Isotype-specific analysis of Ras-subfamily member signalling using xanthine nucleotide selective mutants

Ras鸟嘌呤核苷酸结合蛋白（GTPases）是细胞内信号转导网络的关键调控因子。在其他功能中，它们转导来自活化的细胞表面受体的信号，最终导致生物反应的调节，包括增殖、分化、凋亡、衰老和细胞运动。迄今为止，对Ras功能的研究主要依赖于组成活性或阻断Ras变体的异源表达。同样，下游ras效应通路的功能研究也广泛涉及使用中间蛋白的突变版本。尽管这种方法无疑有助于揭示Ras生物学的一般原理，但此类研究的生理学相关性仍是许多研究人员关注的问题。此外，这些实验提供了很少的信息，个别Ras物种在不同下游效应物的生理激活的特异性。我们的目标是创建和开发一个系统，允许研究野生类型的信号中继，“正常”Ras。该策略是基于生成稳定表达的细胞系。对黄嘌呤核苷酸（XNPs）核苷酸特异性改变的Ras蛋白，但其他功能特性不受影响。用XNPs灌注渗透细胞将使我们能够操纵单个ras亚家族成员的激活状态，并研究它们的下游细胞效应。使用这种方法，我们打算破译单个ras亚家族成员在三种已建立的ras效应物Raf， PI3K和Ral-GDS的激活中的效力。

Ras guanine nucleotide-binding proteins (GTPases) are key regulators in the intracellular signal transduction network. Amongst other functions, they transduce signals from activated cell surface receptors, eventually leading to the modulation of biological responses including proliferation, differentiation, apoptosis, senescence and cell motility. To date, the study of Ras function has mostly relied on the heterologous expression of constitutively active or blocking Ras variants. Similarly, functional investigation of downstream Ras-effector pathways has widely involved use of mutant versions of intermediary proteins. Although this approach has undoubtedly helped unravel general principles of Ras biology, the physiological relevance of such studies is a concern to many investigators. Moreover, such experiments provide little information on the specificity of individual Ras species in the physiological activation of distinct downstream effectors. Our aim is to create and exploit a system allowing the study of signal relay by wild type, "normal" Ras. The strategy is based on the generation of cell lines stably expressing. Ras proteins with altered nucleotide specificity towards xanthine nucleotids (XNPs) but with otherwise unaffected funtional properties. Perfusion of permeabilised cells with XNPs will allow us to manipulate the activation status of individual Ras-subfamily members and study their downstream cellular effects. Using this approach we intend to decipher the potency of individual Ras-subfamily members in the activation of the three established Ras-effectors Raf, PI3K and Ral-GDS.