P75NTR signal transduction suppressed by BFAR and p75NTR interactions

Hongmei Li,Huili Shi,Keke Huo

doi:10.1007/s11427-012-4306-y

Abstract

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.

Highlights

P75NTR is a low-affinity nerve growth factor receptor, which promotes cell proliferation as a positive modulator of high-affinity receptor tyrosine kinase receptor A (TrkA), as well as binds with cell ligands to induce apoptosis and mediate death signals
Nerve growth factor (NGF) has two structurally unrelated receptors: tyrosine kinase receptor A (TrkA), which selectively binds to NGF, and p75 neurotrophin receptor (p75NTR), which binds all members of the neurotrophin family (NGF, BDNF, NT-3, and NT-4/5)
Missouri, USA); DUAL membrane yeast two-hybrid system kit, human embryonic brain cDNA library, and the HTX β-galactosidase assay kit were purchased from Dualsystems Biotech (Schlieren, Switzerland); Isopropyl β-D-1-Thiogalactopyranoside (IPTG), protease inhibitor cocktails, and Phenylmethanesulfonyl fluoride (PMSF) were products of Amresco (Solon, Ohio, USA); Ni-NTA agarose beads were purchased from Qiagen (Hilden, Germany); glutathione sepharose 4B beads were purchased from Pharmacia Biotech (Stockholm, Sweden)

Summary

Introduction

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. P75NTR belongs to the type I single-pass transmembrane glycoprotein, with a molecular weight of 75 kD This protein contains a signal peptide, an extracellular domain, a transmembrane domain, and an intracellular domain. To detect the p75NTR signaling pathway and to analyze nervous disease mechanisms, p75NTR was utilized as a bait to screen a human embryonic brain cDNA library using the split-ubiquitin membrane yeast two-hybrid system. The assay revealed bi-functional apoptosis regulator (BFAR), a novel interaction protein of p75NTR. Results from the study revealed some biological functions of p75NTR and BFAR interactions

Methods

Results

Conclusion