The Rho Family GTPases in Integrin Signaling

描述(申请人提供)：整合素介导的黏附调节生长因子和细胞因子受体下游的许多信号的传递，调节细胞的增殖、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域(或CEMM)，这些微域富含脂质修饰的蛋白质和鞘磷脂。我们以前的工作表明，在正常的、锚定依赖的细胞类型中，作为细胞运动和增殖的关键介质，小GTPase Rac可以独立于黏附而被生化激活，但其靶向质膜和与效应器的相互作用仍然需要整合素的参与。RAC膜结合部位位于CEMms内，整合素控制其定位，使细胞从底物上分离，触发CEMms从质膜快速清除并移动到细胞内隔室。复制细胞触发CEMMs的胞吐作用，恢复质膜定位。CEMMS的内化是由小窝介导的，需要一小部分小窝蛋白-1在酪氨酸14上磷酸化。相反，CEMMS在复制过程中的胞吐需要小GTP酶Ral A。基于这些初步和已发表的数据，我们建议：1.研究RAC激活和膜靶向的机制，以确定CEMMs是否与鸟嘌呤核苷酸交换因子协同作用，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.探讨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作用，研究Ral下游的途径和激活Ral的机制。3.探讨磷酸化Y14小窝蛋白触发小窝细胞内吞作用的机制。将识别和研究与小窝蛋白或小窝细胞膜相互作用的蛋白质。还将研究内吞作用中对动力素的需求。

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.