Binding of the P2Y2 nucleotide receptor to filamin A regulates migration of vascular smooth muscle cells.
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.
Yu
Ningpu
N
Department of Biochemistry, University of Missouri, Columbia, MO 65211-7310, USA.
Erb
Laurie
L
Shivaji
Rikka
R
Weisman
Gary A
GA
Seye
Cheikh I
CI
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001011:Aorta / Q000166:cytology / Q000502:physiology; D001665:Binding Sites / Q000235:genetics / Q000502:physiology; D002465:Cell Movement / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D003285:Contractile Proteins / Q000378:metabolism*; D064448:Filamins; D051379:Mice; D018345:Mice, Knockout; D008840:Microfilament Proteins / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D009924:Organ Culture Techniques; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000235:genetics / Q000502:physiology; D058910:Purinergic P2 Receptor Agonists; D018048:Receptors, Purinergic P2 / Q000235:genetics / Q000378:metabolism*; D058473:Receptors, Purinergic P2Y2; D014162:Transfection; D020798:Two-Hybrid System Techniques; D014544:Uridine Triphosphate / Q000494:pharmacology
D003285:Contractile Proteins; D064448:Filamins; D008840:Microfilament Proteins; C546386:P2ry2 protein, mouse; D058910:Purinergic P2 Receptor Agonists; D018048:Receptors, Purinergic P2; D058473:Receptors, Purinergic P2Y2; D000255:Adenosine Triphosphate; D014544:Uridine Triphosphate