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Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Fusion proteins and antibodies
  5. Precipitation assays
  6. Tissue culture
  7. Immunocytochemistry
  8. Results
  9. The C-terminus of IRSp53 interacts with PDZ2 of PSD-95
  10. Discussion
  11. Acknowledgements
  12. References

The insulin receptor substrate of 53 kDa (IRSp53) is a target of the small GTPase cdc42 which is strongly enriched in the postsynaptic density of excitatory synapses. IRSp53 interacts with the postsynaptic shank1 scaffolding molecule in a cdc42 regulated manner. The functional significance of the cdc42/IRSp53 pathway in postsynaptic sites is however, unclear. Here we identify PSD-95 as a second synaptic interaction partner of IRSp53. Interaction is mediated by a C-terminal PDZ binding motif in IRSp53 and the second PDZ domain of PSD-95. In HEK cells, overexpressed IRSp53 induces filopodia and targets PSD-95 into these processes. Immunoprecipitation and immunocytochemistry experiments demonstrate that the interaction occurs at postsynaptic sites in the brain. By virtue of its PDZ-binding and SH3 domains, IRSp53 is capable of inducing the formation of a triple complex (shank1/IRSp53/PSD-95).

The generation of excitatory synapses in the central nervous system requires a complex assembly process in which elements of the postsynaptic receptor apparatus are assembled at postsynaptic sites on dendrites. In many cases, glutamatergic synapses are localized on the heads of dendritic spines (Harris and Kater 1994; see review by Hering and Sheng 2001). During maturation postsynaptic proteins accumulate at the synapse, as exemplified in several studies by the appearance of clusters of the postsynaptic marker PSD-95 (Friedman et al. 2000; Okabe et al. 2001). Via its PDZ domains, PSD-95 is one of the major anchoring proteins for postsynaptic transmitter receptors and ion channels (Kim et al. 1995; Kornau et al. 1995). Through an intricate network of protein interactions, a large protein complex of up to 100 proteins is assembled at the spine heads around the PSD-95/transmitter receptor complex (Husi et al. 2000; Walikonis et al. 2000; Li et al. 2004) which has been termed the postsynaptic density (PSD). The function of the PSD appears to be to physically link postsynaptic receptors to signalling molecules, and to provide stable attachment of the receptors to the actin-based cytoskeleton of the dendritic spine. Shank proteins (shank1–3, also known as SSTRIP, ProSAP, synamon or CortBP) constitute another group of postsynaptic scaffolding molecules which link transmitter receptors (Kreienkamp et al. 2000; Naisbitt et al. 1999; Yao et al. 1999; Zitzer et al. 1999) to actin binding proteins (Du et al. 1998; Boeckers et al. 2001; Okamoto et al. 2001). Overexpression of shank1 in neurones leads to enhanced maturation of dendritic spines (Sala et al. 2001). We and others have recently identified IRSp53 as an interaction partner for shank1 (Bockman et al. 2002; Soltau et al. 2002). A proline-rich region of shank1 binds to the SH3 domain of IRSp53 in a cdc42-regulated manner (Soltau et al. 2002). These data suggested that shank1 might be an effector molecule of cdc42 in an undefined regulatory pathway. Here, we show that IRSp53, via binding to a PDZ domain of the PSD-95 molecule, mediates the formation of a triple complex consisting of shank1 and PSD-95. Our data suggest that one result of cdc42/IRSp53 signalling is the regulated assembly of a macromolecular complex between shank and PSD-95 proteins.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Fusion proteins and antibodies
  5. Precipitation assays
  6. Tissue culture
  7. Immunocytochemistry
  8. Results
  9. The C-terminus of IRSp53 interacts with PDZ2 of PSD-95
  10. Discussion
  11. Acknowledgements
  12. References

Fusion proteins and antibodies

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Fusion proteins and antibodies
  5. Precipitation assays
  6. Tissue culture
  7. Immunocytochemistry
  8. Results
  9. The C-terminus of IRSp53 interacts with PDZ2 of PSD-95
  10. Discussion
  11. Acknowledgements
  12. References

GST-fusion proteins of PSD-95 or SAP102 domains (Muller et al. 1996) were expressed and purified as described. Rabbit IRSp53 (rbAnti-IRS) and shank antibodies were used as described (Zitzer et al. 1999; Soltau et al. 2002); guinea-pig anti-IRS (gpAnti-IRS) was supplied by T. Böckers (University of Münster, Germany). Monoclonal anti-PSD-95 was obtained from Upstate Biotechnology (Lake Placid, NY, USA).

