AMPA receptor synaptic targeting regulated by stargazin interactions with the Golgi-resident PDZ protein nPIST
AMPA receptor synaptic targeting regulated by stargazin interactions with the Golgi-resident PDZ protein nPIST
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DOI：
10.1523/jneurosci.1255-04.2004
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发表时间：
2004-08-25
影响因子：
5.3
通讯作者：
Chetkovich, DM
中科院分区：
文献类型：
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作者：
Cuadra, AE;Kuo, SH;Chetkovich, DM
突触AMPA受体（AMPARs）的调控在大脑突触强度的改变中起着关键作用。Stargazin是一种与ampar相互作用的蛋白，对突触ampar的聚集和调控至关重要。Stargazin通过其胞外结构域与AMPAR相互作用，并通过其c端PDZ结合基序与PDZ[突触后密度-95 (PSD-95)/大盘(Dlg)/带闭合-1 (ZO-1)]蛋白相互作用，这些相互作用是Stargazin和AMPAR突触靶向所必需的。通过研究stargazin突变体在培养海马神经元中的表达，我们在stargazin胞质C端发现了一个与残基243 - 283对应的新结构域，该结构域也是stargazin和AMPAR突触聚类所必需的。为了鉴定与stargazin突触聚类结构域相互作用的蛋白质，我们进行了酵母双杂交实验，发现该stargazin结构域与nist（与TC10特异性相互作用的蛋白质的神经元异构体）结合，这是一种富含高尔基蛋白的蛋白质，涉及跨膜蛋白的运输。通过原位杂交、免疫组织化学、共免疫沉淀研究和生化分离，我们发现stargazin和nist在大脑中共定位并相互作用。最后，通过研究AMPAR在转染海马神经元中的聚类，我们发现过表达的nist增强了AMPAR突触的聚类，而转染显性阴性的nist则减弱了AMPAR突触的聚类。这些研究发现了AMPAR突触聚集所必需的一个新的stargazin结构域，并表明nist和stargazin相互作用在AMPAR转运到突触中起着关键作用。
Regulation of AMPA receptors (AMPARs) at synapses plays a critical role in alterations of synaptic strength in the brain. Stargazin, an AMPAR-interacting protein, is critical for clustering and regulation of synaptic AMPARs. Stargazin interacts with AMPARs via its extracellular domain and with PDZ [ postsynaptic density-95 (PSD-95)/Discs large (Dlg)/zona occludens-1 (ZO-1)] proteins via its C-terminal PDZ-binding motif, and these interactions are necessary for stargazin and AMPAR synaptic targeting. By studying the expression of stargazin mutant constructs in cultured hippocampal neurons, we identified a novel domain corresponding to residues 243 - 283 within the cytoplasmic C terminus of stargazin that is also required for stargazin and AMPAR synaptic clustering. To identify proteins that interact with this stargazin synaptic clustering domain, we performed a yeast two-hybrid assay and found that this stargazin domain binds to nPIST ( neuronal isoform of protein-interacting specifically with TC10), a Golgi-enriched protein implicated in trafficking of transmembrane proteins. Using in situ hybridization, immunohistochemistry, coimmunoprecipitation studies, and biochemical fractionation, we found that stargazin and nPIST colocalize and interact in the brain. Finally, by studying AMPAR clustering in transfected hippocampal neurons, we found that overexpression of nPIST enhances AMPAR synaptic clustering, whereas transfection of a dominant-negative nPIST construct attenuates AMPAR synaptic clustering. These studies identify a novel stargazin domain necessary for synaptic clustering of AMPARs and suggest that nPIST and stargazin interactions play a critical role in AMPAR trafficking to the synapse.