HRas C-Terminus in Membrane Trafficking and Targeting

膜结合是由脂质修饰启动和维持的过程，它和GTP结合一样有效地控制RAS蛋白的功能。尽管这很重要，但人们对膜系留如何影响信号转导知之甚少。最近的研究表明，HRAs向质膜的转运是通过一条先前未被注意且仍未确定的囊泡内膜途径进行的，该途径需要法尼基修饰和附近的C-末端氨基酸残基。目前还没有确定可能将HRA从这些内膜护送到其工作位置--质膜的特定蛋白质。据报道，当HRAs到达质膜后，它会定位于富含胆固醇和鞘磷脂的亚区(“筏”)。体外方法预测，法尼基和棕榈醇基脂的不同物理性质将使这些脂类在调节HRAs支架插入方面发挥相反的作用，但在完整细胞中对HRAs的研究尚未得出明确的结果。RAFT分区被认为是为了提高信号转导效率，但关于分区是否限制或鼓励与膜相关的特定调节或效应蛋白对HRA的访问，存在相互矛盾的信息。HRAs的新变体已经被制作出来，并表明在完整的细胞中，脂类和C末端残基的变化可以改变HRAs的信号通路。这一新的证据表明，与HRA结合的C末端区域和脂质不仅仅是一个被动的锚定结构域，而且在HRA在质膜上的运输和组织过程中可能发挥比目前模型所设想的更积极的作用。本项目的目的是研究HRAs的C端氨基酸、法尼基和棕榈酸酯是如何分别参与HRAs膜结合的转运和质膜相互作用的。我们将追求三个目标。答：HRAs的变种将被设计用来描述控制其退出内膜的HRAs的特征，并识别将HRAs移动到细胞表面所涉及的囊泡路径和蛋白质。B.法尼基、棕榈酸酯和GTP结合对HRA分配到质膜亚域位置的影响将通过表达具有这些性质的独特和新颖组合的蛋白质来确定。C.HRAs信号的激活和终止所在的膜位置将通过HRAs与调节蛋白和效应器的共定位或共免疫沉淀来确定。这些研究将使用生化免疫共沉淀和膜分离技术，以及膜碎片的直接免疫荧光成像。主要的重点将是完整的细胞，因为这是目前唯一可以发生HRAS棕榈酰化的系统，更重要的是，能够通过监测变异蛋白导致NIH3T3细胞焦点形成的能力来衡量分子性质变化的功能影响。这些结果将使我们更清楚地了解HRAs是如何被结合到细胞膜上的，并使开发出操纵HRAs与膜亚结构域相互作用的方法。这些信息还将识别新的亚域选择性靶向序列，利用这些序列，其他蛋白质，如HRAS效应器，可以被重定向到细胞表面的特定位置。这些研究将帮助我们了解HRAs的膜结合结构域的哪些特性可以影响生物活性，并导致学习如何引导这种相互作用走向有益结果的长期目标。

Membrane binding, a process initiated and sustained by lipid modifications, controls the function of Ras proteins as potently as does GTP binding. Despite this importance, very little is known of how membrane tethering affects signaling. Recent studies indicate that HRas trafficking to the plasma membrane proceeds through a previously unnoticed, and still uncharacterized, vesicular endomembrane pathway that requires farnesyl modification and nearby C-terminal amino acid residues. No specific proteins that might escort HRas from these internal membranes to its working location, the plasma membrane, have been identified. Upon reaching the plasma membrane, HRas is reported to localize into cholesterol-and sphingolipid-rich subdomains ("rafts "). In vitro methods predict that the different physical properties of the farnesyl and palmitoyl lipids will give these lipids opposing roles in regulating HRas raft insertion, but studies with HRas in intact cells have not yielded clear results. Raft partitioning is proposed to increase signaling efficiency, but there is conflicting information on whether partitioning limits or encourages access of particular regulatory or effector proteins to HRas while it is associated with the membrane. Novel variants of HRas have been made, and show that changes in the lipids and residues of the C-terminus can alter HRas signaling pathways in intact cells. This new evidence indicates that the C-terminal region and lipids attached to HRas are more than a passive anchor domain, and are likely to play much more active roles during trafficking and organizing of HRas in the plasma membrane than current models envision. The objective of this project is to examine how the C-terminal amino acids, farnesyl group, and palmitates of HRas individually participate in both the trafficking and plasma membrane interaction phases of HRas membrane binding . Three aims will be pursued. A. Variants of HRas will be designed to characterize features of HRas that control its exit out of endomembranes, and to identify the vesicular paths and proteins involved in moving HRas to the cell surface. B. The influence of the farnesyl group, palmitates and GTP binding on partitioning of HRas into subdomain sites of plasma membrane will be established by expressing proteins with distinct and novel combinations of these properties. C. The membrane locations in which activation and termination of HRas signaling occurs will be identified by co-localization or co-immunoprecipitation of HRas with regulatory proteins and effectors. These studies will use biochemical co-immunoprecipitation and membrane isolation techniques, as well as direct immunofluorescence imaging of membrane fragments. The primary emphasis will be intact cells, as this is the only currently available system where HRas palmitoylation can occur, and more importantly, enables the functional impact of changes in molecular properties to be gauged by monitoring the ability of variant proteins to cause focus formation in NIH3T3 cells. The results will generate a clearer view of how HRas becomes incorporated into cellular membranes and enable development of methods to manipulate the interaction of HRas with membrane subdomains. This information will also identify new subdomain-selective targeting sequences with which other proteins, such as HRas effectors, can be re-directed to specific sites on the cell surface. These studies will help us understand what properties of the membrane-binding domain of HRas can influence biological activity and lead towards the long-term goal of learning how to guide that interaction toward beneficial outcomes.