Gephyrin-mediated γ-Aminobutyric Acid Type A and Glycine Receptor Clustering Relies on a Common Binding Site
Gephyrin-mediated γ-Aminobutyric Acid Type A and Glycine Receptor Clustering Relies on a Common Binding Site
复制标题
DOI：
10.1074/jbc.m111.303412
复制
发表时间：
2011-12-09
影响因子：
4.8
通讯作者：
Schindelin, Hermann
中科院分区：
文献类型：
--
作者：
Maric, Hans-Michael;Mukherjee, Jayanta;Schindelin, Hermann
桥卟啉是抑制性神经递质受体聚集的主要蛋白质决定簇。早期的分析显示桥蛋白与甘氨酸受体β亚基（GlyR β）的残基398-410紧密结合，并且如最近才证实的，还与含有α 1、α 2和α 3亚基的GABA（A）受体（GABA（A）Rs）相互作用。在这里，我们剖析了桥蛋白和GABA（A）Rs之间相互作用的分子基础，并将其与桥蛋白-GlyR β复合物的晶体结构进行了比较。生物物理和生物化学测定显示，与其与GlyR β的紧密相互作用相反，桥蛋白仅与GABAAR α 2松散地相互作用，而其对GABAAR α 1和α 3亚基具有中等亲和力。尽管广泛的亲和力和低的整体序列同源性之间的识别受体亚基的变化，竞争试验证实受体-桥蛋白相互作用是一个相互排斥的过程。选择的桥蛋白点突变体严重削弱了与GlyR β的复合物形成，也消除了GABA（A）R α 1和α 3的相互作用。此外，我们确定了一个共同的结合基序与两个保守的芳香族残基是中心的桥蛋白结合。与生化数据一致，含有α 2亚基的GABA（A）Rs细胞质结构域内相应残基的突变减弱了这些受体在海马神经元突触后位点的聚集。总之，我们的实验提供了关键的见解，关于桥蛋白和GABA（A）R与GlyR相比，在复合物形成的相似性和差异，因此，这些受体在突触后位点的积累。
Gephyrin is the major protein determinant for the clustering of inhibitory neurotransmitter receptors. Earlier analyses revealed that gephyrin tightly binds to residues 398-410 of the glycine receptor beta subunit (GlyR beta) and, as demonstrated only recently, also interacts with GABA(A) receptors (GABA(A)Rs) containing the alpha 1, alpha 2, and alpha 3 subunits. Here, we dissect the molecular basis underlying the interactions between gephyrin and GABA(A)Rs containing these alpha-subunits and compare them to the crystal structure of the gephyrin-GlyR beta complex. Biophysical and biochemical assays revealed that, in contrast to its tight interaction with GlyR beta, gephyrin only loosely interacts with GABAAR alpha 2, whereas it has an intermediate affinity for the GABAAR alpha 1 and alpha 3 subunits. Despite the wide variation in affinities and the low overall sequence homology among the identified receptor subunits, competition assays confirmed the receptor-gephyrin interaction to be a mutually exclusive process. Selected gephyrin point mutants that critically weaken complex formation with GlyR beta also abolished the GABA(A)R alpha 1 and alpha 3 interactions. Additionally, we identified a common binding motif with two conserved aromatic residues that are central for gephyrin binding. Consistent with the biochemical data, mutations of the corresponding residues within the cytoplasmic domain of alpha 2 subunit-containing GABA(A)Rs attenuated clustering of these receptors at postsynaptic sites in hippocampal neurons. Taken together, our experiments provide key insights regarding similarities and differences in the complex formation between gephyrin and GABA(A)Rs compared with GlyRs and, hence, the accumulation of these receptors at postsynaptic sites.