Precipitation assays

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Fusion proteins and antibodies
  5. Precipitation assays
  6. Tissue culture
  7. Immunocytochemistry
  8. Results
  9. The C-terminus of IRSp53 interacts with PDZ2 of PSD-95
  10. Discussion
  11. Acknowledgements
  12. References

Synthetic peptides corresponding to the C-termini of GKAP/SAPAP (sequence IYIPEAQTRL), the rat somatostatin receptor subtype 3 (KASTLSHL), the NMDA receptor 2 A subunit (KKLSSIESDV) and IRSp53 (SGSGTLVSTV) were obtained from Genemed Synthesis Inc., San Francisco, CA, USA; the peptides were coupled to NHS-activated Sepharose (Amersham Pharmarcia Biotech) at a concentration of 3 mg/mL sepharose matrix.

For purification of proteins binding to immobilized C-terminal peptides, a P2 membrane protein fraction was prepared from adult mouse whole brains. Membranes (30 mg total protein) were solubilized in either 2% sodium dodecyl sulfate (SDS; 30 min at room temperature), followed by 20-fold dilution into cold RIPA buffer (final SDS-concentration: 0.2%), or in deoxycholate buffer (50 mm NaF, 50 mm Tris-HCl, pH 9.0, 1% sodium deoxycholate, 5 mm EDTA; 1 h at 4°C). After adsorption to peptide/NHS-sepharose for 2 h at 4°C, the matrices were washed five times with the respective buffers (RIPA or deoxycholate), and eluted by boiling in SDS sample buffer. In some experiments, whole mouse brains were solubilized in deoxycholate buffer (two brains per 20 mL buffer) and used for precipitation after clearing by centrifugation at 30 000 × g for 15 min. After separation by SDS-gel electrophoresis, prominent Coomassie-stained bands were cut out and prepared for digestion with trypsin according to published procedures (Böckers et al. 2001). Tryptic digests were analyzed in an ESI-QTOF2 mass spectrometer (MicroMass, Manchester, UK) by tandem MS/MS sequencing.

Transfected HEK cells were lysed in 1 mL RIPA-lysis buffer (50 mm Tris-HCl, pH 8.0, 150 mm NaCl, 1% NP-40, 0.5% Na-deoxycholate, 5 mm EDTA, 0.1% SDS, 0.2 mm phenylmethylsulfonyl fluoride, 1 µg/mL pepstatin, 10 µg/mL leupeptin, 20 000 kiU/mL trasylol, 100 µg/mL bacitracin) per culture dish on ice for 15 min. Lysates were centrifuged (10 min; 20 000 × g) to remove insoluble matter. For precipitation of shank protein, cleared HEK cell lysates were incubated with GKAP peptide sepharose for 2 h at 4°C. After washing, all precipated complexes were boiled in SDS sample buffer, and analysed on SDS–polyacrylamide gel electrophoresis followed by western blotting.

For precipitation of GST-fusion proteins, these were diluted into RIPA-buffer and incubated with IRS-C-terminal peptide sepharose for 1 h; beads were washed 5 times with RIPA buffer, and bound proteins eluted with SDS sample buffer. After gel electrophoresis of input and bound samples, proteins were stained with Coomassie and quantified using the WinCam gel documentation system (version 2.0; Cybertech, Berlin, Germany).

