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

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.

The Effects of N-terminal HSDCIF Motif of Family B G-Protein-coupled Receptor PAC1 on Its Cell Surface Trafficking and Dimerization

PAC1 is a pituitary adenylate cyclase activating polypeptide (PACAP) receptor to mediate neural protection functions. It belongs to the B family of G protein coupled receptors (GPCRs), and is suggested as an important target for drug development for neurological diseases. The HSDCIF（His- Ser-Asp-Gly-Ile-Phe）functional motif located in the first N-terminal extracellular domain (EC1) of PAC1 has a high homology with PACAP (1-6). A PAC1 HISCIF motif deletion mutant named D-PAC1 was constructed. Vectors of D-PAC1- EYFP with enhanced yellow fluorescent protein (EYFP) and D-PAC1-Rluc were used for the bioluminescent energy transfer (BRET) assay; D-PAC1- EYFP/N and D-PAC1- EYFP/C were used for complements bimolecular fluorescence (BiFC). Immunofluorescence was used to detect D-PAC1 expression and fluorescence confocal microscopy was used for its cellular trafficking observation. Western blot, BRET and BiFC were used for detecting D-PAC1 homologous dimerization. The effects of chemically synthesized oligo peptide HSDCIF on the homologous dimerization of PAC1 were also detected. The results showed that D-PAC1 failed to traffick onto the cell membrane to form dimers, but retentioned in the endoplasmic reticulum. Chemically synthesized HSDCIF peptide competitively inhibited dimerization of PAC1.