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Specificity of Collybistin-Phosphoinositide Interactions

IMPACT OF THE INDIVIDUAL PROTEIN DOMAINS*
  • Michaela Ludolphs
    Affiliations
    From the Institute of Organic and Biomolecular Chemistry, University of Göttingen, Tammannstrasse 2, 37077 Göttingen, Germany,
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  • Daniela Schneeberger
    Affiliations
    Rudolf Virchow Center for Experimental Biomedicine, University of Würzburg, Josef-Schneider-Strasse 2, 97080 Würzburg, Germany,
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  • Tolga Soykan
    Affiliations
    Department of Molecular Neurobiology, Max Planck Institute for Experimental Medicine, Hermann-Rein-Strasse 3, 37075 Göttingen, Germany, and
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  • Jonas Schäfer
    Affiliations
    From the Institute of Organic and Biomolecular Chemistry, University of Göttingen, Tammannstrasse 2, 37077 Göttingen, Germany,
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  • Theofilos Papadopoulos
    Affiliations
    Universitätsmedizin Göttingen, Department of Molecular Biology, Humboldtallee 23, 37073 Göttingen, Germany
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  • Nils Brose
    Affiliations
    Department of Molecular Neurobiology, Max Planck Institute for Experimental Medicine, Hermann-Rein-Strasse 3, 37075 Göttingen, Germany, and
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  • Hermann Schindelin
    Affiliations
    Rudolf Virchow Center for Experimental Biomedicine, University of Würzburg, Josef-Schneider-Strasse 2, 97080 Würzburg, Germany,
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  • Claudia Steinem
    Correspondence
    To whom correspondence should be addressed. Tel.: 49551-3933294; Fax: 49551-3933228; E-mail:
    Affiliations
    From the Institute of Organic and Biomolecular Chemistry, University of Göttingen, Tammannstrasse 2, 37077 Göttingen, Germany,
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  • Author Footnotes
    * This work was supported by the Max Planck Society (to N. B.), the German Research Foundation (a Center of Nanoscale Microscopy and Molecular Physiology of the Brain grant (to N. B.) and Grants PA 2087/1-1 (to T. P.) and SCHI 425/8-1 (to H. S.)) as well as funding through the Rudolf Virchow Center for Experimental Biomedicine (to H. S.), and European Commission Innovative Medicines Initiative FP7-115300 (to N. B.). The authors declare that they have no conflicts of interest with the contents of this article.
Open AccessPublished:November 06, 2015DOI:https://doi.org/10.1074/jbc.M115.673400
      The regulatory protein collybistin (CB) recruits the receptor-scaffolding protein gephyrin to mammalian inhibitory glycinergic and GABAergic postsynaptic membranes in nerve cells. CB is tethered to the membrane via phosphoinositides. We developed an in vitro assay based on solid-supported 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine membranes doped with different phosphoinositides on silicon/silicon dioxide substrates to quantify the binding of various CB2 constructs using reflectometric interference spectroscopy. Based on adsorption isotherms, we obtained dissociation constants and binding capacities of the membranes. Our results show that full-length CB2 harboring the N-terminal Src homology 3 (SH3) domain (CB2SH3+) adopts a closed and autoinhibited conformation that largely prevents membrane binding. This autoinhibition is relieved upon introduction of the W24A/E262A mutation, which conformationally “opens” CB2SH3+ and allows the pleckstrin homology domain to properly bind lipids depending on the phosphoinositide species with a preference for phosphatidylinositol 3-monophosphate and phosphatidylinositol 4-monophosphate. This type of membrane tethering under the control of the release of the SH3 domain of CB is essential for regulating gephyrin clustering.

      Introduction

      The function of neuronal synapses and the dynamic regulation of their efficacy depend on the assembly of the postsynaptic neurotransmitter receptor apparatus. The main scaffolding protein of inhibitory glycinergic and GABAergic postsynapses in mammals is gephyrin (
      • Kneussel M.
      • Betz H.
      Clustering of inhibitory neurotransmitter receptors at developing postsynaptic sites: the membrane activation mode.
      ,
      • Kneussel M.
      • Betz H.
      Receptors, gephyrin and gephyrin-associated proteins: novel insights into the assembly of inhibitory postsynaptic membrane specializations.
      ), whose recruitment to the postsynaptic membrane is controlled by the adaptor protein collybistin (CB)
      The abbreviations used are: CB, collybistin; DH, Dbl homology; OT, optical thickness; PH, pleckstrin homology; PIP, phosphatidylinositol phosphate; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; POPG, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-racemic glycerol); POPS, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine; RIfS, reflectometric interference spectroscopy; SH3, Src homology 3; PI(3,4,5)P3, phosphatidylinositol 3,4,5-trisphosphate; PI(3)P, phosphatidylinositol 3-monophosphate; PI(3,4)P2, phosphatidylinositol 3,4-bisphosphate; PI(4,5)P2, phosphatidylinositol 4,5-bisphosphate; PI(4)P, phosphatidylinositol 4-monophosphate; PI(3,5)P2, phosphatidylinositol 3,5-bisphosphate; SUV, small unilamellar vesicle.
