The colorectal tumour suppressor APC is present in the NMDA-receptor-PSD-95 complex in the brain Hiroyuki Yanai,1,2 Kiyotoshi Satoh,1,2 Akihiko Matsumine2 and Tetsu Akiyama1,2,* 1
Laboratory of Molecular and Genetic Information, Institute for Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113, Japan 2 Department of Oncogene Research, Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565, Japan
Abstract Background: The synaptic protein PSD-95/SAP90 interacts with ion channels such as the N-methyl-Daspartate-receptor (NMDA-R) via its PDZ domain, and is involved in their clustering. Moreover, it interacts with signalling molecules and plays an important role in coupling NMDA-R to pathways that control synaptic plasticity and learning. Results: We report that PSD-95 interacts with the adenomatous polyposis coli (APC) tumour suppressor protein via its PDZ domain. Furthermore, we found that PSD-95, NMDA-R and APC are contained in the same complex in vivo.
Introduction Synapses are highly specialized cell±cell junctions involved in the transmission of signals between neurones and their target cells (Fallon & Hall 1994). The precise organization of synaptic proteins such as neurotransmitter receptors, cytoskeletal proteins and signal transduction molecules is essential for proper synaptic function (Ehlers et al. 1996; Sheng 1996). The postsynaptic membrane has a dense thickening of submembranous cytoskeleton, called postsynaptic density (PSD), that is thought to be an essential component of a synaptic signalling assemblage (Ziff 1997; Craven & Bredt 1998). A number of proteins have been identi®ed as components of PSD, including actin, Ca2/calmodulin-dependent protein kinase II, N-methyl-D-aspartatereceptors (NMDA-R) and PSD-95/SAP90. However, the assembly of individual components to form PSD is not yet well understood. Communicated by: Tadashi Yamamoto *Correspondence: E-mail:
[email protected]. ac.jp q Blackwell Science Limited
PSD-95-NMDA-R±APC association was found to require two cysteine residues conserved in the amino-terminus of PSD-95 that are known to be critical for its multimerization. Conclusion: Our ®ndings suggest that the PSD-95NMDA-R-APC complex forms due to the multimerization of PSD-95 monomers, each of which can associate with either NMDA-R or APC. It is possible that APC is involved in the regulation of ion channel clustering and/or organization of signalling molecules.
PSD-95, one of the major components of PSD, has three tandem PDZ domains, an SH3 domain, and a guanylate kinase homology (GUK) domain. The closely related PSD family of proteins, including PSD95/SAP90, hDLG/SAP97, PSD-93/chapsyn-110 and SAP102, also possesses similar domain structures (Craven & Bredt 1998; Ziff 1997). The PDZ domain binds to the carboxy-terminal S/TXV motif of target proteins (S/T, serine or threonine; X, any amino acid; V, valine) and mediates protein±protein interactions (Ziff 1997; Craven & Bredt 1998). The ®rst and second PDZ domains of PSD-95 binds to the carboxyterminus of ion channels, including the NR2 subunits of NMDA-R and the Shaker-type K channels, thereby inducing the clustering of these ion channels (Kim et al. 1996, 1995; Kornau et al. 1995; Niethammer et al. 1996). Moreover, the PDZ domains of the PSD-95 family of proteins associate with a synaptic Ras-GTPase activating protein (synGAP), CRIPT, neuroligin and neural nitric oxide synthase (nNOS) (Brenman et al. 1996; Irie et al. 1997; Chen et al. 1998; Kim et al. 1998; Niethammer et al. 1998). The GUK domain also functions as the site of interaction with other proteins Genes to Cells (2000) 5, 815±822
