IDENTIFICATION OF NOVEL EFFECTORS FOR THE RAP 1 GTP-ASE

近年来，Rap1 GTPase在信号转导中的作用急剧扩大。Rap1最初是作为Ras信号转导的拮抗剂被分离出来的，现已被发现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。组成性激活的Rap1在人类结节性硬化中的作用，Rap1在60%的人类胶质瘤中过度表达或激活，以及最近发现的egf诱导的Rap1激活剂c3G与乳腺癌细胞中Crk-Cbl复合物的关联表明，Rap1也可能在人类肿瘤发生中发挥作用。本提案的长期目标是确定Rap1的新型下游信号伙伴。Rap1 cDNA将用于利用酵母双杂交试验探测小鼠脑cDNA文库，以寻找编码激活Rap1的潜在结合蛋白的cDNA。PCR和DNA序列分析将用于鉴定潜在Rap1效应蛋白。Rap1突变体与鉴定蛋白的体内外结合研究将用于鉴定真正的效应蛋白。新的Rap1效应物的鉴定和表征将提供Rap1许多不同功能之间的联系，并将有助于阐明Rap1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.