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• W2050546598 abstract "Article9 February 2006free access Activated radixin is essential for GABAA receptor α5 subunit anchoring at the actin cytoskeleton Sven Loebrich Sven Loebrich Zentrum für Molekulare Neurobiologie Hamburg (ZMNH), Universität Hamburg, Hamburg, Germany Search for more papers by this author Robert Bähring Robert Bähring Zentrum für Molekulare Neurobiologie Hamburg (ZMNH), Universität Hamburg, Hamburg, Germany Search for more papers by this author Tatsuya Katsuno Tatsuya Katsuno Department of Cell Biology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto, Japan Search for more papers by this author Sachiko Tsukita Sachiko Tsukita Department of Cell Biology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto, Japan School of Health Sciences, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto, Japan Search for more papers by this author Matthias Kneussel Corresponding Author Matthias Kneussel Zentrum für Molekulare Neurobiologie Hamburg (ZMNH), Universität Hamburg, Hamburg, Germany Search for more papers by this author Sven Loebrich Sven Loebrich Zentrum für Molekulare Neurobiologie Hamburg (ZMNH), Universität Hamburg, Hamburg, Germany Search for more papers by this author Robert Bähring Robert Bähring Zentrum für Molekulare Neurobiologie Hamburg (ZMNH), Universität Hamburg, Hamburg, Germany Search for more papers by this author Tatsuya Katsuno Tatsuya Katsuno Department of Cell Biology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto, Japan Search for more papers by this author Sachiko Tsukita Sachiko Tsukita Department of Cell Biology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto, Japan School of Health Sciences, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto, Japan Search for more papers by this author Matthias Kneussel Corresponding Author Matthias Kneussel Zentrum für Molekulare Neurobiologie Hamburg (ZMNH), Universität Hamburg, Hamburg, Germany Search for more papers by this author Author Information Sven Loebrich1, Robert Bähring1, Tatsuya Katsuno2, Sachiko Tsukita2,3 and Matthias Kneussel 1 1Zentrum für Molekulare Neurobiologie Hamburg (ZMNH), Universität Hamburg, Hamburg, Germany 2Department of Cell Biology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto, Japan 3School of Health Sciences, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto, Japan *Corresponding author. Zentrum für Molekulare Neurobiologie Hamburg (ZMNH), Universität Hamburg, Falkenried 94, 20251 Hamburg, Germany. Tel.: +49 40 42803 6275; Fax: +49 40 42803 7700; E-mail: [email protected] The EMBO Journal (2006)25:987-999https://doi.org/10.1038/sj.emboj.7600995 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Neurotransmitter receptor clustering is thought to represent a critical parameter for neuronal transmission. Little is known about the mechanisms that anchor and concentrate inhibitory neurotransmitter receptors in neurons. GABAA receptor (GABAAR) α5 subunits mainly locate at extrasynaptic sites and are thought to mediate tonic inhibition. Notably, similar as synaptic GABAARs, these receptor subtypes also appear in cluster formations at neuronal surface membranes and are of particular interest in cognitive processing. GABAAR α5 mutation or depletion facilitates trace fear conditioning or improves spatial learning in mice, respectively. Here, we identified the actin-binding protein radixin, a member of the ERM family, as the first directly interacting molecule that anchors GABAARs at cytoskeletal elements. Intramolecular activation of radixin is a functional prerequisite for GABAAR α5 subunit binding and both depletion of radixin expression as well as replacement of the radixin F-actin binding motif interferes with GABAAR α5 cluster formation. Our data suggest radixin to represent a critical factor in receptor localization and/or downstream signaling. Introduction Neurotransmitter receptors are involved in fast neuronal transmission at chemical synapses. In processes of neurotransmitter receptor targeting (Kneussel, 2005), receptors are thought to enter the neuronal surface membrane at extrasynaptic sites, followed by subsequent lateral membrane diffusion. Submembrane scaffold molecules, which locate