Chemokine Receptor Studies: Defining the Dynamics of the Chemosynapse

描述（由申请人提供）：我们提出检验以下假设：当配体结合趋化因子受体时，形成“动态化学突触”，其由受体和衔接蛋白组成，用于使参与肌动蛋白细胞骨架组织和趋化性的小GTP酶和激酶极化、活化、扩增和振荡。此外，我们提出，干扰趋化因子受体与这些衔接子的关联将消融CXCR 2和CXCR 4介导的趋化性、内渗、外渗和转移。有三个具体目标。I）检验AP-2在介导CXCR 2/CXCR 4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的极化，以区分AP-2的这两种功能在CXCR 2/CXCR 4介导的趋化性和转移中的重要性。2)研究IQGAP 1和VASP在连接CXCR 2和CXCR 4化学突触与肌动蛋白细胞骨架中的作用。我们将描述IQGAP 1和VASP与CXCR 2和CXCR 4的相互作用结构域，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对趋化因子的功能反应（包括外渗、内渗、转移）的影响。3)为了检验CXCR 2和CXCR 4“化学突触”处Src家族激酶激活驱动Dock 2依赖性和很大程度上PI 3 K独立性的趋化性的假设。我们将确定Src-p130 Cas-CrkL-Dock 2通路在PI 3 K非依赖性CXC 2/CXCR 4趋化性中的作用。我们将在dHL-60、dU 937、HMEC-1 CXCR 2表达细胞和天然表达CXCR 4的乳腺癌细胞系（高度侵袭性MDA-MB-231、DU 4475、BT-549和非侵袭性MCF-7、MDA-MB- 453和MCF-10A细胞）中研究这些过程。微流体装置和使用双光子成像的真实的时间活体显微镜将分别用于追踪体外趋化性和体内内渗/外渗/转移。趋化因子受体和衔接蛋白，包括化学突触之间的相互作用的功能意义的表征将揭示重要的新的治疗恶性肿瘤的治疗目标。PI 3 K非依赖性趋化性机制的表征将为脓毒症、关节炎和转移的治疗设计提供有价值的信息。公共卫生相关性：本研究旨在验证以下假设：参与调节免疫细胞和肿瘤细胞运动的受体必须与细胞质中的许多蛋白质相互作用，以刺激细胞骨架的适当组织。在这项研究中，我们建议破坏这些蛋白质/蛋白质相互作用，以开发一种新的阻断肿瘤细胞转移和慢性炎症的方法。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，为慢性炎症和转移的药物治疗指明新的靶点。

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.