IDENTIFICATION OF NOVEL EFFECTORS FOR THE RAP 1 GTP-ASE

最近，Rap 1 GTdR在信号转导中的作用已经显著扩大。 Rap 1最初作为Ras信号转导的拮抗剂被分离，现已发现Rap 1参与吞噬细胞的形态发生、IL-2转录、血小板功能和呼吸爆发的产生。 结构性激活Rap 1在人类结节性硬化症中的作用，Rap 1过表达或激活在60%的人类胶质瘤中的存在，以及最近发现EGF诱导的Rap 1激活剂c3 G与乳腺癌细胞中的Crk-Cbl复合物的关联表明Rap 1也可能在人类肿瘤发生中发挥作用。 该提案的长期目标是鉴定Rap 1的新型下游信号伙伴。 Rap 1 cDNA将用于使用酵母双杂交测定来探测小鼠脑cDNA文库，以找到编码激活的Rap 1的潜在结合蛋白的cDNA。 PCR和DNA序列分析将用于识别潜在的Rap 1效应蛋白。 将进行Rap 1突变体和鉴定的蛋白质之间的体外和体内结合研究以鉴定真实的效应蛋白。 新Rap 1效应子的鉴定和表征将提供Rap 1的许多不同功能之间的联系，并将有助于阐明Rap 1在乳腺癌中EGF受体信号通路中的作用。

Recently, the role of the Rap1 GTPase in signal transduction has expanded dramatically. Originally isolated as an antagonist of Ras signal transduction, Rap1 has been found to participate in morphogenesis, IL-2 transcription, platelet function, and the production of the respiratory burst in phagocytic cells. The role of contitutively activated Rap1 in human tuberous sclerosis, the presence of Rap1 overexpression or activation in 60 percent of human gliomas, and the recent finding of the EGF-induced association of the Rap1 activator, c3G, with the Crk-Cbl complex in breast cancer cells suggests that Rap1 may also play a role in human oncogenesis. The long-term objective of this proposal is the identification of novel downstream signaling partners for Rap1. Rap1 cDNAs will be used to probe a mouse brain cDNA library using the yeast two-hybrid assay to find cDNAs that encode potential binding proteins for activated Rap1. PCR and DNA sequence analysis will be used to identify proteins that are potential Rap1 effectors. In vitro and in vivo binding studies between Rap1 mutants and identified proteins will be done to identify authentic effector proteins. Identification and characterization of novel Rap1 effectors will provide links between the many different functions of Rap1 and will help elucidate the role of Rap1 in the EGF receptor signaling pathway in breast cancer.