Anales RANF

S6-03 IN-SITU CROSS-LINKING OF P2X7 COMPLEXES TO IDENTIFY PROTEIN-PROTEIN INTERACTIONS R. Kopp, A. Schmidt, J. Hector, A. Imhof, and A. Nicke Ludwig-Maximilians-Universität München, Munich, Germany P2X receptors are trimeric non-selective cation channels, which are gated by extracellular ATP. The P2X7 subtype differs from all other P2X family members by a particularly long intracellular domain that constitutes about 40% of the protein and is supposed to be crucial for interactions with other proteins. Activation of P2X7 leads to the assembly of the NLRP3 inflammasome, resulting in caspase 1 activation and subsequent release of the mature proinflammatory cytokines IL- 1β and IL-18. Accordingly, P2X7 signaling has been associated with a variety of inflammatory diseases and the P2X7 receptor has gained increasing attention as a promising drug target. Although more than 50 proteins were shown to interact with the P2X7 receptor, only few of these interactions have been verified and P2X7 induced signaling pathways remain largely unclear. This could partly be explained by methodical limitations: Endogenous P2X7 protein expression rate is rather low in native cells, available antibodies lack selectivity and/or sensitivity, and P2X7 interactions seem to have a weak or transient character and might get lost during the purification processes. In this study, a BAC transgenic P2X7-EGFP mouse model in combination with in situ cross- linking of P2X7 complexes was used to analyze covalently cross-linked P2X7 complexes from native mouse tissue. Initial analysis by mass spectrometry identified a number of significantly enriched proteins that are involved in leukocyte migration. Experiments are ongoing to link a previously described, P2X7-mediated leukocyte infiltration to a physical interaction of P2X7 with proteins involved in cell adhesion.