NEUROTROPHIN RECEPTOR INTERACTIONS

研究将探讨75 kDa神经营养素受体p75/NTR的信号机制和功能，以及P75/NTR的几个结构同源物。实验将验证这一假说，即神经营养因子通过一种信号通路激活NF-kappaB，该信号通路涉及p75/NTR依赖的小G蛋白Rac的激活，从而刺激超氧化物的产生。一个可能的p75/NTR新配体将被克隆并进行功能鉴定。通过比较野生型小鼠成纤维细胞和缺乏TACE基因的小鼠成纤维细胞中p75/NTR的处理过程，将评估TACE在细胞表面释放可溶性p75/NTR胞外区的可能参与。P75/NTR的两个结构同源物将被鉴定，通过用合适的表达载体导入不同的哺乳动物细胞系来表达这些蛋白。同系物NRH1将在CHO细胞中表达，并评估其结合神经营养素的能力。将评估NRH1介导的信号激活NF-kappaB和/或RAC的能力，以及NRH1作为异构体p75/NTR受体复合体的亚单位的能力，以及NRH1作为异构体p75/NTR受体复合体的亚单位的能力。通过对文库的筛选，寻找非洲爪哇NRH1可能存在的哺乳动物同源物。NRH2编码一种类似于TACE介导的P75/NTR切割产物的蛋白质。将评估NRH2作为p75rt/NTR信号转导的主要负抑制物的能力。NRH2(以及NRH1，如果在小鼠中存在)的功能将通过小鼠的基因靶向突变来评估。通过文库筛选寻找可能存在的具有p75/NTR样配体结合的NRH2剪接变异体。将研究P75/NTR、NRH1和NRH2在非洲爪哇和小鼠中的发育表达模式，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.