      (
      • Papadopoulos T.
      • Soykan T.
      The role of collybistin in gephyrin clustering at inhibitory synapses: facts and open questions.
      ). Loss of CB results in a strong reduction of gephyrin and GABAA receptor clusters in several regions of the forebrain, which demonstrates the essential role of CB in the assembly and maintenance of GABAergic postsynaptic structures (
      • Papadopoulos T.
      • Korte M.
      • Eulenburg V.
      • Kubota H.
      • Retiounskaia M.
      • Harvey R.J.
      • Harvey K.
      • O'Sullivan G.A.
      • Laube B.
      • Hülsmann S.
      • Geiger J.R.
      • Betz H.
      Impaired GABAergic transmission and altered hippocampal synaptic plasticity in collybistin-deficient mice.
      ).
      CB belongs to the Dbl family of guanine nucleotide exchange factors. In mouse, four differently spliced CB mRNAs are present (CB1SH3+, CB2SH3−, CB2SH3+, and CB3SH3+). All four mRNAs encode a Dbl homology (DH) and a pleckstrin homology (PH) domain. The three major variants (CB1SH3+, CB2SH3+, and CB3SH3+) encode CBs with an additional N-terminal Src homology 3 (SH3) domain but differ in their C termini. A fourth variant (CB2SH3−) encodes a CB2 isoform lacking the SH3 domain (Fig. 1) but is very rare (
      • 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.
      The GDP-GTP exchange factor collybistin: an essential determinant of neuronal gephyrin clustering.
      ) as its protein product is not detectable in mouse brain (
      • Soykan T.
      • Schneeberger D.
      • Tria G.
      • Buechner C.
      • Bader N.
      • Svergun D.
      • Tessmer I.
      • Poulopoulos A.
      • Papadopoulos T.
      • Varoqueaux F.
      • Schindelin H.
      • Brose N.
      A conformational switch in collybistin determines the differentiation of inhibitory postsynapses.
      ). Importantly, the PH domain of the different CBs is required for proper function as indicated by the fact that its deletion abolishes the plasma membrane targeting of gephyrin-CB complexes when cotransfected in HEK293 cells and causes a loss of dendritic gephyrin clusters in dissociated rat cortical neurons (
      • 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.
      The GDP-GTP exchange factor collybistin: an essential determinant of neuronal gephyrin clustering.
      ).
      Figure thumbnail gr1
      FIGURE 1.Domain architectures of the CB variants. Proteins that were used in the present study are marked with an asterisk (*).
      In vitro binding studies utilizing a variety of inositol headgroups, soluble phosphoinositide analogs, and liposomes containing phosphoinositides showed that PH domains bind phosphoinositides with a broad range of selectivity and affinity (
      • Kavran J.M.
      • Klein D.E.
      • Lee A.
      • Falasca M.
      • Isakoff S.J.
      • Skolnik E.Y.
      • Lemmon M.A.
      Specificity and promiscuity in phosphoinositide binding by pleckstrin homology domains.
      • Ferguson K.M.
      • Kavran J.M.
      • Sankaran V.G.
      • Fournier E.
      • Isakoff S.J.
      • Skolnik E.Y.
      • Lemmon M.A.
      Structural basis for discrimination of 3-phosphoinositides by pleckstrin homology domains.
      ,
      • Lietzke S.E.
      • Bose S.
      • Cronin T.
      • Klarlund J.
      • Chawla A.
      • Czech M.P.
      • Lambright D.G.
      Structural basis of 3-phosphoinositide recognition by pleckstrin homology domains.
      • Lemmon M.A.
      Pleckstrin homology (PH) domains and phosphoinositides.
      ). An early membrane activation model suggested that the PH domain of CB binds to phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) (
      • Kneussel M.
      • Betz H.
      Clustering of inhibitory neurotransmitter receptors at developing postsynaptic sites: the membrane activation mode.
      ). In contrast, experiments with immobilized phosphoinositides and purified glutathione S-transferase (GST)-tagged CB variants in overlay assays indicated that the PH domain of CB specifically binds phosphatidylinositol 3-monophosphate (PI(3)P) (
      • Dowler S.
      • Currie R.A.
      • Campbell D.G.
      • Deak M.
      • Kular G.
      • Downes C.P.
      • Alessi D.R.
      Identification of pleckstrin-homology-domain-containing proteins with novel phosphoinositide-binding specificity.
      ,
      • Kalscheuer V.M.
      • Musante L.
      • Fang C.
      • Hoffmann K.
      • Fuchs C.
      • Carta E.
      • Deas E.
      • Venkateswarlu K.
      • Menzel C.
      • Ullmann R.
      • Tommerup N.
      • Dalprà L.
      • Tzschach A.
