Neurotrophin Low Affinity Receptor P75ntr Signaling And Induction Of Apoptosis-related Genes Function

Low affinity neurotrophin receptor, p75NTR is a cell-surface glycoprotein that binds each of the neurotrophins with a similar low nanomoloar affinity. Because the intracellular domain of p75NTR has no kinase domain, the elucidation of the mechanism of its signal transduction is a hotspot. Structurally, p75NTR is a member of the tumor necrosis factor receptor (TNFR) superfamily, that shares unique extracellular cystein clusters and intracellular death domain containing about 80 amino acid residues. Death domains and death domain-like protein interaction motifs in many proteins may bind each other or to some adaptor proteins for signaling. p75NTR could initiate a signal transduction pathway independent of the Trks for its role in apoptosis. During the development of the nervous system, the destination of a considerable body of neurons is to death since not competing for enough prosurvival neurotrophins. Cell apotosis may lead to the markedly loss of neurons in some neurodegenerative diseases such as Alzheimer's disease and Parkinson's syndrome. To illustrate the molecular mechanism of p75NTR signal pathway is of a vital importance.First, our research is focused on the signal transduction of p75NTR. We use the yeast two-hybrid system to identify the interacting protein. The yeast two-hybrid assay is based on the fact that the interaction between a bait protein (fused to the DNA binding domain, BD) and a prey protein (fused to the activation domain, AD) creates a novel transcriptional activator to activate the downstream reporter genes. Results as follow:1, Use PCR technique to amplify the intracellular domain of p75NTR, named p75ICD, and insert it into the yeast expression vector, pAS2-1; Cotransform the pAS2-l-p75ICD and an AD fusion expression library of pACT2 into yeast strain Y190. After screens of triple nutrients(-Trp-Leu-His) and dual reporter genes(His, LacZ) and elimination of false positives, four transformants are to be further tested;2, By sequencing and blast analysis, one clone with a 2.7 kb insert has a highly homology with RanBPM, both of them have the same open reading frame;3, Co-transfecting green monkey kidney COS7 cells with plasmids encoding HA-tagged...