEGAIN, was diffusely distributed, but did not affect the synaptic localization of endogenous SAPAP (Fig. 3Aa,B). A similar result was obtained for Myc-SSCAM-2 containing the GK domain of S-SCAM (Fig. 3Ab,B). The synaptic targeting of PSD-95 or S-SCAM does not depend on the interaction with SAPAP1
Previous reports have revealed that PSD-95 and SSCAM are recruited to synapses by a region other than the GK domain (Craven et al. 1999; Nishimura
Figure 1 Synaptic localization of SAPAP1. (A) Rat primary cultured hippocampal neurones were immunostained with the rabbit anti-SAPAP1 and the mouse antisynaptophysin antibodies. (B) Rat primary cultured hippocampal neurones were infected with Sindbis virus to express GFP-SAPAP1 and were immunostained with the mouse anti-synaptophysin antibody 24 h after infection. (C) Rat primary cultured hippocampal neurones were transfected with pClneo Myc SAPAP1 on day 4 and were immunostained with the mouse antisynaptophysin antibody on day 14. Scale bar = 10 µm.
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Synaptic localization of SAPAP
Figure 2 Various Myc-SAPAP1 proteins expressed in hippocampal neurones. (A) Schematic drawing of various constructs of SAPAP1. The grey and white rectangles indicate the GK-binding region and the proline-rich sequences, respectively. The interactions with neurofilament (NFL), PSD-95, S-SCAM, nArgBP2, dynein light chain (DLC), and Shank are depicted by arrows. The numbers indicate the numbers of amino acid residues of SAPAP1. (B) Various Myc-SAPAP1 proteins in hippocampal neurones. Rat primary cultured hippocampal neurones were transfected with various Myc-tagged SAPAP1 constructs on day 4 and were immunostained with the mouse anti-synaptophysin antibody on day 14. (a) Myc-SAPAP1-VI; (b) MycSAPAP1-IX; and (c) Myc-SAPAP1-X. Scale bar = 10 µm. (C) Quantitative analysis of synaptic localization of various GFPSAPAP1 proteins. Myc-SAPAP1-VI and -IX were diffusely distributed and did not form clusters. The clusters of MycSAPAP1-I and -X were co-localized with synaptophysin similarly and there was no significant difference between groups. NT, not tested. Error bars represent SD.
et al. 2002). These findings argue that the interaction with SAPAP1 is not involved in the synaptic localization of PSD-95 and S-SCAM. To confirm this argument, we over-expressed Myc-SAPAP1-IX in neurones. As described above, Myc-SAPAP1-IX contained the GK domain-binding region and was diffusely distributed. However, Myc-SAPAP1-IX did not disturb the synaptic localization of PSD-95 and S-SCAM (data not shown). © Blackwell Publishing Limited
The C-terminal region of SAPAP1 inhibits the synaptic targeting of Shank
We next sought to determine whether SAPAP1 affected the subcellular localization of Shank. Shank was co-localized with synaptophysin (Fig. 4Aa,B). MycSAPAP1-VI, which contained the PDZ-binding motif and bound Shank, was diffusely distributed and interfered the synaptic localization of Shank (Fig. 4Ab,B). In Genes to Cells (2003) 8, 121– 129
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Figure 3 The GK-domains of PSD-95 or S-SCAM did not affect the synaptic targeting of endogenous SAPAP. (A) Rat primary cultured hippocampal neurones were transfected with either pClneo Myc PSD-95-4 or pClneo Myc S-SCAM-2 on day 4 to express the GK domain of PSD-95 or S-SCAM and were immunostained with the rabbit anti-SAPAP1 and the mouse anti-synaptophysin antibodies on day 14. (a) Endogenous SAPAP in neurones expressing Myc-PSD-95–4. (b) Endogenous SAPAP in neurones expressing Myc-S-SCAM-2. Scale bar = 10 µm. (B) Quantitative analysis of synaptic localization of endogenous SAPAP in neurones expressing Myc-PSD-95-4 or Myc-S-SCAM-2. The co-localization of endogenous SAPAP with synaptophysin was not affected by the overexpression of the GK domain of PSD-95 or S-SCAM. Error bars represent SD.