      • Selicorni A.
      • Lüscher B.
      • Ropers H.H.
      • Harvey K.
      • Harvey R.J.
      A balanced chromosomal translocation disrupting ARHGEF9 is associated with epilepsy, anxiety, aggression, and mental retardation.
      ), and subsequent studies verified the binding of CB to PI(3)P (
      • Soykan T.
      • Schneeberger D.
      • Tria G.
      • Buechner C.
      • Bader N.
      • Svergun D.
      • Tessmer I.
      • Poulopoulos A.
      • Papadopoulos T.
      • Varoqueaux F.
      • Schindelin H.
      • Brose N.
      A conformational switch in collybistin determines the differentiation of inhibitory postsynapses.
      ,
      • Papadopoulos T.
      • Schemm R.
      • Grubmüller H.
      • Brose N.
      Lipid binding defects and perturbed synaptogenic activity of a collybistin R290H mutant that causes epilepsy and intellectual disability.
      ). However, most of the relevant experiments were conducted with lipids spotted on blotting membranes, which have been shown to be less reliable than other techniques (
      • Busse R.A.
      • Scacioc A.
      • Hernandez J.M.
      • Krick R.
      • Stephan M.
      • Janshoff A.
      • Thumm M.
      • Kühnel K.
      Qualitative and quantitative characterization of protein-phosphoinositide interactions with liposome-based methods.
      ). Hence, the question arises as to whether the phosphoinositide specificity of CB observed with overlay assays properly reflects the lipid binding specificity of CB in intact phospholipid bilayers. That this is a critical issue is also illustrated by the fact that PI(3)P, the best characterized CB ligand so far, is mainly concentrated in early endosomes (Fig. 2) (
      • Di Paolo G.
      • De Camilli P.
      Phosphoinositides in cell regulation and membrane dynamics.
      ) and is present at the plasma membrane where CB is ultimately required for GABAergic synapse formation only under specific stimulatory conditions (
      • Lodhi I.J.
      • Bridges D.
      • Chiang S.H.
      • Zhang Y.
      • Cheng A.
      • Geletka L.M.
      • Weisman L.S.
      • Saltiel A.R.
      Insulin stimulates phosphatidylinositol 3-phosphate production via the activation of Rab5.
      ). Instead, PI(3,4)P2, PI(4,5)P2, and PI(3,4,5)P3 are primarily localized to the plasma membrane; PI(4)P is enriched in the Golgi complex and present at the plasma membrane; and PI(3,5)P2 is found in compartments of the late endosomal pathway (Fig. 2) (
      • Saarikangas J.
      • Zhao H.
      • Lappalainen P.
      Regulation of the actin cytoskeleton-plasma membrane interplay by phosphoinositides.
      ,
      • Vicinanza M.
      • D'Angelo G.
      • Di Campli A.
      • De Matteis M.A.
      Function and dysfunction of the PI system in membrane trafficking.
      ).
      Figure thumbnail gr2
      FIGURE 2.Subcellular distribution of PIPs. Shown is the distribution of the predominant PIP species in different cell compartments according to Vicinanza et al. (
      • Vicinanza M.
      • D'Angelo G.
      • Di Campli A.
      • De Matteis M.A.
      Function and dysfunction of the PI system in membrane trafficking.
      ) and modified according to Kong et al. (
      • Kong A.M.
      • Horan K.A.
      • Sriratana A.
      • Bailey C.G.
      • Collyer L.J.
      • Nandurkar H.H.
      • Shisheva A.
      • Layton M.J.
      • Rasko J.E.
      • Rowe T.
      • Mitchell C.A.
      Phosphatidylinositol 3-phosphate [PtdIns3P] is generated at the plasma membrane by an inositol polyphosphate 5-phosphatase: endogenous PtdIns3P can promote GLUT4 translocation to the plasma membrane.
      ) (i), Maffucci et al. (
      • Maffucci T.
      • Brancaccio A.
      • Piccolo E.
      • Stein R.C.
      • Falasca M.
      Insulin induces phosphatidylinositol-3-phosphate formation through TC10 activation.
      ) (ii), Falasca and Maffucci (
      • Falasca M.
      • Maffucci T.
      Role of class II phosphoinositide 3-kinase in cell signalling.
      ) (iii), and Nakatsu et al. (
      • Nakatsu F.
      • Baskin J.M.
      • Chung J.
      • Tanner L.B.
      • Shui G.
      • Lee S.Y.
      • Pirruccello M.
      • Hao M.
      • Ingolia N.T.
      • Wenk M.R.
      • De Camilli P.
      PtdIns4P synthesis by PI4KIIIα at the plasma membrane and its impact on plasma membrane identity.