at postsynaptic specializations, bind and cluster surface membrane receptors opposite to axon terminals that release the appropriate neurotransmitter (Kneussel and Betz, 2000; Moss and Smart, 2001; Kim and Sheng, 2004). In contrast to the localization at synapses, neurotransmitter receptors also locate and concentrate at extrasynaptic surface membrane sites (Hanchar et al, 2004). NMDA-type glutamate receptors at synaptic or extrasynaptic sites activate alternative signaling cascades, leading to either activation or deactivation of the transcription factor CREB, respectively (Hardingham et al, 2002). Inhibitory GABAA receptor (GABAAR) subtypes containing α5 subunits assemble with β and γ subunits (McKernan and Whiting, 1996) and are predominantly expressed in hippocampus (Wisden et al, 1992). Here, they form clusters (Hutcheon et al, 2004) that are mainly found at extrasynaptic locations (Brunig et al, 2002), but to a lesser extent, are also found at synapses (Christie and de Blas, 2002). Extrasynaptic α5-subunit-containing GABAARs are thought to mediate tonic inhibition (Caraiscos et al, 2004). Notably, mutant mice revealed that this GABAAR subpopulation is critically involved in hippocampus-dependent cognitive processes including spatial learning (Collinson et al, 2002) and trace fear conditioning (Crestani et al, 2002). Although GABAAR function has been extensively characterized, the submembrane interactions of this receptor family, including the anchoring at membrane specializations, are largely unknown. Gene depletion of the scaffold protein gephyrin, a main component at inhibitory postsynaptic sites (Kneussel and Betz, 2000), alters the clustering of many GABAAR subtypes (Essrich et al, 1998; Kneussel et al, 1999). However, a direct interaction of gephyrin with any GABAAR subunit has not been reported. It is therefore likely that currently unknown factors exist which anchor GABAARs at gephyrin scaffolds and/or cytoskeletal elements. Notably, GABAAR α5 subunit clusters are among the few subtypes that are completely unaltered upon gephyrin scaffold depletion (Kneussel et al, 2001), although gephyrin has been described at both synaptic and extrasynaptic sites (Danglot et al, 2003). We therefore particularly screened for α5 subunit interacting proteins to shed light on the synaptic/extrasynaptic anchoring of this hippocampal GABAAR subpopulation. Our data identified the ERM-family protein radixin as an essential clustering factor for α5-subunit-containing GABAARs and suggest that a regulated mechanism activates radixin and subsequently bridges respective receptor subtypes with the actin cytoskeleton, which underlies the plasma membrane. Results Radixin interacts with GABAAR α5 subunits To identify novel GABAAR α5 subunit-binding proteins, we performed a yeast two-hybrid screen using the GABAAR α5 subunit intracellular TMIII-TMIV loop (amino acids 342–429) as bait. From 2.8 million clones of an adult rat brain library, three clones were isolated that coded for amino acids 89–185 of radixin, a member of the ERM (ezrin, radixin, moesin) protein family, known to interact with transmembrane proteins (Tsukita et al, 1994) (Figure 1A–C). We mapped the radixin-binding site of GABAAR α5 by generating N-terminal TIII-TMIV-loop deletion mutants based on the bait construct. All mutants lacking residues 342–352 did not interact with radixin (Figure 1A), suggesting for a necessary requirement of this sequence in radixin binding. Consistently, a construct containing residues 342–357 was sufficient to bind radixin. Notably, intracellular TMIII-TMIV loop sequences of GABAAR α1, α3, β2 or γ2 subunits did not interact with radixin in this assay (Figure 1D). The radixin-binding motif of GABAAR α5 is identical in other mammals (man, mouse, cow, dog, monkey) and shares 93% amino acid identity with bird and fish. Moreover, the binding motif is not present in the extrasynaptic GABAAR δ subunit. Figure 1.Identification of radixin as an interactor of GABAAR α5 subunits in the yeast two-hybrid screen. (A) Mapping of the radixin binding site on the large intracellular loop of GABAAR α5 (bait). (B) Radixin domain structure. The fragment isolated from the yeast two-hybrid screen is located in the FERM domain (black