Biochemical and genetic dissection of chemokine receptor CXCR3 signaling

趋化因子是分泌的趋化蛋白的超家族，其通过激活趋化因子受体以递送特异性趋化信号来控制白细胞的运动，尚未明确定义，但已知涉及Gi类异源三聚体G蛋白。然而，Galphai偶联不限于趋化性GPCR，表明产生趋化性信号的特异性不仅仅由Galphai决定，还涉及其他蛋白质。最近的研究表明，趋化因子受体本身，特别是C-末端，在转导趋化信号中是重要的。我们认为趋化性是由一个“趋化体”复合物介导的，该复合物通过与受体的C-尾和Galphai的相互作用被募集到趋化因子受体激活的位点，以介导对细胞的特异性趋化信号。我们将使用趋化因子受体CXCR 3及其配体IP-10、Mig和I-TAC作为趋化因子诱导的信号转导的模型。IP-10、Mig和I-TAC是干扰素-γ诱导的趋化因子，其在吸引T细胞（在区域淋巴结中活化）回到炎症和感染的部位中起重要作用。具体而言，我们建议：1）确定参与IP-10、Mig和I-TAC的差异结合和信号传导的CXCR 3的外部结构域，并确定相同的内部结构域是否将CXCR 3偶联至诱导趋化性的百日咳毒素敏感性G蛋白和诱导受体内化的百日咳毒素不敏感性G蛋白; 2）确定CXCR 3的羧基端是否是配体诱导的信号传导所必需和充分的，并使用酵母双杂交系统鉴定与CXC 43的C端相互作用的蛋白质; 3）确定RACK 1是否是趋化因子受体-Galpha 1 2-β整联蛋白信号通路的关键支架分子;和4）在果蝇中使用基于遗传的体内筛选来鉴定Galpha 1信号通路中的关键调节蛋白。了解CXCR 43信号转导的分子机制是一个重要的问题，相关的吞噬细胞启动的T细胞浸润到病变组织，以及有助于我们了解趋化因子受体信号转导。

Chemokines are a superfamily of secreted chemotactic proteins that control the movement of leukocytes by activating chemokine receptors to deliver a specific chemotactic signal are not well defined but are known to involve the Gi class of heterotrimeric G proteins. However, Galphai coupling is not restricted to chemotactic GPCRs indicating that the specificity in generating a chemotactic signal is not solely determine by Galphai and that additional proteins are involved. Recent studies have demonstrated that the chemokine receptor itself, and in particular the C- terminus, is important in transducing the chemotactic signal. We propose that chemotaxis is mediated by a "chemotaxisome" complex that is recruited to the site of chemokine receptor activation via interactions w2ith the C-tail of the receptor and Galphai to mediate the specific chemotactic signal to the cell. We will use the chemokine receptor CXCR3 and its ligands, IP-10, Mig and I-TAC, as a model for chemokine-induced signal transduction. IP-10, Mig and I-TAC are interferon-gamma inducible chemokines that play important roles in attracting T cells, activated in regional lymph nodes, back to sties of inflammation and infection. Specifically we propose: 1) To determine the external domains of CXCR3 involved in the differential binding and signaling of IP-10, Mig and I-TAC and to determine if the same internal domain couples CXCR3 to pertussis toxin-sensitive G proteins which induce chemotaxis and pertussis toxin-insensitive G proteins which induce receptor internalization; 2) To determine if the carboxyl-terminus of CXCR3 is necessary and sufficient for ligand induced signaling and to identify proteins that interact withy the C-terminus of CXC43 using a yeast two-hybrid system; 3) To establish whether RACK1 is a key scaffolding molecule of the chemotaxisome and is a component of the chemokine receptor-Galphai2-beta integrin signaling pathway; and 4) To use a genetic based in vivo screen in Drosophila to identify key regulatory proteins in the Galphai signaling pathway. Understanding the molecular mechanism of CXCR43 signal transduction is an important question relevant to phagocyte initiated T cell infiltration into diseased tissue as well as contributing to our understanding of chemokine receptor signal transduction.