The regulatory protein collybistin (CB) recruits the receptorscaffolding 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 (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 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.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 (1, 2), whose recruitment to the postsynaptic membrane is controlled by the adaptor protein collybistin (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 structures (4). CB belongs to the Dbl family of guanine nucleotide exchange factors. In mouse, four differently spliced CB mRNAs are present (CB1 SH3ϩ , CB2 SH3Ϫ , CB2 SH3ϩ , and CB3 SH3ϩ ). All four mRNAs encode a Dbl homology (DH) and a pleckstrin homology (PH) domain. The three major variants (CB1 SH3ϩ , CB2 SH3ϩ , and CB3 SH3ϩ ) encode CBs with an additional N-terminal Src homology 3 (SH3) domain but differ in their C termini. A fourth variant (CB2 SH3Ϫ ) encodes a CB2 isoform lacking 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 cotransfected 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 headgroups, soluble phosphoinositide analogs, and liposomes containing phosphoinositides showed that PH domains bind phosphoinositides with a broad range of selectivity and affinity (7-10). An early membrane acti...

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