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such as DLGAP (DAP/SAPAP/GKAP) (Kim et al. 1997; Satoh et al. 1997; Takeuchi et al. 1997). Therefore, the PSD-95 family of proteins appears to serve as scaffolds for ion channels and associated signalling molecules. Indeed, a study of mice lacking PSD-95 has demonstrated that PSD-95 plays an important role in coupling NMDA-R to pathways that control synaptic plasticity and learning (Migaud et al. 1998). It has also been reported that PSD-95 is required for the ef®cient coupling of NMDA-R activity to nitric oxide toxicity (Sattler et al. 1999). The ion channel clustering activity of PSD-95 has been reported to depend on both its amino-terminal domain and PDZ domain. (Hsueh et al. 1997) The amino-terminal domain contains two conserved cysteine residues, Cys-3 and -5, which are able to form intermolecular disulphide linkages, mediating the assembly of homo- or hetero-multimers among PSD-95 family members (Hsueh et al. 1997; Hsueh & Sheng 1999). On the other hand, another group has reported that PSD-95 is palmitoylated on Cys-3 and -5 and that this palmitoylation is essential for its membrane targeting, association with the ion channel Kv1.4 and ion channel clustering activity (El-Husseini et al. 2000; Topinka & Bredt 1998). We previously demonstrated that hDLG interacts with the adenomatous polyposis coli (APC) protein (Matsumine et al. 1996), the mutation of which is responsible for familial adenomatous polyposis (FAP) and many sporadic colorectal tumours (Kinzler & Vogelstein 1996; Polakis 1997). It is well known that APC interacts with and induces the down-regulation of b-catenin, a key component of the Wnt signalling transduction pathway (Rubinfeld et al. 1993; Su et al. 1993; Miller & Moon 1996; Cadigan & Nusse 1997). On the other hand, APC is highly expressed in the central nervous system and co-localizes with hDLG at the synaptic sites in hippocampal neurones (Matsumine et al. 1996; Senda et al. 1998). In the present study, we have shown that PSD-95 also interacts via its PDZ domain with APC. Furthermore, we have shown that PSD-95, NMDA-R and APC are contained within the same complex in vivo, presumably as a result of the disulphide linkage-mediated multimerization of PSD95 monomers, each of which can associate with NMDA-R or APC.
Results PSD-95 interacts with APC We performed a two-hybrid screen of a human foetal brain library using the carboxy-terminal region of APC as bait, and isolated cDNAs containing sequences 816
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encoding not only hDLG (Matsumine et al. 1996) but also PSD-95. A full-length PSD-95 cDNA clone was subsequently obtained from a human cDNA library and found to encode a protein of 724 amino acids of 100% identity to rat PSD-95. However, the amino-terminal 10 amino acids of the predicted protein are different from those of the human PSD-95, which consists of 723 amino acids and lacks the amino-terminal conserved cysteine residues, Cys-3 and -5 (Stathakis et al. 1997). This difference may be due to alternative splicing. Consistent with the results of the two-hybrid screen, PSD-95 generated by in vitro translation was found to interact speci®cally with a carboxy-terminal fragment of APC (amino acids 2475±2843) (APC-C-ter) fused to glutathione-S-transferase (GST), but not with a variant lacking the S/TXV motif (Thr-Ser-Val) (APCC-terDTSV) (Fig. 1A). Conversely, pull-down assays using a series of PSD-95 fragments fused to GST showed that the second PDZ domain (PDZ2) is necessary for interaction with APC-C-ter translated in vitro (Fig. 1B,C). Neither PDZ1 nor PDZ3 showed a detectable binding activity. These results suggest that PSD-95 interacts with the carboxy-terminal S/TXV motif of APC via its PDZ domain.
Kinetics of PSD-95±APC interaction We further characterized the PSD-95±APC interaction using surface plasmon resonance technology. A synthetic peptide APC-C15, which corresponds to the carboxyterminal 15 amino acids of APC, was immobilized on the surface of a sensor chip, and GST-fusion fragments containing each of the three PDZ domains of PSD-95 (PDZ1, 2, 3) were injected in a constant ¯ow. APCC15 bound readily to GST-PDZ2, but very weakly to GST-PDZ1 and -PDZ3 (Fig. 2). Binding curves obtained at different concentrations of the GST-fusion proteins enabled us to estimate the af®nity of GSTPDZ1 and -PDZ2 to APC-C15. The KD value of APC-C15 for GST-PDZ2 was calculated to be 3.45 ´ 10 8 M, based on the association (ka 2.19 ´ 104 M 1s 1) and dissociation (kd 7.56 ´ 10 4s 1) constants. The KD values for GST-PDZ1 were calculated to be 1.53 ´ 10 5 M, based on the association (ka 1.47 ´ 102 M 1s 1) and dissociation (kd 2.26 ´ 10 3 s 1) constants.
