HRas C-Terminus in Membrane Trafficking and Targeting

膜结合是一个由脂质修饰启动和维持的过程，与GTP结合一样有效地控制Ras蛋白的功能。尽管这很重要，但我们对膜系固如何影响信号传导知之甚少。最近的研究表明，HRas转运到质膜是通过一个以前未被注意到的、尚未被表征的囊泡膜途径进行的，该途径需要法尼基修饰和附近的c端氨基酸残基。没有特定的蛋白质可能护送HRas从这些内部膜到其工作位置，即质膜，已被确定。据报道，到达质膜后，HRas定位于富含胆固醇和鞘脂的亚结构域（“筏”）。体外方法预测，法尼脂和棕榈酰脂的不同物理性质将使这些脂质在调节HRas筏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 partitioning被提出用于提高信号传导效率，但是当它与膜相关时，关于分区是否限制或促进特定调节或效应蛋白进入HRas，存在相互矛盾的信息。HRas的新变体已经被制造出来，并表明脂质和c端残基的变化可以改变完整细胞中的HRas信号通路。这一新证据表明，附着在HRas上的c端区域和脂质不仅仅是一个被动的锚定结构域，而且在质膜中HRas的运输和组织过程中可能发挥比当前模型想象的更积极的作用。本项目的目的是研究HRas的c端氨基酸、法尼基和棕榈酸酯如何单独参与HRas膜结合的转运和质膜相互作用阶段。将实现三个目标。A.将设计HRas的变体，以表征HRas控制其离开内膜的特征，并确定将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HRas进入质膜亚结构域位点的影响将通过表达具有这些特性的独特和新颖组合的蛋白质来确定。C. HRas信号发生激活和终止的膜位置将通过HRas与调节蛋白和效应物的共定位或共免疫沉淀来确定。这些研究将使用生化共免疫沉淀和膜分离技术，以及膜片段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用的系统，HRas棕榈酰化可以发生，更重要的是，通过监测变异蛋白在NIH3T3细胞中引起病灶形成的能力，可以测量分子特性变化的功能影响。这些结果将使我们更清楚地了解HRas是如何被纳入细胞膜的，并使我们能够开发出操纵HRas与膜子域相互作用的方法。该信息还将确定新的亚域选择性靶向序列，其他蛋白质，如HRas效应物，可以被重定向到细胞表面的特定位点。这些研究将帮助我们了解HRas膜结合结构域的哪些特性会影响生物活性，并朝着学习如何引导这种相互作用产生有益结果的长期目标前进。

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.