The Rho Family GTPases in Integrin Signaling

描述（由申请人提供）：整合素介导的粘附调节生长因子和细胞因子受体下游许多信号的传递，这些信号调节细胞增殖、迁移、分化和存活。这些信号事件中的许多依赖于富含胆固醇的膜微区（或CEMM），其富含脂质修饰的蛋白质和鞘脂。我们以前的工作表明，在正常的贴壁依赖性细胞类型中，小的GTCRac，细胞运动和增殖的关键介质，可以独立于粘附被生化激活，但其靶向质膜和与效应器的相互作用仍然需要整合素参与。Rac膜结合位点在CEMM内，并且整联蛋白控制它们的定位，使得细胞从基质的脱离触发CEMM从质膜的快速清除并移动到细胞内区室。再接种细胞触发CEMM的胞吐作用以恢复质膜定位。CEMM的内化由小窝介导，并且需要酪氨酸14上的小部分小窝蛋白-1的磷酸化。相反，再铺板期间CEMM的胞吐作用需要小GTP酶Ral A。基于这些初步的和已发表的数据，我们建议：1。研究Rac激活和膜靶向的机制，以确定CEMM是否与鸟嘌呤核苷酸交换因子合作，RhoGDI在Rac激活中的作用，以及体内动力学是否需要加速Rac和RhoGDI解离的“置换因子”。2.研究RalA在CEMM胞吐中的作用。我们将进一步测试Ral在整合素刺激的胞吐中的参与，研究Ral下游的途径和Ral活化的机制。3.研究磷酸化Y14小窝蛋白触发小窝内吞作用的机制。与小窝蛋白或小窝膜相互作用的蛋白质将被鉴定和研究。还将研究内吞作用对发动蛋白的需求。

DESCRIPTION (provided by applicant): Integrin-mediated adhesion regulates the transmission of many signals downstream from growth factor and cytokine receptors that regulate cell proliferation, migration, differentiation and survival. Many of these signaling events depend on cholesterol-enriched membrane microdomains (or CEMMs) that are enriched in lipid-modified proteins and sphingolipids. Our previous work showed that in normal, anchorage-dependent cell types, the small GTPase Rac, a critical mediator of cell motility and proliferation, can be biochemically activated independent of adhesion but its targeting to the plasma membrane and interaction with effectors still require integrin engagement. Rac membrane binding sites are within CEMMs and integrins control their localization such that detachment of cells from the substratum triggers rapid clearance of CEMMs from the plasma membrane and movement to an intracellular compartment. Replating cells triggers exocytosis of CEMMs to restore plasma membrane localization. Internalization of CEMMs is mediated by caveolae and requires phosphorylation of a small fraction of caveolin-1 on tyrosine 14. Conversely, exocytosis of CEMMs during replating requires the small GTPases Ral A. Based on this preliminary and published data, we propose to: 1. Investigate the mechanism of Rac activation and membrane targeting to determine whether CEMMs cooperate with guanine nucleotide exchange factors, what is the role of RhoGDI in Rac activation, and whether in vivo dynamics requires "displacement factors" that accelerate dissociation of Rac and RhoGDI. 2. Investigate the role of RalA in CEMM exocytosis. We will further test the involvement of Ral in integrin- stimulated exocytosis, investigate pathways downstream of Ral and mechanisms of Ral activation. 3. Investigate mechanisms by which phospho-Y14 caveolin triggers caveolar endocytosis. Proteins that interact with caveolin or caveolar membranes will be identified and studied. The requirement for dynamin in endocytosis will also be investigated.