State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai 200433, China.
Sci China Life Sci. 2012 Apr;55(4):367-74. doi: 10.1007/s11427-012-4306-y. Epub 2012 May 9.
p75NTR is a low-affinity nerve growth factor receptor, which promotes cell proliferation as a positive modulator of high-affinity receptor TrkA, as well as binds with cell ligands to induce apoptosis and mediate death signals. To analyze the regulatory mechanisms of p75NTR, the present study utilized a new membrane yeast two-hybrid system to screen a human fetal brain cDNA library. Results identified BFAR, a novel protein that interacts with p75NTR. Interaction specificity was verified by membrane yeast two-hybrid co-transformation assays, in vitro GST pull-down assays, and in vitro co-immunoprecipitation assays. The fluorescent subcellular localization assay revealed that the two proteins co-localized within the cytoplasm. BFAR overexpression in PC-12 and HEK293T cells inhibited the NFκB and JNK signaling pathway, as determined with the luciferase test. Co-transfected p75NTR and BFAR in HEK293T or PC-12 cells, respectively, increased the percentage of cells in the G2/M phase, decreased the number of S-phase cells, and did not change the number of G0/G1-phase cells.
p75NTR 是一种低亲和力的神经生长因子受体，作为高亲和力受体 TrkA 的正调节剂，促进细胞增殖，以及与细胞配体结合诱导细胞凋亡和传递死亡信号。为了分析 p75NTR 的调节机制，本研究利用新型膜酵母双杂交系统筛选人胎脑 cDNA 文库。结果鉴定出 BFAR，这是一种与 p75NTR 相互作用的新型蛋白。通过膜酵母双杂交共转化实验、体外 GST 下拉实验和体外共免疫沉淀实验验证了相互作用的特异性。荧光亚细胞定位实验显示这两种蛋白在细胞质中共定位。在 PC-12 和 HEK293T 细胞中转染 BFAR 过表达可通过荧光素酶实验抑制 NFκB 和 JNK 信号通路。分别在 HEK293T 或 PC-12 细胞中转染共表达的 p75NTR 和 BFAR，可增加 G2/M 期细胞的百分比，减少 S 期细胞的数量，而不改变 G0/G1 期细胞的数量。
