Chiou Tzu-Ting, Long Philip, Schumann-Gillett Alexandra, Kanamarlapudi Venkateswarlu, Haas Stefan A, Harvey Kirsten, O'Mara Megan L, De Blas Angel L, Kalscheuer Vera M, Harvey Robert J
Department of Physiology and Neurobiology, University of Connecticut, Storrs, CT, United States.
Department of Pharmacology, UCL School of Pharmacy, London, United Kingdom.
Front Mol Neurosci. 2019 Mar 12;12:60. doi: 10.3389/fnmol.2019.00060. eCollection 2019.
The recruitment of inhibitory GABA receptors to neuronal synapses requires a complex interplay between receptors, neuroligins, the scaffolding protein gephyrin and the GDP-GTP exchange factor collybistin (CB). Collybistin is regulated by protein-protein interactions at the N-terminal SH3 domain, which can bind neuroligins 2/4 and the GABAR α2 subunit. Collybistin also harbors a RhoGEF domain which mediates interactions with gephyrin and catalyzes GDP-GTP exchange on Cdc42. Lastly, collybistin has a pleckstrin homology (PH) domain, which binds phosphoinositides, such as phosphatidylinositol 3-phosphate (PI3P/PtdIns3P) and phosphatidylinositol 4-monophosphate (PI4P/PtdIns4P). PI3P located in early/sorting endosomes has recently been shown to regulate the postsynaptic clustering of gephyrin and GABA receptors and consequently the strength of inhibitory synapses in cultured hippocampal neurons. This process is disrupted by mutations in the collybistin gene (), which cause X-linked intellectual disability (XLID) by a variety of mechanisms converging on disrupted gephyrin and GABA receptor clustering at central synapses. Here we report a novel missense mutation (chrX:62875607C>T, p.R356Q) in that affects one of the two paired arginine residues in the PH domain that were predicted to be vital for binding phosphoinositides. Functional assays revealed that recombinant collybistin CB3 was deficient in PI3P binding and was not able to translocate EGFP-gephyrin to submembrane microaggregates in an clustering assay. Expression of the PI3P-binding mutants CB3 and CB3 in cultured hippocampal neurones revealed that the mutant proteins did not accumulate at inhibitory synapses, but instead resulted in a clear decrease in the overall number of synaptic gephyrin clusters compared to controls. Molecular dynamics simulations suggest that the p.R356Q substitution influences PI3P binding by altering the range of structural conformations adopted by collybistin. Taken together, these results suggest that the p.R356Q mutation in is the underlying cause of XLID in the probands, disrupting gephyrin clustering at inhibitory GABAergic synapses loss of collybistin PH domain phosphoinositide binding.
抑制性γ-氨基丁酸（GABA）受体募集到神经元突触需要受体、神经连接蛋白、支架蛋白桥连蛋白和GDP-GTP交换因子结肠直肠癌缺失蛋白（CB）之间复杂的相互作用。结肠直肠癌缺失蛋白受N端Src同源结构域3（SH3结构域）处的蛋白质-蛋白质相互作用调节，该结构域可结合神经连接蛋白2/4和GABARα2亚基。结肠直肠癌缺失蛋白还含有一个Rho鸟嘌呤核苷酸交换因子（RhoGEF）结构域，介导与桥连蛋白的相互作用并催化Cdc42上的GDP-GTP交换。最后，结肠直肠癌缺失蛋白有一个普列克底物蛋白同源（PH）结构域，可结合磷酸肌醇，如磷脂酰肌醇3-磷酸（PI3P/PtdIns3P）和磷脂酰肌醇4-单磷酸（PI4P/PtdIns4P）。最近研究表明，位于早期/分拣内体中的PI3P可调节桥连蛋白和GABA受体的突触后聚集，进而调节培养的海马神经元中抑制性突触的强度。结肠直肠癌缺失蛋白基因的突变会破坏这一过程，这些突变通过多种机制导致X连锁智力障碍（XLID），这些机制都集中在中枢突触处桥连蛋白和GABA受体聚集的破坏上。在这里，我们报告了一个新的错义突变（chrX:62875607C>T，p.R356Q），该突变影响PH结构域中两个成对的精氨酸残基之一，据预测这两个残基对结合磷酸肌醇至关重要。功能分析表明，重组结肠直肠癌缺失蛋白CB3缺乏PI3P结合能力，并且在一项聚集试验中无法将增强绿色荧光蛋白（EGFP）-桥连蛋白转运至亚膜微聚集体。在培养的海马神经元中表达PI3P结合突变体CB3和CB3表明，与对照相比，突变蛋白没有在抑制性突触处积累，反而导致突触桥连蛋白簇的总数明显减少。分子动力学模拟表明，p.R356Q取代通过改变结肠直肠癌缺失蛋白所采用的结构构象范围来影响PI3P结合。综上所述，这些结果表明，该错义突变是先证者XLID的潜在病因，通过破坏结肠直肠癌缺失蛋白PH结构域磷酸肌醇结合导致抑制性GABA能突触处桥连蛋白聚集缺失。