      ) (iv). PIP-metabolizing enzymes and other proteins involved in the synthesis, degradation, and trafficking of PIPs (indicated by arrows) are not shown for simplicity. Of note, many of the PIP-metabolizing enzymes are present in more than one cellular compartment, and their overall distribution does not completely fit to the PIP distribution. PM, plasma membrane; EE, early endosome; RE, recycling endosome; LY, lysosome; MVB/LE, multivesicular body/late endosome; PAS, preautophagosomal structure; PH, phagosome; TGN, trans-Golgi network; GC, Golgi complex; ER, endoplasmic reticulum; N, nucleus; SE, sorting endosome.
      In the present study, we assessed the lipid binding specificity of CB in a more natural phospholipid bilayer environment that reflects the situation in live cells more closely. For that purpose, we used fluid planar lipid bilayers composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) doped with different phosphoinositides. Immobilized on a silicon/silicon dioxide substrate, these membranes enabled us to monitor the specific interaction of CB variants with receptor lipids in a time-resolved and label-free manner by means of reflectometric interference spectroscopy (RIfS) (Fig. 3A) (
      • Gauglitz G.
      • Brecht A.
      • Kraus G.
      • Nahm W.
      Chemical and biochemical sensors based on interferometry at thin (multi-)layers.
      ,
      • Roth G.
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      • Wöllner K.
      • Brünjes J.
      • Gauglitz G.
      • Wiesmüller K.H.
      • Jung G.
      Ubiquitin binds to a short peptide segment of hydrolase UCH-L3: a study by FCS, RIfS, ITC and NMR.
      • Stephan M.
      • Kramer C.
      • Steinem C.
      • Janshoff A.
      Binding assay for low molecular weight analytes based on reflectometry of absorbing molecules in porous substrates.
      ). RIfS is a well established technique to quantitatively monitor protein-receptor interactions at solid-supported membranes (
      • Krick R.
      • Busse R.A.
      • Scacioc A.
      • Stephan M.
      • Janshoff A.
      • Thumm M.
      • Kühnel K.
      Structural and functional characterization of the two phosphoinositide binding sites of PROPPINs, a β-propeller protein family.
      • Braunger J.A.
      • Kramer C.
      • Morick D.
      • Steinem C.
      Solid supported membranes doped with PIP2: influence of ionic strength and pH on bilayer formation and membrane organization.
      ,
      • Vilardi F.
      • Stephan M.
      • Clancy A.
      • Janshoff A.
      • Schwappach B.
      WRB and CAML are necessary and sufficient to mediate tail-anchored protein targeting to the ER membrane.
      • Schütte O.M.
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      • Ries A.
      • Römer W.
      • Werz D.B.
      • Steinem C.
      2-Hydroxy fatty acid enantiomers of Gb3 impact Shiga toxin binding and membrane organization.
      ).
      Figure thumbnail gr3
      FIGURE 3.Setup of the RIfS experiments. A, schematic drawing of the RIfS setup. B, scheme of membrane preparation and protein binding on the silicon dioxide surface.

      Discussion

      Based on an in vitro phospholipid bilayer membrane system, the present study provides important new insights into the specificity of CB2 binding to phosphoinositides and elucidates the mechanism of autoinhibition of CB2 at the molecular level. We made use of solid-supported planar bilayers doped with different phosphoinositides to provide a membrane system that partially resembles, but at the same time simplifies, the natural situation at cellular membranes. It allowed for a quantitative analysis of the binding properties of CB2 in a label-free manner by means of reflectometric interference spectroscopy. Previous studies on the specificity of CB used pure phosphoinositides spotted on synthetic membranes (protein-lipid overlay assays) (
      • Soykan T.
      • Schneeberger D.
      • Tria G.
      • Buechner C.
      • Bader N.
      • Svergun D.
      • Tessmer I.
      • Poulopoulos A.
      • Papadopoulos T.
      • Varoqueaux F.
      • Schindelin H.
      • Brose N.
      A conformational switch in collybistin determines the differentiation of inhibitory postsynapses.
      ,
      • Kalscheuer V.M.
      • Musante L.
      • Fang C.
      • Hoffmann K.
      • Fuchs C.
      • Carta E.
      • Deas E.
      • Venkateswarlu K.
      • Menzel C.
      • Ullmann R.
      • Tommerup N.
      • Dalprà L.
      • Tzschach A.
      • Selicorni A.
      • Lüscher B.
      • Ropers H.H.
      • Harvey K.
      • Harvey R.J.
      A balanced chromosomal translocation disrupting ARHGEF9 is associated with epilepsy, anxiety, aggression, and mental retardation.
      ,
      • Papadopoulos T.
      • Schemm R.
      • Grubmüller H.
      • Brose N.
      Lipid binding defects and perturbed synaptogenic activity of a collybistin R290H mutant that causes epilepsy and intellectual disability.
      ). In these assays, phosphoinositides are not embedded in a lipid membrane, and thus the headgroup positions of the phosphorylated inositols are not defined and aligned, which can alter the binding specificity (
      • Busse R.A.
      • Scacioc A.
      • Hernandez J.M.
      • Krick R.
      • Stephan M.
      • Janshoff A.
