Analysis of Trk receptor signalosome in neuronal cells

为了阐明神经营养因子包括神经生长因子和神经营养因子的促分化和促存活作用的信号转导机制，我们建立了酵母双杂交系统，分别以神经生长因子和神经营养因子的高亲和力受体TrkA和TrkB的胞内结构域为诱饵蛋白。我们从大鼠脑文库和小鼠脑文库中分离到与TrkA或TrkB胞内区结合的独立克隆20-30个。从与这些克隆的核苷酸序列的数据库搜索中，我们发现了七个与Trk受体信号有关的新蛋白质。这7个蛋白分别是：Geporrin、Rho GTP酶激活蛋白(RhoGAP)、核苷二磷酸激酶B、核孔蛋白153、核孔蛋白p58、κB基序结合磷蛋白和105kD蛋白样蛋白。Geporrin、Rho GTP酶激活蛋白(RhoGAP)和核苷二磷酸激酶B都能与TrkA和TrkB的胞内区结合，表明这三种蛋白都结合到TrkA和TrkB的同源区。Rho GTP酶激活蛋白(RhoGAP)和两个核孔蛋白可与野生型TrkA和激酶缺陷型TrkA的胞内区结合。κB基序结合的磷酸蛋白和105kD的类似蛋白只能结合到蛋白激酶缺陷的TrkA的胞内区。我们正在研究这七种蛋白在神经细胞中的结合活性及其在信号转导机制中的功能。

In order to clarify the signal transduction mechanisms underlying the differentiation-promoting and survival-promoting effects of neurotrophins including NGF and BDNF, we performed the yeast two-hybrid system using the intracellular domains of TrkA and TrkB, the high-affinity receptors of NGF and BDNF, respectively, as the bait protein. We isolated independent 20-30 clones which bind to the intracellular region of TrkA or TrkB, from rat brain cDNA library or mouse, brain cDNA library. From the database search with nucleotide sequences of these clones, we identified seven new proteins involved in the Trk receptor signal. The seven proteins are gephyrin, Rho GTPase activating protein(RhoGAP), nucleoside diphosphate kinase B, nucleoporin 153, nucleoporin p58, κB motif-binding phosphoprotein, and 105kD kinase like protein. Gephyrin, Rho GTPase activating protein(RhoGAP) and nucleoside diphosphate kinase B could bind to the intracellular regions of both TrkA and TrkB, indicating that these three proteins bind to the homology region between TrkA and TrkB. Rho GTPase activating protein(RhoGAP) and two nucleoporins could bind to the intracellular regions of both wild-type TrkA and kinase-defective TrkA. κB motif-binding phosphoprotein and 105kD kinase like protein could bind only to the intracellular regions of kinase-defective TrkA. We are now investigating the binding activities and the functions in the signal transduction mechanism of the seven proteins in neuronal cells.