论文部分内容阅读
G蛋白偶联受体(G-protein couple receptors,GPCRs)是最大的超家族膜受体,其中它的B家族成员垂体腺苷酸环化酶激活肽1(PAC1)是垂体腺苷酸环化酶激动多肽(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。二聚化或寡聚化是GPCRs普遍存在的现象,但是目前尚没有关于PAC1形成同源二聚体或寡聚体的报道。为了验证PAC1也能进行同源二聚化,该文采用生物发光能量转移(bioluminescence resonance energy transfer,BRET)方法进行检测,结果显示不同浓度梯度共转染中国仓鼠卵巢细胞(Chinese hamster ovary,CHO)的PAC1-Rluc与PAC1-EYFP重组载体,在底物腔肠素h(coelenterazine h)作用下呈现明显的BRET信号。双分子荧光互补(BiFC)检测显示,带有EYFP N端基因标记的PAC1与带有EYFP C端基因标记的PAC1共转染CHO细胞,能呈现完整的EYFP荧光信号。同时,Western blot检测也显示,高表达PAC1的细胞中可检测到PAC1二聚体的大分子。因此,PAC1是能够进行正常同源二聚化的,这个发现将为后续神经损伤药物的开发奠定全新的理论基础,同时也为其他GPCRs同源二聚化的研究起到启发和借鉴作用。
G-protein couple receptors (GPCRs) are the largest superfamily membrane receptors in which pituitary adenylate cyclase activating peptide 1 (PAC1), a member of the B family, is a pituitary adenylate cyclase The specific receptor of PACAP mediates the functions of PACAP neuroprotection and is one of the important targets of drug development in nervous system diseases. Dimerization or oligomerization is a common phenomenon of GPCRs, but no reports have been reported on the formation of homodimers or oligomers of PAC1. In order to verify that PAC1 can also perform homodimerization, this paper uses the method of bioluminescence resonance energy transfer (BRET) for detection. The results showed that Chinese hamster ovary (CHO) PAC1-Rluc and PAC1-EYFP recombination vector showed obvious BRET signal under the action of substrate coelenterazine h. Bimolecular Fluorescence Complementation (BiFC) assay showed that PAC1 with EYFP N-terminal gene and PAC1 with EYFP C-terminal gene were co-transfected into CHO cells, showing a complete EYFP fluorescence signal. In the meantime, Western blot also showed that PAC1 dimer was detected in PAC1-overexpressing cells. Therefore, PAC1 is capable of normal homodimerization, and this discovery will lay a new theoretical basis for the development of subsequent nerve injury drugs. At the same time, it will also serve as inspiration and reference for the research on homodimerization of other GPCRs.