Post-phosphorylation prolyl isomerisation of gephyrin represents a mechanism to modulate glycine receptors function
2007; Springer Nature; Volume: 26; Issue: 7 Linguagem: Inglês
10.1038/sj.emboj.7601625
ISSN1460-2075
AutoresM Moretto Zita, Ivan Marchionni, Elisa Bottos, Massimo Righi, Giannino Del Sal, Enrico Cherubini, Paola Zacchi,
Tópico(s)Signaling Pathways in Disease
ResumoArticle8 March 2007free access Post-phosphorylation prolyl isomerisation of gephyrin represents a mechanism to modulate glycine receptors function M Moretto Zita M Moretto Zita International School for Advanced Studies, Neuroscience Programme, Area Science Park, Trieste, Italy Search for more papers by this author Ivan Marchionni Ivan Marchionni International School for Advanced Studies, Neuroscience Programme, Area Science Park, Trieste, Italy Search for more papers by this author Elisa Bottos Elisa Bottos International School for Advanced Studies, Neuroscience Programme, Area Science Park, Trieste, Italy Search for more papers by this author Massimo Righi Massimo Righi International School for Advanced Studies, Neuroscience Programme, Area Science Park, Trieste, Italy Search for more papers by this author Giannino Del Sal Giannino Del Sal Laboratorio Nazionale CIB, AREA Science Park, Trieste, Italy Dipartimento di Biochimica Biofisica Chimica delle Macromolecole, Trieste, Italy Search for more papers by this author Enrico Cherubini Enrico Cherubini International School for Advanced Studies, Neuroscience Programme, Area Science Park, Trieste, Italy Search for more papers by this author Paola Zacchi Corresponding Author Paola Zacchi International School for Advanced Studies, Neuroscience Programme, Area Science Park, Trieste, Italy Search for more papers by this author M Moretto Zita M Moretto Zita International School for Advanced Studies, Neuroscience Programme, Area Science Park, Trieste, Italy Search for more papers by this author Ivan Marchionni Ivan Marchionni International School for Advanced Studies, Neuroscience Programme, Area Science Park, Trieste, Italy Search for more papers by this author Elisa Bottos Elisa Bottos International School for Advanced Studies, Neuroscience Programme, Area Science Park, Trieste, Italy Search for more papers by this author Massimo Righi Massimo Righi International School for Advanced Studies, Neuroscience Programme, Area Science Park, Trieste, Italy Search for more papers by this author Giannino Del Sal Giannino Del Sal Laboratorio Nazionale CIB, AREA Science Park, Trieste, Italy Dipartimento di Biochimica Biofisica Chimica delle Macromolecole, Trieste, Italy Search for more papers by this author Enrico Cherubini Enrico Cherubini International School for Advanced Studies, Neuroscience Programme, Area Science Park, Trieste, Italy Search for more papers by this author Paola Zacchi Corresponding Author Paola Zacchi International School for Advanced Studies, Neuroscience Programme, Area Science Park, Trieste, Italy Search for more papers by this author Author Information M Moretto Zita1, Ivan Marchionni1, Elisa Bottos1, Massimo Righi1, Giannino Del Sal2,3, Enrico Cherubini1 and Paola Zacchi 1 1International School for Advanced Studies, Neuroscience Programme, Area Science Park, Trieste, Italy 2Laboratorio Nazionale CIB, AREA Science Park, Trieste, Italy 3Dipartimento di Biochimica Biofisica Chimica delle Macromolecole, Trieste, Italy *Corresponding author. International School for Advanced Studies, Neuroscience Programme, Area Science Park, Basovizza SS14 Km 163.5, 34012 Trieste, Italy. Tel.: +39 403756510; Fax:+39 403756502; E-mail: [email protected] The EMBO Journal (2007)26:1761-1771https://doi.org/10.1038/sj.emboj.7601625 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The microtubule binding protein gephyrin plays a prominent role in establishing and maintaining a high concentration of inhibitory glycine receptors juxtaposed to presynaptic releasing sites. Here, we show that endogenous gephyrin undergoes proline-directed phosphorylation, which is followed by the recruitment of the peptidyl-prolyl isomerase Pin1. The interaction between gephyrin and Pin1 is strictly dependent on gephyrin phosphorylation and requires serine–proline consensus sites encompassing the gephyrin proline-rich