Tissue culture

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Fusion proteins and antibodies
  5. Precipitation assays
  6. Tissue culture
  7. Immunocytochemistry
  8. Results
  9. The C-terminus of IRSp53 interacts with PDZ2 of PSD-95
  10. Discussion
  11. Acknowledgements
  12. References

HEK cells were cultivated and transfected as described (e.g. Zitzer et al. 1999). Hippocampal neurones were prepared at embryonic day 19 and plated at a density of about 500 cells/mm2. Cultures were grown in neurobasal medium (Gibco BRL) supplemented with B27 (Gibco BRL) and 0.5 mm glutamine. 12.5 µm glutamate was included for the first 4 days in culture.

Immunocytochemistry

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Fusion proteins and antibodies
  5. Precipitation assays
  6. Tissue culture
  7. Immunocytochemistry
  8. Results
  9. The C-terminus of IRSp53 interacts with PDZ2 of PSD-95
  10. Discussion
  11. Acknowledgements
  12. References

Transfected HEK cells growing on glass cover slips were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) and permeabilized with 0.1% Triton X-100 in PBS for 2 min at room temperature. After blocking [2% bovine serum albumin (BSA) in PBS] for 1 h at room temperature cells were incubated with rbAnti-IRS (diluted 1 : 5000 in blocking solution) for 1 h at room temperature, followed by 1 h of incubation with Cy3-conjugated anti-mouse secondary antibody (diluted 1 : 1000 in PBS).

After 16 days in culture hippocampal neurones were prepared for immunofluorescence staining by fixation in ice-cold methanol; after blocking in 2% horse serum/PBS (1 h), neurones were incubated with affinity purified rbAnti-IRS (diluted 1 : 100) and monoclonal anti-PSD-95 (diluted 1 : 25) for 4 h. Cy2-labelled goat anti-rabbit and Cy3-labelled goat anti-mouse were used as secondary antibodies. Immunostaining was visualized by confocal microscopy using a Zeiss LSM 410 microscope (63 × objective; pinhole setting: 20) as described (Roth et al. 1997); images were processed using Adobe Photoshop version 4.0.

The C-terminus of IRSp53 interacts with PDZ2 of PSD-95

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Fusion proteins and antibodies
  5. Precipitation assays
  6. Tissue culture
  7. Immunocytochemistry
  8. Results
  9. The C-terminus of IRSp53 interacts with PDZ2 of PSD-95
  10. Discussion
  11. Acknowledgements
  12. References

The C-terminus of one splice variant of IRSp53, termed IRSp53.1, contains a sequence which conforms to the consensus for PDZ binding domains (Fig. 1a), i.e. a tSXV-COOH motif which is found also in NMDA receptor subunits known to bind to PDZ domains of PSD-95 (e.g. Kornau et al. 1995; Sheng and Sala 2001). This particular splice variant was also detected in several clones obtained from a human brain cDNA library (unpublished data), clearly demonstrating that IRSp53.1 is prominently expressed in the central nervous system (see also Okamura-Oho et al. 2001). In order to identify a possible binding partner for the C-terminus of IRSp53.1, we used a synthetic decapeptide corresponding to the last 10 amino acid residues (IRS-C), covalently coupled it to a sepharose matrix, and purified binding proteins from mouse brain membranes by affinity chromatography. When membranes were solubilized in deoxycholate at pH 9, as described by Husi et al. (2000), two main bands at molecular weights 95 and 100 kDa were specifically precipitated by the IRS-C matrix, together with some lower intensity bands at 180 kDa and 50 kDa (Fig. 1b). In the method described by Husi et al. (2000), the NMDA receptor/PSD-95 complex remains intact, and binding of the two main proteins to the peptide sepharose could be mediated indirectly via other proteins. To exclude this, we also solubilized the membranes in 2% SDS, which should disrupt most endogenous protein interactions, followed by dilution into RIPA buffer for subsequent adsorption to the sepharose matrix. In this case, only the two main proteins at 95 and 100 kDa were precipitated (Fig. 1b), clearly identifying these two as direct interaction partners of the IRSp53.1 C-terminus. These two bands were absent when purification was performed with the GKAP C-terminal peptide in a control reaction (Fig. 1b). In contrast, when purification was performed with a peptide corresponding to the NMDA receptor C-terminus, an almost identical picture was obtained in both experimental settings (SDS or deoxycholate solubilization). By tryptic digestion of the excised bands, followed by mass spectroscopic sequencing of individual peptides, the two proteins were identified as PSD-95/SAP90 (95 kDa), and PSD-93/chapsyn (100 kDa band) in the IRS-C and in the NMDA-receptor experiment (Fig. 1c).