      • Thumm M.
      • Kühnel K.
      Qualitative and quantitative characterization of protein-phosphoinositide interactions with liposome-based methods.
      ).
      Here we first tested the specificity of binding of the PH domain of CB2 to different phosphoinositides, which is of key relevance as the PH domain of CB2 is functionally essential (
      • 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.
      The GDP-GTP exchange factor collybistin: an essential determinant of neuronal gephyrin clustering.
      ,
      • Kalscheuer V.M.
      • Musante L.
      • Fang C.
      • Hoffmann K.
      • Fuchs C.
      • Carta E.
      • Deas E.
      • Venkateswarlu K.
      • Menzel C.
      • Ullmann R.
      • Tommerup N.
      • Dalprà L.
      • Tzschach A.
      • Selicorni A.
      • Lüscher B.
      • Ropers H.H.
      • Harvey K.
      • Harvey R.J.
      A balanced chromosomal translocation disrupting ARHGEF9 is associated with epilepsy, anxiety, aggression, and mental retardation.
      ,
      • Reddy-Alla S.
      • Schmitt B.
      • Birkenfeld J.
      • Eulenburg V.
      • Dutertre S.
      • Böhringer C.
      • Götz M.
      • Betz H.
      • Papadopoulos T.
      PH-domain-driven targeting of collybistin but not Cdc42 activation is required for synaptic gephyrin clustering.
      ). In general, PH domains are best known for their ability to bind phosphoinositides and to be targeted to cellular membranes (
      • Lemmon M.A.
      Pleckstrin homology (PH) domains and phosphoinositides.
      ,
      • Hyvönen M.
      • Macias M.J.
      • Nilges M.
      • Oschkinat H.
      • Saraste M.
      • Wilmanns M.
      Structure of the binding site for inositol phosphates in a PH domain.
      ). Most lipid-binding PH domains show a preference for one or several phosphoinositides, such as the PH domain of phospholipase C-δ1, which binds specifically to PI(4,5)P2 (
      • Lemmon M.A.
      • Ferguson K.M.
      • O'Brien R.
      • Sigler P.B.
      • Schlessinger J.
      Specific and high-affinity binding of inositol phosphates to an isolated pleckstrin homology domain.
      ). For the isolated PH domain of CB2, the dissociation constants for the phosphoinositides tested were in the 0.1–0.8 μm range with an up to 8 times higher affinity for monophosphorylated PI(3)P and PI(4)P. In the literature, quite a large range of KD values can be found for PH domain binding to phosphoinositides, ranging from about 30 μm in the case of the interaction of the pleckstrin PH domain with PI(4,5)P2 (
      • Kavran J.M.
      • Klein D.E.
      • Lee A.
      • Falasca M.
      • Isakoff S.J.
      • Skolnik E.Y.
      • Lemmon M.A.
      Specificity and promiscuity in phosphoinositide binding by pleckstrin homology domains.
      ) to 10 nm values for the PH domains of phospholipase C-δ1 and Grp1-PH (
      • Ferguson C.G.
      • James R.D.
      • Bigman C.S.
      • Shepard D.A.
      • Abdiche Y.
      • Katsamba P.S.
      • Myszka D.G.
      • Prestwich G.D.
      Phosphoinositide-containing polymerized liposomes: stable membrane-mimetic vesicles for protein-lipid binding analysis.
      ,
      • Knight J.D.
      • Falke J.J.
      Single-molecule fluorescence studies of a PH domain: new insights into the membrane docking reaction.
      ). Another important aspect in this regard is the binding capacity of the membrane as a function of different phosphoinositides. We used 10 mol % of the corresponding receptor lipid, which is roughly 10 times higher than the concentrations of phosphoinositides found in native cellular membranes and was chosen to optimize the signal-to-noise ratio in our assay. If the receptor lipids were homogeneously distributed in the POPC membranes and assuming a 1:1 stoichiometry of PIP-CB2 binding as has been shown for other PH domain-containing proteins (
      • Hyvönen M.
      • Macias M.J.
      • Nilges M.
      • Oschkinat H.
      • Saraste M.
      • Wilmanns M.
      Structure of the binding site for inositol phosphates in a PH domain.
      ,
      • Garcia P.
      • Gupta R.
      • Shah S.
      • Morris A.J.
      • Rudge S.A.
      • Scarlata S.
      • Petrova V.
      • McLaughlin S.
      • Rebecchi M.J.
      The pleckstrin homology domain of phospholipase C-δ1 binds with high affinity to phosphatidylinositol 4,5-bisphosphate in bilayer membranes.
      ,
      • Rameh L.E.
      • Arvidsson A.-k.
      • Carraway 3rd, K.L.
      • Couvillon A.D.
      • Rathbun G.
      • Crompton A.
      • VanRenterghem B.
      • Czech M.P.
      • Ravichandran K.S.
      • Burakoff S.J.
      • Wang D.S.
      • Chen C.S.
      • Cantley L.C.
