Vertebrate-specific sequences in the gephyrin E-domain regulate cytosolic aggregation and postsynaptic clustering
Vertebrate-specific sequences in the gephyrin E-domain regulate cytosolic aggregation and postsynaptic clustering
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DOI：
10.1242/jcs.003905
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发表时间：
2007-04-15
影响因子：
4
通讯作者：
Schwarz, Guenter
中科院分区：
文献类型：
--
作者：
Lardi-Studler, Barbara;Smolinsky, Birthe;Schwarz, Guenter
桥卟啉是一种多功能蛋白质，有助于钼辅因子（Moco）的合成和甘氨酸和GABAA受体的突触后聚集。它包含三个主要的功能域（G-C-E），并通过未知的机制形成胞质聚集体和突触后簇。在这里，通过转染EGFP标记的缺失和突变型桥蛋白构建体的神经元，研究桥蛋白聚集和聚集的结构决定因素。EGFP-桥蛋白形成含有内源性桥蛋白和GABA（A）-受体的突触后簇。孤立的GC-或E-结构域未能聚集和发挥显性负效应的内源性桥蛋白聚类。干扰分子间E结构域二聚化的构建体容易自聚集，但显示受损的突触后聚类。最后，用同源细菌MoeA序列取代脊椎动物特异性E结构域序列的两个突变构建体发现了一个对桥蛋白聚集至关重要的区域。一个构建体未能聚集，但保留了Moco生物合成能力，证明了桥蛋白酶活性和聚集的独立性。重新插入两个脊椎动物特异性残基恢复了桥蛋白聚集并增加了含有GABA（A）受体的突触后簇的形成，而PSD- 95簇是突触能突触的标志物.这些结果强调了与已知的二聚化界面不同的特定E-结构域区域在控制桥蛋白聚集和突触后聚集中的关键作用，并表明桥蛋白簇的形成影响抑制性和兴奋性突触之间的稳态平衡。
Gephyrin is a multifunctional protein contributing to molybdenum cofactor ( Moco) synthesis and postsynaptic clustering of glycine and GABAA receptors. It contains three major functional domains (G-C-E) and forms cytosolic aggregates and postsynaptic clusters by unknown mechanisms. Here, structural determinants of gephyrin aggregation and clustering were investigated by neuronal transfection of EGFP-tagged deletion and mutant gephyrin constructs. EGFP-gephyrin formed postsynaptic clusters containing endogenous gephyrin and GABA(A)-receptors. Isolated GC- or E- domains failed to aggregate and exerted dominant- negative effects on endogenous gephyrin clustering. A construct interfering with intermolecular E-domain dimerization readily auto- aggregated but showed impaired postsynaptic clustering. Finally, two mutant constructs with substitution of vertebrate- specific E-domain sequences with homologue bacterial MoeA sequences uncovered a region crucial for gephyrin clustering. One construct failed to aggregate, but retained Moco biosynthesis capacity, demonstrating the independence of gephyrin enzymatic activity and aggregation. Reinserting two vertebrate- specific residues restored gephyrin aggregation and increased formation of postsynaptic clusters containing GABA(A) receptors at the expense of PSD- 95 clusters - a marker of glutamatergic synapses. These results underscore the key role of specific E- domain regions distinct from the known dimerization interface for controlling gephyrin aggregation and postsynaptic clustering and suggest that formation of gephyrin clusters influences the homeostatic balance between inhibitory and excitatory synapses.