image

Figure 1. The C-terminus of IRSp53.1 binds to PSD-95. (a) C-terminal sequences of IRSp53 splice variants. IRSp53.1 contains a C-terminal PDZ domain binding motif (underlined). A synthetic peptide corresponding to residues 512–521 of this variant was used for affinity purification of putative binding proteins. (b) Affinity purification. C-terminal decapeptides of GKAP/SAPAP (GKAP), the NMDA receptor 2 A subunit (NMDA) and IRSp53.1 were coupled to NHS-sepharose. After solubilization of mouse brain membranes in desoxycholate buffer (D) or in 2% SDS, followed by dilution in RIPA (R), interacting proteins were purified by binding to the immobilized peptides. After extensive washing, bound proteins were eluted in SDS sample buffer, separated by SDS-gel electrophoresis, and stained with Coomassie brilliant blue. The bands indicated by arrowheads were cut out, subjected to tryptic digestion and subsequent mass spectroscopy. (c) List of peptides that were identified by mass spectroscopy sequencing from the IRSp53.1-purified samples (upper band: PSD-93 peptide sequences; lower band: PSD-95 peptide sequences). (d) Pulldown assay. GST fusion proteins of the various PDZ domains were prepared as indicated. Equal amounts were subjected to either SDS-gel electrophoresis, followed by Coomassie staining (upper panel), or diluted 50-fold in RIPA buffer and precipitated with IRS-C coupled peptide, followed by washing and SDS-gel electrophoresis (lower panel). The amount of precipitated protein was estimated for the largest band in the GST-fusion protein preparation, in comparison with the respective band in the input for each of the fusion proteins. Quantification of bands was performed using the WinCam gel documentation software.

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As we assumed that interaction is mediated by one of the PDZ domains of the PSD-95 protein, we made use of a set of GST fusion proteins of various PDZ domains of either PSD-95 itself or one of its closest homologues, SAP102, that was available to us. Both PSD-95 and SAP102 are largely equivalent with respect to their binding abilities towards C-terminal PDZ domain ligands, as shown in overlay assays and coprecipitation experiments (e.g. Müller et al. 1996). When GST fusions of the first and second or only the second PDZ domain of SAP102 as well as PSD-95 were incubated with the IRS-C sepharose matrix, they were efficiently precipitated (Fig. 1d). In contrast, a fusion protein containing additionally the third PDZ domain of SAP102, or the full length open reading frame of PSD-95 (with all three PDZ domains, the SH3 and the GK domain), was precipitated much less efficiently. Interestingly, in the SAP102 experiment with PDZ domains 1–3, an apparent proteolytic breakdown product presumably containing only the first two domains, bound better than the full-length product. GST alone was included as a negative control and did not bind to the IRS-C-sepharose. Thus our data indicate that IRS-C may bind to the second and possibly the first PDZ domains, whereas the presence of the third domain (or the sequence intervening between the second and third domain) obviously interferes with accessibility of the second domain.

