Chemokine receptors and signal transduction pathways in leukocytes
During the last funding period we started to investigate the orphan G-protein coupled receptor RDC1, which was proposed by phylogentic analyses to be a chemokine receptor. Preliminary studies from the laboratory suggested a potential relation with CXCR4. We could indeed show that RDC1 binds CXCL12, the ligand of CXCR4. However, we also observed that activated plasmacytoid dendritic cells produce an activity that rapidly and selectively internalizes RDC1 expressed on primary B cells, which is clearly distinct from known chemokines. Thus, this novel biological cross-talk between a specific subset of antigen presenting cells and B cells will be investigated in detail. The biochemical characterization of the putative ligand will be the first milestone.Afterwards we propose to investigate the cellular responses that can be stimulated with the putative ligand, including chemotaxis, cell proliferation and differentiation. Activation of intracellular signal transduction pathways by RDC1 will be compared with the known events stimulated by other chemokine receptors such as CXCR4. Next, the biological importance of RDC1 will be explored. We will investigate the expression of RDC1 and of the putative ligand in health and pathologies.Deletion of the gene in mice encoding the ligand is considered. We reason that the proposed experiments will unveil the physiological role of this evolutionary highly conserved receptor. The meager notion on RDC1 and the complete lack of information about the putative ligand makes it difficult to anticipate the outcome of the studies, but our newly generated reagents and the preliminary data represent a promising starting point to successfully demonstrate the function of RDC1 and its putative ligand.During the course of investigating the coupling of chemokine receptor to the actin-dependent migration we observed that the RhoGTPase specific GTP exchange factor P-Rex1 becomes rapidly and transiently phosphorylated. The kinetics of P-Rex1 phosphorylation are in agreement with the protein being involved in rapid RhoGTPase activation and actin polymerization. We propose to characterize the phosphorylation of P-Rex1 at the molecular level. Preliminary data suggest multiple phosphorylation sites which could affect activity and localization of the protein. Thus, it is important to reveal the physiological role of the different phosphorylation events and to delineate the pathways which lead to the posttranslational modifications.