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P.03 IDENTIFYING REGIONS OF P2X7 RECEPTOR INTRACELLULAR DOMAINS INVOLVED IN DOWNSTREAM SIGNALING Ahmad Q Jaradat and Mark T Young School of Biosciences, Cardiff University, Sir Martin Evans Building, Museum Avenue, Cardiff, CF10 3AX UK The P2X7 receptor is an ATP-gated cation ion channel which displays a widespread expression in mammals, and has been implicated in several pathologies, including immune disorders and cancer. Unique among the P2X receptor family, P2X7 possesses a long (240 amino acid) intracellular C-terminal domain (CTD) which is thought to couple cation channel activation by extracellular ATP to signaling pathways including cell blebbing, pore formation and ERK phosphorylation. Truncation mutants of the CTD have been demonstrated to show impaired ATP-induced membrane blebbing, and impaired ATP-induced uptake of the cationic dye Yo-Pro1. Removal of the N-terminal domain (NTD) has been demonstrated to abolish ATP-induced ERK phosphorylation. P2X7-dependent cell blebbing has been proposed to arise from the ATP-induced dissociation of P2X7 and non-muscle myosin, and P2X7-dependent Yo-Pro1 uptake has been proposed to be cholesterol-dependent, with a cysteine-rich, palmitoylated portion of the CTD proximal to the second transmembrane domain (CRR) able to sequester cholesterol to prevent inhibition of dye uptake in the full-length receptor. We set out to investigate which portions of the intracellular domains are involved in downstream signaling following P2X7 receptor activation, and whether or not the different signaling phenomena are coupled, by constructing a series of truncations, deletions and point mutants in the well-characterized rat P2X7 receptor fused to a C- terminal GFP-His tag. Constructs were transfected into HEK293 cells, and cell surface protein expression, ATP-induced cell blebbing, calcium uptake, Yo-Pro1 uptake, and ERK phosphorylation were measured. We found that any mutant which impaired blebbing also impaired Yo-Pro1 uptake, implying that these signaling phenomena are intrinsically coupled. Our data for ERK phosphorylation was more complex, but imply a role for both the NTD and CTD, and suggest that this pathway is distinct from the blebbing/dye uptake pathway. While both non-muscle myosin IIA and myosin V were expressed in HEK cells, we found no evidence for a physical interaction of either protein with P2X7, and so the molecular pathway for P2X7-dependent blebbing in these cells remains unclear. Cholesterol depletion by Methyl- β -cyclodextrin (MCD) was found to enhance ATP-induced Yo-Pro1 uptake in some CTD mutants, and further investigation of the CRR by deletion and multiple point mutagenesis revealed an essential role for this region for P2X7 dye uptake function.