To verify that this interaction between both proteins occurs in a cellular system, HEK cells were transfected with expression constructs for PSD-95 only, or together with IRSp53.1. By immunoprecipitation with rbAnti-IRS, PSD-95 could be specifically precipitated only when IRSp53.1 was coexpressed (Fig. 2a). In order to demonstrate interaction between both proteins in vivo in the central nervous system, solubilized brain membranes were subjected to immunoprecipitation analysis. After precipitation with rbAnti-IRS, but not with an unrelated control serum, both IRSp53 (in this case detected with gpAnti-IRS) and PSD-95 could be detected in the precipitate (Fig. 2b), clearly demonstrating that a complex between IRSp53 and PSD-95 is formed in vivo.

image

Figure 2. Interaction of IRSp53.1 and PSD-95 in vivo. (a) HEK cells were transfected with an EGFP-PSD-95 expression construct alone (left panels) or IRSp53.1 and PSD-95 (right). After lysis in RIPA buffer, IRSp53 was precipitated with a rbAnti-IRS; input (5%) and immunoprecipitates (IP) were analyzed by western blotting (WB) using rbAnti-IRS (upper panels) or a monoclonal anti-PSD-95 antibody (lower panels). Note that several endogenous IRSp53 immunoreactive bands can be detected in HEK cells not transfected with IRSp53 cDNA. The position of the recombinantly expressed IRSp53.1 is indicated by an arrowhead. Detection of IRSp53.1 in precipitates is obscured by the heavy chain of the precipitating antibody. (b) Coprecipitation from rat brain. Adult rat brains were solubilized in RIPA buffer at 37°C; cleared lysate was subjected to immunoprecipitation using rbAnti-IRS or a-non-related control antiserum (control). Input (5%) and immunoprecipitates (IP) were analyzed by western blotting (WB) using gpAnti-IRS (upper panel) or mouse anti-PSD95 (lower panel).

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IRSp53 is a major target of the small G-protein cdc42, and mediates the effect of cdc42 on the formation of filopodia in fibroblast cells (Krugmann et al. 2001). As shown previously, overexpression of IRSp53 in HEK cells induces strong outgrowth of filopodia. IRSp53.1, while strongly present in the cytosol, is also localized to these processes (Soltau et al. 2002 and Fig. 3a, left panels). PSD-95, when expressed as a GFP-fusion protein in HEK cells, is diffusely distributed throughout the cytosol of the cells (Fig. 3a, right panels). Coexpression of both proteins induces localization of both proteins to the cell periphery (as illustrated in sections taken through the center of cells; Fig. 3b, upper row); more importantly, IRSp53.1 induces localization of PSD-95 to the filopodial processes (evident in sections taken at the base of the same cells, Fig. 3b, lower row).

image

Figure 3. Colocalization of IRSp53.1 and PSD-95 in transfected cells and cultured primary hippocampal neurones. (a) HEK cells transfected with IRSp53.1 (left) or EGFP-PSD-95 (right) were stained with a rbAnti-IRS, followed by Cy3 labelled secondary goat antirabbit antibody (red fluorescence). PSD-95 was visualized using the green EGFP autofluorescence (GFP-af). (b) HEK cells were cotransfected with IRSp53.1 and EGFP-PSD-95, and expressed proteins were detected as in (a). Confocal sections of the same cells were taken either through the center of the cells (upper row) or close to the glass cover slip (lower row). The right panel shows the merge of the two fluorescent images obtained from the green and red channels. Note the extensive formation of filopodia-like processes in cells transfected with IRSp53.1 in (a) and (b). (c) Cultivated rat hippocampal neurones (16 days in vitro) were stained with affinity-purified rbAnti-IRS (diluted 1 : 100) and monoclonal anti-PSD-95 (1 : 25) antibodies, followed by Cy2-anti rabbit and Cy3-anti-mouse secondary antibodies. Shown is the staining along an individual dendrite, as viewed by confocal microscopy. Bars, 5 µm.

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Using rbAnti-IRS and mouse monoclonal anti-PSD-95 antibodies, both proteins were codetected in primary cultured hippocampal neurones (Fig. 3c). Both PSD-95 (red in Fig. 3c) and IRSp53 (green fluorescence) exhibit a punctate staining consistent with an entirely synaptic localization. Abundant colocalization of both proteins is evident by the yellow staining in the merged picture. Synaptic enrichment of IRSp53 has also been observed in a previous study (Abbott et al. 1999).

