Specificity of Collybistin-Phosphoinositide Interactions

Abstract

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...

Endosomal Phosphatidylinositol 3-Phosphate Promotes Gephyrin Clustering and GABAergic Neurotransmission at Inhibitory Postsynapses

“…Based on these data, one might speculate that PI3P may not be the only signaling lipid involved in the Cb-dependent accumulation of gephyrin at postsynapses. In agreement with this idea, a recent study indicates broader PIP specificities for the short Cb splice variant, which lacks the N-terminal SH3 domain, than those determined for an open-conformation mutant (W24A/E262A) of the SH3 domain-containing isoform (12). Furthermore, Vps34 has been shown to exist in two complexes in both yeast and mammalian cells, which not only have opposing effects but can potentially negatively regulate one another (18).…”

“…The following single-stranded DNA oligonucleotides encoding the pre-miRNAs of PI3K-C3, PI3K-C2␣, and PI3K-C2␤ used in this study were purchased from Invitrogen, Germany: PI3K-C3: forward, 5Ј-TGCTGTCAAGAAGGTACAAAGATCCTGTT-TTGGCCACTGACTGACAGGATCTTTACCTTCTTGA-3Ј, and reverse, 5-CCTGTCAAGAAGGTAAAGATCCTGT-CAGTCAGTGGCCAAAACAGGATCTTTGTACCTTCTT-GAC-3Ј; PI3K-C2␣, forward, 5Ј-TGCTGTACAAATGATGG-TTCAAGGTGGTTTTGGCCACTGACTGACCACCTTGA-CATCATTTGTA-3Ј, and reverse, 5Ј-CCTGTACAAATGAT-GTCAAGGTGGTCAGTCAGTGGCCAAAACCACCTTGA- (7,44,59). In addition, Cb binds in its open conformation, via its PH domain, to PI3P and further PIPs located at the plasma membrane (9,12,15). B, particularly at early developmental stages and at extrasynaptic sites, GABA A Rs undergo constitutive endocytosis and are either rapidly recycled back to the cell surface or targeted to lysosomal degradation (5,60).…”

PX-RICS-deficient mice mimic autism spectrum disorder in Jacobsen syndrome through impaired GABAA receptor trafficking

“…In this context, it is notable that collybistin, a brain-specific GDP/GTP exchange factor for Cdc42, is present in the scaffold protein complex for synaptic GABA A Rs and regulates gephyrin-dependent GABA A R anchoring and clustering 49 . Collybistin binds to phosphatidylinositols (PIs), with a preference for PI3P and PI4P, through its pleckstrin homology domain 50 , as PX-RICS does 20 . Thus, the balance between the activities of the Cdc42 GDP/GTP exchange factor and GAP (that is, collybistin and PX-RICS) might control the synaptic expression and localization of GABA A Rs.…”

Mode of Ezrin-Membrane Interaction as a Function of PIP 2 Binding and Pseudophosphorylation

“…To see this figure in color, go online. of a protein layer that is determined by the height of the layer as well as the surface coverage (27,31,32,42). For ezrin bound to DOGS-Ni-NTA or PIP 2 , the change in OT as a result of protein surface coverage is reflected by the increase in DOT as a function of increasing receptor lipid content in the membrane.…”