      A comparative analysis of the phosphoinositide binding specificity of pleckstrin homology domains.
      ), a lower phosphoinositide concentration should have been sufficient. However, we found that a larger PIP concentration was required to obtain a good signal-to-noise ratio as the overall protein surface coverage was, depending on the phosphoinositide, rather low. Assuming that at 10 mol % the maximum protein surface is reached (
      • Janke M.
      • Herrig A.
      • Austermann J.
      • Gerke V.
      • Steinem C.
      • Janshoff A.
      Actin binding of ezrin is activated by specific recognition of PIP2-functionalized lipid bilayers.
      ), the maximum protein layer thickness obtained by RIfS experiments is a measure of the binding capacity of the membrane. Interestingly, a large protein layer thickness for CB2PH was obtained for phosphoinositides with a phosphate group at the 5-position with the largest dprotein-max found for the most strongly phosphorylated form, PI(3,4,5)P3. The monophosphorylated phosphoinositols showed a lower binding capacity. This might be explained by a non-homogeneous, clustered distribution of the receptor lipids prior to and after protein binding (
      • Saarikangas J.
      • Zhao H.
      • Lappalainen P.
      Regulation of the actin cytoskeleton-plasma membrane interplay by phosphoinositides.
      ), respectively, resulting in more than one phosphoinositide bound to CB and would require further investigations.
      The submicromolar affinity of the isolated PH domain of CB2 was also seen with the DH/PH tandem domain of CB2. The slightly lower binding affinity of CB2SH3− for monophosphorylated phosphoinositols as compared with the isolated PH domain of CB2 is likely due to the multiple interactions between the PH and DH domains, which influence the interactions between the DH/PH domains and membrane lipids (
      • Papadopoulos T.
      • Schemm R.
      • Grubmüller H.
      • Brose N.
      Lipid binding defects and perturbed synaptogenic activity of a collybistin R290H mutant that causes epilepsy and intellectual disability.
      ,
      • Xiang S.
      • Kim E.Y.
      • Connelly J.J.
      • Nassar N.
      • Kirsch J.
      • Winking J.
      • Schwarz G.
      • Schindelin H.
      The crystal structure of Cdc42 in complex with collybistin II, a gephyrin-interacting guanine nucleotide exchange factor.
      ). The maximum protein layer thickness was largest for PI(4,5)P2 and PI(3,4,5)P3, which is similar to the results obtained for CB2PH and fits to the dimensions estimated from the crystal structure of CB2SH3− (long axis) (
      • Soykan T.
      • Schneeberger D.
      • Tria G.
      • Buechner C.
      • Bader N.
      • Svergun D.
      • Tessmer I.
      • Poulopoulos A.
      • Papadopoulos T.
      • Varoqueaux F.
      • Schindelin H.
      • Brose N.
      A conformational switch in collybistin determines the differentiation of inhibitory postsynapses.
      ). Based on the results obtained with POPC membranes containing either phosphatidylinositol or phosphatidylserine, we conclude that the molecular recognition of the OH groups of the inositol headgroup and the specific phosphorylation state are more relevant for the specific recognition of CB than an interaction driven purely by electrostatics.
      In mouse brain, CB2SH3− exists only in trace amounts, and the majority of CB isoforms contain an additional SH3 domain (
      • Soykan T.
      • Schneeberger D.
      • Tria G.
      • Buechner C.
      • Bader N.
      • Svergun D.
      • Tessmer I.
      • Poulopoulos A.
      • Papadopoulos T.
      • Varoqueaux F.
      • Schindelin H.
      • Brose N.
      A conformational switch in collybistin determines the differentiation of inhibitory postsynapses.
      ). This SH3 domain causes CB to adopt a closed and autoinhibited conformation in which the SH3 domain interacts with the DH/PH tandem domain (
      • Soykan T.
      • Schneeberger D.
      • Tria G.
      • Buechner C.
      • Bader N.
      • Svergun D.
      • Tessmer I.
      • Poulopoulos A.
      • Papadopoulos T.
      • Varoqueaux F.
      • Schindelin H.
      • Brose N.
      A conformational switch in collybistin determines the differentiation of inhibitory postsynapses.
      ), thus preventing its membrane recruitment (Fig. 8). To analyze the phosphoinositide binding characteristics of the biologically relevant CB variants, i.e. the ones with an N-terminal SH3 domain, we studied CB2SH3+ and a constitutively activated variant in which the autoinhibitory effect of the SH3 domain is eliminated (CB2SH3+/W24A/E262A). With CB2SH3+, the amount of bound protein is greatly diminished as compared with CB2PH and CB2SH3−, independently of the phosphoinositide used. Taking into account the more compact structure of CB2SH3+, which has a smaller radius of gyration (Rg = 26.3 Å) than CB2SH3− (Rg = 28.3 Å) (
      • Soykan T.
      • Schneeberger D.
      • Tria G.
      • Buechner C.
