Biochemical and Genetic Dissection of Chemokine Receptor CXCR3 Signaling

趋化因子受体 CXCR3 信号传导的生化和遗传学剖析

• 批准号: 7651313
• 负责人: ANDREW D LUSTER
• 金额: $ 34.4万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-01 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7651313
• 关键词: Binding; Biochemical Genetics; Biological Process; CXCL10 gene; CXCL11 gene; CXCL9 gene; CXCR3 gene; Cells; Chemotactic Factors; Chemotaxis; Complex; Disease; Dissection; Family; Funding; G-Protein-Coupled Receptors; Health; Immune; In Vitro; Inflammatory; Leukocyte Trafficking; Leukocytes; Ligands; Mass Spectrum Analysis; Mediating; Mediator of activation protein; Modeling; Molecular; Movement; Pathway interactions; Physiological; RNA Interference; Receptor Signaling; Role; Scaffolding Protein; Signal Transduction; Signal Transduction Pathway; Signaling Molecule; T-Lymphocyte; Tail; Technology; Testing; cell motility; chemokine; chemokine receptor; in vivo; migration; novel; novel strategies; receptor; receptor internalization; scaffold; trafficking

Chemokines are a super-family of chemotactic cytokines that control the movement of leukocytes by activating G protein-coupled receptors. The signaling domains of these receptors, and the signaling molecules with which they interact, have yet to be elucidated, and have been the focus of our studies in the prior funding period. We have focused our studies on the chemokine receptor CXCR3 and its ligands CXCL10, CXCL9, and CXCL11 as a model for chemokine-induced signal transduction, and have investigated which CXCR3 domains are involved in transducing two chemokine-induced functions in vitro, chemotaxis and receptor internalization. CXCR3 is expressed on effector T cells, where it mediates the trafficking of these cells in vivo. Chemokine receptors induce directed cell migration by promoting a transient localization of an active signaling complex at the leading edge of the cell. Cells thus sense direction by spatially regulating the activity of the signal transduction pathway. In the prior funding period, we initiated studies to test the hypothesis that chemotaxis is mediated by a scaffolding complex called the "chemotaxisome" that serves to hold relevant downstream mediators in close proximity to the activating receptor via interactions with the chemokine receptor itself. We have found that the C-tail of the CXCR3 is required for inducing chemotaxis and have identified two important domains within the C-tail. One domain contains a PDZ binding domain, which we have found is required for CXCRS-induced chemotaxis in vitro. In addition, we have found that a candidate PDZ domain scaffold protein, GIPC, interacts with this CXCR3 PDZ binding domain and is also important for CXCRS-induced chemotaxis. In the present proposal, we will determine the significance of the CXCR3 PDZ binding domain and the PDZ scaffold protein GIPC in CXCR3-mediated T cell recruitment in vivo. With regard to receptor internalization, we have found that CXCL11 is the physiological inducer of CXCR3 internalization, and have found that distinct domains of CXCR3 mediate CXCL11 vs. CXCL9/10 CXCR3 internalization. This raises the possibility that differences in CXCR3 internalization underlie some of the different biological functions of these 3 CXCR3 ligands. However, the role of receptor internalization in chemokine directed leukocyte trafficking in vivo is not well understood. In the present proposal, we will determine the role of receptor internalization in CXCRS-induced T cell trafficking in vivo by investigating the roles of the CXCR3 domains we have determined to be important for internalization in vitro. In addition to studies defining the roles of CXCR3 signaling domains in T cell trafficking in vivo, we will use RNAi technology and mass spectroscopy to continue our studies to define novel components of the CXCR3 signal transduction pathway. The molecular pathway that regulates chemoattractant signal transduction that results in the directed migration of leukocytes has important implications for health and disease. Once defined, the modulation of this pathway may allow for novel approaches to modulate immune and inflammatory diseases.

趋化因子是趋化性细胞因子的超家族，其通过激活G蛋白偶联受体来控制白细胞的运动。这些受体的信号结构域以及与它们相互作用的信号分子尚未阐明，并且一直是我们在先前资助期间研究的重点。我们的研究集中在趋化因子受体CXCR 3及其配体CXCL 10，CXCL 9和CXCL 11作为趋化因子诱导的信号转导的模型，并研究了CXCR 3结构域参与体外转导两种趋化因子诱导的功能，趋化性和受体内化。CXCR 3在效应T细胞上表达，在那里它介导这些细胞在体内的运输。趋化因子受体通过促进短暂的细胞迁移来诱导定向细胞迁移。 在细胞的前缘定位活性信号复合物。因此，细胞通过在空间上调节信号转导途径的活性来感知方向。在之前的资助期间，我们启动了研究来测试趋化性由称为“趋化体”的支架复合物介导的假设，该支架复合物通过与趋化因子受体本身的相互作用将相关下游介质保持在激活受体附近。我们已经发现CXCR 3的C-尾是诱导趋化性所必需的，并且已经鉴定了C-尾内的两个重要结构域。一个域 含有PDZ结合结构域，我们发现这是CXCRS诱导的体外趋化性所必需的。此外，我们还发现了一个候选的PDZ结构域支架蛋白GIPC与CXCR 3 PDZ结合结构域相互作用，并且对于CXCRS诱导的趋化性也很重要。在本提案中，我们将确定CXCR 3 PDZ结合结构域和PDZ支架蛋白GIPC在体内CXCR 3介导的T细胞募集中的意义。关于受体内化，我们已经发现CXCL 11是CXCR 3内化的生理诱导剂，并且已经发现CXCR 3的不同结构域介导CXCL 11与CXCL 9/10 CXCR 3内化。这就提出了CXCR 3基因的差异 内化是这3种CXCR 3配体的一些不同生物学功能的基础。然而，受体内化在趋化因子介导的体内白细胞运输中的作用尚不清楚。在本提案中，我们将通过研究我们已经确定对体外内化重要的CXCR 3结构域的作用来确定受体内化在CXCRs诱导的T细胞体内运输中的作用。除了研究CXCR 3信号结构域在体内T细胞运输中的作用外，我们还将使用RNAi技术和质谱继续我们的研究，以确定CXCR 3信号转导途径的新组分。 调节导致白细胞定向迁移的化学引诱物信号转导的分子途径对健康和疾病具有重要意义。一旦确定，这种途径的调节可能允许新的方法来调节免疫和炎症性疾病。