Biochemical and genetic dissection of chemokine receptor CXCR3 signaling

趋化因子是一个分泌的趋化蛋白超家族，通过激活趋化因子受体来传递特定的趋化信号来控制白细胞的运动，目前还没有很好的定义，但已知涉及Gi类异三聚体G蛋白。然而，Galphai偶联并不局限于趋化gpcr，这表明产生趋化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端，在转导趋化信号中起重要作用。我们提出趋化性是由一种“趋化酶体”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，从而介导特定的趋化信号到细胞。我们将使用趋化因子受体CXCR3及其配体IP-10、Mig和I-TAC作为趋化因子诱导的信号转导模型。IP-10， Mig和I-TAC是干扰素诱导的趋化因子，在吸引T细胞回到炎症和感染的区域淋巴结中发挥重要作用。具体来说，我们建议：1)确定CXCR3的外部结构域参与IP-10、Mig和I-TAC的差异结合和信号转导，并确定相同的内部结构域是否将CXCR3与诱导趋化的百日咳毒素敏感G蛋白和诱导受体内化的百日咳毒素不敏感G蛋白偶联；2)利用酵母双杂交系统确定CXCR3的羧基端对于配体诱导的信号传导是否必要和充分，并鉴定与cxcr43的c端相互作用的蛋白；3)确定RACK1是否是趋化酶体的关键支架分子，是否是趋化因子受体- galphai2 - β整合素信号通路的组成部分；4)利用果蝇体内基因筛选技术鉴定Galphai信号通路的关键调控蛋白。了解CXCR43信号转导的分子机制是与吞噬细胞启动的T细胞浸润病变组织相关的重要问题，也有助于我们对趋化因子受体信号转导的理解。

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.