NEUROTROPHIN RECEPTOR INTERACTIONS

研究将检查75 kDa神经营养因子受体p75/NTR和P75/NTR的几种结构同源物的信号传导机制和功能。实验将检验神经营养因子通过涉及刺激超氧化物产生的小G蛋白Rac的p75/NTR依赖性激活的信号传导途径激活NF-κ B的假设。p75/NTR的一个推定的新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基因的小鼠成纤维细胞中的p75/NTR加工，评估蛋白酶TACE可能参与可溶性p75/NTR胞外域从细胞表面释放。将表征p75/NTR的两种结构同源物，通过用合适的表达载体转染各种哺乳动物细胞系来表达这些蛋白。同源物NRH 1将在CHO细胞中表达，并评估其结合神经营养因子的能力。评估NRH 1介导的信号传导激活NF-κ B和/或Rac的能力，评估NRH 1作为异聚体p75/NTR受体复合物亚基的能力，评估NRH 1作为异聚体p75/NTR受体复合物亚基的能力。可能存在的哺乳动物同源爪蟾NRH 1将寻求cDNA文库筛选。NRH 2编码类似于推定的TACE介导的P75/NTR切割产物的蛋白质。将评估NRH 2作为p75 rt/NTR信号传导的显性负性抑制剂的能力。将通过小鼠中的基因靶向突变评估NRH 2（和NRH 1，如果存在于小鼠中）的功能。通过cDNA文库筛选寻找可能存在的具有p75/NTR样配体结合的NRH 2剪接变体。将在非洲爪蟾和小鼠中检查P75/NTR、NRH 1和NRH 2的发育表达模式，以评估是否存在功能性相互作用的机会。

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.