      • Bader N.
      • Svergun D.
      • Tessmer I.
      • Poulopoulos A.
      • Papadopoulos T.
      • Varoqueaux F.
      • Schindelin H.
      • Brose N.
      A conformational switch in collybistin determines the differentiation of inhibitory postsynapses.
      ), the protein size does not explain this significant decrease in protein layer thickness. Instead, the results indicate that a large fraction of CB2SH3+ is not capable of binding to the PIP-containing membranes. This provides key support for the notion that CB2SH3+ adopts an autoinhibited conformation that is stabilized by contacts between the SH3 domain and the DH/PH tandem domain (Fig. 8). Furthermore, our data show that a main consequence of the SH3 domain-mediated autoinhibition of CB is an inhibition of phosphoinositide binding and membrane tethering of CB (
      • Soykan T.
      • Schneeberger D.
      • Tria G.
      • Buechner C.
      • Bader N.
      • Svergun D.
      • Tessmer I.
      • Poulopoulos A.
      • Papadopoulos T.
      • Varoqueaux F.
      • Schindelin H.
      • Brose N.
      A conformational switch in collybistin determines the differentiation of inhibitory postsynapses.
      ), whereas the guanine nucleotide exchange factor activity does not seem to be affected (
      • Soykan T.
      • Schneeberger D.
      • Tria G.
      • Buechner C.
      • Bader N.
      • Svergun D.
      • Tessmer I.
      • Poulopoulos A.
      • Papadopoulos T.
      • Varoqueaux F.
      • Schindelin H.
      • Brose N.
      A conformational switch in collybistin determines the differentiation of inhibitory postsynapses.
      ,
      • Xiang S.
      • Kim E.Y.
      • Connelly J.J.
      • Nassar N.
      • Kirsch J.
      • Winking J.
      • Schwarz G.
      • Schindelin H.
      The crystal structure of Cdc42 in complex with collybistin II, a gephyrin-interacting guanine nucleotide exchange factor.
      ,
      • Mayer S.
      • Kumar R.
      • Jaiswal M.
      • Soykan T.
      • Ahmadian M.R.
      • Brose N.
      • Betz H.
      • Rhee J.S.
      • Papadopoulos T.
      Collybistin activation by GTP-TC10 enhances postsynaptic gephyrin clustering and hippocampal GABAergic neurotransmission.
      ,
      • Tyagarajan S.K.
      • Ghosh H.
      • Harvey K.
      • Fritschy J.M.
      Collybistin splice variants differentially interact with gephyrin and Cdc42 to regulate gephyrin clustering at GABAergic synapses.
      ). This is different in Asef, the closest CB homologue, where the SH3 domain-mediated autoinhibition of Asef affects the enzymatic guanine nucleotide exchange factor activity of the DH domain (
      • Mitin N.
      • Betts L.
      • Yohe M.E.
      • Der C.J.
      • Sondek J.
      • Rossman K.L.
      Release of autoinhibition of ASEF by APC leads to CDC42 activation and tumor suppression.
      ).
      Figure thumbnail gr8
      FIGURE 8.Schematic drawing of the proposed modes of protein binding. Proteins bind to solid-supported POPC membranes doped with 10 mol % of the respective PIP.
      To further assess the autoinhibitory influence of the SH3 domain on phosphoinositide binding of CB2, we made use of the CB2SH3+/W24A/E262A variant in which two amino acids in the SH3-DH/PH interface are exchanged. The corresponding mutation leads to an open conformation exhibiting a larger flexibility and a more elongated protein shape in solution. In lipid overlay assays, the mutant protein binds more strongly to PI(3)P as compared with wild-type CB2SH3+ albeit not as well as CB2SH3− (
      • Soykan T.
      • Schneeberger D.
      • Tria G.
      • Buechner C.
      • Bader N.
      • Svergun D.
      • Tessmer I.
      • Poulopoulos A.
      • Papadopoulos T.
      • Varoqueaux F.
      • Schindelin H.
      • Brose N.
      A conformational switch in collybistin determines the differentiation of inhibitory postsynapses.
      ). A similar behavior of CB2SH3+/W24A/E262A was observed with solid-supported POPC membranes containing PI(3)P. Although the binding capacity of CB2SH3+ was estimated to be about 3 times lower than that of CB2SH3− (Fig. 7A), it was regained to about 90% in the case of the CB2SH3+/W24A/E262A mutant. Interestingly, this regain in activity is a function of the phosphoinositide. Although the binding capacity of the mutant for the monophosphorylated phosphoinositols and for PI(3,5)P2 is large, it remains partially diminished for the other PIPs, and the mutant protein does not significantly bind to phosphoinositides with two juxtaposed phosphate groups, i.e. PI(3,4)P2 and PI(4,5)P2.
      The present results provide the first insights into the phosphoinositide binding specificity of CB in a phospholipid bilayer context. Of most biological relevance are the data on CB2SH3+ and its open and disinhibited CB2SH3+/W24A/E262A variant because essentially all CB isoforms expressed in murine brain carry an N-terminal SH3 domain (
      • Soykan T.
