MOLECULAR MECHANISMS IN POLARIZED CELL MIGRATION

极化细胞迁移的分子机制

基本信息

批准号：
7020707
负责人：
JOSE VAZQUEZ-PRADO
金额：
$ 4.88万
依托单位：
CINVESTAV-IPN
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-02-01 至 2009-01-31
项目状态：
已结题

项目摘要

Revised Abstract: DESCRIPTION (provided by applicant): Polarized cell migration (PCM) is key for cell defense against infections, wound healing, axonal growth, embryonic development, tumor cell metastasis and angiogenesis. Our hypothesis postulates that P-REX1, an exchange factor for Rac, establishes critical protein-protein interactions with receptors, G proteins and novel elements participating in PCM. Our specific aims are: 1) To define molecular links leading to the activation of Rac by G proteins. 2) To determine if P-REX1, identified as a Rac activator responding to Gbetagamma and PI3K (Welch et al, 2002, Cell 108:809), establishes protein interactions relevant for PCM. 3) To determine the role of G betagamma- and P-REXl-interacting proteins in the activity of Rac and PCM. Our studies consider the modular architecture exhibited by P-REX1, which contains two DEP and two PDZ domains, suggestive of modulation by direct protein-protein interactions. Our long term goal is to understand the molecular aspects of signal transduction required for PCM of endothelial cells and to identify molecular antiangiogenic tools. The role of Gbetagamma in PCM will be monitored in the presence of phosducin-like protein- and GRK2- derived peptides. P-REX1 deletion mutants lacking the different structural domains will be prepared to identify the structural domains in P-REX1 recognized by Gbetagamma and PI3K and to reveal their role in the activation of Rac and PCM. To identify novel elements able to modulate endothelial cell migration, Gbetagamma- and P-REXl interacting proteins will be cloned by yeast two hybrid and, in parallel, will be identified by proteomic approaches. It will be determined if CXCR1 and CXCR2 GPCRs containing PDZ-interacting motives in their carboxyl terminal domain are able to establish stable interactions with P-REX1. CXCR4 that does not contain that motif will be used as a control. Human HEK293T cells will be the model to study molecular interactions. Those cells will be transfected with the diverse DNA constructs. Recombinant molecules will be expressed fused to GST, EGFP, Myc or HA tags that will facilitate their study. Endothelial cells will be used to determine the role of Gbetagamma, P-REX1 and their interacting proteins, on the activation of Rac and polarized migration responding to the activation of angiogenic G protein coupled receptors. Discovery of molecular elements that might impede unwanted endothelial cell migration will provide elements for the design of antiangiogenic treatments.

修订后的摘要：描述（由申请人提供）：极化细胞迁移（PCM）是细胞防御感染、伤口愈合、轴突生长、胚胎发育、肿瘤细胞转移和血管生成的关键。我们的假设假设 P-REX1（Rac 的交换因子）与受体、G 蛋白和参与 PCM 的新元素建立了关键的蛋白质-蛋白质相互作用。我们的具体目标是： 1) 定义导致 G 蛋白激活 Rac 的分子联系。 2) 确定 P-REX1（被鉴定为响应 Gbetagamma 和 PI3K 的 Rac 激活剂）是否建立与 PCM 相关的蛋白质相互作用。 3)确定Gβ-和P-REX1-相互作用蛋白在Rac和PCM的活性中的作用。我们的研究考虑了 P-REX1 所展示的模块化架构，其中包含两个 DEP 和两个 PDZ 结构域，表明通过直接蛋白质-蛋白质相互作用进行调节。我们的长期目标是了解内皮细胞 PCM 所需的信号转导的分子方面，并确定分子抗血管生成工具。 Gbetagamma 在 PCM 中的作用将在磷蛋白样蛋白和 GRK2 衍生肽存在的情况下进行监测。将制备缺乏不同结构域的 P-REX1 缺失突变体，以鉴定 P-REX1 中被 Gbetagamma 和 PI3K 识别的结构域，并揭示它们在 Rac 和 PCM 激活中的作用。为了鉴定能够调节内皮细胞迁移的新元件，将通过酵母二杂交体克隆Gbetagamma-和P-REX1相互作用蛋白，并同时通过蛋白质组学方法进行鉴定。将确定在其羧基末端结构域中含有 PDZ 相互作用基序的 CXCR1 和 CXCR2 GPCR 是否能够与 P-REX1 建立稳定的相互作用。不包含该基序的 CXCR4 将用作对照。人类 HEK293T 细胞将成为研究分子相互作用的模型。这些细胞将被不同的 DNA 构建体转染。重组分子将与 GST、EGFP、Myc 或 HA 标签融合表达，以促进其研究。内皮细胞将用于确定 Gbetagamma、P-REX1 及其相互作用蛋白对 Rac 激活和响应血管生成 G 蛋白偶联受体激活的极化迁移的作用。可能阻止不需要的内皮细胞迁移的分子元件的发现将为抗血管生成治疗的设计提供元件。