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(Circulation Research. 2008;102:581.)
© 2008 American Heart Association, Inc.

Cellular Biology

Binding of the P2Y₂ Nucleotide Receptor to Filamin A Regulates Migration of Vascular Smooth Muscle Cells

Ningpu Yu, Laurie Erb, Rikka Shivaji, Gary A. Weisman, Cheikh I. Seye

From the Department of Biochemistry, University of Missouri, Columbia.

Correspondence to Cheikh I. Seye, PhD, Department of Biochemistry, 540c Life Sciences Center, 1201 Rollins Rd, University of Missouri, Columbia, MO 65211-7310. E-mail seyec{at}missouri.edu

The functional expression of the G protein–coupled P2Y₂ nucleotide receptor (P2Y₂R) has been associated with proliferation and migration of vascular smooth muscle cells (SMCs), two processes involved in atherosclerosis and restenosis. Activation of the P2Y₂R causes dynamic reorganization of the actin cytoskeleton, which transmits biochemical signals and forces necessary for cell locomotion, suggesting that P2Y₂Rs may be linked to the actin cytoskeleton. Here, we identified filamin A (FLNa) as a P2Y₂R-interacting protein using a yeast 2-hybrid system screen with the C-terminal region of the P2Y₂R as bait. The FLNa binding site in the P2Y₂R is localized between amino acids 322 and 333. Deletion of this region led to selective loss of FLNa binding to the P2Y₂R and abolished Tyr phosphorylation of FLNa induced by the P2Y₂R 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; P0.01) and migration (3.6 fold; P0.01) 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 P2Y₂R knockout mice. Expression of the full-length P2Y₂R in SMCs isolated from P2Y₂R knockout mice restored both UTP-induced spreading and migration. In contrast, UTP-induced spreading and migration did not occur in SMCs isolated from P2Y₂R knockout mice transfected with a mutant P2Y₂R that does not bind FLNa. Furthermore, ex vivo studies showed that both ATP and UTP (10 µmol/L) promoted migration of SMCs out of aortic explants isolated from wild-type but not P2Y₂R knockout mice. Thus, this study demonstrates that P2Y₂R/FLNa interaction selectively regulates spreading and migration of vascular SMCs.

Key Words: cytoskeleton • smooth muscle cell • migration • nucleotide receptor

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