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Molecular Basis of the γ-Aminobutyric Acid A Receptor α3 Subunit Interaction with the Clustering Protein Gephyrin*

  • Verena Tretter
    Correspondence
    To whom correspondence may be addressed. Tel.: 43-1-40160-34350
    Affiliations
    Department of Biochemistry and Molecular Biology, Center for Brain Research, Medical University Vienna, Spitalgasse 4, 1090 Vienna, Austria
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  • Bernd Kerschner
    Affiliations
    Department of Biochemistry and Molecular Biology, Center for Brain Research, Medical University Vienna, Spitalgasse 4, 1090 Vienna, Austria
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  • Ivan Milenkovic
    Affiliations
    Department of Biochemistry and Molecular Biology, Center for Brain Research, Medical University Vienna, Spitalgasse 4, 1090 Vienna, Austria
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  • Sarah L. Ramsden
    Affiliations
    Department of Pharmacology, School of Pharmacy, University of London, 29-39 Brunswick Square, WC1N 1AX London, United Kingdom
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  • Joachim Ramerstorfer
    Affiliations
    Department of Biochemistry and Molecular Biology, Center for Brain Research, Medical University Vienna, Spitalgasse 4, 1090 Vienna, Austria
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  • Leila Saiepour
    Footnotes
    Affiliations
    Department of Pharmacology, School of Pharmacy, University of London, 29-39 Brunswick Square, WC1N 1AX London, United Kingdom
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  • Hans-Michael Maric
    Affiliations
    Rudolf Virchow Center, University of Würzburg, Josef-Schneider-Strasse 2, 97080 Würzburg, Germany
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  • Stephen J. Moss
    Affiliations
    Tufts University School of Medicine, Boston, Massachusetts 02111

    Department of Neuroscience, Physiology, and Pharmacology, University College London, London WC1E 6BT, United Kingdom
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  • Hermann Schindelin
    Affiliations
    Rudolf Virchow Center, University of Würzburg, Josef-Schneider-Strasse 2, 97080 Würzburg, Germany
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  • Robert J. Harvey
    Affiliations
    Department of Pharmacology, School of Pharmacy, University of London, 29-39 Brunswick Square, WC1N 1AX London, United Kingdom
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  • Werner Sieghart
    Affiliations
    Department of Biochemistry and Molecular Biology, Center for Brain Research, Medical University Vienna, Spitalgasse 4, 1090 Vienna, Austria
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  • Kirsten Harvey
    Correspondence
    To whom correspondence may be addressed. Tel.: 44-207-753-5888
    Affiliations
    Department of Pharmacology, School of Pharmacy, University of London, 29-39 Brunswick Square, WC1N 1AX London, United Kingdom
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  • Author Footnotes
    * This work was supported by a Medical Research Council New Investigator Award G0501258 (to K. H.), the EC-FP7 integrated project “Neurocypres,” grant agreement (to W. S.), HEALTH-F4-2008-202088 grant, the Institute of Neurological Disorders and Stroke grants (NS047478, NS48045, NS051195, NS054900, NS056359, and NS065725, to S. J. M.), and the Deutsche Forschungsgemeinschaft Rudolf Virchow Center for Experimental Biomedicine Grants FZ 82 and SFB487 C7 (to H. S.).