domain. Upon binding, Pin1 triggers conformational changes in the gephyrin molecule, thus enhancing its ability to bind the beta subunit of GlyRs. Consistently, a downregulation of GlyR clusters was detected in hippocampal neurons derived from Pin1 knockout mice, which was paralleled by a reduction in the amplitude of glycine-evoked currents. Our results suggest that phosphorylation-dependent prolyl isomerisation of gephyrin represents a mechanism for regulating GlyRs function. Introduction The reversible phosphorylation of proteins on serine and threonine residues preceding proline represents a key signalling pathway for the control of various cellular processes. Extensively characterised as pivotal mechanism controlling cell proliferation and differentiation (Blume-Jensen and Hunter, 2001; Lu et al, 2002), this signalling cascade has been recently involved in the regulation of synaptic structure and function. At excitatory synapses, mass spectrometric analysis performed on isolated postsynaptic density proteins (PSD) has led to the identification of a number of novel serine/proline phosphorylation sites (Jaffe et al, 2004). Interestingly, three scaffolding molecules, namely PSD-95, PSD-93 and Shank3, are shown to undergo proline-directed phosphorylation and to share a similar phosphorylation motif. Moreover, PSD-95, the central organising element of the PSD that links NMDA receptors to the cytoskeleton, has been shown to be phosphorylated by the serine/threonine kinase Cdk-5, and this event appears to negatively regulate the clustering of NMDA receptors (Morabito et al, 2004). At inhibitory synapses, antisense (Kirsch et al, 1993) and knockout (Feng et al, 1998) experiments have clearly highlighted the involvement of the scaffolding molecule gephyrin in the proper localisation of glycine receptors (GlyRs) and selected gamma-aminobutyric acid A receptors (GABAARs) (Essrich et al, 1998; Kneussel et al, 1999a, 2001). Gephyrin has been shown to bind with high affinity to an amphipathic amino-acid sequence in the large cytoplasmic loop of the β subunit of GlyR (Meyer et al, 1995; Kneussel et al, 1999b), whereas it only functionally associates with individual GABAA receptor subtypes (Meyer et al, 1995). Gephyrin interacts with the actin- and microtubule-based cytoskeleton (Mammoto et al, 1998; Giesemann et al, 2003) and these interactions are thought not only to provide the physical constraints required to maintain receptors at synapses, but also to regulate the constant flux of receptor and scaffolding elements in and out postsynaptic sites (Choquet and Triller, 2003; Hanus et al, 2006). Receptor accumulation at synapses has been proposed to rely on the ability of gephyrin to reversibly multimerise into a submembraneous hexagonal protein lattice, which accommodates a high number of receptor binding sites (Schwarz et al, 2001; Sola et al, 2001, 2004; Xiang et al, 2001). Finally, an early association of gephyrin with GlyR clusters along the biosynthetic pathway has been documented, which seems to modify receptor trafficking and delivery to synapses (Hanus et al, 2004; Maas et al, 2006). However, the precise molecular mechanisms underlying these dynamic processes are still largely unknown. Given the emerging role of proline-directed phosphorylation in the regulation of glutamatergic synapses (Jaffe et al, 2004), in the present study we have investigated whether a similar type of modulation occurs at inhibitory synapses. Compelling evidence has shown that proline-directed phosphorylation acts through the induction of conformational changes onto the target proteins (Yaffe et al, 1997; Zhou et al, 1999). These modifications are catalysed by a peptidyl-prolyl isomerase, Pin1 (Peptidyl-prolyl Isomerase NIMA interacting protein 1) (Lu et al, 1996), which specifically binds phosphorylated serine or threonine residues immediately preceeding proline (pSer/Thr-Pro motifs) and promotes the cis/trans isomerisation of the peptide bond (Ranganathan et al, 1997; Shen et al, 1998). Such conformational changes have been shown to have profound effects on the function of Pin1 substrates as they can modulate catalytic activity, phosphorylation status, protein–protein interactions, subcellular localisation and protein stability (Lu, 