NIDD, a Novel DHHC-containing Protein, Targets Neuronal Nitric-oxide Synthase (nNOS) to the Synaptic Membrane through a PDZ-dependent Interaction and Regulates nNOS Activity*
NIDD, a Novel DHHC-containing Protein, Targets Neuronal Nitric-oxide Synthase (nNOS) to the Synaptic Membrane through a PDZ-dependent Interaction and Regulates nNOS Activity*
复制标题
DOI：
10.1074/jbc.m401471200
复制
发表时间：
2004-07
影响因子：
4.8
通讯作者：
Fuminori Saitoh;Q. Tian;A. Okano;H. Sakagami;H. Kondo;Tatsuo Suzuki
中科院分区：
文献类型：
--
作者：
Fuminori Saitoh;Q. Tian;A. Okano;H. Sakagami;H. Kondo;Tatsuo Suzuki
神经元型一氧化氮合酶（nNOS）在细胞内定位于适当位点是通过其与PDZ结构域的相互作用来介导的，这在确定中枢神经系统中一氧化氮（NO）的反应位点方面发挥着重要作用。在此，我们报告一种新的与nNOS相互作用、含DHHC结构域且树突状mRNA存在的蛋白（NIDD）（GenBank™登录号AB098078）的鉴定与特性分析。该蛋白通过以PDZ结构域依赖的方式将nNOS定位于突触质膜，从而增强nNOS的酶活性。推导得出的NIDD蛋白由392个氨基酸残基组成，具有5个跨膜片段、一个锌指DHHC结构域以及在其C末端尾部的一个PDZ结合基序（-EDIV）。体外蛋白质相互作用实验表明，NIDD的C末端尾部区域与nNOS的PDZ结构域特异性相互作用。利用缺失突变体进行的实验证实了这种对PDZ结构域的依赖性，并且通过对转染了NIDD和nNOS的COS - 7细胞进行共沉降实验进一步证实了这种相互作用。在大鼠大脑中，NIDD和nNOS在突触体和突触质膜组分中均有富集，并且存在于脂筏和突触后致密物组分中。通过对培养的皮质神经元中的蛋白质进行双重染色，也观察到了这些蛋白质的共定位。因此，尽管从它们mRNA表达的分布情况来看，共定位区域有限，但NIDD和nNOS在大脑中确实存在共定位。最重要的是，NIDD和nNOS的共转染增加了产生NO的nNOS活性。这些结果表明，NIDD通过将nNOS定位于突触后膜，在突触后位点对NO信号通路的调节中发挥着重要作用。
Targeting of neuronal nitric-oxide synthase (nNOS) to appropriate sites in a cell is mediated by interactions with its PDZ domain and plays an important role in specifying the sites of reaction of nitric oxide (NO) in the central nervous system. Here we report the identification and characterization of a novel nNOS-interacting DHHC domain-containing protein with dendritic mRNA (NIDD) (GenBank™ accession number AB098078), which increases nNOS enzyme activity by targeting the nNOS to the synaptic plasma membrane in a PDZ domain-dependent manner. The deduced NIDD protein consisted of 392 amino acid residues and possessed five transmembrane segments, a zinc finger DHHC domain, and a PDZ-binding motif (-EDIV) at its C-terminal tail. In vitro pull-down assays suggested that the C-terminal tail region of NIDD specifically interacted with the PDZ domain of nNOS. The PDZ dependence was confirmed by an experiment using a deletion mutant, and the interaction was further confirmed by co-sedimentation assays using COS-7 cells transfected with NIDD and nNOS. Both NIDD and nNOS were enriched in synaptosome and synaptic plasma membrane fractions and were present in the lipid raft and postsynaptic density fractions in the rat brain. Co-localization of these proteins was also observed by double staining of the proteins in cultured cortical neurons. Thus, NIDD and nNOS were co-localized in the brain, although the colocalizing regions were restricted, as indicated by the distribution of their mRNA expression. Most important, co-transfection of NIDD and nNOS increased NO-producing nNOS activity. These results suggested that NIDD plays an important role in the regulation of the NO signaling pathway at postsynaptic sites through targeting of nNOS to the postsynaptic membrane.