    2 Present address: Medicines and Healthcare Products Regulatory Agency, Market Towers, 1 Nine Elms Lane, London, U. K.
Open AccessPublished:August 31, 2011DOI:https://doi.org/10.1074/jbc.M111.291336
      The multifunctional scaffolding protein gephyrin is a key player in the formation of the postsynaptic scaffold at inhibitory synapses, clustering both inhibitory glycine receptors (GlyRs) and selected GABAA receptor (GABAAR) subtypes. We report a direct interaction between the GABAAR α3 subunit and gephyrin, mapping reciprocal binding sites using mutagenesis, overlay, and yeast two-hybrid assays. This analysis reveals that critical determinants of this interaction are located in the motif FNIVGTTYPI in the GABAAR α3 M3–M4 domain and the motif SMDKAFITVL at the N terminus of the gephyrin E domain. GABAAR α3 gephyrin binding-site mutants were unable to co-localize with endogenous gephyrin in transfected hippocampal neurons, despite being able to traffic to the cell membrane and form functional benzodiazepine-responsive GABAARs in recombinant systems. Interestingly, motifs responsible for interactions with GABAAR α2, GABAAR α3, and collybistin on gephyrin overlap. Curiously, two key residues (Asp-327 and Phe-330) in the GABAAR α2 and α3 binding sites on gephyrin also contribute to GlyR β subunit-E domain interactions. However, isothermal titration calorimetry reveals a 27-fold difference in the interaction strength between GABAAR α3 and GlyR β subunits with gephyrin with dissociation constants of 5.3 μm and 0.2 μm, respectively. Taken together, these observations suggest that clustering of GABAAR α2, α3, and GlyRs by gephyrin is mediated by distinct mechanisms at mixed glycinergic/GABAergic synapses.

      Introduction

      Inhibitory transmission in the central nervous system is mediated by GABAA and glycine receptors (GlyRs)
      The abbreviations used are: GlyR
      glycine receptor
      GABAAR
      GABAA receptor
      ITC
      isothermal titration calorimetry
      XR
      Xenopus Ringer solution.
      consisting of pentameric combinations of subunits (α1–6, β1–3, γ1–3, δ, ϵ, θ, and π for GABAARs and α1–4 and β for GlyRs), each comprising an extracellular domain, four membrane-spanning helices (M1–M4) and a large intracellular loop between M3 and M4. Cell type-specific gene expression patterns, subunit stoichiometry, and interplay between presynaptic and postsynaptic specializations are thought to underlie the spatial and temporal localization of these receptors. In particular, a postsynaptic matrix of receptor-associated proteins is essential for the dynamic localization of both GABAARs and GlyRs and also recruits components of specific signaling cascades to synapses (
      • Fritschy J.M.
      • Harvey R.J.
      • Schwarz G.
      ). The multifunctional protein gephyrin (
      • Prior P.
      • Schmitt B.
      • Grenningloh G.
      • Pribilla I.
      • Multhaup G.
      • Beyreuther K.
      • Maulet Y.
      • Werner P.
      • Langosch D.
      • Kirsch J.
      • Betz H.
      ) is a key player in the clustering of both GlyRs and GABAARs. For example, studies using gephyrin knock-out mice or mRNA knockdown (
      • Feng G.
      • Tintrup H.
      • Kirsch J.
      • Nichol M.C.
      • Kuhse J.
      • Betz H.
      • Sanes J.R.
      ,
      • Kneussel M.
      • Brandstätter J.H.
      • Laube B.
      • Stahl S.
      • Müller U.
      • Betz H.
      ,
      • Fischer F.
      • Kneussel M.
      • Tintrup H.
      • Haverkamp S.
      • Rauen T.
      • Betz H.
      • Wässle H.
      ,
      • Kneussel M.
      • Brandstätter J.H.
      • Gasnier B.
      • Feng G.
      • Sanes J.R.
      • Betz H.
      ,
      • Lévi S.
      • Logan S.M.
      • Tovar K.R.
      • Craig A.M.
      ,
      • Yu W.
      • Jiang M.
      • Miralles C.P.
      • Li R.W.
      • Chen G.
      • de Blas A.L.
      ) have shown a loss of postsynaptic clustering of GlyRs as well as GABAARs containing α2 and γ2 subunits. However, GABAAR α1 and α5 subunit clustering is unaltered in gephyrin knock-out mice (
      • Kneussel M.
      • Brandstätter J.H.
      • Laube B.
      • Stahl S.
      • Müller U.
      • Betz H.
      ,
      • Fischer F.
      • Kneussel M.
      • Tintrup H.
      • Haverkamp S.
      • Rauen T.
      • Betz H.
      • Wässle H.
      ,
      • Kneussel M.
      • Brandstätter J.H.
      • Gasnier B.
      • Feng G.
      • Sanes J.R.
      • Betz H.
      ,
      • Lévi S.
      • Logan S.M.
      • Tovar K.R.
      • Craig A.M.
      ), demonstrating that gephyrin-independent clustering mechanisms also exist in vivo. GABAARs containing the α4, α5, and α6 subunits are located preferentially at extrasynaptic sites (
      • Belelli D.