2004; Wulf et al, 2005). Interestingly, gephyrin purified from GlyR preparations has been found to be phosphorylated at the serine and threonine residues (Langosch et al, 1992). Moreover, from the analysis of gephyrin amino-acid sequence, several putative Pin1 consensus sites have been identified, suggesting an involvement of this post-phosphorylation regulatory mechanism in the modulation of gephyrin scaffolding functions. Here, we provide evidence that endogenous gephyrin undergoes proline-directed phosphorylation. Gephyrin, upon phosphorylation, interacts with the peptidyl-prolyl cis/trans isomerase Pin1, which in turn induces a conformational change in gephyrin. Interestingly, gephyrin binding to the large cytoplasmic loop of the GlyR β subunit (GlyR β loop), a functional surrogate for full-length GlyRs (Meier and Grantyn, 2004), is strongly reduced in Pin1−/− cells. Moreover, hippocampal neurons isolated from Pin1 knockout mice show a reduced number of GlyR clusters, which is associated with a significant decrease in the peak amplitude of glycine-evoked currents. Results Recombinant and endogenous gephyrin undergo proline-directed phosphorylation Previous experiments have identified a kinase activity tightly associated with affinity-purified GlyR preparations that phosphorylates gephyrin mainly on serine and, to a lesser extent, on threonine residues (Langosch et al, 1992). In order to test whether some of these serine and threonine residues precede a proline (pSer/Thr-Pro), we took advantage of the phospho-dependent antibody mitotic phosphoprotein monoclonal 2 (MPM-2) (Davis et al, 1983). To this aim, FLAG-tagged gephyrin was overexpressed in HEK 293 cells and half of the cell lysate was treated with calf intestine phosphatase (CIP) before performing immunoprecipitation experiments with a monoclonal antibody specific for the FLAG epitope or with anti-myc 9E10 monoclonal antibody as negative control. Western blotting using the MPM-2 antibody revealed that a protein of about 100 kDa molecular weight was recognised only in the anti-FLAG immunoprecipitate obtained from CIP-untreated cell lysate (Figure 1A, lane 4). When the same membrane was stripped and reprobed with a polyclonal anti-gephyrin antibody, it was found that gephyrin-FLAG was efficiently immunoprecipitated from both cell lysates and, most importantly, that the MPM-2 reactive band was also specifically recognised by the anti-gephyrin antibody (Figure 1A, lane 10). Altogether, these findings indicate that gephyrin is indeed an MPM-2 antigen. Figure 1.Recombinant and endogenous gephyrin contains consensus sequences for proline-directed phosphorylation. (A) HEK 293 cells transfected with gephyrin-FLAG were lysed, half of the lysate was treated with CIP and immunoprecipitated with anti-FLAG monoclonal antibody (lanes 4 and 6) and with a non-related antibody (anti-MYC) as negative control (lane 2). Immunoprecipitates were analysed by Western blotting using the MPM-2 antibody (left panel). The same membrane was stripped and reprobed with the anti-gephyrin antibody (right panel). (B) Endogenous gephyrin was immunoprecipitated from mouse brain lysate with an anti-gephyrin polyclonal antibody (lanes 4 and 6) and with normal rabbit serum (NRS) as negative control (lane 2). Half of the cell homogenate was treated with CIP before immunoprecipitation. Immunoprecipitates were analysed as described in (A). Download figure Download PowerPoint To see whether endogenous gephyrin is phosphorylated on Ser/Thr-Pro residues, we immunoprecipitated gephyrin from mouse brain homogenates using the affinity-purified polyclonal antibody raised against the full-length protein or with preimmune serum as negative control and immunoblotted with the MPM-2 antibody (Figure 1B, left panel). Also in this case, half of the homogenate was dephosphorylated as described above. As shown in Figure 1B (left panel), the MPM-2 reactive band was observed only in the anti-gephyrin immunoprecipitate from CIP-untreated brain lysate (lane 4). Upon stripping and reprobing the membrane with a monoclonal anti-gephyrin antibody, it was found that endogenous gephyrin was efficiently immunoprecipitated from both CIP-treated and -untreated homogenates (Figure 1B, lanes 10 and 12). These data