bar). (C) Alignment of the amino-acid sequence of rat radixin (NM_001005889) with the sequence encoded by the positive clone from the screen. (D) Intracellular TMIII-TMIV loops of the GABAA receptor subunits α1, α3, β2 and γ2 were tested for radixin interaction. Download figure Download PowerPoint Radixin–GABAAR α5 interaction requires radixin activation To substantiate these interaction data biochemically, we performed co-immunoprecipitation experiments using rat brain extracts. As detected with a radixin-specific antibody, endogenous radixin specifically co-precipitated with endogenous GABAAR α5 subunit protein (Figure 2A). Co-precipitation resulted in a 2.5-fold increase in radixin band intensity, as compared to the input material, indicating a physiological relevance of the interaction. Radixin can exist in both an inactive closed conformation (Ishikawa et al, 2001), due to intramolecular interaction, and an active open conformation, known to depend on Rho-family GTPase signaling-mediated phosphorylation (Matsui et al, 1998) (Figure 2B). To investigate whether active radixin could bind to the GABAAR α5 intracellular TMIII–TMIV loop, we performed GST-pulldown assays using rat brain extracts (Figure 2C), and probed them with radixin-specific or anti-ERM C-terminal phospho-threonine-specific antibodies. Radixin displayed specific binding to immobilized GST-GABAAR α5 loop fusion protein but not to GST alone. Also, the phospho-ERM antibody recognized a band at this size in the pulldown fraction. Together with the data from the yeast two-hybrid system, these results indicate that radixin, including its active conformation, is a direct GABAAR α5 subunit interactor. Figure 2.Intramolecular activation of radixin is required for GABAAR α5 subunit binding. (A) Co-immunoprecipitation of radixin and GABAAR α5 from rat brain extract. (B) Schematic representation of radixin intramolecular activation upon phosphorylation of threonine 564. Arrows: sites of mutagenesis; K: lysine; T: threonine. (C) Pulldown assays from rat brain extract with immobilized GABAAR α5 loop fused to GST. (D) Neuronal localization of wild-type and mutant radixin proteins and quantification of clusters per 30 μm dendrite. Arrows depict prominent membrane localization. (E) GST-pulldown assays with myc-tagged radixin wild-type and mutant proteins upon heterologous expression in HEK293 cells. (F) Combinatorial application of kinase activators and the phosphatase inhibitor Calyculin A leads to radixin activation in COS cells. The form of radixin, which binds to the GABAAR α5 loop is phosphorylated at threonine 564, as detected with a phospho ERM-specific antibody. (G) Intracellular TMIII-TMIV loops of the GABAAR subunits α1, α3, β2 and γ2 are not able to bind the active radixin mutant in GST-pulldown assays. Scale bars: 20 μm. Download figure Download PowerPoint Since the open conformation of ERM proteins is able to bind both membrane proteins and/or F-actin (Tsukita et al, 1994; Simons et al, 1998; Vaiskunaite et al, 2000), we applied site-directed mutagenesis to functionally address whether the subcellular localization of radixin might depend on its activation state. The generation of radixin mutants was based on a wild-type radixin-GFP fusion protein, which displayed similar subcellular localization as endogenous radixin (Supplementary Figure 1A). First, we introduced a negative charge in the radixin polypeptide by replacing threonine 564 (Figure 2B and D), a modification known to mimic phosphorylation and to keep ERM proteins in the open and active conformation (Huang et al, 1999; Ishikawa et al, 2001). This mutant (Radixin-GFP T564D) should be able to bind both actin filaments and phosphatidylinositol-4,5-bisphosphate (PIP2) and consistently displays a strong tendency to locate at the plasma membrane. In fact, both its localization at the neuronal plasma membrane and the number of puncta per 30 μm dendrite were even more prominent, as compared to wild-type radixin-GFP (Figure 2D). We also generated a FERM-domain group mutant (Radixin-GFP (K253,254,262,263N)), in which surface membrane localization and subsequent intramolecular activation is prevented and which is restricted to the cytoplasm (Figure 2D and Supplementary Figure 5A and B). This