The P2Y2 nucleotide receptor mediates vascular cell adhesion molecule-1 expression through interaction with VEGF receptor-2 (KDR/Flk-1).
UTP stimulates the expression of pro-inflammatory vascular cell adhesion molecule-1 (VCAM-1) in endothelial cells through activation of the P2Y(2) nucleotide receptor P2Y(2)R. Here, we demonstrated that activation of the P2Y(2)R induced rapid tyrosine phosphorylation of vascular endothelial growth factor receptor (VEGFR)-2 in human coronary artery endothelial cells (HCAEC). RNA interference targeting VEGFR-2 or inhibition of VEGFR-2 tyrosine kinase activity abolishes P2Y(2)R-mediated VCAM-1 expression. Furthermore, VEGFR-2 and the P2Y(2)R co-localize upon UTP stimulation. Deletion or mutation of two Src homology-3-binding sites in the C-terminal tail of the P2Y(2)R or inhibition of Src kinase activity abolished the P2Y(2)R-mediated transactivation of VEGFR-2 and subsequently inhibited UTP-induced VCAM-1 expression. Moreover, activation of VEGFR-2 by UTP leads to the phosphorylation of Vav2, a guanine nucleotide exchange factor for Rho family GTPases. Using a binding assay to measure the activity of the small GTPases Rho, we found that stimulation of HCAEC by UTP increased the activity of RhoA and Rac1 (but not Cdc42). Significantly, a dominant negative form of RhoA inhibited P2Y(2)R-mediated VCAM-1 expression, whereas expression of dominant negative forms of Cdc42 and Rac1 had no effect. These data indicate a novel mechanism whereby a nucleotide receptor transactivates a receptor tyrosine kinase to generate an inflammatory response associated with atherosclerosis.
Seye
Cheikh I
CI
Department of Biochemistry, University of Missouri-Columbia, Columbia, Missouri 65212, USA. seyec@missouri.edu
Yu
Ningpu
N
González
Fernando A
FA
Erb
Laurie
L
Weisman
Gary A
GA
D000595:Amino Acid Sequence; D000818:Animals; D001161:Arteriosclerosis / Q000209:etiology / Q000378:metabolism; D001665:Binding Sites; D002460:Cell Line; D003331:Coronary Vessels / Q000502:physiology; D004730:Endothelium, Vascular / Q000502:physiology*; D006801:Humans; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011817:Rabbits; D018048:Receptors, Purinergic P2 / Q000737:chemistry / Q000378:metabolism*; D058473:Receptors, Purinergic P2Y2; D015398:Signal Transduction / Q000187:drug effects; D013329:Structure-Activity Relationship; D014544:Uridine Triphosphate / Q000494:pharmacology; D019010:Vascular Cell Adhesion Molecule-1 / Q000096:biosynthesis*; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000819:agonists / Q000737:chemistry / Q000378:metabolism*
C546385:P2RY2 protein, human; D018048:Receptors, Purinergic P2; D058473:Receptors, Purinergic P2Y2; D019010:Vascular Cell Adhesion Molecule-1; D040301:Vascular Endothelial Growth Factor Receptor-2; D014544:Uridine Triphosphate