indicate that the scaffolding protein gephyrin undergoes proline-directed phosphorylation in mouse brain, thus representing a newly identified MPM-2 antigen. Gephyrin interacts in vitro with the peptidyl-prolyl isomerase Pin1 in a phosphorylation-dependent manner The significance of proline-directed phosphorylation as a signalling mechanism relies on the ability of phosphorylated Ser/Thr-Pro motifs to recruit the prolyl isomerase Pin1 (Lu, 2004). Pin1's phosphoserine- and phosphotreonine-binding activity is mediated by its N-terminal WW domain, a compact protein-interacting module characterised by the presence of two highly conserved tryptophan (W) residues (Lu et al, 1999). As the MPM-2 antibody recognises phosphorylated Ser/Thr-Pro epitopes on several important Pin1 substrates, we investigated whether gephyrin also represents a Pin1 target. To this aim, lysates of HEK 293 cells transfected with gephyrin-FLAG were subjected to pull-down assay with beads loaded with GST-Pin1 or with GST alone as negative control. Proteins bound to beads were separated on SDS-containing gels and immunoblotted using the anti-FLAG antibody. As shown in Figure 2A, only GST-Pin1 beads precipitated with high efficiency the ectopically expressed gephyrin-FLAG. Similar pull-down experiments were then performed to assay the ability of endogenous gephyrin present on neuroblastoma SH-SY5Y cells to interact with Pin1 (Figure 2B). Also in this case, gephyrin was detected only when associated with GST-Pin1 fusion protein. In this case, immunoblot analysis was performed using the monoclonal antibody raised against the C-terminal domain of gephyrin protein. Finally, to examine whether the WW domain of Pin1 exhibited a phosphorylation-dependent binding activity on endogenous gephyrin from SH-SY5Y, as shown for many other Pin1 interactors, we employed the Tyr23 to Ala Pin1 (Pin1Y23A), a mutant that contains a single alanine substitution at the critical Tyr23 in the WW domain resulting in a loss of the phosphoserine/threonine-binding activity (Lu et al, 1999). As shown in Figure 2C, the mutant expressed and purified as GST fusion protein completely abrogated the interaction between Pin1 and gephyrin. In contrast, inactivation of the prolyl isomerase activity in the mutant Cys113 to Ala (Pin1C113A) (Winkler et al, 2000) did not affect the binding of Pin1 to endogenous gephyrin. Figure 2.Gephyrin interacts with Pin1 in a phosphorylation-dependent manner in vitro. (A) Lysates of HEK 293 cells transfected with gephyrin FLAG were subjected to GST (lane 2) or GST-Pin1 (lane 3) pull-down followed by Western blot with anti-FLAG antibody. (B) Endogenous gephyrin from lysates of a neuroblastoma cell line (SH-SY5Y) was subjected to GST pull-down as described in (A). Western blot analysis was performed with anti-gephyrin antibody. (C) Endogenous gephyrin from SH-SY5Y cells was subjected to pull-down using GST (lane 2), GST-Pin1 (lane 3), GST-Pin1 Y23A (lane 4) and GST-Pin1 C113A (lane 5). Western blot analysis was performed with anti-gephyrin antibody. Download figure Download PowerPoint These results demonstrate that the WW domain of Pin1 is responsible for binding the phosphorylated form of gephyrin. Pin1 binding to gephyrin requires Ser-Pro epitopes contained within the proline-rich domain Phosphoproteins known to recruit Pin1 in a phosphorylation-dependent manner are commonly phosphorylated on multiple Ser/Thr residues clustered at critical regulatory domains. In this context, the intervening region of gephyrin, which contains several potential protein interaction domains, harbors the majority of Ser/Thr-Pro epitopes that are organised in two clusters. Interestingly, one of these clusters containing three serine–proline epitopes encompasses a proline-rich region of gephyrin, making it an attractive candidate for Pin1 interaction. To explore this possibility, we initially constructed a gephyrin mutant devoid of the proline-rich region (Δ174–243) encoded by exon 8 and tested it using the previously described GST-Pin1 pull-down assay. As shown in Figure 3A, gephyrin mutant displayed a greatly reduced binding to Pin1 as compared with full length gephyrin, suggesting that Ser188, Ser194 and Ser200-Pro epitopes are involved in Pin1 recruitment. It is possible