BACKGROUND Autism spectrum disorder (ASD) has a complex genetic etiology. Some symptoms and mutated genes, including neuroligin (NLGN), neurexin (NRXN), and SH3 and multiple ankyrin repeat domains protein (SHANK), are shared by schizophrenia and ASD. Little is known about the molecular pathogenesis of ASD. One of the possible molecular pathogenesis is an imbalance of excitatory and inhibitory receptors linked with the NLGN-PSD-95-SHANK complex via postsynaptic density protein/Drosophila disc large tumor suppressor/zonula occludens-1 protein (PDZ) binding. In the present study, we focused on GPR85 as a candidate gene for ASD because the C-terminal amino acid sequence of GPR85 [Thr-Cys-Val-Ile (YCVI)] is classified as a type II PDZ-binding motif, and GPR85 is a risk factor for schizophrenia. GPR85 is an orphan receptor that regulates neural and synaptic plasticity and modulates diverse behaviors, including learning and memory. While searching for molecules that associate with GPR85, we found that GPR85 was associated with postsynaptic density protein (PSD)-95 linked with NLGN in the brain. METHODS We examined the proteins that associate with the C-terminal sequence of GPR85 by pull-down assay and immunoblot analysis and searched for a mutation of the GPR85 gene in patients with ASD. We used immunostaining to examine the intracellular localization of mutated GPR85 and its influence on the morphology of cells and neurons. RESULTS The C-terminal sequence of GPR85 interacted with PSD-95 at PDZ1, while NLGN interacted with PSD-95 at PDZ3. Two male patients with ASD from independent Japanese families possessed inherited missense mutations at conserved sites in GPR85: one had T1033C (M152T) and the other had G1239T (V221L). These mutations were located in a domain related to G protein interaction and signal transduction. In contrast to wild-type GPR85, mutated GPR85 was more preferentially accumulated, causing endoplasmic reticulum stress, and disturbed the dendrite formation of hippocampal neurons. CONCLUSIONS GPR85 associated with the PSD-95 linked with NLGN, which is related to ASD. GPR85 carrying the mutations detected in ASD patients disturbed dendrite formation that could be the candidate for molecular pathogenesis of ASD through the associated NLGN-PSD-95 receptor complex.

中文翻译：

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GPR85与PSD-95-神经白蛋白复合体和自闭症谱系障碍的关联：分子分析。

背景技术自闭症谱系障碍（ASD）具有复杂的遗传病因。精神分裂症和ASD共有一些症状和突变的基因，包括神经胶蛋白（NLGN），神经毒素（NRXN）和SH3以及多个锚蛋白重复域蛋白（SHANK）。关于ASD的分子发病机理知之甚少。可能的分子发病机制之一是通过突触后密度蛋白/果蝇圆盘大肿瘤抑制物/闭合小带-1蛋白（PDZ）结合与NLGN-PSD-95-SHANK复合物连接的兴奋性受体和抑制性受体失衡。在本研究中，我们将GPR85作为ASD的候选基因，因为GPR85的C末端氨基酸序列[Thr-Cys-Val-Ile（YCVI）]被归为II型PDZ结合基序，而GPR85是精神分裂症的危险因素。GPR85是一种孤儿受体，可调节神经和突触可塑性并调节包括学习和记忆在内的多种行为。在寻找与GPR85相关的分子时，我们发现GPR85与大脑中与NLGN相关的突触后密度蛋白（PSD）-95相关。方法我们通过下拉测定和免疫印迹分析检查了与GPR85 C端序列相关的蛋白质，并寻找ASD患者GPR85基因的突变。我们使用免疫染色检查了突变的GPR85在细胞内的定位及其对细胞和神经元形态的影响。结果GPR85的C端序列在PDZ1与PSD-95相互作用，而NLGN在PDZ3与PSD-95相互作用。两名来自日本独立家庭的ASD男性患者在GPR85的保守位点具有遗传的错义突变：一名患有T1033C（M152T），另一名患有G1239T（V221L）。这些突变位于与G蛋白相互作用和信号转导相关的域中。与野生型GPR85相反，突变的GPR85更优先积累，引起内质网应激，并干扰海马神经元的树突形成。结论GPR85与与NLGN相关的PSD-95相关，这与ASD有关。携带在ASD患者中检测到的突变的GPR85干扰了树突的形成，该树突可能通过相关的NLGN-PSD-95受体复合物成为ASD分子发病机制的候选者。这些突变位于与G蛋白相互作用和信号转导相关的域中。与野生型GPR85相反，突变的GPR85更优先积累，引起内质网应激，并干扰海马神经元的树突形成。结论GPR85与与NLGN相关的PSD-95相关，这与ASD有关。携带在ASD患者中检测到的突变的GPR85干扰了树突的形成，该树突可能通过相关的NLGN-PSD-95受体复合物成为ASD分子发病机制的候选者。这些突变位于与G蛋白相互作用和信号转导相关的域中。与野生型GPR85相反，突变的GPR85更优先积累，引起内质网应激，并干扰海马神经元的树突形成。结论GPR85与与NLGN相关的PSD-95相关，这与ASD有关。携带在ASD患者中检测到的突变的GPR85干扰了树突的形成，该树突可能通过相关的NLGN-PSD-95受体复合物成为ASD分子发病机制的候选者。结论GPR85与与NLGN相关的PSD-95相关，这与ASD有关。携带在ASD患者中检测到的突变的GPR85干扰了树突的形成，该树突可能通过相关的NLGN-PSD-95受体复合物成为ASD分子发病机制的候选者。结论GPR85与与NLGN相关的PSD-95相关，这与ASD有关。携带在ASD患者中检测到的突变的GPR85干扰了树突的形成，该树突可能通过相关的NLGN-PSD-95受体复合物成为ASD分子发病机制的候选者。