finding is in line with a previously described corresponding ezrin mutant, deficient in PIP2 binding (Barret et al, 2000). In addition, respective myc-tagged radixin mutants were heterologously expressed in both HEK293 or COS cells and lysates were tested for interaction with the GABAAR α5 subunit intracellular loop in a GST-pulldown assay (Figure 2E and data not shown). Notably, wild-type radixin-myc, expressed in these cell lines, did not interact with α5 subunit loops. The antibody specific for the phosphorylated form of the C-terminal threonine residue did not detect phosphorylated ERM proteins in the cell lysate. In contrast, the active radixin T564D mutant displayed specific interaction, whereas the nonactivatable (K(253,254,262,263)N) mutant did not bind to α5 subunit loops (Figure 2E). These data show that, in contrast to neurons, HEK293 and COS cells are unable to sufficiently activate wild-type radixin and further indicate that an open and active conformation of radixin, as caused by T564D replacement, is a requirement for GABAAR α5 interaction. We then applied a combination of kinase activators and the phosphatase inhibitor Calyculin A to HEK293 and COS cells, expressing wild-type radixin-myc. Remarkably, under these conditions, specific interaction with the GABAAR α5 subunit intracellular loop was observed (Figure 2F and data not shown). However, in COS cells, the pulldown experiment revealed a myc-specific signal of lower molecular weight, as compared to the input band. As a control, detection with the phospho-ERM-specific antibody revealed that the band of lower molecular weight indeed represents the phosphorylated form of radixin-myc (Figure 2F), suggesting that slight differences in SDS–PAGE are due to posttranslational modifications of the phosphorylated protein in this cell type. These data show that radixin phosphorylation, leading to an open and active conformation of the molecule, is an essential requirement for GABAAR α5 interaction. In addition to the analysis in the yeast system, we tested in a GST-pulldown assay whether the activated full-length radixin T564D mutant would bind other GABAAR subunit loops (Figure 2G). However, in accordance with our previous observations, experiments with different GABAAR subunit TMIII-TMIV loops indicated that the open conformation, represented by Radixin T564D-myc, exclusively bound GABAAR α5 but not GABAAR α1, α3, β2 or γ2 loops. Radixin colocalizes with GABAAR α5 subunits in neurons We then investigated whether both proteins colocalize in neurons. Immunostaining of mature hippocampal cultures revealed a significant number of colocalized puncta in neuronal dendrites (Figure 3), indicating that endogenous radixin is localized to positions that harbour endogenous GABAAR α5 subunit clusters (Figure 3, A3). A quantitative evaluation upon immunostaining of endogenous proteins revealed that approximately 57% of total α5-subunit-immunoreactive puncta merged with radixin immunoreactivity. Vice versa 63% of total radixin immunoreactive puncta merged with GABAAR α5 immunoreativity (Figure 3B). Figure 3.Colocalization of radixin (green) and GABAAR α5 (red) in neuronal dendrites. (A) Coimmunostaining of cultured hippocampal neurons expressing endogenous (A3) or tagged versions of radixin and GABAAR α5 (A1 and A2). As shown in A1 and A2, prior to fixation, neurons were incubated with anti-HA antibody to ensure surface staining of the receptor. (B) Quantification of endogenous GABAAR α5 subunit colocalization with endogenous radixin and vice versa. Scale bars: 20 μm (A1); 3 μm (A2, A3). Download figure Download PowerPoint We also tested whether the observed clusters contain the active radixin conformation by application of coimmunostaining with the phospho-specific ERM antibody, indicative of active ERM proteins, and a cell membrane marker (Supplementary Figure 1D). The results indicate that indeed, in neurons radixin is activated and that radixin clusters actually locate at the plasma membrane. They are further in conclusion with the observation that active radixin (Radixin-GFP T564D) is detected at membrane locations (Figure 2, D2) and in the P2 plasma membrane fraction upon differential centrifugation (Supplementary Figure 5A). To control the