Co-immunoprecipitation of APC with PSD-95 and NMDA-R To examine whether PSD-95 is associated with APC in vivo, we subjected a lysate from mouse brain to q Blackwell Science Limited
APC is present in the NMDA-R-PSD-95 complex
NR 2A and 2B was inhibited by preincubation of antiAPC antibodies with the antigen used for immunization. Thus, PSD-95, APC and NMDA-R seem to be present in the same complex in vivo.
Requirement of amino-terminal conserved cysteine residues for APC-PSD-95-NMDA-R complex formation
Figure 1 PSD-95 interacts with the S/TXV motif of APC via its PDZ domain. (A) The carboxy-terminal domain of APC interacts with PSD-95. The in vitro-translated, 35S-labelled PSD-95 indicated in the ®gure (IVT PSD-95) was incubated with APC fragments containing or lacking the carboxy-terminal S/TXV motif (APC-C-ter or APC-C-terDTSV, respectively) that were produced as GST fusion proteins and immobilized to glutathione-Sepharose. The bound proteins were analysed by SDS-PAGE and autoradiography. Schematic representations of APC-C-ter and APC-C-terDTSVand their corresponding PSD95 binding activities are also shown. () strong activity; ( ) no detectable activity. (B) Fragments of PSD-95 containing the second PDZ domain interact with APC-C-ter. In vitrotranslated, 35S-labelled APC-C-ter (IVT APC-C-ter) was incubated with a series of deletion mutants of PSD-95 fused to GST. GST-clone 1 was generated from the cDNA fragment obtained by the two-hybrid screen of a human brain library. (C) Schematic representation of PSD-95 deletion mutants and corresponding APC binding activities. () detectable activity; ( ) no detectable activity.
immunoprecipitation with anti-APC antibodies, and then immunoblotted the precipitates with anti-PSD-95 antibodies. As shown in Fig. 3(A,B), PSD-95 was found to co-precipitate with APC. Furthermore, immunoblot analysis of APC immunoprecipitates with antibodies to the NR 2A (Fig. 3A) or 2B (Fig. 3B) subunits revealed that both are present. Co-precipitation of APC with q Blackwell Science Limited
Since NR2A and 2B interact with PSD-95 but not directly with APC (Fig. 4), we attempted to elucidate the mechanism by which the APC-PSD-95-NMDA-R complex is formed. As described above, APC was found to interact with the PDZ2 domain of PSD-95, while NMDA-R was reported to interact with the PDZ1 and 2 domains of PSD-95 (Kornau et al. 1995; Niethammer et al. 1996). It may therefore be possible that NMDA-R and APC bind simultaneously to the PDZ1 and 2 domains of the same PSD-95 monomer. Alternatively, it is also possible that PSD-95 monomers associated with NMDA-R or APC oligomerize to form the APCPSD-95-NMDA-R complex. To discriminate between these possibilities, we generated a mutant PSD-95 (PSD-95-C3,5S) which is de®cient in oligomer formation (Hsueh et al. 1997), due to the substitution of a conserved pair of cysteine residues, Cys-3 and -5, with serine residues. When COS-7 cells were transfected with PSD-95-C3,5S, NR2A and EGFP-tagged APC-C-ter (EGFP-APC) and extracts were subjected to immunoprecipitation with anti-PSD-95 antibodies, the immunoprecipitates were found to contain PSD95-C3,5S, NR2A and EGFP-APC (Fig. 4). However, immunoprecipitates obtained with anti-GFP antibodies were found to contain EGFP-APC and PSD-95-C3,5S, but not NR2A. On the other hand, when COS-7 cells were transfected with wild-type PSD-95 in addition to with NR2A and EGFP-APC, NR2A was found to coprecipitate with PSD-95 and EGFP-APC. These results suggest that the presence of PSD-95, NMDA-R and APC within the same complex is due to the multimerization of PSD-95 monomers individually associated with either NMDA-R or APC.