      • Harrison N.L.
      • Maguire J.
      • Macdonald R.L.
      • Walker M.C.
      • Cope D.W.
      ) and are likely to be localized by other clustering factors, such as the actin-binding protein radixin (
      • Loebrich S.
      • Bähring R.
      • Katsuno T.
      • Tsukita S.
      • Kneussel M.
      ). Hence, although the majority of GlyRs are likely to be clustered by gephyrin, only certain GABAAR subtypes are subject to gephyrin-dependent clustering. Curiously, the subcellular localization of gephyrin is in turn dependent on the presence of certain GABAAR subtypes. For example, targeted deletion of the GABAAR α1, α3, and γ2 subunit genes results in a loss of synaptic gephyrin clusters (
      • Schweizer C.
      • Balsiger S.
      • Bluethmann H.
      • Mansuy I.M.
      • Fritschy J.M.
      • Mohler H.
      • Lüscher B.
      ,
      • Alldred M.J.
      • Mulder-Rosi J.
      • Lingenfelter S.E.
      • Chen G.
      • Lüscher B.
      ,
      • Li R.W.
      • Yu W.
      • Christie S.
      • Miralles C.P.
      • Bai J.
      • Loturco J.J.
      • De Blas A.L.
      ,
      • Kralic J.E.
      • Sidler C.
      • Parpan F.
      • Homanics G.E.
      • Morrow A.L.
      • Fritschy J.M.
      ,
      • Studer R.
      • von Boehmer L.
      • Haenggi T.
      • Schweizer C.
      • Benke D.
      • Rudolph U.
      • Fritschy J.M.
      ,
      • Winsky-Sommerer R.
      • Knapman A.
      • Fedele D.E.
      • Schofield C.M.
      • Vyazovskiy V.V.
      • Rudolph U.
      • Huguenard J.R.
      • Fritschy J.M.
      • Tobler I.
      ), resulting in nonsynaptic dendritic gephyrin aggregates.
      The interaction of the GlyR β subunit with the gephyrin E domain has been characterized in detail (
      • Meyer G.
      • Kirsch J.
      • Betz H.
      • Langosch D.
      ,
      • Harvey K.
      • Duguid I.C.
      • Alldred M.J.
      • Beatty S.E.
      • Ward H.
      • Keep N.H.
      • Lingenfelter S.E.
      • Pearce B.R.
      • Lundgren J.
      • Owen M.J.
      • Smart T.G.
      • Lüscher B.
      • Rees M.I.
      • Harvey R.J.
      ,
      • Sola M.
      • Bavro V.N.
      • Timmins J.
      • Franz T.
      • Ricard-Blum S.
      • Schoehn G.
      • Ruigrok R.W.
      • Paarmann I.
      • Saiyed T.
      • O'Sullivan G.A.
      • Schmitt B.
      • Betz H.
      • Weissenhorn W.
      ,
      • Schrader N.
      • Kim E.Y.
      • Winking J.
      • Paulukat J.
      • Schindelin H.
      • Schwarz G.
      ,
      • Kim E.Y.
      • Schrader N.
      • Smolinsky B.
      • Bedet C.
      • Vannier C.
      • Schwarz G.
      • Schindelin H.
      ). By contrast, a direct interaction of gephyrin with GABAAR subunits has proven elusive, perhaps because of the number of individual GABAAR subunits, splice variants, accessory proteins, and post-translational modifications that could influence these interactions. However, recent studies (
      • Tretter V.
      • Jacob T.C.
      • Mukherjee J.
      • Fritschy J.M.
      • Pangalos M.N.
      • Moss S.J.
      ,
      • Saiepour L.
      • Fuchs C.
      • Patrizi A.
      • Sassoè-Pognetto M.
      • Harvey R.J.
      • Harvey K.
      ) have demonstrated that the GABAAR α2 subunit interacts directly with both gephyrin and the RhoGEF collybistin (
      • Harvey K.
      • Duguid I.C.
      • Alldred M.J.
      • Beatty S.E.
      • Ward H.
      • Keep N.H.
      • Lingenfelter S.E.
