人类GABARAPL2基因的亚细胞定位

为了对GABARAPL2（GABAA受体相关蛋白相似蛋白2）基因的功能进行初步分析，首先通过同源比较的方法将序列与其同源物进行比较，发现GABARAPL2的氨基酸序列与GABARAP（GABAA受体相关蛋白）高度同源，而GABARAP已证实通过结合细胞骨架的微蛋白，使GABAA受体聚集，定位在细胞膜上，本文采用PCR法从人脑组织的cDNA文库中扩增出GABARAPL2的cDNA，克隆至T质粒载体中进行测序验证，然后以此为模板引物中引入酶切位点再次PCR，扩增出GABARAPL2的开放阅读框，并将其插入到加强型绿色荧光蛋白融合表达载体EGFP中，将绿色荧光蛋白标记的GABARAPL2和GABARAP分别转染HLF细胞株，结果两种蛋白的分布情况基本一致，在细胞质内和核内均有分布，而且核内的分布较胞质为多，结构功能域分析表明，GABARAPL2含有蛋白激酶C磷酸化位点和酪氨酸激酶磷酸化位点，可能通过磷酸化参与细胞骨架的变化，结论 GABARAPL2和GABARAP不仅在胞质中作为受体相关蛋白协助受体的聚集、定位，还参与体内许多其它重要的生理过程。

Subcellular localization of GABARAPL2

In order to study the function of human GABARAPL2,homologous comparison was done by multiple alignment of amino acid sequence.It showed that GABARAPL2 was highly homology to GABARAP(GABA A-receptor-associated protein),which was recently reported to mediate the localization of GABA A receptor to cytoskeleton by binding to both GABA A receptor and microtubule.GABARAPL2 was predicated to contain two protein kinase C phosphorylation sites and one tyrosine kinase phosphorylation site.Phosphorylated GABARAPL2 may be involved in the cytoskeleton changes and consequent modulation of the function of GABA receptors.To construct fusion vector,the cDNA of GABARAPL2 was amplified by PCR,which was then clone into T vector and was sequenced afterwards.To elucidate the subcellular distrbution of GABARAPL2 and GABARAP,their open reading frame were amplified dy PCR and then subcloned into expression vector EGFP.No matter where the proteins were fused to ,C-terminus or N-terminus of EGFP,bright green fluorescent signals of the tagged proteins could be visualized by confocal laser-scanning microscope when they were expressed in HLF cells.The results showed that GABARAP and GABARAPL2 were mostly distributed in nuclear,and a little in cytoplasm of HLF cell.This new finding indicated that GABARAPL2 might exert other functions except localizing GABA A receptor in cellular membrane by linking the receptor to microtubules,which may be also true for GABARAP.