APC and NMDA-R competitively bind to the PDZ domain of PSD-95 To con®rm the results obtained with the PSD-95C3,5-S mutant, we next examined the in vitro interaction of the PDZ domain of PSD-95 with APC and NR2A. In Fig. 5A, GST-PDZ1-3 was ®rst incubated with 35S-labelled APC-C-ter, and then incubated with an amylose resin to which the amino-terminal 100 Genes to Cells (2000) 5, 815±822
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Figure 2 Interaction between APC-C15 and GST-PDZ1, 2 and 3. The APC-C15 peptide was immobilized on a sensor chip and GST-PDZ1, 2 or 3 fusion proteins were passed over the chip at a concentration of 5 mM. Binding activities (in resonance units) were monitored in a BIAcore 1000 instrument (Pharmacia Biosensor). The sensograms represent the real time binding of APC-C15 to the indicated GST fusion proteins.
Figure 3 Co-immunoprecipitation of APC with the PSD-95-NMDA-R complex from mouse brain. Mouse brain lysates were subjected to immunoprecipitation with antibodies to APC or PSD-95. Antigen , antibodies were preincubated with antigen before use in immunoprecipitation. Precipitated proteins were separated by SDS-PAGE and subjected to immunoblotting analysis with antibodies to APC, PSD-95, NR2A (A) or NR2B (B).
Figure 4 Requirement of two conserved cysteine residues for APC-PSD-95NMDA-R complex formation. COS-7 cells were transfected with NR2A, EGFP-tagged APC-C-ter, and PSD-95 or PSD-95-C3,5S. PSD-95-C3,5S is an multimerization-de®cient mutant, in which Cys-3 and -5, are replaced with Ser residues. Cell lysates were subjected to immunoprecipitation with antibodies to GFP or PSD-95. Precipitated proteins were separated by SDS-PAGE and subjected to immunoblotting analysis with antibodies to NR2A, PSD-95 and GFP as indicated.
amino acids of NR2A fused to MBP (MBP-NR2AC100) was immobilized. No detectable amount of APC-C-ter co-precipitated with an MBP-NR2Aamylose resin, although GST-PDZ1-3 was detected in the precipitate (data not shown). On the other hand, 818
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APC-C-ter co-precipitated with GST-PDZ1-3 when incubated with GSH-Sepharose. In Fig. 5B, 35Slabelled PSD-95 and an MBP-NR2A-C100-amylose resin were incubated in the presence of various amounts of APC-C15 peptide. APC-C15 was found to compete q Blackwell Science Limited
APC is present in the NMDA-R-PSD-95 complex
Figure 5 APC and NR2A competitively bind to PSD-95. (A) GST-PDZ1-3 (2 mg or 20 mg) was ®rst incubated with in vitro translated, 35S-labelled APC-C-ter, and then incubated with amylose resin to which the amino-terminal 100 amino acids (amino acids 1365±1464) of NR2A fused to MBP (MBPNR2A-C100) (1 mg) were immobilized. [35S]APC-C-ter precipitated with MBP-NR2A-C100-amylose resin was detected by SDS-PAGE followed by autoradiography. In parallel experiments, GSH-Sepharose was used to precipitate [35S]APC-C-ter. (B) In vitro translated, 35S-labelled PSD-95 and MBP-NR2AC100-amylose resin were incubated in the presence of various amounts of APC-C15 peptide (5 ng, 50 ng, 500 ng or 5 mg). 35Slabelled PSD-95 precipitated with an MBP-NR2A-amylose resin was detected by SDS-PAGE followed by autoradiography.
with the interaction between PSD-95 and NR2A-C100 in a dose-dependent manner. These results suggest that GST-PDZ1-3 associated with APC is not able to interact with NR2A. Since APC only interacts with PDZ2, these results suggest that APC binding to the PDZ2 region of PSD-95 somehow interferes with the concomitant binding of NR2A to the PDZ1 region.