the reverse experiment, we over-expressed the middle region of Shank1/synamon containing the PDZ domain (Myc-synamon-PDZ). Myc-synamon-PDZ was diffusely distributed but did not affect the synaptic localization of endogenous SAPAP (data not shown). Synaptic targeting of GFP-SAPAP1 does not depend on NMDA receptor or AMPA receptor activity
Our previous study revealed that the synaptic localization of GFP-BEGAIN depends on NMDA receptor activity (Yao et al. 2002). We performed similar experiments for GFP-SAPAP1 using various blockers. We examined the subcellular localization of GFP-SAPAP1 in primary cultured hippocampal neurones treated with 124
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L(+)-2-amino-5-phosphonopentanoic acid, 6-cyano-7nitroquinoxaline-2,3-dione, or tetrodotoxin. None of these treatments affected the co-localization of GFPSAPAP1 and synaptophysin (Fig. 5). GFP-SAPAP1 is immobile in comparison to GFP-BEGAIN, GFP-PSD-95 and GFP-S-SCAM at synapses
In the next set of experiments, we compared the mobilities of SAPAP1 and other PSD components at synapses in a FRAP assay. GFP-BEGAIN recovered to 40% at 150 s after the bleaching. The recovery of GFP-PSD-95 and GFP-S-SCAM was more remarkable (Fig. 6). In contrast, the recovery of GFP-SAPAP1 was almost negligible. We treated neurones with a medium containing © Blackwell Publishing Limited
Synaptic localization of SAPAP
Figure 4 The over-expression of the C-terminal region of SAPAP1 inhibited the synaptic targeting of Shank. (A) Effect of MycSAPAP1-VI on the co-localization of Shank with synaptophysin. Rat primary cultured hippocampal neurones were transfected with pClneo Myc SAPAP1-VI on day 4 to express the C-terminal region of SAPAP1 and were immunostained with the rabbit antisynamon and the mouse anti-synaptophysin antibodies on day 14. (a) Shank was co-localized with synaptophysin. (b) The co-localization of Shank with synaptophysin was disturbed by Myc SAPAP1-VI. Scale bar = 10 µm. (B) Quantitative analysis of synaptic localization of Shank in neurones expressing Myc-SAPAP1-VI. About 60% of Shank clusters were co-localized with synaptophysin under the overexpression of Myc SAPAP1-VI. The student’s paired t-test was used to find the statistical significance of the difference between groups. Error bars represent SD.
300 µm glutamate and 10 µm glycine. However, under this treatment, GFP-SAPAP1 was still immobile (data not shown).
Discussion PSD at excitatory synapses has a specific set of proteins, including glutamate receptors, receptor-associated proteins and cytoskeletons (Kennedy 1997; O’Brien et al. 1998; Scannevin & Huganir 2000). PSD has been considered to be a highly organized and stable subcellular microdomain. However, recent studies have revealed that AMPA receptors are inserted or removed dynamically at synapses (Carroll et al. 1999; Liao et al. 1999; Shi et al. 1999). NMDA receptors are also mobile, and move © Blackwell Publishing Limited
between synaptic and extrasynaptic pools (Tovar & Westbrook 2002). Dendritic spines alter morphology depending on synaptic activities (Fischer et al. 2000). Thereby, the emerging picture of PSD is that components of a stable basic structure form the core, and other components are assembled around it in a dynamic and regulated manner. To understand the molecular organization of PSD, we have attempted to determine how each component is recruited to PSD and which molecule belongs to the stable basic structure or the dynamic regulated structure. In the previous study, we reported that BEGAIN, a protein interacting with the GK domain, depends on PSD-95 and NMDA receptor activity to be precisely localized at synapses (Yao et al. 2002). In contrast, the synaptic targeting of MAGUIN, Genes to Cells (2003) 8, 121– 129
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Figure 5 Effect of various blockers on the synaptic targeting of GFP-SAPAP1. (A) Rat primary cultured hippocampal neurones were infected with Sindbis virus to express GFP-SAPAP1. 6 h later, various inhibitors were added to the medium to the indicated final concentration. Neurones were fixed 24 h later and were immunostained with the mouse anti-synaptophysin. (a) Treated with 100 µm of L(+)-2-amino-5-phosphonopentanoic acid (APV); (b) treated with 100 µm of cyano-7-nitroquinoxaline-2,3dione (CNQX); and (c) treated with 1 µm of tetrodotoxin (TTX). Scale bar = 10 µm. (B) Quantitative analysis of synaptic localization of GFP-SAPAP1 in neurones treated with various blockers. Co-localization of GFP-SAPAP1 with synaptophysin was not affected by APV, CNQX or TTX. Error bars represent SD.
