The P2Y2 Nucleotide Receptor in Vascular Inflammation and Angiogenesis - Extracellular ATP and Adenosine as Regulators of Endothelial Cell Function - page 57

stimulate U937 monocyte and Jurkat lymphocyte adherence to HCAECs and human
submandibular gland cells, respectively [6, 75]. The P2Y 2 R contains the integrin
binding domain arginine-glycine-aspartic acid (RGD) in its first extracellular loop
[21]. The RGD motif has been shown to be the core recognition sequence for
many integrins including

α v β 5 [39]. Recent studies have indicated that the
RGD sequence in the P2Y 2 R mediates its interaction with

α v β 3 and

β 5 integrins, and
is required for G o - and G 12/13 -mediated signal transduction [5, 21, 47]. It has been
reported previously that the

α v β 3 /

α v β 3 integrin on monocytes mediates their adherence to
endothelial and epithelial cells, an early event in the acute inflammatory response
[8]. Thus, protein complex formation between

β 5 integrins and P2Y 2 Rs also
may be involved in the regulation of monocyte binding to endothelial cells mediated
by P2Y 2 Rs.
Our studies also have focused on the mechanisms whereby P2Y 2 R activation
increases the expression of VCAM-1 [74]. VEGF, a pleiotropic factor that regulates
multiple biological phenomena, including endothelial cell survival, proliferation,
and migration, also stimulates endothelial cell expression of adhesion molecules,
including VCAM-1, ICAM-1, and selectins [44]. These multiple responses are
mediated through the interaction of VEGF with two receptor tyrosine kinases, Flt-1
or VEGF receptor-1 and Flk-1/KDR or VEGF receptor-2 [44]. It is widely accepted
that VEGFR-2 mediates the growth and migration of endothelial cells, the perme-
ability of blood cells to ions and small molecules, and the expression of VCAM-1
in human umbilical vein endothelial cells [91]. It is increasingly apparent that
VEGFR-2 serves as a point of convergence for multiple signals arising from diverse
stimuli. VEGFR-2 transactivation follows activation of G protein-coupled receptors,
including the endothelial differentiation gene product and the bradykinin receptor
[81, 83]. We hypothesized that transactivation of VEGFR-2 may be involved in
P2Y 2 R-mediated VCAM-1 expression in endothelial cells.
We demonstrated for the first time that activation of the P2Y 2 R in human
coronary artery endothelial cells induces the rapid tyrosine phosphorylation of
VEGFR-2 and the activation of Vav2, a Rho guanine nucleotide exchange factor,
leading to increased expression of VCAM-1 [74]. Two lines of evidence demon-
strate the involvement of VEGFR-2 in this process. First, SU1498, a specific
VEGFR-2 tyrosine kinase inhibitor, diminished UTP-induced VCAM-1 expres-
sion [74]. Second, selective inhibition of VEGFR-2 expression by siRNA inhibited
UTP-induced VCAM-1 expression [74]. Ligand-independent activation of recep-
tor tyrosine kinases is involved in many biological responses initiated by G
protein-coupled receptors. It has been reported that activation of the endothelial
differentiation gene product in response to sphingosine 1-phosphate stimulates
endothelial nitric oxide synthase through activation of Flk-1/KDR [81]. EGF recep-
tor transactivation by G protein-coupled receptor ligands may involve activation of
matrix metalloproteinases, release of membrane heparin-bound EGF, and binding
of heparin to the EGF receptor [83]. At the structural level, we have identified two
SH3-binding sites (PXXP motifs) in the intracellular C terminus of the P2Y 2 R that
enable P2Y 2 R interaction with Src and the regulation of EGF and PDGF recep-
tor activity [50]. We demonstrated that PP2, the Src kinase inhibitor, decreased

α v β 3 /