that this difference reflects major structural changes owing to the loss of the entire gephyrin proline-rich region. Therefore, we performed a serine to alanine scan mutagenesis throughout gephyrin exon 8 and assayed the mutant proteins for Pin1 binding. As shown in Figure 3B, sequential disruption of Ser-Pro sites almost completely abolished the interaction of gephyrin to Pin1, as the triple mutant (Mut-C) retained a very low residual binding similar to the deletion mutant initially tested. These findings prompted us to expand our site-directed mutagenesis to all the remaining Pin1 putative consensus sites. The following gephyrin mutants were therefore generated and their contributions studied in GST-Pin1 pull-down assays: Thr96Ala–Thr123Ala (Mut-E); Thr266Ala–Ser270Ala–Thr286Ala (Mut-F) and Ser319Ala–Thr337Ala (Mut-G). Moreover, a Ser/Thr-Ala gephyrin mutant comprehensive of all seven consensus sites, namely Thr96Ala–Thr132Ala–Thr266Ala–Ser270Ala–Thr286Ala–Ser319Ala–Thr337Ala (Mut-D), was also included. As shown in Figure 3C, all gephyrin variants tested showed similar levels of binding as the gephyrin wild-type (WT) protein to GST-Pin1 beads, thus strongly confirming the importance of serine residues present within the proline-rich domain of gephyrin for Pin1 interaction. Figure 3.Pin1 binding requires Ser-Pro epitopes contained within the proline-rich domain of gephyrin. (A) GST-Pin1 pull-down assay using lysates of HEK 293 cells transfected with gephyrin WT (lanes 1–3) compared with gephyrin depleted of exon 8 (lanes 4–6). (B) (upper panel) Schematic representation of gephyrin domains (G, N-terminal domain; L, linker or intervening region; E, C-terminal domain) together with the positions of putative Pin1 consensus motifs. The proline-rich domain (PP) contains three Pin1 consensus sites at residues 188, 194 and 200. Mut-A, Mut-B and Mut-C refer to gephyrin S188A, S188A-S194A and S188A-S194A-S200A mutants, respectively. (Lower panel) GST-Pin1 pull-down assay using gephyrin WT (lanes 1–3), Mut-A (lanes 4–6), Mut-B (lanes 7–9) and Mut-C (lanes 10–12). The triple mutant retained a low residual binding similar to the deletion mutant initially tested in (A). (C) (upper panel) GST-Pin1 pull-down assay performed on gephyrin WT (lanes 1–3), Mut-C (lanes 4–6) and Mut-D (T96A-T132A-T266A-S270A-T286A-S319A-T337A) (lanes 7–9). (Lower panel) GST-Pin1 pull-down on Mut-E (T96A-T123A) (lanes 1–3), Mut-F (T266A-S270A-T286A) (lanes 4–6) and Mut-G (S319A-T337A) (lanes 7–9). Download figure Download PowerPoint Gephyrin associates with Pin1 in HEK 293 cells and in the mouse brain To characterise the potential interaction between Pin1 and gephyrin in intact mammalian cells, we initially expressed both proteins in HEK 293 cells and examined their subcellular distribution. Under these conditions, ectopically expressed gephryin produces large cytoplasmic aggregates characterised by their ability to actively sequester several gephyrin interaction partners (Figure 4A, left panel). In single transfections, Pin1-FLAG showed a diffuse distribution both in the nucleus and the cytoplasm (Figure 4A, middle panel). When gephyrin-GFP and Pin1-FLAG were cotransfected, a fraction of Pin1-FLAG was clearly relocalised to intracytoplasmic gephyrin aggregates (Figure 4B), thus indicating colocalisation of the two proteins. We also expressed gephyrin Mut C-GFP in HEK 293 cells. This triple mutant formed cytosolic aggregates in a manner indistinguishable from that generated by WT gephyrin (Figure 4A, right panel). Consistent with the pull-down data, this gephyrin mutant was strongly impaired in recruiting Pin1-FLAG immunoreactivity upon cotransfection (Figure 4C). Figure 4.Gephyrin interacts with Pin1 in vitro. (A) Immunofluorescence assay to determine the subcellular distribution of gephyrin WT, Mut-C (see above) and Pin1-FLAG ectopically expressed in HEK 293 cells. In single transfection experiments, gephyrin-GFP and Mut-C-GFP were revealed by the intrinsic green fluorescence of GFP. Pin1-FLAG was visualised by anti-FLAG antibody, followed by TRITC-conjugated secondary antibody. Scale bar, 10 μm. (B) Cotransfection experiments with gephyrin-GFP and Pin1-FLAG. (C) Cotransfection experiments with gephyrin Mut-C-GFP and Pin1-FLAG. (D) Lysates