Abstract 732: The P2Y2 Receptor Selectively Binds to Filamin A to Regulate Spreading and Migration of Vascular Smooth Muscle Cells
The functional expression of the G protein-coupled P2Y2 nucleotide receptor has been associated with the development of intimal lesions. Activation of this receptor also stimulates actin cytoskeleton reorganization and migration of vascular smooth muscle cells (SMCs). Since cell migration has been linked to the dynamic reorganization of the actin cytoskeleton, which transmits biochemical signals and forces necessary for cell locomotion, the aim of the present study was to identify cytoskeletal proteins that bind to the P2Y2 receptor and potentially regulate SMC migration. Using the yeast two-hybrid system, we isolated filamin A, a filamentous actin-cross linking protein that interacts with the C-terminal domain of the P2Y2 receptor and we used deletion mapping to identify amino acid deletion in the P2Y2 receptor that led to selective loss of filamin A binding. Ex-vivo treatment of aortic explants with the P2Y2 receptor agonist UTP (10 μmol/L) significantly promoted migration of SMCs in wild type but not in P2Y2 receptor−/− mice. Likewise, using a Transwell migration assay we showed that UTP increased migration of SMCs in wild type (3.5 folds, P<0.01 vs. untreated cells) but not P2Y2 receptor −/− mice (P<0.4 vs. untreated cells). Adenoviral infection of the full length P2Y2 receptor restored UTP-mediated cell migration in P2Y2 receptor −/− mice whereas infection of a mutant P2Y2 receptor that does not bind filamin A did not. UTP-induced migration was preceded by a rapid phosphorylation of filamin A that was not observed either in P2Y2 receptor−/− SMCs or in P2Y2 receptor−/− SMCs infected with the mutant P2Y2 receptor that does not bind filamin A. Treatment of SMCs from wild type mice with UTP (10 μmol/L) caused a 4-fold increase in spreading to collagen I as compared to unstimulated cells. The UTP-mediated increase of SMC spreading was not found in P2Y2 receptor−/− SMCs but was restored by adenoviral infection of the full length P2Y2 receptor cDNA into these cells. However, adenoviral delivery of a mutant receptor −/−which does not bind to filamin A did not restore UTP-mediated spreading of P2Y2 receptor SMCs to collagen. This study demonstrates that P2Y2-dependent modulation of the actin cytoskeleton selectively regulates spreading and migration of SMC.