NEUROTROPHIN RECEPTOR INTERACTIONS

研究将研究75 kDa神经营养因子受体p75/NTR和p75/NTR的几个结构同源物的信号传导机制和功能。实验将验证神经营养因子通过信号通路激活NF-kappaB的假设，该信号通路涉及p75/NTR依赖性小G蛋白Rac的激活，从而刺激超氧化物的产生。一个新的p75/NTR配体将被克隆和功能表征。通过比较野生型小鼠成纤维细胞和缺乏TACE基因小鼠成纤维细胞的p75/NTR加工过程，将评估蛋白酶TACE可能参与细胞表面可溶性p75/NTR外结构域的释放。p75/NTR的两个结构同源物将被表征，通过转染不同的哺乳动物细胞系，用合适的表达载体表达这些蛋白。同源NRH1将在CHO细胞中表达，并评估其结合神经营养因子的能力。将评估NRH1介导的信号激活NF-kappaB和/或Rac的能力，评估NRH1作为异质p75/NTR受体复合物亚基的能力，评估NRH1作为异质p75/NTR受体复合物亚基的能力。通过cDNA文库筛选，寻找可能存在的非洲爪蟾NRH1的哺乳动物同源基因。NRH2编码一种类似于假定的tace介导的P75/NTR裂解产物的蛋白质。NRH2作为p75rt/NTR信号的显性负性抑制剂的能力将被评估。NRH2（如果小鼠中存在NRH1）的功能将通过小鼠基因靶向突变来评估。通过cDNA文库筛选，寻找可能存在的具有p75/NTR样配体结合的NRH2剪接变体。我们将在爪蟾和小鼠中检测P75/NTR、NRH1和NRH2的发育表达模式，以评估是否存在功能相互作用的机会。

Studies will examine the signaling mechanisms and function of the 75 kDa neurotrophin receptor, p75/NTR and several structural homologs of P75/NTR. Experiments will test the hypothesis that neurotrophins activate NF-kappaB by a signaling pathway involving p75/NTR- dependent activation of the small G protein Rac, which stimulates superoxide production. A putative novel ligand for p75/NTR will be cloned and functionally characterized. The possible involvement of t he protease TACE in release of a soluble p75/NTR ectodomain from the cell surface will be assessed, by comparing p75/NTR processing in wildtype mouse fibroblasts and fibroblasts from mice lacking the TACE gene. Two structural homologs of p75/NTR will be characterized, expressing these proteins by transfection of various mammalian cell lines with suitable expression vectors. Homolog NRH1 will be expressed in CHO cells and assessed for ability to bind neurotrophins. The capacity of NRH1- mediated signaling to activate NF-kappaB and/or Rac will be assessed,, and the capacity of NRH1 to act as a subunit of a heteromeric p75/NTR receptor complex will be assessed, and the capacity of NRH1 to act as a subunit of a heteromeric p75/NTR receptor complex will be assessed. The possible existence of a mammalian homolog of Xenopus NRH1 will be sought by cDNA library screening. NRH2 encodes a protein resembling the product of the putative TACE-mediated cleavage of P75/NTR. The capacity of NRH2 to act as a dominant negative inhibitor of p75rt/NTR signaling will be assessed. Functions of NRH2 (and NRH1 if present in mice) will be assessed by gene-targeted mutations in mice. The possible existence of a NRH2 splice variant possessing a p75/NTR- like ligand binding will be sought by cDNA library screening. The patterns of developmental expression of P75/NTR, NRH1 and NRH2 will be examined in Xenopus and mice, to assess whether the opportunity for functional interactions exist.