Chemokine Receptor Studies: Defining the Dynamics of the Chemosynapse

趋化因子受体研究：定义趋化突触的动力学

基本信息

批准号：
7915941
负责人：
Ann Richmond
金额：
$ 21.94万
依托单位：
VANDERBILT UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-08-01 至 2011-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7915941
关键词：
1-Phosphatidylinositol 3-Kinase Actins Adaptor Signaling Protein Affect Arrestins Arthritis Back Binding Biological Breast Cancer Cell CXCR4 gene Cancer cell line Cell membrane Cells Chemotaxis Chronic Clathrin Clathrin-Coated Vesicles Complex Cytokinesis Cytoplasm Cytoskeleton DOCK1 protein Data Development Dissociation Dynamin Early Endosome Endocytic Vesicle Endosomes Endothelial Cells Event Extravasation G-Protein-Coupled Receptors GTP-Binding Proteins GTPase-Activating Proteins Guanosine Triphosphate Guanosine Triphosphate Phosphohydrolases Heat-Shock Response Heterotrimeric G Protein Subunit IL8 gene IL8RB gene Image Immune In Vitro Inflammation Leukocyte Chemotaxis Leukocytes Ligand Binding Ligands Link Lysosomes MCF7 cell Maintenance Malignant Neoplasms Mediating Membrane Microfluidic Microchips Modeling Monomeric GTP-Binding Proteins Neoplasm Metastasis Pathway interactions Pharmacotherapy Phosphatidylinositol 4,5-Diphosphate Phosphotransferases Photons Process Protein Binding Protein Phosphatase 2A Regulatory Subunit PR53 Proteins Recruitment Activity Recycling Resistance Role Sepsis Serine Signal Transduction Testing Time Tumor Angiogenesis Vesicle arrestin 2 cdc42 GTP-Binding Protein cell motility chemokine chemokine receptor clathrin A coated pit human BCAR1 protein in vivo insight intravital microscopy kinase inhibitor late endosome neoplastic cell neutrophil new therapeutic target numb protein perlecan protein protein interaction public health relevance receptor response src-Family Kinases therapy design trafficking tumor growth vasodilator-stimulated phosphoprotein wortmannin

项目摘要

DESCRIPTION (provided by applicant): We propose to test the hypothesis that when ligand binds chemokine receptors, a "dynamic chemosynapse" forms, comprised of receptor and adaptor proteins that serve to enable polarization, activation, amplification, and oscillation of small GTPases and kinases involved in organization of the actin cytoskeleton, and chemotaxis. Moreover, we propose that interference of chemokine receptor association with these adaptors will ablate CXCR2- and CXCR4-mediated chemotaxis, intravasation, extravasation, and metastasis. There are three specific aims. I) To test the hypothesis that a major role of AP-2 in mediating CXCR2/CXCR4 chemotaxis and metastasis is to orchestrate polarization and amplification of intracellular signals. We will separately interfere with AP-2 mediated receptor trafficking versus polarization of intracellular signals to distinguish the importance of these two functions of AP-2 in CXCR2/CXCR4 mediated chemotaxis and metastasis. 2) To characterize the role of IQGAP1 and VASP in linking the CXCR2 and CXCR4 chemosynapse to the actin cytoskeleton. We will characterize the interacting domains of IQGAP1 and VASP with CXCR2 and CXCR4 and determine the effects of altering these interactions on the functional responses to chemokine (including extravasation, intravasation, metastasis) of leukocytes, endothelial cells and breast cancer cells. 3) To test the hypothesis that Src-family kinase activation at the CXCR2 and CXCR4 `chemosynapse' drives chemotaxis that is Dock2 dependent and largely PI3K independent. We will determine the role of the Src-p130Cas-CrkL-Dock2 pathway in PI3K independent CXC2/CXCR4 chemotaxis. We will study these processes in dHL-60, dU937, HMEC-1 CXCR2 expressing cells and in the breast cancer cell lines naturally expressing CXCR4 (highly invasive MDA-MB-231, DU4475, BT-549 and non-invasive MCF-7, MDA-MB- 453 and MCF-10A cells). Microfluidic devices and real time intravital microscopy using 2 photon imaging will be used to track chemotaxis in vitro and intravasation/extravasation /metastasis in vivo, respectively. Characterization of the functional significance of this interaction between chemokine receptors and adaptor proteins that comprise the chemosynapse will unveil important new therapeutic targets for the treatment of malignancies. Characterization of the mechanism for PI3K independent chemotaxis will provide valuable information for design of therapies for sepsis, arthritis, and metastasis. PUBLIC HEALTH RELEVANCE: This study aims to test the hypothesis that receptors involved in regulating the motility of immune cells and tumor cells must interact with a number of proteins in the cytoplasm of the cell to stimulate proper organization of the cytoskeleton. In this study we propose to disrupt these protein/protein interactions to develop a new way of blocking tumor cell metastasis and chronic inflammation. We also will interrupt specific intracellular signals initiated by these protein/protein interactions to point the way to new targets for drug therapy for chronic inflammation and metastasis.