which is a synaptic membrane-associated protein interacting with both PSD-95 and S-SCAM, does not depend on the interaction with PSD-95 or S-SCAM (Yao et al. 1999a; Iida et al. 2002). It does not require NMDA receptor activity, either. Here, we have characterized the synaptic localization of SAPAP1. SAPAP1 is targeted to synapses by its N-terminal region, suggesting that the synaptic targeting of SAPAP1 is not mediated by the interaction with PSD-95 or SSCAM. Consistent with this, over-expression of the GK domain of PSD-95 or S-SCAM did not affect the localization of endogenous SAPAP. Our previous studies have indicated that SAPAP1 recruits PSD-95 and S-SCAM to Triton X-100-insoluble structures (Takeuchi et al. 1997; Hirao et al. 2000). Thereby, we have tested whether SAPAP1 determines the synaptic localization of PSD-95 and S-SCAM. However, over-expression of the GK domain-binding region of SAPAP1 did not interfere the synaptic localization of PSD-95 or S-SCAM. This result is consistent with previous reports that the GK domain is not involved in the synaptic localization of PSD-95 or S-SCAM (Craven et al. 1999; Nishimura et al. 2002). These findings imply that the synaptic membraneassociated guanylate kinases, PSD-95 and S-SCAM, come to synapses independently of SAPAP and then interact 126
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with SAPAP. It remains to be identified which molecule recruits SAPAP1 to the synapses. Shank is a key molecule that regulates actin cytoskeletons and links NMDA receptors with metabotropic glutamate receptors (Boeckers et al. 1999; Naisbitt et al. 1999; Tu et al. 1999; Yao et al. 1999b). The C-terminal region of SAPAP1 partially inhibits the synaptic targeting of Shank. This finding supports that SAPAP1 may recruit Shank to synapses. We cannot, however, conclusively remark that SAPAP1 recruits Shank to synapses. Moreover, the middle region of Shank1/synamon, which covers the PDZ domain, is diffusely distributed, suggesting two possibilities. The first is that the PDZ domain is not sufficient for the synaptic accumulation and the other region is also required. The second is that the endogenous target of the PDZ domain is limited and the exogenously expressed PDZ domain overflows. In the latter case, the role of tethering Shank to PSD may be attributable to SAPAP. The chronic treatment with NMDA inhibitor does not affect the localization of PSD-95 (Rao & Craig 1997). However, PSD-95 has a dynamic turnover at synapses, and the mobility of PSD-95 is suppressed by blocking of NMDA and AMPA receptors (Okabe et al. 1999). It is likely that synapses acquire the machinery to © Blackwell Publishing Limited
Synaptic localization of SAPAP
Figure 6 FRAP of various PSD components. (A) Time course of fluorescence recovery of GFP-SAPAP1, GFP-BEGAIN, GFP-S-SCAM and GFP-PSD-95. Data were indicated as mean ± SD of eight records. (B) GFP-PSD-95, GFP-S-SCAM and GFPBEGAIN recovered after bleaching, whereas GFP-SAPAP1 did not. Scale bar = 5 µm.
regulate PSD-95 in an activity-dependent manner. We have raised a question as to whether SAPAP1 shows a dynamic behaviour regulated by receptor activities at mature synapses. First, we examined the subcellular localization of GFP-SAPAP1 in neurones treated with various blockers. GFP-SAPAP1 is properly targeted to synapses under the NMDA blockade. Furthermore, we performed a FRAP study. GFP-PSD-95 is mobile at synapses. GFP-BEGAIN and GFP-S-SCAM are also mobile. In contrast, GFP-SAPAP1 is static. The treatment with glutamate and glycine to activate NMDA receptors has no effect on the mobility of GFP-SAPAP1. These findings lead us to the conclusion that SAPAP1 is a component of the static core structure of PSD. © Blackwell Publishing Limited
Experimental procedures Antibodies The rabbit antibodies against SAPAP1 and S-SCAM were raised against the C-terminal region (amino acid residues, 604–992) of SAPAP1 and the N-terminal region (amino acid residues, 1–422) of S-SCAM, respectively. The rabbit anti-synamon and the sheep anti-Myc antibodies were previously described (Yao et al. 1999b). Mouse monoclonal anti-synaptophysin and anti-PSD-95 (K28/ 86.2) antibodies were obtained from Roche Molecular Biochemicals and Upstate Biotechnology Inc., respectively. The secondary antibodies for dual labelling were purchased from Chemicon International Inc. Our anti-SAPAP1 antibody recognizes not only SAPAP1, but also other isoforms of SAPAP. The anti-synamon antibody is also likely to react with all the isoforms of Shank. Genes to Cells (2003) 8, 121– 129
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Plasmid construction Various expression vectors were constructed by conventional molecular biology techniques and PCR method. pSindGFP, pSindGFP PSD-95-1, pClneo Myc PSD-95-4, pSindGFP SSCAM-1, pClneo Myc S-SCAM-2, and pSindGFP BEGAIN-1 were as previously described (Hirao et al. 1998; Nishimura et al. 2002; Yao et al. 2002). pSindGFP and pClneo Myc SAPAP1-I contain the amino acid residues 1–992 of SAPAP1. pClneo Myc SAPAP1-VI, -IX and -X contain the amino acid residues 549– 992, 337–560 and 1–342 of SAPAP1, respectively. pClneo Myc synamon-PDZ contains the amino acid residues 558–1202 of Shank1/synamon.