      • Pearce B.R.
      • Lundgren J.
      • Owen M.J.
      • Smart T.G.
      • Lüscher B.
      • Rees M.I.
      • Harvey R.J.
      ,
      • Kins S.
      • Betz H.
      • Kirsch J.
      ). Here, we report a detailed characterization of the interaction between the GABAAR α3 subunit and gephyrin in recombinant systems and neuronal cultures, revealing overlapping binding determinants on gephyrin for GABAAR α2, GABAAR α3, and collybistin that are distinct from the E domain GlyR β-subunit binding site.

      DISCUSSION

      It is becoming increasingly apparent that GABAAR α subunits not only influence GABAAR physiology, pharmacology, and biological function, but also mediate the synaptic versus extrasynaptic localization of these receptor subtypes via distinct protein-protein interactions. For example, the GABAAR α2 subunit co-localizes with gephyrin in vivo and interacts with both gephyrin and the RhoGEF collybistin via overlapping binding sites (
      • Tretter V.
      • Jacob T.C.
      • Mukherjee J.
      • Fritschy J.M.
      • Pangalos M.N.
      • Moss S.J.
      ,
      • Saiepour L.
      • Fuchs C.
      • Patrizi A.
      • Sassoè-Pognetto M.
      • Harvey R.J.
      • Harvey K.
      ). For this reason, we considered that other GABAAR α subunits might also be clustered at synapses by these molecules. The α3 subunit was selected for analysis because (i) GABAARs containing this subunit co-localize with gephyrin, e.g. in cerebellar cortex (
      • Sassoè-Pognetto M.
      • Panzanelli P.
      • Sieghart W.
      • Fritschy J.M.
      ), the thalamic reticular nucleus (
      • Winsky-Sommerer R.
      • Knapman A.
      • Fedele D.E.
      • Schofield C.M.
      • Vyazovskiy V.V.
      • Rudolph U.
      • Huguenard J.R.
      • Fritschy J.M.
      • Tobler I.
      ), or at perisomatic synapses in the globus pallidus (
      • Gross A.
      • Sims R.E.
      • Swinny J.D.
      • Sieghart W.
      • Bolam J.P.
      • Stanford I.M.
      ) and (ii) gephyrin is mislocalized in GABAAR α3 subunit knock-out mice (
      • Studer R.
      • von Boehmer L.
      • Haenggi T.
      • Schweizer C.
      • Benke D.
      • Rudolph U.
      • Fritschy J.M.
      ,
      • Winsky-Sommerer R.
      • Knapman A.
      • Fedele D.E.
      • Schofield C.M.
      • Vyazovskiy V.V.
      • Rudolph U.
      • Huguenard J.R.
      • Fritschy J.M.
      • Tobler I.
      ).
      Using a combination of deletion mutagenesis, overlay, and yeast two-hybrid assays we have determined that GABAAR α3 specifically interacts with gephyrin via a critical motif (FNIVGTTYPI) that overlaps with sequences that bind gephyrin and collybistin in GABAAR α2. Curiously, very few amino acids are conserved between the equivalent regions of the α2 and α3 subunits, suggesting that either the nature of the amino acids in these motifs is crucial or that conserved amino acids within (Tyr-375) or directly flanking these minimal motifs (e.g. Asn-378) are crucial determinants of gephyrin binding. Certainly, deletion of the minimal gephyrin binding motif prevented synaptic clustering and co-localization of recombinant GABAAR α3 with endogenous gephyrin in cultured neurons.
      Using the yeast-two hybrid system, we also mapped crucial determinants of GABAAR α3 binding to gephyrin to the start of the E domain. These residues overlap with previously characterized determinants of GABAAR α2 binding on gephyrin and show partial overlap with key determinants (Asp-327 and Phe-330) of GlyR β binding to gephyrin (
      • Kim E.Y.
      • Schrader N.
      • Smolinsky B.
      • Bedet C.
      • Vannier C.
      • Schwarz G.
      • Schindelin H.