Discussion In the present study we found that PSD-95 interacts with the tumour suppressor APC. The human PSD-95 identi®ed in this study is 100% identical to rat PSD-95, but its amino-terminal 10 amino acids are different from those of the previously reported human PSD-95 (Stathakis et al. 1997). This difference is probably due to alternative splicing. The interaction between q Blackwell Science Limited
PSD-95 and APC was shown to require the PDZ domain of PSD-95 and the carboxy-terminal S/TXV motif of APC. Furthermore, we demonstrated that PSD-95, APC and NMDA-R are co-precipitated from a mouse brain lysate, suggesting that these molecules are present in the same complex in vivo. We previously reported that APC interacts with hDLG (Matsumine et al. 1996). Thus, it is highly possible that APC also binds to other members of the PSD-95 family of proteins, such as PSD-93/chapsyn110 and SAP102. Some of these proteins heterodimerize with one another and are found within the same NMDA-R complex (Kim et al. 1996). Furthermore, we previously demonstrated that DLGAP (DAP/SAPAP/ GKAP), which is associated with the GUK domain of hDLG and PSD-95 (Kim et al. 1997; Satoh et al. 1997; Takeuchi et al. 1997), co-immunoprecipitates with PSD-95, NMDA-R and APC (Satoh et al. 1997). Thus, APC appears to be present as a member of a membraneassociated multicomponent complex consisting of the PSD-95 family of proteins, DLGAP and NMDA-R. The PSD-95 family of proteins are differentially distributed at cellular and subcellular levels in the central nervous system. PSD-95, chapsyn-110 and SAP-102 are detected mainly in the postsynaptic and presynaptic nerve terminals (Kistner et al. 1993; Hunt et al. 1996), while hDLG is found mainly in the presynaptic nerve terminals and unmyelinated axons (Muller et al. 1995). We previously found that PSD-95, APC and DLGAP are co-localized in the postsynaptic regions and nerve ®bres in the mouse cerebellum (Satoh et al. 1997). Further analysis is required to ascertain if different members of the PSD-95 family of proteins are associated with APC at different subcellular sites. Both in vitro pull-down assay and surface plasmon resonance measurements revealed that the PDZ2 region of PSD-95 interacts with the carboxy-terminal S/TXV motif of APC. In contrast, the PDZ1 and 3 regions exhibit barely detectable APC binding activity. The interaction between hDLG and APC also requires the PDZ2 region of hDLG and the carboxy-terminal S/TXV motif of APC. The af®nity of the PDZ2 region of PSD-95 for the carboxy-terminus of APC (KD 3.45 ´ 10 8 M) is comparable to the af®nity of this region for NMDA-R and Shaker-type K channels (KD in the order of 10 7,10 8 M) (Marfatia et al. 1996; Muller et al. 1996). However, interaction with NMDA-R and Shaker-type K channel is known to involve not only PDZ2 but also the PDZ1 region of PSD-95 (Kim et al. 1995; Kornau et al. 1995; Niethammer et al. 1996). Furthermore, SAP102 has been shown to interact with NMDA-R via its PDZ1, 2 and 3 regions (Lau et al. Genes to Cells (2000) 5, 815±822
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1996; Muller et al. 1996). On the other hand, the PDZ3 region of PSD-95 has been reported to be responsible for its interaction with CRIPT and neuroligin (Irie et al. 1997; Niethammer et al. 1998). Thus, PDZ domains have different af®nities to different target proteins, depending on the amino acid sequences of the corresponding S/TXV motif, and may therefore have different functions. Although APC and NMDA-R do not interact directly with each other (Fig. 4), both proteins coprecipitated from rat brain lysates. As described above, APC interacts with PDZ2, whereas NMDA-R is able to bind to PDZ1 in addition to PDZ2 (Kornau et al. 1995; Niethammer et al. 1996). It therefore seemed theoretically possible that both NMDA-R and APC could bind at the same time to the PDZ1 and 2 regions of a single PSD-95 monomer, thus forming a APCPSD-95-NMDA-R complex. However, when we exogenously expressed an multimerization-de®cient mutant of PSD-95 (PSD-95-C3,5S) in COS-7 cells, APC and NMDA-R did not co-precipitate. This ®nding suggests that the association of PSD-95, NMDA-R and APC within the same complex is due to the multimerization of PSD-95 monomers to which NMDA-R and APC are individually bound. Previous studies have suggested that Cys-3 and -5 in PSD-95 are potential sites for disulphide bonding and/ or palmitoylation (Hsueh et al. 1997; Topinka & Bredt 1998; Hsueh & Sheng 1999; El-Husseini et al. 2000). Topinka and Bredt reported that palmitoylation of these cysteine residues facilitates PSD-95 membrane association and binding to the potassium channel Kv1.4 (Topinka & Bredt 1998). El-Husseuni et al. further demonstrated that palmitoylation of PSD-95 facilitates its vesiculotubular sorting, postsynaptic targeting and ion channel clustering (El-Husseini et al. 2000). On the other hand, Hsueh et al. reported that Cys-3 and -5 form intermolecular disulphide linkages and mediate the multimerization of PSD-95. More recently, however, Hsueh and Sheng reported that PSD-95 mutants lacking these cysteine residues retain their ability to associate with membranes and to bind to Kv1.4 (Hsueh & Sheng 1999), but they lose the ability to form a ternary complex along with Kv1.4 to the cell adhesion molecule Fasciclin II. We also found that PSD-95C3,5S is able to bind to NMDA-R, but is unable to form a ternary complex with NMDA-R and APC. Whatever the chemical modi®cation of Cys-3 and -5, these cysteine residues are required for multimerization of PSD-95 and for formation of ternary complexes with two distinct PDZ-binding proteins. Consistent with the results obtained with the mutant 820
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PSD-95-C3,5S, the in vitro binding experiments shown in Fig. 5 revealed that APC and NMDA-R competitively bind to the PDZ domain of PSD-95, i.e. GSTPDZ1-3 associated with APC was not able to interact with NR2A. Similarly, NR2A may not be able to interact with the PDZ1 region of PSD-95 if its PDZ2 domain is already occupied by APC. Additionally, it is interesting to note that the human PSD-95 identi®ed in this study contains a conserved Cys-3 and -5, whereas the human PSD-95 clone identi®ed previously by Stathakis et al. does not contain these residues (Stathakis et al. 1997). Thus, the latter may be a splice variant that does not oligomerize and that possesses a different function. PSD-95 is thought to play an important role in the clustering and immobilization of NMDA-R at the postsynaptic membrane (Kornau et al. 1995; Kim et al. 1996). PSD-95 is also associated with CRIPT, which may link PSD-95 to the tublin-based cytoskeleton (Niethammer et al. 1998). Furthermore, PSD-95 interacts with signalling molecules such as ras-GTPase activating protein, synGAP and tyrosine kinase Fyn (Chen et al. 1998; Kim et al. 1998; Tezuka et al. 1999). Characterization of mice lacking PSD-95 has revealed that NMDA-R-mediated synaptic plasticity is dramatically altered in these mice; NMDA-R-dependent long-term potentiation (LTP) is enhanced and spatial learning is impaired (Migaud et al. 1998). More recently, it has been reported that PSD-95 is required for ef®cient coupling of NMDA-R activity to nitric oxide toxicity (Sattler et al. 1999). It is therefore possible that APC plays a role in the organization and regulation of NMDA-R, cytoskeletal proteins and signal transduction molecules, and is involved in synaptic plasticity and nitric oxide toxicity. It remains to be seen whether APC function in neuronal cells involves its ability to induce the degradation of b-catenin.
Experimental procedures Plasmids construction cDNAs encoding the carboxy-terminal regions of APC [amino acids 2475±2844 (APC-C-ter) and 2475±2841] and NR2A (amino acids 1365±1464), full length PSD-95 and its derivatives (amino acids 41±356, 60±164, 146±356, 146±251, 315±395 and 60±395) were ampli®ed by PCR. The resulting products were subcloned into the pGEX vector (Pharmacia), except for the NR2A carboxy-terminal region fragments, which were inserted into the pMALp2 vector (New England BioLabs). For the in vitro translation, PSD-95 and APC (amino acids 2475±2844) were subcloned into pBluescript II (Stratagene) containing the Kozak sequence. For expression in COS-7 cells, the NR2A and PSD-95 q Blackwell Science Limited
APC is present in the NMDA-R-PSD-95 complex cDNAs were subcloned into the mammalian expression vector pRcCMV (Invitrogen). The APC fragments were subcloned into a modi®ed pEGFPc2 vector (Clontech) to express EGFP-tagged APC fragments. Mutant PSD-95 C3,5S was generated by PCR ampli®cation using a 50 primer in which a G to C substitution at the second position of the cysteine-3 and -5 had been introduced. The resulting PCR products were subcloned into pRcCMV.