Hippocampal neurone culture and transfection All procedures related to the care and treatment of animals were in accordance with institutional and National Institutes of Health guidelines. Hippocampal neurone cultures were taken from E18 embryos as previously described (Takeuchi et al. 1997; Goslin et al. 1998). Neurones were transfected at day 4, after plating with calcium phosphate precipitation method.
Sindbis virus production and infection Sindbis virus production was performed as previously described (Nishimura et al. 2002). Neurones (1 × 104/18 mm cover glass) were infected using 3 – 5 µL of the virus stock per 1 mL of the culture medium 10 days after plating. In most experiments, we obtained about 10 –30 infected neurones.
image was averaged four times and taken setting the confocal aperture at 3. Measurements were performed using NIHimage 1.61. As in our previous study, we defined signals with the following properties as clusters: (i) with peak fluorescence levels 50% greater than the maximal fluorescence levels of diffuse dendritic signals in the vicinity; (ii) with 10–100 pixels in size; and (iii) located between 10 µm and 100 µm from the soma.We examined clusters from 700 µm of dendrites for each neurone (Yao et al. 2002).
FRAP FRAP experiment was carried out with an Olympus FV300-BX confocal microscope using a 60× (Numerical Aperture = 1.25) oil immersion objective. Images were taken at 488 nm (2% power) and then a brief, intense pulse of laser light (100% power) was rendered to bleach the fluorescent molecules in a defined region (1– 2 µm in radius).The recovery of fluorescence was monitored at 3 s intervals for 60 s and at 10 s intervals for 100 s by scanning at attenuated laser light (2% power). The relative fluorescence intensity was calculated as (It /Ib)/(Ct /Cb). It and Ct are the average intensities of the bleached region and the control region at various time points. Ib and Cb are the average intensities of the bleached region and the control region before bleaching. The fluorescence intensity immediately after photobleaching (t = 0) was regarded as the background and all intensities were background subtracted.
Acknowledgements This study was supported by grants-in-aids for Scientific Research on Priority Areas and Scientific Research B, Exploratory Research, and Special Coordination Funds for Promoting Science and Technology from the Ministry of Education, Culture, Sports, Science and Technology of Japan.We thank Ms Masako MiyaharaTenkatsu and Ms Chiaki Rokukawa for skilful technical assistance.
References Immunocytochemistry Hippocampal neurones were fixed with 4% (w/v) paraformaldehyde, 4% (w/v) sucrose in PBS for 15 min, blocked with 50 mm glycine in PBS for 30 min, and permeabilized with 0.25% (w/v) Triton X-100 in PBS for 5 min. After blocking with PBS containing 10% (w/v) BSA, the cells were incubated with the first antibody in PBS containing 3% (w/v) BSA overnight, washed with PBS, and incubated with the second antibody in PBS containing 3% (w/v) BSA for 1 h. After washing with PBS, the samples were embedded in 95% (w/v) glycerol in PBS. The images were obtained by a confocal microscopy (Olympus FV300-BX).
Image analyses Images were prepared for presentation using Adobe Photoshop software. Confocal images of neurones were obtained using an Olympus FV300-BX or Zeiss LSM 510 40× objective with sequential acquisition at 1024 × 1024 pixels resolution. Each
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