of HEK 293 cells transfected with Pin1WT in the presence of gephyrin FLAG or with the vector alone (as a negative control) were immunoprecipitated with monoclonal antibodies anti-FLAG (lanes 4 and 5). Immunoprecipitates were analysed by Western blotting using anti-gephyrin and anti-Pin1 antibodies. Efficient dephosphorylation of gephyrin upon CIP treatment was verified by Western blot on total lysates and on immunoprecipitated gephyrin-FLAG with MPM-2 antibody (7–8 and 10–11, respectively.) (E) Co-immunoprecipitation experiment on mouse brain lysates using a polyclonal anti-gephyrin antibody and NRS as negative control (lanes 2 and 3, respectively). Immunoprecipitates were analysed by Western blotting using a monoclonal antibody anti-gephyrin and a polyclonal antibody against Pin1. Download figure Download PowerPoint We then performed immunoprecipitation experiments to investigate the presence of Pin1/gephyrin complexes in vitro. HEK 293 cells were cotransfected with plasmids encoding for Pin1WT and gephyrin-FLAG, or Pin1WT and vector alone as negative control, and cell lysates were immunoprecipitated with the anti-FLAG monoclonal antibody. The bound protein complexes were analysed by Western blotting using anti-gephyrin and anti-Pin1 polyclonal antibodies for gephyrin and Pin1 detection, respectively. As shown in Figure 4D, not only Pin1 was specifically immunoprecipitated from cells expressing gephyrin-FLAG (lane 5), but also its gephyrin-dependent immunoprecipitation was completely abolished upon dephosphorylation of gephyrin-FLAG by phosphatase treatment (lane 6). The efficient dephosphorylation of Pin1 binding sites upon CIP addition was confirmed by the lack of MPM-2 immunoreactivity on immunoprecipitated gephyrin-FLAG (Figure 4D, right panel, lane 10). This latter result is in agreement with our findings with the Pin1-binding-defective mutant (Pin1Y23A) and further supports the phosphorylation-dependent interaction of Pin1 with gephyrin. In addition, endogenous Pin1 and gephyrin were found in complex upon co-immunoprecipitation from mouse brain homogenates (Figure 4E), indicating that gephyrin is phosphorylated on Pin1 consensus sites and it interacts with the prolyl isomerase in neuronal cells. Pin1 elicits conformational changes in gephyrin To test whether Pin1 can induce a conformational change in gephyrin, a partial proteolysis assay was carried out. To this aim, His-tagged gephyrin full length was overexpressed in fibroblasts obtained from the Pin1 knockout mouse embryo (Pin1−/− mouse embryo fibroblasts, MEFs). This allows phosphorylation of ectopically expressed gephyrin in the absence of Pin1-mediated rearrangement. After transfection (48 h), His-tagged gephyrin was efficiently purified from cell extracts on nickel column and incubated with either GST-Pin1, the catalytically inactive mutants GST-Pin1C113A and GST-Pin1S67E (Zhou et al, 2000) or GST alone. Finally, all the reactions were incubated with the protease subtilisin under identical conditions. After limited digestion, the corresponding protease fragments were visualised by SDS–PAGE followed by Western blot analysis using a pool of anti-gephyrin antibodies. The different pattern of digestion products obtained highlights a more efficient gephyrin protection from subtilisin cleavage promoted by GST-Pin1 as compared with GST or GST-Pin1C113A and GST-Pin1S67E (Figure 5). This effect is not due to steric hindrance exerted by GST or Pin1 binding, but is dependent only on Pin1 isomerase activity as both Pin1 mutants catalytically impaired but fully competent to bind their substrate were completely ineffective. Figure 5.Pin1 induces conformational changes of gephyrin. HIS-tagged gephyrin was overexpressed in Pin1−/− MEFs and purified from cell extracts on a nickel column (lane 2). The purified protein was incubated with GST (lane 3), GST-Pin1 (lane 4), GST-Pin1 C113A (lane 5) and GST-Pin1 S67E (lane 6). Each sample was incubated with subtilisin and the corresponding protease fragments were visualised by SDS–PAGE followed by Western blot analysis using a pool of anti-gephyrin antibodies. Lane 7 represents an experiment performed in the absence of HIS-tagged gephyrin to exclude the possibility that the pool of anti-gephyrin antibodies may