Two-hybrid screening Two-hybrid screening was performed as previously described (Matsumine et al. 1996). A full-length PSD-95 cDNA was obtained from a human brain library.
In vitro binding assay [35S]methionine labelled PSD-95 and APC-C-ter were synthesized by in vitro transcription-translation using the TNTTMcoupled reticulocyte lysate system (Promega). Proteins fused to glutathione-S-transferase (GST) or maltose-binding protein (MBP) were synthesized in E. coli and isolated by absorption to glutathione-Sepharose (GSH-Sepharose) (Pharmacia) or amylose resin (New England Biolabs). GST- or MBP-fusion proteins immobilized to beads were incubated with in vitro translation products in buffer A (1% Triton X-100, 20 mM Tris-HCl, 140 mM NaCl, 1 mM EGTA, 10% (v/v) glycerol, 1.5 mM MgCl2, 1 mM DTT, 1 mM sodium vanadate, 50 mM NaF, 1 mg/mL aprotinin, 10 mg/mL p-amidinophenyl methane sulphonyl ¯uoride hydrochloride, pH 8.0) and then washed extensively with buffer B (20 mM Tris-HCl, 150 mM NaCl, 1% NP-40, pH 8.0). Proteins adhering to the beads were analysed by SDSPAGE followed by autoradiography.
Cell culture and transfection COS-7 cells were maintained in Dulbecco's modi®ed Eagle's medium (DMEM) supplemented with 10% foetal bovine serum. COS7 cells were transiently transfected with expression constructs at 40±60% con¯uency using the lipofectamine method (Gibco BRL).
Antibodies Antibodies to APC were prepared as previously described (Matsumine et al. 1996). Antibodies to PSD-95, NMDA-R and GFP were obtained from Transduction Laboratories, Chemicon and Clontech, respectively.
Immunoprecipitation and immunoblotting Cell lysates from COS-7 cells transfected with expression constructs were prepared by solubilization in buffer A containing 10 mM N-ethylmaleimide (NEM), included to inactivate free sulfhydryl groups (Hsueh & Sheng 1999). The Triton X-100 insoluble fractions from mouse whole brain were solubilized in q Blackwell Science Limited
SDS buffer (20 mM Tris-HCl, 150 mM NaCl, 5 mM EDTA, 5 mM EGTA, 50 mM NaF, 1 mM sodium vanadate, 1 mM PMSF, 1 mg/ mL aprotinin, 1 mM DTT) containing 1% SDS and then diluted with 5 volumes of buffer A. The lysates were incubated with primary antibodies for 1 h at 4 8C. Immunocomplexes were precipitated with protein A-Sepharose (Pharmacia), washed with buffer B, eluted with SDS sample buffer and processed for immunoblotting analysis. The blots were probed with antibodies against APC, NR2A, PSD-95 and GFP, and then visualized with alkaline phosphatase-conjugated secondary antibodies (Promega).
Surface plasmon resonance measurements A synthetic peptide corresponding to the carboxy-terminal 15 amino acids of APC (APC-C15) was immobilized on the Fc2 surface of a CM5 research grade sensor chip with the amine coupling kit (Pharmacia). Analytes were perfused (20 mL/min) over the Fc1 (control, no peptides immobilized) and Fc2 (capture) surfaces in running buffer (10 mM Hepes, 150 mM NaCl, 3 mM EDTA, 0.05% Tween 20, pH 7.4). Binding activities (in resonance units) were monitored in a BIAcore 1000 instrument (Pharmacia Biosensor) and measured as the difference between Fc2 and Fc1 binding curves. All experiments were performed at 25 8C. The data obtained were analysed with the BIA Evaluation program 3.0 (Pharmacia).
Acknowledgements This work was supported by Grants-in-Aid for Scienti®c Research on Priority Areas and the Organization for Pharmaceutical Safety and Research.
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Received: 5 June 2000 Accepted: 23 June 2000
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