The P2Y2 Nucleotide Receptor in Vascular Inflammation and Angiogenesis - Extracellular ATP and Adenosine as Regulators of Endothelial Cell Function - page 58

P2Y 2 R-mediated VEGFR-2 tyrosine phosphorylation, consistent with the involve-
ment of Src in EGF and PDGF receptor transactivation by P2Y 2 Rs [50]. We also
found that the P2Y 2 R and VEGFR-2 co-localize in cells upon UTP stimulation
and demonstrated for the first time a direct interaction between these receptors in
a UTP-dependent manner [74]. Deletion or mutation of the SH3-binding domains
prevented UTP-induced expression of VCAM-1 [74]. Because expression of domi-
nant negative Src inhibited the P2Y 2 R/VEGFR-2 interaction, we concluded that Src
binding to the SH3-binding domains in the P2Y 2 R mediates both the UTP-induced
P2Y 2 R and VEGFR-2 interaction and the transactivation of VEGFR-2 by UTP that
regulates VCAM-1 expression.
Since leukocyte infiltration and migration are key processes involved in
atherosclerosis, these findings suggest that P2Y 2 receptors represent a novel target
for reducing arterial inflammation associated with cardiovascular disease. Recent
advances in the field of cell migration have identified many cellular events link-
ing a motility signal to remodeling of the actin cytoskeleton that enables a cell to
move [54]. Briefly, these events include receptor-mediated activation of Rac GTPase
that, in turn, activates WASP protein binding to allow a complex of proteins called
Arp2/3 to nucleate actin and facilitate formation of lamellipodia or branched actin
filaments at the leading edge of a cell. A capping protein terminates actin poly-
merization while another protein (cofilin), a downstream target of RhoA GTPase,
begins severing actin filaments so that the cell can continue to move. We found that
actin stress fiber formation induced by UTP occurred in 1321N1 cells expressing
the RGD-containing wild type P2Y 2 R but not in cells expressing an RGE mutant
P2Y 2 R [47]. Furthermore, stress fiber formation and cytoskeletal rearrangements
mediated by P2Y 2 R/ α V β 3/5 interactions required the activation of G i/o and G 12 pro-

teins, and not the activation of phospholipase C, intracellular calcium mobilization
or protein kinase C (PKC) that occurs concomitantly due to the coupling of the
P2Y 2 RtoG q protein [88]. Our data indicate that mutation of the RGD domain
in the P2Y 2 R to RGE (arginine-glycine-glutamate), which does not bind integrins,
inhibits RhoA GTPase and cofilin activities [5, 47], consistent with the involve-
ment of

α V β 3/5 integrins in these responses. Figure 4.1 summarizes the signaling
pathways that are coupled to P2Y 2 receptor activation.

4.1.3 Potential Role of Nucleotides and P2Y 2 Receptors
in Neuroinflammation

ATP and UTP induce cytokine-like effects in astrocytes that promote neuroinflam-
matory responses [55, 56, 87]. These nucleotides also induce cell-cell adhesion
in the monocyte/macrophage lineage and neutrophil adherence to endothelial cell
monolayers [58, 85], supporting the notion that released ATP and UTP lead to
endothelial cell activation by autocrine/paracrine mechanisms. It also has been
found that ATP and UTP induce cell migration [14, 31, 41, 62] and chemotaxis
of microglial cells [38] and primary rat cortical astrocytes [87].