B族G蛋白偶联受体PAC1的N端基序HSDCIF对其二聚化和上膜运输的影响

发布时间：2018-05-15 18:30

本文选题：B族G蛋白偶联受体(GPCRs) + PAC　；参考：《中国生物化学与分子生物学报》2013年09期

【摘要】：B族G蛋白偶联受体(G protein-coupled receptors,GPCRs)PAC1是垂体腺苷酸环化酶激活多肽(pituitary adenylate cyclase activating polypeptide,PACAP)的特异受体,介导PACAP神经保护等功能,是神经系统疾病药物开发的重要靶点之一.HSDCIF(His-Ser-Asp-Cys-Ile-Phe)为位于PAC1的N端胞外1区(extracellar domain 1,EC1)的一段短肽序列,与特定负责激活PAC1受体的激动域PACAP(1-6)具有极高的同源性.利用基因敲除技术构建出缺陷HSDCIF基序的PAC1突变体(简称D-PAC1);利用基因工程原理和技术构建系列真核表达重组载体,包括融合了增强型黄色荧光蛋白(enhanced yellow fluorescent protein,EYFP)的表达载体D-PAC1-EYFP;用于生物发光能量转移(bioluminescence resonance energy transfer,BRET)检测的D-PAC1-Rluc;以及用于双分子荧光互补(bimolecular fluorescence complementation,BiFC)实验的D-PAC1-EYFP/N和D-PAC1-EYFP/C.免疫荧光检测(immunofluorescence assay)测定D-PAC1的表达;荧光共聚焦显微观察D-PAC1的细胞运输,然后通过Western印迹、BRET与BiFC方法来检测D-PAC1的二聚化情况,综合评价HSDCIF基序对PAC1二聚化和在细胞中定位的影响.检测结果显示,缺陷HSDCIF基序的突变体D-PAC1不能发生二聚化,也不能正常的进行上膜运输,而是滞留在内质网中,同时外源化学合成的寡肽HSDCIF可以竞争性地抑制正常PAC1的二聚化.
[Abstract]:The B G protein coupled receptor (G protein-coupled receptors, GPCRs) PAC1 is a specific receptor for pituitary adenylate cyclase activating polypeptide (pituitary adenylate cyclase activating polypeptide), which mediates the function of neuroprotection, which is one of the most important targets in the development of neurologic diseases. A short peptide sequence of the 1 region (Extracellar domain 1, EC1) in the N terminal of PAC1 is highly homologous to the specific PACAP (1-6) responsible for activating the PAC1 receptor in the PACAP (1-6). Using gene knockout technique to construct a PAC1 mutant of the defective HSDCIF based sequence (referred to as D-PAC1), and to construct a series of eukaryotic expression restructures using genetic engineering principles and techniques. The carrier, including the expression vector D-PAC1-EYFP that combines the enhanced yellow fluorescent protein (EYFP), and the D-PAC1-Rluc for the detection of the bioluminescence energy transfer (bioluminescence resonance energy transfer, BRET); and for the fluorescence complementation of the bioluminescence. D-PAC1-EYFP/N and D-PAC1-EYFP/C. immunofluorescence assay (immunofluorescence assay) were used to determine the expression of D-PAC1; fluorescence confocal microscopy was used to observe the cell transport of D-PAC1, and then the dimerization of D-PAC1 was detected by Western blotting, BRET and BiFC methods, and a comprehensive evaluation of HSDCIF sequence on PAC1 dimerization and localization in cells. The results show that the mutant D-PAC1 of the defective HSDCIF based sequence can not produce dimerization, nor does it normally carry on the upper membrane transport, but is detained in the endoplasmic reticulum, and the exogenous chemical synthesized oligopeptide HSDCIF can competitively inhibit the dimerization of normal PAC1.

【作者单位】：暨南大学生命科学技术学院生物工程研究所;广东医学院医学遗传学教研室;
【基金】：国家自然科学基金项目(No.31100545,No.31200679) 广东省自然科学基金项目(No.S2011010002931) 广东省科技计划项目(No.2011B031800388)资助~~
【分类号】：R363

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