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中的关键调节因子。在其他功能中，它们从活化的细胞表面受体传递信号，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，Ras功能的研究主要依赖于组成型活性或阻断Ras变体的异源表达。类似地，下游Ras效应通路的功能研究广泛涉及中间蛋白的突变形式的使用。虽然这种方法无疑有助于解开Ras生物学的一般原理，但这些研究的生理相关性是许多研究人员关注的问题。此外，这样的实验提供了很少的信息，在不同的下游效应物的生理激活的具体Ras物种。我们的目标是创建和开发一个系统，允许研究野生型，“正常”Ras的信号中继。该策略基于稳定表达的细胞系的产生。对黄嘌呤核苷酸（XNP）具有改变的核苷酸特异性但其他功能特性不受影响的Ras蛋白。用XNP灌注透化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其下游细胞效应。使用这种方法，我们打算破译单个Ras亚家族成员在激活三个已建立的Ras效应子Raf、PI 3 K和Ral-GDS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.