The SH3 domain of IRSp53 binds to the proline-rich region of shank1 (Soltau et al. 2002); therefore we analyzed if IRSp53.1 is capable to act as a bridge between shank and PSD proteins. For this purpose, various combinations of the three proteins were coexpressed in HEK cells; after lysis of cells in RIPA buffer, precipitation of shank1 was achieved with immobilized GKAP C-terminal peptide, which targets the PDZ domain of shank proteins (see scheme in Fig. 4). PSD-95 is not precipitated by this method in the absence of shank1 (Fig. 4a). Whereas small amounts of PSD-95 could be coprecipitated with shank1 in the absence of IRSp53.1, this amount was strongly increased when IRSp53.1 was coexpressed, demonstrating that IRSp53 is indeed capable of bridging shank1 and PSD-95 molecules. The weak coprecipitation of PSD-95 by shank1 in the absence of IRSp53 may be explained by the presence of endogenous IRSp53.1, or SAPAP/GKAP in HEK cells. SAPAP/GKAP proteins constitute a second class of proteins, which bridge shank and PSD-95 (Boeckers et al. 1999; Naisbitt et al. 1999; Yao et al. 1999). To further show that all three proteins exist in one complex in the rodent brain, we precipitated PSD-95 from solubilized mouse brain using the NMDA-receptor C-terminal peptide immobilized on NHS sepharose. A similar peptide derived from the rat somatostatin receptor subtype 3 (which binds neither shank nor PSD-95) was used as a negative control. As seen before, the NMDA receptor peptide is able to precipitate PSD-95 and PSD-93 rather efficiently, making both proteins detectable by Coomassie staining (arrows in Fig. 4c, upper panel). In the precipitate of the NMDA receptor matrix, but not of the SSTR3 matrix, both IRSp53 and, to a lesser degree, shank can be detected by western blotting. This is consistent with the notion that all three proteins are part of the same NMDA-receptor associated complex. The reverse experiment, precipitation of shank and associated proteins from solubilized brain using GKAP-sepharose, is not possible as shank is precipitated efficiently only when solubilization is performed in 2% SDS, which disrupts associations between postsynaptic proteins (not shown). We assume that shank can not be precipitated efficiently in deoxycholate buffer because it is buried deeply within the postsynaptic complex and its PDZ domain is occupied by endogenous ligands, including GKAP and various G-protein-coupled receptors.

image

Figure 4. IRSp53.1 promotes formation of a shank1-IRSp53.1-PSD-95 triple complex. (a) HEK cells were transfected with shank1, IRSp53 and PSD-95 in the combinations indicated. After lysis in RIPA buffer, shank1-containing protein complexes were precipitated with GKAP-C-terminal peptide coupled to NHS-sepharose. Isolated protein complexes were subjected to western blotting using rbAnti-IRS and rabbit anti-shank1, and mouse anti-PSD-95 antibodies. In, input; pr, precipitate. (b) The scheme explains the various protein–/protein interactions (bold double arrows) that lead to precipitation of the triple complex. Ank, CRIB (cdc42/rac interactive binding site), GK (guanylate kinase domain), PDZ, SAM (sterile alpha motif) and SH3 indicate the location of the various protein interaction domains. P, putative tyrosine phosphorylation sites in IRSp53. (c) Mouse brain was solubilized in deoxycholate buffer; after centrifugation precipitation was performed using either immobilized NMDA-receptor C-terminal peptide (N) or rat SSTR3 C-terminal peptide (S). Samples were analyzed by Coomassie staining (upper panel) and western blotting (WB) using rbAnti-IRS and anti-shank antisera, as indicated. The positions of PSD-95 and PSD-93, as identified already in Fig. 1, are indicated by arrows. In, input.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Fusion proteins and antibodies
  5. Precipitation assays
  6. Tissue culture
  7. Immunocytochemistry
  8. Results
  9. The C-terminus of IRSp53 interacts with PDZ2 of PSD-95
  10. Discussion
  11. Acknowledgements
  12. References

