title: Specificity of Collybistin-Phosphoinositide Interactions: IMPACT OF THE INDIVIDUAL PROTEIN DOMAINS.
abstract: 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.
authors.affiliation: From the Institute of Organic and Biomolecular Chemistry, University of Göttingen, Tammannstrasse 2, 37077 Göttingen, Germany.
authors.forename: Michaela
authors.initials: M
authors.lastname: Ludolphs
authors.affiliation: Rudolf Virchow Center for Experimental Biomedicine, University of Würzburg, Josef-Schneider-Strasse 2, 97080 Würzburg, Germany.
authors.forename: Daniela
authors.initials: D
authors.lastname: Schneeberger
authors.affiliation: Department of Molecular Neurobiology, Max Planck Institute for Experimental Medicine, Hermann-Rein-Strasse 3, 37075 Göttingen, Germany, and.
authors.forename: Tolga
authors.initials: T
authors.lastname: Soykan
authors.affiliation: From the Institute of Organic and Biomolecular Chemistry, University of Göttingen, Tammannstrasse 2, 37077 Göttingen, Germany.
authors.forename: Jonas
authors.initials: J
authors.lastname: Schäfer
authors.affiliation: Universitätsmedizin Göttingen, Department of Molecular Biology, Humboldtallee 23, 37073 Göttingen, Germany.
authors.forename: Theofilos
authors.initials: T
authors.lastname: Papadopoulos
authors.affiliation: Department of Molecular Neurobiology, Max Planck Institute for Experimental Medicine, Hermann-Rein-Strasse 3, 37075 Göttingen, Germany, and.
authors.forename: Nils
authors.initials: N
authors.lastname: Brose
authors.affiliation: Rudolf Virchow Center for Experimental Biomedicine, University of Würzburg, Josef-Schneider-Strasse 2, 97080 Würzburg, Germany.
authors.forename: Hermann
authors.initials: H
authors.lastname: Schindelin
authors.affiliation: From the Institute of Organic and Biomolecular Chemistry, University of Göttingen, Tammannstrasse 2, 37077 Göttingen, Germany, csteine@gwdg.de.
authors.forename: Claudia
authors.initials: C
authors.lastname: Steinem
mesh_terms: D000327:Adsorption; D008051:Lipid Bilayers / Q000737:chemistry; D008567:Membranes, Artificial; D010716:Phosphatidylinositols / Q000378:metabolism*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D064067:Rho Guanine Nucleotide Exchange Factors / Q000737:chemistry* / Q000378:metabolism*; D013057:Spectrum Analysis; D013329:Structure-Activity Relationship; D013347:Subcellular Fractions / Q000378:metabolism; D013696:Temperature
chemical_list: D008051:Lipid Bilayers; D008567:Membranes, Artificial; D010716:Phosphatidylinositols; D064067:Rho Guanine Nucleotide Exchange Factors
keywords: GABA receptor; inositol phospholipid; lipid bilayer; lipid-protein interaction; membrane function