Ligand binding of the second PDZ domain regulates clustering of PSD-95 with the Kv1.4 potassium channel
Ligand binding of the second PDZ domain regulates clustering of PSD-95 with the Kv1.4 potassium channel
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DOI：
10.1074/jbc.m106940200
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发表时间：
2002-02-01
影响因子：
4.8
通讯作者：
Fujiyoshi, Y
中科院分区：
文献类型：
--
作者：
Imamura, F;Maeda, S;Fujiyoshi, Y
突触连接处蛋白质组装的分子机制被认为对神经功能很重要。PSD-95是一种主要的突触后密度蛋白，由PDZ1、PDZ2和PDZ3三个结构域、SH3结构域和GK结构域组成。它通过PDZ1或PDZ2结构域与N-甲基-D-天冬氨酸受体NR2亚基或Shaker型K+通道Kv1.4结合，而PDZ3则与不同的伙伴结合。这些多个结构域的分子内相互作用被认为是有效的蛋白质聚类的基础。我们在全长PSD-95的PDZ1(PDZ1mDelta)和/或PDZ2(PDZ2mDelta)中引入错义和缺失突变，以破坏每个结构域与目标蛋白的结合，同时保留整体结构。在共表达突变体和Kv1.4的COS-1细胞中分析PSD-95突变体的离子通道聚集活性。携带功能异常的PDZ2(PSD-95：1-2mDelta)的突变体的聚集率显著降低，而含有功能异常的PDZ1(P`SD-95：1mDelta-2)的突变体表现出与野生型相当的活性。此外，我们还通过构建一系列PDZ1-PDZ2转化突变体，研究了PDZ2在全长PSD-95中的位置要求。令人惊讶的是，PSD-95：2-1mDelta的集群活动存在严重缺陷。综上所述，这些发现表明，与Kv1.4亲和力最高的PDZ2是有效结合配体所必需的。此外，全长PSD-95中第二个PDZ结构域位置上的配体结合是有效和典型簇形成的先决条件。这项研究表明，正确定位全长PSD-95蛋白中的多个结构域是获得最佳蛋白活性所必需的。
The molecular mechanisms underlying the protein assembly at synaptic junctions are thought to be important for neural functions. PSD-95, one of the major postsynaptic density proteins, is composed of three PDZ domains (PDZ1, PDZ2, and PDZ3), an SH3 domain, and a GK (guanylate kinase) domain. It binds to the N-methyl-D-aspartate glutamate receptor NR2 subunit or to the Shaker-type K+ channel, Kv1.4, via the PDZ1 or PDZ2 domain, whereas PDZ3 binds to distinct partners. The intramolecular interaction of these multiple domains has been implicated in efficient protein clustering. We introduced missense and deletion mutations into PDZ1 (PDZ1mDelta) and/or PDZ2 (PDZ2mDelta) of the full-length PSD-95 to disrupt the association of each domain with the target proteins, while preserving the overall structure. The ion channel clustering activities of the PSD-95 mutants were analyzed in COS-1 cells coexpressing each mutant and Kv1.4. The mutant bearing the dysfunctional PDZ2 (PSD-95:1-2mDelta) showed significantly reduced clustering efficiency, whereas the mutant with the dysfunctional PDZ1 (P`SD-95:1mDelta-2) exhibited activity comparable with the wild-type activity. Furthermore, we also examined the requirements for the position of PDZ2 in full-length PSD-95 by constructing a series of PDZ1-PDZ2 inversion mutants. Surprisingly, the clustering activity of PSD-95:2-1mDelta, was severely defective. Taken together, these findings show that PDZ2, which is endowed with the highest affinity for Kv1.4, is required for efficient ligand binding. In addition, the ligand binding at the position of the second PDZ domain in full-length PSD-95 is prerequisite for efficient and typical cluster formation. This study suggests that the correct placement of the multiple domains in the full-length PSD-95 protein is necessary for the optimal protein activity.