We have used a peptide-based affinity purification technique to identify proteins interacting with the putative PDZ-binding motif at the C-terminus of IRSp53.1. This technique proves in our hands to be a rapid and rigorous purification method, as it can be applied under rather stringent conditions such as solubilization in 2% SDS as shown in Fig. 1. We identify an interaction of IRSp53.1 with PDZ2 of the PSD-95 molecule. Recently, Hori et al. (2003) have demonstrated an interaction between the C-terminus of IRSp53 and the PDZ domain of MALS which occurs at cell-cell contacts in epithelial cells, thus confirming the affinity of IRSp53.1 for type I PDZ domains. Though MALS is also prominently expressed in the rodent brain, we did not purify a protein of the appropriate molecular weight, presumably because either MALS may be involved in other interactions within the brain, or because under the competitive conditions of a whole brain lysate, the peptide purification approach used here selects for those binding partners with the highest affinity.

Our data indicate that IRSp53.1 can link two prominent proteins of the postsynaptic NMDA-receptor complex, namely shank1 and PSD-95. The SH3 domain of IRSp53 binds to part of the proline rich region of shank1 (Soltau et al. 2002), whereas its C-terminus associates with PDZ domain 2 of PSD-95. Both shank1 and PSD-95 possess several protein interaction motifs, which mediate multiple interactions (e.g. Boeckers et al. 2002; Lim et al. 2002). Given the complexity of protein interactions within the PSD, it is conceivable that within any two postsynaptic scaffold proteins, there may be more than one direct or indirect link. Thus, there is already another family of proteins (GKAP/SAPAP1-4) which can bind to PSD-95 (at the GK domain) and shank1 (via the PDZ domain of shank; Naisbitt et al. 1999). IRSp53.1 and GKAP/SAPAPs would therefore act in concert to provide a stable double bridge between the PSD-95 proteins, which anchor transmitter receptors, and shanks, which are more deeply buried within the PSD (Valtschanoff and Weinberg 2001) and link the whole complex to cytoskeletal associated proteins such as cortactin (Du et al. 1998) and fodrin (Böckers et al. 2001). An important difference between IRSp53.1 and GKAP/SAPAPs, however, is that the interaction of IRSp53.1 with shank1 is regulated by the small G-protein cdc42 (Soltau et al. 2002). So far it is unclear which signals lead to macromolecular assembly within the postsynaptic receptor complex; time lapse video imaging experiments suggest that synaptic PSD-95 clusters for example are assembled opposite presynaptic nerve terminals from a diffuse cytoplasmic pool of PSD-95 (Bresler et al. 2001). Thus, scaffolding molecules such as those discussed here may be held in a ‘closed’ conformation by intramolecular interactions, and interaction domains may become accessible for intermolecular interaction only upon a certain stimulus. Our data indicate such a pathway as activation of IRSp53.1 by cdc42 allows docking of a preassembled IRSp53.1/PSD-95 complex to the proline-rich region of shank1, demonstrating how protein interactions may be triggered by the activity of small G-proteins. Interestingly, Park et al. (2003) have shown recently that βPIX, an exchange factor for rac and cdc42, is tethered to the PDZ domain of shank; thus shank appears to accumulate several components of small G-protein signalling pathways at postsynaptic sites which might play a role in the coordination of complex assembly.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Fusion proteins and antibodies
  5. Precipitation assays
  6. Tissue culture
  7. Immunocytochemistry
  8. Results
  9. The C-terminus of IRSp53 interacts with PDZ2 of PSD-95
  10. Discussion
  11. Acknowledgements
  12. References

We thank Hans-Hinrich Hönck, Birgit Schwanke and Sönke Harder for excellent technical assistance. gpAnti-IRS was supplied by Professor T. Böckers (University of Ulm, Germany), GST fusion constructs by Professor Craig Garner (Stanford University, CA, USA and Marcus Christenn (UKE Hamburg, Germany). Financial support from Deutsche Forschungsgemeinschaft (SFB545/B7 to DR and H-JK), the European commission (QLG3-CT-1999–00908 to DR) and the HFSP (RG 0120/1999-B, to SK) is gratefully acknowledged.



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