人缓激肽受体（B₂R）和内皮细胞分化因子受体（edg-1）在酵母中的功能表达及定位研究

【作者】 杨光孝；

【导师】 沈大棱；

【作者基本信息】 复旦大学 ， 遗传学， 2004， 博士

【摘要】 G蛋白偶联受体（GPCRs）在制药领域中占有极其重要的地位，市售西药中有1／2的作用靶点是GPCR，在中药中以GPCRs为靶点进行药物筛选和分子机理研究尚未见报道。在遗传背景清楚的宿主中表达GPCR并纯化其蛋白不仅可以用作受体亚型、嵌合受体、突变受体等药理和生化分析的研究中，而且还可以分析6PCR与G蛋白的相互作用以及由此所引起的信号传导，寻找可以作为药物的先导物、孤儿GPCR的配体等。甲醇酵母表达系统是发展迅速、应用广泛的一种真核表达系统与大肠杆菌等原核表达系统相比，它能对表达的蛋白进行翻译后的修饰和加工；与杆状病毒、哺乳动物细胞表达系统相比，它又具有培养成本低、产量高、便于产物的分离和纯化等优点。甲醇酵母中以可调控的AOX1强启动子控制下表达异源基因，其产物较酿酒酵母表达的更接近真核生物甚至人类。因此利用甲醇酵母表达G蛋白偶联受体，不但可以进行快速、高通量的配体和药物先导物的筛选；而且可以提供足量的GPCR蛋白进行蛋白结构、受体亚型、嵌合受体、突变受体的药理和生化分析。因此，以甲醇酵母为宿主的表达体系具有其他系统不可比拟的优点。内皮细胞分化因子（edg-1）和缓激肽受体（B₂R）同属于G蛋白偶联的受体超家族，都与内皮型一氧化氮合成酶（eNOS）等多种信号途径偶联。其中edg-1与心血管疾病、免疫系统疾病密切相关：而B₂R与心血管疾病及各种炎症密切相关。edg-1和B₂R都尚未在酵母细胞中表达。为了开发一种操作简便，成本低廉的以edg-1和B₂R为靶点的药物筛选系统，以期获得抗心血管疾病的药物或其他用途的药物。我们用RT-PCR方法从人脐静脉内皮细胞中克隆了edg-1受体和B_R基因，首次利用甲醇酵母Pichia pastoris来表达edg-1和B₂R受体，并对表达受体的细胞定位和功能进行了研究。在B₂R基因的C-端接上绿色荧光蛋白基因（EGFP）作报告基因，构建了EGFP融合的B₂R-EGFP基因，将B₂R-EGFP融合基因克隆到甲醇酵母整合型表达载体pPIC9k上。利用pPIC9k的α信号肽序列促进膜受体的跨膜分布，电击转化P．pastoris GS115菌株，抗生素G418浓度梯度筛选高抗性转化子，重组菌株用甲醇诱导表达。流式细胞仪分析表明B₂R-EGFP在GS115中获得了很强的表达；Western印迹杂交结果证实了表达产物为B₂R-EGFP融合受体蛋白；在激光共聚焦显微镜下重组菌株观察到很强的绿色荧光；虽然表达的受体蛋白也有内膜分布，但免疫荧光分析证实表达的受体蛋白大量定位到了细胞膜上。免疫荧光分析还证明了表达的B₂R受体蛋白具有同配体结合的功能。同样构建pPIC9k-edg-1-EGFP表达载体，电转化P．pastoris GS115菌株，但不筛选高G418抗性转化子，甲醇诱导表达。诱导的转化子流式细胞仪检测表明edg-1-EGFP在GS115中获得了很强的表达；Western印迹杂交结果证实了表达产物为edg-1-EGFP融合受体蛋白；在激光共聚焦显微镜下重组菌株发出很强的绿色荧光；免疫荧光分析证实表达的重组受体大量定位于酵母细胞膜上。鞘氨醇-1-磷酸是edg-1受体的高亲和性配体，克隆并原核表达了鞘氨醇激酶基因（SphK），利用纯化的鞘氨醇激酶制备γ-³²P标记的鞘氨醇-1-磷酸(S-1-³²P)，放射性标记的配体结合分析表明重组edg-1受体具有配体结合功能。鞘氨醇-1-磷酸明显降低重组酵母GS115：：pPIC9K-edg-1-EGFP的生长速度，表明重组edg-1受体和酵母细胞内信号途径偶联，受体具有生物学功能。我们的研究为进一步利用B₂R和edg-1受体建立药物筛选系统打下了基础。更多还原

【Abstract】 G protein-coupled receptors（GPCRs） are the largest class of targets for modern drugs by virtue of their roles in the regulation of cellular functions. Heterologous production of GPCRs in a defined host background has been established as an important and valuable tool for the pharmacological and biochemical analysis of defined receptor subtypes, as well as chimeric or mutant receptors. Furthermore, the analysis of G protein interaction and subsequent signal transduction, the detection of ’lead’ compounds in the search for new drugs and of ligands for orphan GPCRs by high-throughput screens, as well as the purification of receptor protein suitable for biochemical, biophysical and/or structural studies are important goals. Here, due to their ability of high level production, easy manipulation, and low costs, yeast-based expression systems have proven very attractive. Unlike Escherichia coli, yeasts have the potential to perform eukaryotic post-translational modifications, such as N-glycosylation, which may affect receptor function. The methylotropic yeast strain, Pichia pastoris, has a strong inducible promoter and it is less prone to hyperglycosylation than Saccharomyces cerevisiae. Therefore, recombinant P. pastoris has been developed as an excellent host for the expression of foreign GPCRs.The human endothelial differentiation gene l（edg-l） and bradykinin B2 receptor （B₂R） belong to the large family of G-protein-coupled receptors. In order to develop a convenient and rapid assay for testing compounds which might be effective as edg-1 and B₂R receptors agonists or antagonists, the gene of edg-1 and B₂R receptors were expressed in the methylotrophic yeast Pichia pastoris.The expression plasmids were constructed in which the edg-1 or B₂R gene is under the control of the highly inducible promoter of P. pastoris alcohol oxidase 1 gene, the green fluorescent protein（GFP） gene were introduced at the C-terminal of the edg-1 or B₂R gene to permit easy detection.Fluorescence activated cell sorting （FACS） analysis and Western blotanalysis revealed that the edg-1 and B₂R recombinant receptor proteins were distinctly expressed. The results were further confirmed by Confocal microscopy which produced bright green fluorescence.The localization of the recombinant edg-1 or B₂R receptor proteins were proved by immunofluorescence microscopy which indicated a distinct expression of the edg-1 or B₂R in the plasma membrane of the transformed yeast cells.The binding of bradykinin ligand to the B₂R recombinant receptor proteins were also proved by immunofluorescence microscopy which exhibited a high affinities of bradykinin to the GS115-B2-EGFP cell membrane.In radioligand binding analysis, the edg-1 recombinant receptor proteins also showed high affinities of S— 1 -³²P.Exogenous SPP treatment, inhibited GS115-edg-1-EGFP cells proliferation.In conclusion, we provide compelling evidence that the edg-1 and B₂R receptor were functionally expressed in P. pastoris and localization on the plasma membrane for the first time. These results strongly suggest that the GS115-B2-EGFP or the GS115-edg-1-EGFP cell line is a useful tool to study the effect of potential pharmacological agents that may modulate the function of edg-1 or B₂R receptor.更多还原