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.
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