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I. Background of brain-derived neurotrophic factor (brain-derived neurotrophic factor, BDNF) is a neurotrophic factor important member of the family, in the vertebrate nervous system plays an important role. BDNF receptors can be divided into two categories: low-affinity neurotrophin receptor p75 (p75NTR) and the high affinity tyrosine kinase receptor TrkB. Research shows, BDNF biological effects on the body mainly through TrkB receptors implementation. BDNF and TrkB receptor binding, receptor intracellular domain tyrosine residues are activated, which in turn activates mitogen-activated protein kinase (mitogen-activated protein kinase, MAPK) signaling pathway, phosphatidylinositol 3 - kinase (phosphatidylinositol 3-kinase, PI3K) signaling pathway and phospholipase CY (phospholipase Cy.PLC-γ) signaling pathway and a series of intracellular signal transduction pathways, thereby BDNF-mediated biological functions. RanBPM (Ran-binding protein in the microtubule-organizing center, also known as RanBP9) is widely distributed in almost all mammalian tissues, especially the heart, brain and kidney has a relatively high expression levels. RanBPM having the characteristics with a variety of molecules, is considered a stand molecules. Furthermore, RanBPM cell surface receptors capable of MET, LFA-1, L1, Axl / Sky and Plexin-A so integration in various intracellular signaling pathways. Second, the research purposes despite RanBPM have been shown to NGF receptor TrkA and p75NTR and binding, but RanBPM in neurotrophin-mediated effects on biological function is unclear. Therefore, in this study, we mainly solve the three levels of problems: First, RanBPM whether expressed in the mammalian central nervous system, the most abundant neurotrophin receptor TrkB binding? Secondly, if the two can be combined, RanBPM whether able to participate in BDNF-mediated TrkB downstream signaling pathways? Finally, RanBPM is able to participate in BDNF / TrkB-mediated biological functions, such as neuronal morphogenesis and survival? Third, experimental methods 1. various expression plasmid 2. Rat RanBPM siRNA plasmid and efficient determination of three knockout rat hippocampal neurons in primary culture and immunofluorescence staining 4. immunoprecipitation and Western blot5.PC12 cell lines stably expressing TrkB (PC1210 cell lines ) build 6.BDNF mediated TrkB, TrkBY515 phosphorylation and signaling molecules MAPK, Akt activation detector 7. assessment PC1210 hippocampal neuronal cell differentiation and dendritic morphogenesis detection methods 8. assessment PC1210 Apoptosis Detection means of four experimental results a confirmed RanBPM is a newly discovered protein binding with TrkB we first constructed with HA tag human RanBPM cDNA eukaryotic expression plasmid (pcDNA3.1-HA-hRanBPM) and with Flag label rat TrkB cDNA eukaryotic expression plasmid (pcDNA3.1-Flag-rTrkB). Then through the co-immunoprecipitation and immunofluorescence cytochemistry method proved RanBPM and TrkB can be combined, and both at the subcellular level consistent positioning. 2, confirmed that TrkB tyrosine kinase region combined with RanBPM we first constructed in different regions of TrkB deletion mutants: ΔCT (missing the C-terminus) and ΔTK (missing tyrosine kinase domain and the C-terminus), through co-immunoprecipitation method Reference RanBPM binding with TrkB △ CT, but not combined with TrkB △ TK. Subsequently, we constructed a mutant TrkB.T1 (presence in the body of a truncated form of TrkB, intracellular membrane-proximal region containing only a little) and T1TK (the tyrosine kinase domain grafted on to TrkB.T1 ), and confirmed that RanBPM can not TrkB.T1 binding, but can be combined with T1TK. 3, confirmed that the kinase activity of TrkB on the importance of a combination of both, NT4 and BDNF and TrkB are able to promote the combination of Trk inhibitor K252a RanBPM can weaken RanBPM with TrkB combination. RanBPM and loss of kinase activity TrkB.KD weaker degree of integration. However, TrkB two ligands NT4 (30ng/ml), and BDNF (30 ng / ml) are able to significantly increase the TrkB / RanBPM binding capacity. 4, RanBPM does not affect the BDNF-mediated phosphorylation of TrkB and TrkBY515, but involved in regulating BDNF mediated MAPK and PI3K signal transduction studies confirm: Compared with the corresponding control groups, and overexpression of RanBPM not affect BDNF knockout RanBPM mediated phosphorylation of TrkB and TrkBY515. Transfected with empty vector pcDNA3.1 control group, over-expression of RanBPM can promote BDNF-mediated signaling molecules MAPK and Akt activation, and this effect was dose-dependent RanBPM; knocked out after endogenous RanBPM , compared with control siRNA, BDNF-mediated activation of MAPK and Akt are reduced; addition, RanBPM involved in regulating MAPK short duration and long-duration two kinds of signal transduction way. 5, RanBPM promote BDNF-mediated neuronal morphogenesis transfected with the empty vector pcDNA3.1 control group, can promote BDNF overexpression RanBPM PC1210 mediated cell differentiation, including the differentiation of the increase in the proportion and the average longest projection length increases; addition, overexpression of RanBPM can increase BDNF mediated hippocampal neuron dendrites total number and total neurite length. After removal of endogenous RanBPM knock, BDNF-mediated PC1210 cell differentiation and morphogenesis extent hippocampal neurons are inhibited. 6, RanBPM enhanced BDNF in the serum deprivation induced apoptosis protective effect in regard to join BDNF case, pcDNA3.1 empty vector transfected control group, the group RanBPM overexpression induced apoptosis in serum deprivation number significantly reduced; while endogenous RanBPM knockout after the number of apoptotic cells was significantly increased compared with control siRNA, indicating that RanBPM able to participate in BDNF serum deprivation induced apoptosis aspects of protection. V. Conclusion The scaffold protein RanBPM experiment with TrkB tyrosine kinase involved in regulating the interaction region BDNF mediated TrkB signaling pathways downstream of two main-MAPK and PI3K signaling pathway, and thus participate in BDNF / TrkB-mediated neural Yuan morphogenesis and cell survival and other biological functions.
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