colocalization of endogenous proteins, tagged constructs of both radixin and GABAAR α5 were generated (Supplementary Figure 1A, B and E), which enabled us to perform live cell staining of the receptor in order to visualize its surface localization. Upon neuronal expression of radixin-myc together with HA-GABAAR α5, similar staining patterns were observed (Figure 3A, A1 and A2), confirming that both interactors colocalize and further revealing that interaction mainly occurs at the cell surface. In contrast, radixin-GFP fluorescence did not colocalize with GluR2-containing AMPA receptors at excitatory synapses (Supplementary Figure 1C). Also, no significant colocalization was obtained upon neuronal coexpression of either ezrin-myc (8%) or moesin-myc (4%) together with HA-GABAAR α5 (Supplementary Figure 2). We therefore conclude that GABAAR α5 is a specific interactor of radixin within the ERM family. In accordance with the literature (Brunig et al, 2002), synaptic clusters of GABAAR α5 were rarely detected. Quantitative analysis revealed that 25% of GABAAR α5 clusters locate at synapses with 75% remaining at extrasynaptic sites, as judged by triple immunostaining of endogenous receptor subunits together with radixin and the synaptic marker SV2 (Figure 4A and B). Immunoreactive puncta of endogenous radixin were found to localize at synapses with similar frequencies. Here, 87% of radixin puncta were extrasynaptic (Figure 4A and B). For coclusters of GABAAR α5 and radixin, we calculated a synaptic localization of 7.5%, a finding which indicates that more than 90% of total coclusters are found at extrasynaptic positions (Figure 4A and B). Figure 4.Synaptic and extrasynaptic localization of radixin, GABAAR α5 and radixin/GABAAR α5 coclusters. (A) Triple immunostaining of endogenous radixin (green), endogenous GABAAR α5 (red) and the synaptic marker SV2 (blue) on mature cultured hippocampal neurons. Colored arrows depict examples of colocalization. (B) Quantification of extrasynaptic radixin, GABAAR α5 and radixin/ GABAAR α5 coclusters. (C) Neuronal expression of radixin-GFP and myc-tagged GABAAR α5 (red). Prior to fixation, neurons were incubated with anti-myc antibody to ensure surface detection of the receptor. Radixin/GABAAR α5 coclusters are occasionally found at VIAAT-positive inhibitory synapses (white, arrow). Scale bars: 15 μm (C1); 4 μm (A, C3). Download figure Download PowerPoint Since this experiment was performed upon fixation of neurons with subsequent immunostaining, our quantification also includes intracellular receptor molecules on their way to or from the surface membrane. We therefore performed live cell staining, labelling surface membrane receptors only. Neuronal expression of radixin-GFP (Supplementary Figure 1A and B) together with myc-GABAAR α5 also displayed synaptic coclusters, as detected by counterstaining against synaptophysin (data not shown) or the vesicular inhibitory amino-acid transporter VIAAT, selective for inhibitory synapses (Figure 4C). The ratio of synaptic/extrasynaptic coclusters was similar between live cell staining and analysis on fixed cells (data not shown), confirming our previous observation (Figure 3A) that radixin–receptor interactions mainly occur at the plasma membrane. Furthermore, these data show that radixin-GABAAR α5 coclusters occasionally occur at inhibitory synapses. Radixin and gephyrin represent independent systems Immunostaining detected very little colocalization of radixin with postsynaptic gephyrin, which is known to be critical for the clustering of GABAARs other than those containing the α5 subunit (Kneussel et al, 1999, 2001) (Figure 5A). Furthermore, triple expression of YFP-gephyrin together with radixin-myc and HA-GABAAR α5 revealed that GABAAR α5 does not simultaneously colocalize with both overexpressed gephyrin and overexpressed radixin. However, HA-GABAAR α5 subunit immunoreactivity either merged with radixin-myc or, to a lesser extent, with YFP-gephyrin fluorescence (Figure 5B, B2). Quantification revealed that about 60% of HA-GABAAR α5 colocalize with radixin-myc, whereas only 34% of HA-GABAAR α5 clusters were positive for YFP-gephyrin (Figure 5B), although co-immunoprecipitation of endogenous gephyrin and endogenous GABAAR α5 