      • Schneeberger D.
      • Tria G.
      • Buechner C.
      • Bader N.
      • Svergun D.
      • Tessmer I.
      • Poulopoulos A.
      • Papadopoulos T.
      • Varoqueaux F.
      • Schindelin H.
      • Brose N.
      A conformational switch in collybistin determines the differentiation of inhibitory postsynapses.
      ). Our corresponding data nicely corroborate the notion that CB2SH3+ is autoinhibited with respect to phosphoinositide binding and that this autoinhibition is relieved upon introduction of the W24A/E262A mutation, which “opens” the CB2SH3+ conformation and exposes the PH domain for proper lipid binding. In neurons expressing wild-type CB, this conformational activation of phosphoinositide binding of CB is mediated by neuroligin-2 (
      • Poulopoulos A.
      • Aramuni G.
      • Meyer G.
      • Soykan T.
      • Hoon M.
      • Papadopoulos T.
      • Zhang M.
      • Paarmann I.
      • Fuchs C.
      • Harvey K.
      • Jedlicka P.
      • Schwarzacher S.W.
      • Betz H.
      • Harvey R.J.
      • Brose N.
      • Zhang W.
      • Varoqueaux F.
      Neuroligin 2 drives postsynaptic assembly at perisomatic inhibitory synapses through gephyrin and collybistin.
      ), TC10 (
      • Mayer S.
      • Kumar R.
      • Jaiswal M.
      • Soykan T.
      • Ahmadian M.R.
      • Brose N.
      • Betz H.
      • Rhee J.S.
      • Papadopoulos T.
      Collybistin activation by GTP-TC10 enhances postsynaptic gephyrin clustering and hippocampal GABAergic neurotransmission.
      ), and neuroligin-4 (
      • Hoon M.
      • Soykan T.
      • Falkenburger B.
      • Hammer M.
      • Patrizi A.
      • Schmidt K.F.
      • Sassoè-Pognetto M.
      • Löwel S.
      • Moser T.
      • Taschenberger H.
      • Brose N.
      • Varoqueaux F.
      Neuroligin-4 is localized to glycinergic postsynapses and regulates inhibition in the retina.
      ), likely along with other interactors of the SH3 and PH domains of CB, to promote CB-dependent gephyrin clustering at nascent GABAergic synapses. Furthermore, our data show that CB2SH3+ can bind to several phosphoinositide variants but show a significant preference for PI(3)P and PI(4)P in bilayer membranes. This partial selectivity, which has been observed with other assay systems as well (
      • Soykan T.
      • Schneeberger D.
      • Tria G.
      • Buechner C.
      • Bader N.
      • Svergun D.
      • Tessmer I.
      • Poulopoulos A.
      • Papadopoulos T.
      • Varoqueaux F.
      • Schindelin H.
      • Brose N.
      A conformational switch in collybistin determines the differentiation of inhibitory postsynapses.
      ,
      • Kalscheuer V.M.
      • Musante L.
      • Fang C.
      • Hoffmann K.
      • Fuchs C.
      • Carta E.
      • Deas E.
      • Venkateswarlu K.
      • Menzel C.
      • Ullmann R.
      • Tommerup N.
      • Dalprà L.
      • Tzschach A.
      • Selicorni A.
      • Lüscher B.
      • Ropers H.H.
      • Harvey K.
      • Harvey R.J.
      A balanced chromosomal translocation disrupting ARHGEF9 is associated with epilepsy, anxiety, aggression, and mental retardation.
      ,
      • Papadopoulos T.
      • Schemm R.
      • Grubmüller H.
      • Brose N.
      Lipid binding defects and perturbed synaptogenic activity of a collybistin R290H mutant that causes epilepsy and intellectual disability.
      ,
      • Reddy-Alla S.
      • Schmitt B.
      • Birkenfeld J.
      • Eulenburg V.
      • Dutertre S.
      • Böhringer C.
      • Götz M.
      • Betz H.
      • Papadopoulos T.
      PH-domain-driven targeting of collybistin but not Cdc42 activation is required for synaptic gephyrin clustering.
      ), is also seen with the isolated PH domain of CB (CB2PH) but only to a smaller degree with the N-terminally truncated CB2SH3− variant, which lacks the SH3 domain. In view of these findings and given that neurons almost exclusively express SH3 domain-containing CB isoforms, future studies on the regulation of CB function will have to focus on PI(3)P and PI(4)P signaling toward CB.

      Author Contributions

      M. L. and J. S. isolated the proteins and performed the RIfS experiments. D. S. and H. S. designed the protein constructs and helped with protein purification. T. S. and T. P. performed lipid overlay assays and critically reviewed the manuscript. C. S. and N. B. designed the experiments and wrote the manuscript. All authors reviewed the results and approved the final version of the manuscript.

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