Cuadra Adolfo E, Kuo Sheng-Han, Kawasaki Yoshimi, Bredt David S, Chetkovich Dane M
Davee Department of Neurology and Clinical Neurosciences, Northwestern University Medical School, Chicago, Illinois 60611-3008, USA.
J Neurosci. 2004 Aug 25;24(34):7491-502. doi: 10.1523/JNEUROSCI.1255-04.2004.
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.
突触处α-氨基-3-羟基-5-甲基-4-异恶唑丙酸受体（AMPARs）的调节在大脑突触强度改变中起关键作用。Stargazin是一种与AMPAR相互作用的蛋白质，对突触AMPARs的聚集和调节至关重要。Stargazin通过其细胞外结构域与AMPARs相互作用，并通过其C末端的PDZ[突触后致密物95（PSD-95）/盘状大蛋白（Dlg）/紧密连接蛋白1（ZO-1）]结合基序与PDZ蛋白相互作用，这些相互作用对于Stargazin和AMPAR的突触定位是必需的。通过研究Stargazin突变体构建体在培养的海马神经元中的表达，我们在Stargazin的细胞质C末端鉴定出一个对应于243-283位氨基酸残基的新结构域，该结构域对于Stargazin和AMPAR的突触聚集也是必需的。为了鉴定与这个Stargazin突触聚集结构域相互作用的蛋白质，我们进行了酵母双杂交实验，发现这个Stargazin结构域与nPIST（与TC10特异性相互作用的蛋白质的神经元异构体）结合，nPIST是一种富含高尔基体的蛋白质，与跨膜蛋白的运输有关。通过原位杂交、免疫组织化学、免疫共沉淀研究和生化分级分离，我们发现Stargazin和nPIST在大脑中共同定位并相互作用。最后，通过研究转染的海马神经元中AMPAR的聚集，我们发现nPIST的过表达增强了AMPAR的突触聚集，而转染显性负性nPIST构建体则减弱了AMPAR的突触聚集。这些研究鉴定出一个对AMPAR突触聚集必需的新的Stargazin结构域，并表明nPIST与Stargazin的相互作用在AMPAR向突触的运输中起关键作用。