failed under our experimental conditions (Figure 5B). Triple detection against endogenous radixin, endogenous gephyrin and the inhibitory synapse marker VIAAT consistently showed that gephyrin mainly locates at synaptic sites, while radixin is rarely found in association with inhibitory presynaptic terminals (Figure 5C). In fact, only about 13% of radixin puncta colocalized with VIAAT, representing similar values as obtained for counterstaining with SV2 (Figure 4B). Furthermore, we employed biochemical approaches to investigate whether any association of radixin with gephyrin could be detected. Immunoprecipitation with a gephyrin-specific antibody displayed no specific association with radixin (Figure 5D). Vice versa, immunoprecipitation with a radixin-specific antibody did not lead to co-precipitation of gephyrin (Figure 5E). We therefore conclude that radixin and gephyrin represent independent systems. Figure 5.Radixin and gephyrin represent independent systems. (A) Immunostaining of endogenous radixin (red) and endogenous gephyrin (green) on a dendritic region. (B) Triple expression of YFP-gephyrin (green), radixin-myc (red) and HA-GABAAR α5 (blue) in cultured hippocampal neurons. Note that GABAAR α5 subunits colocalize either with radixin (arrowheads) or with gephyrin (arrows). Quantification of HA-GABAAR α5 colocalization with radixin-myc and YFP-gephyrin (upper right). Endogenous gephyrin does not co-immunoprecipitate with endogenous GABAAR α5 under our experimental conditions (lower right). (C) Triple immunostaining of endogenous gephyrin, radixin and VIAAT. Only about 14% of radixin clusters are found at VIAAT-positive inhibitory synapses. Arrows: gephyrin-positive synapses. (D) Immunoprecipitation of gephyrin from rat brain lysate. (E) Immunoprecipitation of radixin from rat brain lysate. Scale bars: 2 μm (A), 12 μm (B1) 1 μm (C). Download figure Download PowerPoint Radixin is an essential clustering factor for α5 subunit-containing GABAARs To investigate the role of radixin–GABAAR α5 subunit interactions, we performed loss-of-function experiments. We interfered with radixin expression as well as with radixin binding either to actin or the GABAAR α5 subunit. Based on a previously described antisense oligonucleotide protocol against radixin (Takeuchi et al, 1994; Paglini et al, 1998) known to significantly knockdown radixin expression, a mixture of two oligonucleotide probes was microinjected into mature cultured hippocampal neurons. To visualize injected neurons, either GFP vector or lucifer yellow were added. Notably, fluorescent neurons displayed a drastic decrease in α5 subunit cluster number with remaining diffuse background immunoreactivity, as compared to untreated control neurons in the same culture or to sense-treated neurons (Figure 6A). In fact, the average number of clusters per 30 μm of dendrite dropped from 18.6 in control cells to 1.5 in antisense-treated cells (P<0.001; Figure 6B). The same treatment led to a reduction in radixin immunoreactivity but did not affect clustering of GABAAR α1 subunits (Supplementary Figure 3A and B). Figure 6.Knockdown or depletion of radixin expression leads to loss of GABAAR α5 clustering. (A) Top: An antisense oligonucleotide injected (green) and a noninjected cell are compared. Synapses are represented by SV2 (blue). The immunoreactivity for GABAAR α5 is diffuse in antisense-treated cells (A2), as compared to normal receptor clusters in untreated (A1) or sense oligonucleotide-treated cells (A3). Arrowheads and arrows depict extrasynaptic and synaptic clusters, respectively. (B) Quantification of GABAAR α5 cluster density in sense- and antisense-injected cells. (C) Whole-cell extracts from cultured hippocampal neurons confirm that radixin expression is efficiently downregulated upon antisense treatment, while total GABAAR α5 expression is unaltered. (D) Western Blot analysis of biotinylated surface GABAAR α5 in sense- or antisense-transfected neurons. (E) Western Blot analysis of P2 plasma membrane preparations from wild-type or radixin knockout mice. (F) Immunohistochemical analysis of wild-type and radixin knockout hippocampal slices. Left: TOTO staining to visualize overall tissue structure. Middle and right: GABAAR α5 staining reveals