Specificity of Collybistin-Phosphoinositide Interactions IMPACT OF THE INDIVIDUAL PROTEIN DOMAINS * Received for publication, June 18, 2015, and in revised form, November 2, 2015 Published, JBC Papers in Press, November 6, 2015, DOI 10.1074/jbc.M115.673400 Michaela Ludolphs ‡ , Daniela Schneeberger § , Tolga Soykan ¶ , Jonas Scha ¨fer ‡ , Theofilos Papadopoulos**, Nils Brose ¶ , Hermann Schindelin § , and Claudia Steinem ‡1 From the ‡ Institute of Organic and Biomolecular Chemistry, University of Go ¨ttingen, Tammannstrasse 2, 37077 Go ¨ttingen, Germany, § Rudolf Virchow Center for Experimental Biomedicine, University of Wu ¨rzburg, Josef-Schneider-Strasse 2, 97080 Wu ¨rzburg, Germany, ¶ Department of Molecular Neurobiology, Max Planck Institute for Experimental Medicine, Hermann- Rein-Strasse 3, 37075 Go ¨ttingen, Germany, and **Universita ¨tsmedizin Go ¨ttingen, Department of Molecular Biology, Humboldtallee 23, 37073 Go ¨ttingen, Germany The regulatory protein collybistin (CB) recruits the receptor- scaffolding protein gephyrin to mammalian inhibitory glyciner- gic and GABAergic postsynaptic membranes in nerve cells. CB is tethered to the membrane via phosphoinositides. We devel- oped 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 sub- strates 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 (CB2 SH3 ) 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” CB2 SH3 and allows the pleck- strin homology domain to properly bind lipids depending on the phosphoinositide species with a preference for phosphatidyl- inositol 3-monophosphate and phosphatidylinositol 4-mono- phosphate. This type of membrane tethering under the control of the release of the SH3 domain of CB is essential for regulating gephyrin clustering. The function of neuronal synapses and the dynamic regula- tion 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 (1, 2), whose recruitment to the post- synaptic membrane is controlled by the adaptor protein colly- bistin (CB) 2 (3). Loss of CB results in a strong reduction of gephyrin and GABA A receptor clusters in several regions of the forebrain, which demonstrates the essential role of CB in the assembly and maintenance of GABAergic postsynaptic struc- tures (4). CB belongs to the Dbl family of guanine nucleotide exchange factors. In mouse, four differently spliced CB mRNAs are present (CB1 SH3+ , CB2 SH3- , CB2 SH3+ , and CB3 SH3+ ). All four mRNAs encode a Dbl homology (DH) and a pleckstrin homo- logy (PH) domain. The three major variants (CB1 SH3+ , CB2 SH3+ , and CB3 SH3+ ) encode CBs with an additional N-ter- minal Src homology 3 (SH3) domain but differ in their C ter- mini. A fourth variant (CB2 SH3- ) encodes a CB2 isoform lack- ing the SH3 domain (Fig. 1) but is very rare (5) as its protein product is not detectable in mouse brain (6). 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 cotrans- fected in HEK293 cells and causes a loss of dendritic gephyrin clusters in dissociated rat cortical neurons (5). In vitro binding studies utilizing a variety of inositol head- groups, soluble phosphoinositide analogs, and liposomes con- taining phosphoinositides showed that PH domains bind phos- phoinositides with a broad range of selectivity and affinity (7–10). An early membrane activation model suggested that the PH domain of CB binds to phosphatidylinositol 3,4,5-trisphos- phate (PI(3,4,5)P 3 ) (1). In contrast, experiments with immobi- lized 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) (11, 12), and subsequent studies verified the binding of CB to PI(3)P (6, 13). 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 (14). 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 * 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 Cen- ter 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. 1 To whom correspondence should be addressed. Tel.: 49551-3933294; Fax: 49551-3933228; E-mail: csteine@gwdg.de. 2 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-palmi- toyl-2-oleoyl-sn-glycero-3-phospho-(1'-racemic glycerol); POPS, 1-palmi- toyl-2-oleoyl-sn-glycero-3-phospho-L-serine; RIfS, reflectometric interfer- ence spectroscopy; SH3, Src homology 3; PI(3,4,5)P 3 , phosphatidylinositol 3,4,5-trisphosphate; PI(3)P, phosphatidylinositol 3-monophosphate; PI(3,4)P 2 , phosphatidylinositol 3,4-bisphosphate; PI(4,5)P 2 , phosphatidylinositol 4,5-bisphosphate; PI(4)P, phosphatidylinositol 4-monophosphate; PI(3,5)P 2 , phosphatidylinositol 3,5-bisphosphate; SUV, small unilamellar vesicle. crossmark THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 291, NO. 1, pp. 244 –254, January 1, 2016 © 2016 by The American Society for Biochemistry and Molecular Biology, Inc. 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