      ) (Fig. 7) This suggests that binding of GABAARs containing α2, α3, and GlyRs to gephyrin could be mutually exclusive. However, given that mutation of Asp-327 and Phe-330 in mutants A5 and A6 did not appear to disrupt GlyR β-E domain interactions in the YTH system, key differences may exist between GABAAR and GlyR binding to gephyrin. Consistent with this hypothesis, ITC revealed that the GABAAR α3 bound to the gephyrin E-domain in a 1:1 ratio and displayed a lower affinity (KD = 5.3 ± 1.5 μm) than previously observed for GlyR β (two binding sites with KD values of 0.2 μm and 11 μm) (
      • Schrader N.
      • Kim E.Y.
      • Winking J.
      • Paulukat J.
      • Schindelin H.
      • Schwarz G.
      ,
      • Kim E.Y.
      • Schrader N.
      • Smolinsky B.
      • Bedet C.
      • Vannier C.
      • Schwarz G.
      • Schindelin H.
      ). Collectively, these observations may explain why GABAAR-gephyrin interactions have been difficult to characterize by immunoprecipitation and are prone to the effects of detergents (
      • Tretter V.
      • Jacob T.C.
      • Mukherjee J.
      • Fritschy J.M.
      • Pangalos M.N.
      • Moss S.J.
      ).
      Figure thumbnail gr7
      FIGURE 7Structural representations of the GlyR β and GABAAR α3 loop binding sites on the gephyrin E domain. Color-coded surface of the gephyrin E domain dimer (
      • Kim E.Y.
      • Schrader N.
      • Smolinsky B.
      • Bedet C.
      • Vannier C.
      • Schwarz G.
      • Schindelin H.
      ) shows one monomer colored according to the subdomain architecture and the other in gray. Residues involved in GlyR β subunit binding (
      • Kim E.Y.
      • Schrader N.
      • Smolinsky B.
      • Bedet C.
      • Vannier C.
      • Schwarz G.
      • Schindelin H.
      ) are shown in pink, residues implicated in GABAAR α3 subunit binding by alanine scanning mutagenesis are shown in purple. Amino acids Phe-330 and Asp-327 of gephyrin, which are involved in the binding of both receptor types, are marked in orange.
      GABAARs containing the α3 subunit co-localized with gephyrin in both dendritic and perisomatic locations in different brain regions (
      • Winsky-Sommerer R.
      • Knapman A.
      • Fedele D.E.
      • Schofield C.M.
      • Vyazovskiy V.V.
      • Rudolph U.
      • Huguenard J.R.
      • Fritschy J.M.
      • Tobler I.
      ,
      • Sassoè-Pognetto M.
      • Panzanelli P.
      • Sieghart W.
      • Fritschy J.M.
      ,
      • Gross A.
      • Sims R.E.
      • Swinny J.D.
      • Sieghart W.
      • Bolam J.P.
      • Stanford I.M.
      ). This may reflect different roles for the α3 subunit in mediating dendritic inhibition, affecting the efficacy and plasticity of excitatory synaptic inputs of principal cells, versus perisomatic inhibition, controlling output by synchronizing action potential firing of larger groups of principal cells. Certainly, although GABAA receptors containing the α3 subunit are thought to represent only 10–15% of all GABAA receptors, they are the major GABAA receptor subtype expressed in brain stem monoaminergic nuclei (
      • Fiorelli R.
      • Rudolph U.
      • Straub C.J.
      • Feldon J.
      • Yee B.K.
      ,
      • Corteen N.L.
      • Cole T.M.
      • Sarna A.
      • Sieghart W.
      • Swinny J.D.
      ). Consistent with these findings, GABAARs containing α3 have been linked to sensorimotor gating and affective and cognitive functions phenotypes (
      • Fiorelli R.
      • Rudolph U.
      • Straub C.J.
      • Feldon J.
      • Yee B.K.
      ,
      • Yee B.K.
      • Keist R.
      • von Boehmer L.
      • Studer R.
      • Benke D.
      • Hagenbuch N.
      • Dong Y.
      • Malenka R.C.
      • Fritschy J.M.
      • Bluethmann H.
      • Feldon J.
      • Möhler H.
      • Rudolph U.
      ). A molecular understanding of the basis of synaptic localization of GABAAR α3-containing receptors by gephyrin adds to our knowledge of this interesting receptor subtype.

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