Biochemical and genetic dissection of chemokine receptor CXCR3 signaling

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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.