Cloning and expression of ligand-gated ion-channel receptor L2 in central nervous system
Cloning and expression of ligand-gated ion-channel receptor L2 in central nervous system
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DOI：
10.1016/j.bbrc.2005.07.079
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发表时间：
2005-09-23
影响因子：
3.1
通讯作者：
Sugimoto, T
中科院分区：
文献类型：
--
作者：
Houtani, T;Munemoto, Y;Sugimoto, T
通过计算机辅助搜索程序在人类基因组数据库中鉴定了配体门控离子通道型孤儿受体（L2，根据锌离子的存在也称为ZAC）。L2蛋白与5-羟色胺受体5 HT 3A和5 HT 313具有部分同源性。我们从人尾状核中克隆了L2 cDNA，并对其外显子-内含子结构进行了如下表征：（1）L2蛋白有四个跨膜区（M1 -M4），在M3和M4之间有一个长的胞质环。(2)该序列在包括黑猩猩、狗、牛和负鼠在内的物种中是保守的。(3)9个外显子形成其蛋白质编码区，特别是外显子5对应于氨基末端侧的二硫键区。我们的分析使用多个组织cDNA面板显示，至少有两个剪接变体的L2 mRNA的存在。cDNA PCR扩增结果显示，L2 mRNA在脑、胰腺、肝、肺、心、肾和骨骼肌等组织中均有表达，而5 HT 3A mRNA在脑、心、胎盘、肺、肾、胰腺和骨骼肌中均有表达，5 HT 3B mRNA在脑、肾和骨骼肌中均有表达，表明这些受体在组织中的表达具有不同的意义。L2 mRNA和蛋白在脑内的区域表达。RT-PCR研究证实L2 mRNA在成年大脑的海马、纹状体、杏仁核和丘脑中表达。L2蛋白通过使用抗肽抗体进行免疫定位。免疫组织化学染色显示L2样免疫反应阳性细胞主要表达于海马CA 3区锥体细胞和齿状回多形层。我们用GFP融合蛋白报告系统分析了L2蛋白在HEK 293细胞中的表达。Western印迹显示L2蛋白在胞外侧赋予糖链。在转染的HEK 293细胞中，细胞膜和细胞内斑点被GFP密集地标记，表明选择性地分派到最终目的地。(c)2005年爱思唯尔公司All rights reserved.
An orphan receptor of ligand-gated ion-channel type (L2, also termed ZAC according to the presence of zinc ion for channel activation) was identified by computer-assisted search programs on human genome database. The L2 protein shares partial homology with serotonin receptors 5HT3A and 5HT313. We have cloned L2 cDNA derived from human caudate nucleus and characterized the exon-intron structure as follows: (1) The L2 protein has four transmembrane regions (M1 -M4) and a long cytoplasmic loop between M3 and M4. (2) The sequence is conserved in species including chimpanzee, dog, cow, and opossum. (3) Nine exons form its protein-coding region and especially exon 5 corresponds to a disulfide bond region on the amino-terminal side. Our analysis using multiple tissue cDNA panels revealed that at least two splicing variants of L2 mRNA are present. The cDNA PCR amplification study revealed that L2 mRNA is expressed in tissues including brain, pancreas, liver, lung, heart, kidney, and skeletal muscle while 5HT3A mRNA could be detected in brain, heart, placenta, lung, kidney, pancreas, and skeletal muscle, and 5HT3B mRNA in brain, kidney, and skeletal muscle, suggesting different significance in tissue expression of these receptors. Regional expression of L2 mRNA and protein was examined in brain. The RT-PCR studies confirmed L2 mRNA expression in hippocampus, striatum, amygdala, and thalamus in adult brain. The L2 protein was immunolocalized by using antipeptide antibodies. Inummostained tissue sections revealed that L2-like immunoreactivity was dominantly expressed in the hippocampal CA3 pyramidal cells and in the polymorphic layer of the dentate gyrus. We analyzed the expression of L2 protein in HEK293 cells using GFP fusion protein reporter system. Western blots revealed that L2 protein confers sugar chains on the extracellular side. In transfected HEK293 cells, cellular membranes and intracellular puncta were densely labeled with GFP, suggesting selective dispatch to the final destination. (c) 2005 Elsevier Inc. All rights reserved.