REGULATION OF INTEGRIN GLYCOSYLATION AND FUNCTION BY RAS

β 1整合素受体的异常功能在肿瘤的发生发展中起着关键作用。 炎症性疾病如类风湿性关节炎的发展。在 因此，密集的研究集中在定义分子 调节整合素活性的途径。在许多情况下，整合素 整合素的功能是由整合素对配体的亲和力改变引起的，而不是由 受体表达的变化。最近的研究表明， 细胞内信号分子，如ras，可以调节整合素 构象，导致改变的细胞粘附性和/或运动性。的 指导β 1整联蛋白构象变化的ras分子效应物 还没有很好的定义。作为本研究提案的一部分提供的数据 支持RAS在β 1整联蛋白调节中的作用。异位 上皮细胞中显性负性ras亚型（N17 ras）的表达 诱导β 1整联蛋白的异常糖基化。相应地， 表达异常糖基化的β 1整合素显示出许多缺陷， 整合素介导的细胞应答包括细胞与基质的附着、细胞 铺展、粘着斑形成、细胞运动和酪氨酸激酶 介导的信号转导。因此，提出了一个中心假设，即ras 调节高尔基体介导的β 1整联蛋白的糖基化，反过来， 糖基化修饰直接影响整联蛋白功能。这 假设包括三个主要的调查领域。第一，什么是 ras活性的改变诱导细胞内 整合素糖基化？在目的1中，凝集素亲和印迹和碳水化合物 测序将用于确定N17 ras介导的细胞内的变化。 β 1整合素的碳水化合物组成。这样的实验很可能 鉴定作为ras调节候选物的高尔基体糖基转移酶， 从而阐明Ras依赖性整联蛋白所涉及的分子事件 糖基化其次，整合素糖基化的变化是否直接影响 功能？ 在目标2中，将开发允许表达 N17 ras的表达与变体整合素糖型的表达解偶联。 预期这些实验将证实糖基化的差异， 而不是NI 7 ras的其他下游效应，直接负责 整合素信号传导的缺陷。最后，是否存在介导的胰岛素水平变化？ 糖基化和功能参与炎症？ 在目标3中，N17 ras将 被引入T淋巴细胞和单核细胞系， 确定ras活性的变化是否与上皮细胞相似， 整合素糖基化和功能。T细胞的β 1整合素和 单核细胞代表通过糖基化调节的良好候选者， 已知这些细胞在体内表达变体整联蛋白糖型。 此外，这些变体糖型的表达与 细胞表型的变化。例如，高尔基体介导的 β 1整合素的糖基化在两种T细胞成熟过程中均被观察到， 和单核细胞活化。总的来说，在具体实施方案中提出的实验 目的1-3预期定义和表征一种新的，ras依赖的， 一种信号转导途径，通过调节 选择的高尔基体糖基转移酶的活性。对这样一个 通路可以提供对变体整合素的生理功能的了解 在T细胞成熟期间表达的糖型，单核细胞和 角质形成细胞活化和转移。

The abnormal function of beta1 integrin receptors plays a key role in the development of inflammatory diseases such as rheumatoid arthritis. In consequence, intense investigation has centered on defining the molecular pathways that regulate integrin activity. In many instances, changes in integrin function arise from altered integrin affinity for ligand, rather than from changes in receptor expression. Recent studies have suggested that intracellular signaling molecules, such as ras, can modulate integrin conformation, leading to altered cell adhesiveness and/or motility. The molecular effectors of ras that direct conformational changes in beta1 integrins have not been well-defined. Data presented as part of this research proposal supports a role for ras in the regulation of beta1 integrins. The ectopic expression of a dominant negative ras isoform (N17ras) in epithelial cells induces aberrant glycosylation of beta1 integrins. Correspondingly, cells that express aberrantly glycosylated beta1 integrins display numerous deficits in integrin-mediated cellular responses including cell attachment to matrix, cell spreading, focal adhesion formation, cell motility and tyrosine-kinase mediated signal transduction. A central hypothesis is thus proposed that ras regulates the Golgi -mediated glycosylation of beta1 integrins and that, in turn, modifications in glycosylation directly affect integrin function. This hypothesis encompasses three principal areas of inquiry. First, what is the molecular mechanism by which alterations in ras activity induce changes in integrin glycosylation? In Aim l, lectin affinity blotting and carbohydrate sequencing will be used to define the N17ras-mediated changes in the carbohydrate composition of beta1 integrins. Such experiments will likely identify Golgi glycosyltransferases that are candidates for regulation by ras, thus elucidating the molecular events involved in ras-dependent integrin glycosylation. Secondly, do changes in integrin glycosylation directly affect function? In Aim 2, methods will be developed that will allow the expression of N17ras to be uncoupled from the expression of variant integrin glycoforms. These experiments are expected to confirm that differences in glycosylation, rather than other downstream effects of NI 7ras, are directly responsible for deficiencies in integrin signaling. Finally, are rasmediated changes in in te grin glycosylation and function involved in inflammation? In Aim 3, N17ras will be introduced into T lymphocyte and monocytic cell lines in order to determine if, similar to epithelial cells, changes in ras activity can modulate integrin glycosylation and function. The beta1 integrins of T cells and monocytes represent good candidates for regulation by glycosylation because these cells are known to express variant integrin glycoforms in vivo. Moreover, the expression of these variant glycoforms correlates well with changes in cell phenotype. For example, modifications in Golgi--mediated glycosylation of beta1 integrins are observed during both T cell maturation and monocyte activation. Collectively, the experiments proposed in Specific Aims 1-3 are expected to define and characterize a novel, ras dependent, signal transduction pathway that modulates integrin function by regulating the activity of selected Golgi glycosyltransferases. The elucidation of such a pathway may provide insight into the physiologic function of variant integrin glycoforms that are expressed during T cell maturation, monocyte and keratinocyte activation, and metastasis.