荧光互补与光能量共振转移检测B类G蛋白偶联受体PAC1二聚化

Researching Homologous Dimerization of G Protein Coupling Receptor PAC1 through BRET and BiFC

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。

Gprotein couple receptors （GPCRs） are the biggest super family membrane receptors. PAC 1 belongs to the B family of GPCRs and is pituitary adenosine acid cyclization enzyme excited peptide （PACAP） spe cific receptors, mediating PACAP neural protection function, which is one of the important targets for drug devel opment to diseases of the nervous system. Dimerization or oligomerization is a common phenomenon to GPCRs. But there is no report homologus dimerization or oligomerization for PAC 1 at present. In order to verify PAC 1 dimerization, we use BRET to test CHO cells which are cotransfected PAC 1Rluc and PAC 1EYFP with different density gradient. The result presents obviously BRET signal by adding coelenterazine h. While BiFC test shows that CHO cells which are cotransfecte PAC 1EYFP/N and PAC 1EYFP/C appear complete EYFP fluorescent sig nal. Western blot test also shows that cells which high expressing PAC1 contain macromolecules ofPAC1 dimer. So PAC 1 can normally form homologous dimerization. This discovery will lay the novel theoretical foundation for the subsequent drug development, and offer illumination and reference for researching other GPCRs.