描述（由申请人提供）：我们建议测试以下假设：当配体结合趋化因子受体时，会形成“动态趋化突触”，由受体和接头蛋白组成，用于实现小 GTP 酶和激酶的极化、激活、放大和振荡参与肌动蛋白细胞骨架的组织和趋化性。此外，我们提出干扰趋化因子受体与这些接头的关联将消除CXCR2和CXCR4介导的趋化性、内渗、外渗和转移。具体目标有三个。 I) 检验 AP-2 在介导 CXCR2/CXCR4 趋化性和转移中的主要作用是协调细胞内信号的极化和放大的假设。我们将分别干扰 AP-2 介导的受体运输与细胞内信号极化，以区分 AP-2 的这两种功能在 CXCR2/CXCR4 介导的趋化和转移中的重要性。 2) 表征 IQGAP1 和 VASP 在连接 CXCR2 和 CXCR4 化学突触与肌动蛋白细胞骨架中的作用。我们将表征 IQGAP1 和 VASP 与 CXCR2 和 CXCR4 的相互作用域，并确定改变这些相互作用对白细胞、内皮细胞和乳腺癌细胞趋化因子功能反应（包括外渗、内渗、转移）的影响。 3) 测试以下假设：CXCR2 和 CXCR4“趋化突触”处的 Src 家族激酶激活驱动趋化性，该趋化性依赖于 Dock2，很大程度上不依赖于 PI3K。我们将确定 Src-p130Cas-CrkL-Dock2 通路在 PI3K 独立的 CXC2/CXCR4 趋化作用中的作用。我们将在 dHL-60、dU937、HMEC-1 CXCR2 表达细胞和自然表达 CXCR4 的乳腺癌细胞系（高度侵袭性 MDA-MB-231、DU4475、BT-549 和非侵袭性 MCF-7， MDA-MB-453 和 MCF-10A 细胞）。微流体装置和使用2光子成像的实时活体显微镜将分别用于追踪体外趋化性和体内渗入/外渗/转移。趋化因子受体和构成趋化突触的衔接蛋白之间这种相互作用的功能意义的表征将揭示治疗恶性肿瘤的重要新治疗靶点。 PI3K 独立趋化作用机制的表征将为脓毒症、关节炎和转移疗法的设计提供有价值的信息。公共健康相关性：本研究旨在检验以下假设：参与调节免疫细胞和肿瘤细胞运动的受体必须与细胞细胞质中的许多蛋白质相互作用，以刺激细胞骨架的正确组织。在这项研究中，我们建议破坏这些蛋白质/蛋白质相互作用，以开发一种阻止肿瘤细胞转移和慢性炎症的新方法。我们还将中断由这些蛋白质/蛋白质相互作用引发的特定细胞内信号，为慢性炎症和转移药物治疗的新靶标指明道路。