Binding of the P2Y2 nucleotide receptor to filamin A regulates migration of vascular smooth muscle cells

Abstract

The functional expression of the G protein-coupled P2Y(2) nucleotide receptor (P2Y(2)R) has been associated with proliferation and migration of vascular smooth muscle cells (SMCs), two processes involved in atherosclerosis and restenosis. Activation of the P2Y(2)R causes dynamic reorganization of the actin cytoskeleton, which transmits biochemical signals and forces necessary for cell locomotion, suggesting that P2Y(2)Rs may be linked to the actin cytoskeleton. Here, we identified filamin A (FLNa) as a P2Y(2)R-interacting protein using a yeast 2-hybrid system screen with the C-terminal region of the P2Y(2)R as bait. The FLNa binding site in the P2Y(2)R is localized between amino acids 322 and 333. Deletion of this region led to selective loss of FLNa binding to the P2Y(2)R and abolished Tyr phosphorylation of FLNa induced by the P2Y(2)R agonist UTP. Using both time-lapse microscopy and the Transwell cell migration assay, we showed that UTP significantly increased SMC spreading on collagen I (6.8 fold; P < or = 0.01) and migration (3.6 fold; P < or = 0.01) of aortic SMCs isolated from wild-type mice, as compared with unstimulated SMCs. UTP-induced spreading and migration of aortic SMCs did not occur with cells isolated from P2Y(2)R knockout mice. Expression of the full-length P2Y(2)R in SMCs isolated from P2Y(2)R knockout mice restored both UTP-induced spreading and migration. In contrast, UTP-induced spreading and migration did not occur in SMCs isolated from P2Y(2)R knockout mice transfected with a mutant P2Y(2)R that does not bind FLNa. Furthermore, ex vivo studies showed that both ATP and UTP (10 micromol/L) promoted migration of SMCs out of aortic explants isolated from wild-type but not P2Y(2)R knockout mice. Thus, this study demonstrates that P2Y(2)R/FLNa interaction selectively regulates spreading and migration of vascular SMCs.

Figure 1

FLNa binds to the P2Y₂R CTD and coimmunoprecipitates with the HA-P2Y₂R. A, CASMC lysates were analyzed by SDS-PAGE and immunoblotted by goat anti-FLNa antibody. M2 melanoma cells (kindly provided by Dr J.H. Hartwig, Harvard Medical School, Boston, Mass), which are deficient in FLNa, were used in parallel assays as a negative control, and FLNa transfected cells (A7 cells) were used as a positive control. Results are representative of 3 independent experiments. B, Immobilized GST-P2Y₂RCTD or GST was incubated with CASMC lysates (10₇ cells) overnight at 4°C, bound proteins were eluted with 100 mmol/L glutathione, and Western analysis was performed with anti-FLNa antibody. Results are representative of 3 independent experiments. C, Human 1321N1 cells transfected with cDNA encoding the HA-tagged P2Y₂R or with the pLXSN vector alone were lysed and immunoprecipitated (IP) with anti-HA affinity matrix or normal IgG, and immunoprecipitates were analyzed by immunoblotting (IB) with anti-FLNa antibody.FLNa levels in whole cell lysates are shown (bottom). Results are representative of 3 independent experiments.

Figure 2

Identification of the FLNa binding domain of the P2Y₂R. A, WT and progressive C-terminal truncation mutants of the HA-tagged P2Y₂R were expressed in human 1321N1 astrocytoma cells. B,Lysates from transfected cells were immunoprecipitated (IP) with anti-HA affinity matrix, andimmunoprecipitates were analyzed by immunoblotting (IB) with anti-FLNa antibody. FLNa levels in whole cell lysates were used to normalize FLNa-binding data (means±SEM) from 3 independent experiments (bar graphs). **P<0.01 as compared with WT control, with a representative blot shown on the left.

