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* 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.
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.
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 (
). 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 (
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 (
). 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 (
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 (
). 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) (
). 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) (
). 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) (
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) (
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) (
). 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 (
). 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 (
). 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 (
), 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 (
), 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 (
), 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 (
). 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) (
). 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 (
), 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) (
), 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 (
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− (
). 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 (
). 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 (
), 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 (
), 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.
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.
Clustering of inhibitory neurotransmitter receptors at developing postsynaptic sites: the membrane activation mode.