A类清道夫受体N端第1～27位氨基酸胞浆结构域缺失对功能的影响

The Truncation 1 to 27 Amino Acid Residues of the N-Terminal Cytoplasmic Domain Mediate the Function of Scavenger Receptor A

目的确定A类清道夫受体胞浆域中的信号序列,探讨其对于受体功能的影响。方法构建A类清道夫受体N端第1～27位氨基酸胞浆结构域缺失体的表达质粒,并将其经脂质体介导瞬时转染入CHO细胞中。用Westernblot检测A类清道夫受体在细胞中的表达,并用流式细胞仪定量检测转染后的蛋白表达效率。用DiI标记的乙酰化低密度脂蛋白与转染的细胞共孵育,激光共聚焦显微镜下观察乙酰化低密度脂蛋白在细胞内的分布,并进行荧光定量分析。结果A类清道夫受体N端第1～27位氨基酸胞浆结构域缺失体可大量表达于转染细胞膜和细胞浆。在高浓度乙酰化低密度脂蛋白刺激下,A类清道夫受体N端第1～27位氨基酸胞浆结构域缺失体的细胞内定位发生变化,也可存在于转染细胞核内。A类清道夫受体N端第1～27位氨基酸胞浆结构域缺失体摄取DiI标记乙酰化低密度脂蛋白的能力较全长相比下降约60%,细胞粘附能力较全长A类清道夫受体相比则下降约8.9%。结论A类清道夫受体的N端第1～27位氨基酸结构对于A类清道夫受体在细胞内的定位表达、结合和摄取配体以及调节细胞粘附的功能具有重要的作用。

Aim To elucidate the role of the motifs in the cytoplasmic domain of scavenger receptor A （SR-A）. Methods A SR-A DNA mutant （ SR-AΔ1-27 ） was constructedted dy truncating the 1 to 27 amino acid residues of the N-terminal cytoplasmic domain. SR-A and SR-AΔ1-27 were transfected into CHO cells by Lipofectamine 2000. Western blot, laser confocal microscopy, flow cytometry and cell adhesion assay were used additionally. Results Compared with SR-A, expression of the SR-AΔ1-27 was increased and amount of cell-associated Dil-labeled ac-LDL in SR-AΔ1-27 expressing cells were almost reduced 80%. SR-AΔ1-27 was able to assemble in the plasma membrane and cytoplasm. SR-AΔ1-27 could bind its lipoprotein ligands but decline to internalize the bound ligands greatly. Incited by Ac-LDL, SR-AΔ1-27 located not only in membrane and plasma, but also in the nuclear. The SR-AΔ1-27 display decreased adhesion. Conclusion The 1 to 27 amino acid residues of the N-terminal cytoplasmic domain mediate SR-A expresion and location, lipid uptake and cell adhesion.