Figure 3

P2Y₂R/FLNa interaction is required for UTP-induced phosphorylation of FLNa and ERK1/2. SMCs from aortas of P2Y₂R KO mice were infected with adenoviruses expressing the WT or truncation mutant 5 P2Y₂R (Figure 2A). Infected cells were serum starved for 48 hours and stimulated with UTP (10 µmol/L) for the indicated time. A, Cell lysates were immunoprecipitated (IP) with anti–phospho-tyrosine antibody, and immunoprecipitates were analyzed by immunoblotting (IB) with anti-FLNa antibody. B, Cell lysates were immunoblotted with anti–phospho-FLNa (Ser2152) antibody (Millipore Corp). Total FLNa levels in cell lysates were used for normalization. C, Lysates were immunoblotted with antibody against phospho-ERK1 (Millipore Corp). Total ERK1/2 levels in cell lysates were used for normalization. Data represent means±SEM of results from 3 independent experiments. *P<0.05, **P<0.01 as compared with WT P2Y₂R at 0 min.

Figure 4

Spreading of SMCs on collagen-coated coverslips. Aortic SMCs from WT or P2Y₂R KO mice were plated on collagen I–coated coverslips and then were incubated for 1 hour in serum-free medium with or without UTP (10 µmol/L). A, Cell spreading was visualized with an Axiovert 200M inverted microscope, and a representative micrograph is shown with arrows indicating spreading cells. B, Aortic SMCs from WT or P2Y₂R KO mice were treated as above, and cell spreading was visualized by time-lapse microscopy. Also, aortic SMCs from P2Y₂R KO mice were transfected with cDNA encoding WT or truncation mutant 5 P2Y₂R. Then, cell transfectants were incubated with or without UTP (10 µmol/L) for 1 hour, and the percentage of cells exhibiting UTP-induced cell spreading was determined by time-lapse microscopy. Data represent means±SEM for 3independent experiments performed in triplicate. **P<0.01, as compared with the indicated condition.

Figure 5

Loss of FLNa/P2Y₂R interaction affects UTP-induced SMC migration. A, Aortic SMCs from WT or P2Y₂RKO mice were added to the upper chamber of Transwells. The lower chamber contained serum-free DMEM with or without UTP (10 µmol/L). Cell migration expressed as the number of cells migrating across the membrane was evaluated after 8 hours. B, Aortic SMCs from P2Y₂R KO mice expressing WT or truncation mutant 5 P2Y₂R, or vector alone were assayed for UTP-induced cell migration, as described above. Incubation for 8 hours with10% FBS in the lower chamber was used as a positive control. Data with UTP and FBS are expressed as fold increase over untreated control and represent means±SEM of results from 3 independent experiments. **P<0.01.

Figure 6

FLNa siRNA suppresses UTP-induced SMC spreading and motility. SMCs from aortas of WT mice were transfected with siRNA targeting FLNa or scrambled siRNA. A, The lysates of cell transfectants were immunoblotted with anti-FLNa antibody. B and C,Cell spreading and motility of SMCs in response to UTP or FBS. Data represent means≤SEM for 3 independent experiments. **P<0.01 as compared with the indicated condition.

Figure 7

Migration of SMCs out of mouse aortic explants. Aortic explants were obtained from WT and P2Y₂R KO mice and cultured for 4 hours in DMEM with or without ATP or UTP (10 µmol/L) or 5% (vol/vol) FBS. Some explants were also cultured for 4 hours with a nucleotide regenerating system (CPK). Then, medium was replaced daily with DMEM alone and after 5 days, and cells growing out of the aortic explants were visualized with an Olympus IX70 inverted microscope. A, Micrographs from a representative experiment are shown. Arrows indicate the direction of cell migration. Magnification, ×20. B, SMCs migrating from aortic explants treated as above were removed after 5 days by incubation with trypsin–EDTA solution. The number of cells migrating from explants was determined in 3 independent experiments performed in triplicate. Data with ATP, UTP, FBS, and CPK are expressed as fold increases over untreated control and represent means±SEM of results from 3 independent experiments. **P<0.01.