| Neurotrophic factors is a family of secreted polypeptide growth factors which could promote the development and survival of certain neuronal populations both in the peripheral and in the central nervous system. It is a target-derived polypeptide, essential for development and maintenance of peripheral sympathetic and neural crest-derived sensory neurons as well as adult neurons in the brain. Some of the neurotrophic factors may be therapeutically beneficial for patients with nerve injury or some neurological disorders such as Alzheimer's disease and Parkinson's disease.The biological effects of neurotrophic factors are mediated by their high-affinity ligand-receptor which present on the surface of those responsive neurons. According to the distinct components of receptors and different chemeical properties of ligands, neurotrophic factors could be classified to several families, for example GDNF and NGF family. The biological effects of GDNF, NGF and EGF were mediated by their corresponding receptor RET, TrkA and EGFR respectively. Although the ligands are totally different, all of these receptors belong to the family of receptor tyrosine kinases(RTKs). After binding with ligands, the RTKs would dimerize, simultaneously autophosphorylated and activated and then trigger the downstream signaling pathway. Since distinct signal transduction pathways are determined by the specific interaction between RTKs and their downstream effectors or regulators, it will be a possibly good strategy to investigate on the downstream molecules to better understand thecomplexities of receptor signaling networks.In present study, the human brain cDNA library was screened by using yeast two-hybrid system to search for new intracellular substrates or regulatory proteins of RET,EGFR and TrkA.The main results of our research are as follows:1. The intracellular part of RET receptor was fused to LexA and used as a bait to screen a human brain LexA two-hybrid cDNA library, and 274 positive clones were obtained. Among them, 21 plamids were selected and identified to be SH2-B and some other gene fragments. The interaction between RET and SH2-B was confirmed by using p-galactosidase activity assay in yeast. When overexpressed in PC 12 cells, wild type SH2-B could co-immunoprecipitated with RET in response to GDNF while SH2-B mutant R555E could not. It was observed that overexpression of wild type SH2-B enhanced GDNF-induced neurite growth while overexpression of SH2-B mutant R555E could inhibit it. Our results suggest that SH2-B could interact with RET in PC 12 cells stimulated with GDNF, and the interaction may be involved with the differentiation of PC 12 cells induced by GDNF.2. The intracellular part of EGFR was fused to LexA and used as a bait to screen a human brain LexA two-hybrid cDNA library, and 112 positive clones were obtained. Among them, 6 plamids were selected and identified to be dokl and some other gene fragments. The interaction between EGFR and dokl was confirmed by using p-galactosidase activity assay in yeast. Moreover, we found that the interaction of EGFR with dokl was mediated by the PTB domain since the dokl PTB could bind with EGFR while the dokl A PTB could not. After alanine mutagenesis scanning study, together with yeast two-hybrid analysis, our results demonstrated that the amino acid residue 73 Y, 76L, 77R and 92R in the dokl PTB domain may be critical forthe interactaction of doklwith and EGFR.3. The intracellular part of TrkA was fused to LexA and used as a bait to screen a human brain LexA two-hybrid cDNA library, and 269 positive clones were obtained. Among them, 11 plamids were selected and identified to be TrkAlc-BPl and some other gene fragments. The interaction between TrkAIC-BPl and TrkAIC was confirmed by using p-galactosidase activityassay and co-immunoprecipitation experiment in yeast. Our results suggest that TrkA -BP1 is a new TrkA-binding protein and it may be a new member of rho-GAP family since the protein sequence analysis revealed that it contains a...
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