recognise some other antigen absorbed aspecifically on a nickel column. Download figure Download PowerPoint Pin1 regulates gephyrin ability to interact with the β subunit of GlyRs Pin1-dependent conformational rearrangement of gephyrin may affect the ability of this protein to bind the β subunit of GlyRs. To address this question, MEFs derived from Pin1 knockout and WT mice were cotransfected with gephyrin-FLAG and GFP-tagged intracellular loop of the β subunit of GlyRs (GFP-β loop). After transfection (48 h), gephyrin-FLAG solubilised from both cell lines was immunoprecipitated using either the anti-FLAG monoclonal antibody or, as negative control, the anti-myc 9E10 monoclonal antibody. The bound protein complexes were analysed by Western blotting using the anti-gephyrin polyclonal antibody, for gephyrin detection, or anti-GFP polyclonal antibody for the GFP-β loop. Regardless of Pin1 expression, the anti-FLAG antibody immunoprecipitated comparable amounts of gephyrin-FLAG (Figure 6). However, in the absence of endogenous Pin1, the amount of GFP-β loop co-immunoprecipitated by gephyrin-FLAG was drastically reduced (compare lanes 7–5). Interestingly, the impairment of binding of GFP-β loop to gephyrin was fully rescued when Pin1−/− MEFs were cotransfected with Pin1WT (lane 9). These results provide evidence that Pin1-induced conformational changes of gephyrin influence the ability of this protein to interact with the functional surrogate of GlyR β subunit. Figure 6.Lack of Pin1 impairs the ability of gephyrin to interact efficiently with GlyRs. Gephyrin-FLAG and GFP-β loop were transfected in MEFs Pin1+/+ (1), MEFs Pin1−/− (2) and MEFs Pin1−/− plus Pin1 (3). Lysates were immunoprecipitated with anti-FLAG (lanes 5, 7 and 9) or anti-MYC as negative control (lanes 4, 6 and 8). Immunoprecipitates were analysed by Western blotting using polyclonal antibodies against gephyrin, GFP and Pin1. Download figure Download PowerPoint GlyRs punctae and the amplitude of glycine-evoked currents are reduced in hippocampal neurons from Pin1 knockout mice To further assess the involvement of Pin1 in gephyrin-dependent GlyRs function(s), we initially investigated the cellular distribution of GlyRs in primary hippocampal neurons derived from WT and Pin1 knockout mice (Atchison et al, 2003). In several staining experiments, we consistently found a loss of GlyRs immunoreactive punctae on the membranes of Pin1−/− neurons as compared with WT neurons (Figure 7A and B). By contrast, immunocytochemical staining for intracellular gephyrin revealed a similar distribution of its immunoreactive clusters. As a consequence, a reduced level of colocalisation between GlyRs and gephyrin immunoreactivities was detected. Figure 7.Hippocampal neurons from Pin−/− mice express a reduced number of GlyRs punctae associated with decreased peak amplitude of glycine-evoked currents. (A) Hippocampal neurons derived from Pin1 WT and (B) knockout mice were labelled with monoclonal antibody (mAb4a) recognising the GlyRs (green), polyclonal anti-gephyrin antibody (red) and the neuronal marker β3-tubulin (blue). Side panels are magnification of the boxed areas. Scale bars, 10 μm. (C) Superimposed current responses evoked at −40 mV by different concentrations of glycine (bar) in WT (PIN+/+, upper traces) and knockout (PIN−/−, lower traces) mice. The numbers near the current traces refer to the concentrations of glycine used (in μM). (D) Dose–response curve for glycine-evoked currents obtained in WT (filled symbols) and knockout mice (open symbols). Each point is the average of 8–16 individual responses (*P<0.05 and **P<0.001). Download figure Download PowerPoint To examine whether the reduced number of GlyR clusters observed in mice lacking Pin1 expression induces modifications in GlyR function, whole-cell patch-clamp recordings (in voltage clamp configuration) were performed at −40 mV from WT and Pin1−/− neurons. Recordings were routinely performed in the presence of the GABAA antagonist picrotoxin (50 μM), the GABAB receptor antagonist CGP 55485 (1 μM) and the broad-spectrum ionotropic glutamate receptor antagonist kynurenic acid (1 mM). All cells tested (n=84) responded to glycine application with an inwa
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