loss of GABAAR α5 receptor clusters with remaining diffuse signals upon radixin deficiency. (G) Quantification of GABAAR α5 subunit clusters between radixin +/+ and −/− genotypes (P<0.05). Scale bars: 10 μm in (A), 200 μm in (F) (middle) and 10 μm in (F) (right). Download figure Download PowerPoint A loss of receptor clusters raises the question whether this effect is due to a defect in trafficking or anchoring. Since unclustered, diffuse receptors are barely, if at all detectable using light microscopy (Feng et al, 1998), we also treated neuron cultures with antisense oligonucleotides and performed biochemical analysis on total and surface membrane proteins to check whether unclustered receptors remain at the cell surface. Western blots of the respective samples showed that the total amount of GABAA receptor α5 was unchanged in cultures in which radixin was downregulated (Figure 6C). This observation could be confirmed applying biotinylation of total surface protein, followed by streptavidin precipitation and GABAAR α5-specific Western blot detection (Figure 6D). Consistent with results obtained upon antisense oligonucleotide knockdown of radixin expression, hippocampal slices derived from radixin knockout mice (Kikuchi et al, 2002) genetically confirmed that radixin is an essential clustering factor for α5 subunit containing GABAARs. Here, receptor clusters were reduced to background levels in radixin-deficient tissue, as compared to wild-type tissue (P<0.05; Figure 6F and G). Specificity of the GABAAR α5-specific signal was controlled in a competition experiment using the GST-GABAAR α5-loop as blocking peptide (Supplementary Figure 4A). Staining of nuclei with the fluorescent dye TOTO confirmed that the anatomy of the hippocampal formation was unaltered between the genotypes (Figure 6F). Furthermore, control stainings confirmed the absence of radixin protein in slices derived from −/− mice, whereas immunostaining against the unrelated GABAAR α3 subunit was unaltered between the genotypes (Supplementary Figure 4B and C) In addition, P2 plasma membrane fractions of brain lysates derived from radixin knockout mice did not display major differences in the amount of surface GABAAR α5 (Figure 6E), indicating that unclustered receptors remain at the neuronal surface membrane upon the loss of radixin. Radixin and its ERM-family homologs ezrin and moesin display a number of structural and functional similarities (Tsukita and Yonemura, 1997; Bretscher et al, 2002). Based on a described ezrin mutant (Allenspach et al, 2001), we also generated a GFP-fused deletion construct (Radixin-(1–468)-GFP), in which the C-terminal F-actin-binding domain of radixin is replaced by GFP (Figure 7A). Over-expression of Radixin-(1–468)-GFP induced severe loss of GABAAR α5 subunit clustering, while control cells within the same field of vision (Figure 7B) and cells expressing the noninterfering cytoplasmic mutant Radixin-GFP (K253,254,262,263N) (Supplementary Figure 5B) displayed normal GABAAR α5 subunit clusters. The average cluster number per 30 μm of dendrite was reduced to 6.4 in cells expressing the truncated radixin construct, as compared to 26.4 in control cells (P<0.001; Figure 7C). Consistently, this C-terminal deletion mutant Radixin-(1–468)-GFP does not interact with F-actin in a cosedimentation assay, as judged from its absence in the pellet fraction. In contrast, the active full-length form represented by Radixin-GFP T564D cosediments with F-actin and is therefore enriched in the pellet (Figure 7D). Also, wild-type radixin-GFP, expressed in neurons, highly colocalizes with rhodamine phalloidine-stained actin (Supplementary Figure 1F). However, since the N-terminal FERM domain is intact, Radixin-(1–468)-GFP is still able to associate with the plasma membrane as suggested by its prominent abundance in the P2 pellet upon differential centrifugation (Supplementary Figure 5A). Consequently, this mutant is likely to compete with endogenous radixin for GABAAR α5 interaction" @default.
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• W2050546598 title "Activated radixin is essential for GABAA receptor α5 subunit anchoring at the actin cytoskeleton" @default.
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