Expression of functional G protein-coupled receptors in photoreceptors of transgenic Xenopus laevis.
Expression of functional G protein-coupled receptors in photoreceptors of transgenic Xenopus laevis.
复制标题
转基因非洲爪蟾光感受器中功能性 G 蛋白偶联受体的表达。
DOI：
10.1021/bi051386z
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发表时间：
2005
期刊：
影响因子：
--
通讯作者：
Li,Ning
中科院分区：
文献类型：
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作者：
Zhang,Li;Salom,David;He,Jianhua;Okun,Alex;Ballesteros,Juan;Palczewski,Krzysztof;Li,Ning
G蛋白偶联受体（GPCR）是最大的跨膜信号转导蛋白超家族，然而，唯一已知的GPCR晶体结构是视紫红质。这种差异反映了难以产生足够数量和质量的纯化气相化学还原样品。视紫红质是视网膜视杆神经元的光受体，在脊椎动物的视网膜中大量产生。我们使用转基因非洲爪蟾将这些视网膜视杆细胞转化为生物反应器，成功地生产了20个模型GPCR。受体聚集在视杆外节，并均匀糖基化。配体和[35 S]GTPγS结合试验证实了5 HT 1A和EDG 1GPCR正确折叠并具有功能。通过利用所有GPCR构建体共有的视紫红质C-末端免疫亲和标签高度纯化5 HT 1AR。我们还开发了一种自动化系统，每天可以产生数百只转基因蝌蚪。这种表达方法可以扩展到其他动物模型系统，并成为生产大量GPCR和其他膜蛋白用于药理学和结构研究的通用方法。
G protein-coupled receptors (GPCRs) constitute the largest superfamily of transmembrane signaling proteins; however, the only known GPCR crystal structure is that of rhodopsin. This disparity reflects the difficulty in generating purified GPCR samples of sufficient quantity and quality. Rhodopsin, the light receptor of retinal rod neurons, is produced in large amounts of homogeneous quality in the vertebrate retina. We used transgenicXenopus laevisto convert these retina rod cells into bioreactors to successfully produce 20 model GPCRs. The receptors accumulated in rod outer segments and were homogeneously glycosylated. Ligand and [35S]GTPγS binding assays of the 5HT1Aand EDG1GPCRs confirmed that they were properly folded and functional. 5HT1AR was highly purified by taking advantage of the rhodopsin C-terminal immunoaffinity tag common to all GPCR constructs. We have also developed an automated system that can generate hundreds of transgenic tadpoles per day. This expression approach could be extended to other animal model systems and become a general method for the production of large numbers of GPCRs and other membrane proteins for pharmacological and structural studies.