Abstract
Vascular endothelial cadherin (VE-cadherin) mediates homophylic adhesion between endothelial cells and is an important regulator of angiogenesis, blood vessel permeability and leukocyte trafficking. Rac1, a member of the Rho family of GTPases, controls VE-cadherin adhesion by acting downstream of several growth factors, including angiopoietin-1 and vascular endothelial growth factor (VEGF). Here we show that UTP-induced activation of the Gq protein-coupled P2Y2 nucleotide receptor (P2Y2R) in human coronary artery endothelial cells (HCAECs) activated Rac1 and caused a transient complex to form between P2Y2R, VE-cadherin and VEGF receptor-2 (VEGFR-2). Knockdown of VE-cadherin expression with siRNA did not affect UTP-induced activation of extracellular signal-regulated kinases 1/2 (ERK1/2) but led to a loss of UTP-induced Rac1 activation and tyrosine phosphorylation of p120 catenin, a cytoplasmic protein known to interact with VE-cadherin. Activation of the P2Y2R by UTP also caused a prolonged interaction between p120 catenin and vav2 (a guanine nucleotide exchange factor for Rac) that correlated with the kinetics of UTP-induced tyrosine phosphorylation of p120 catenin and VE-cadherin. Inhibitors of VEGFR-2 (SU1498) or Src (PP2) significantly diminished UTP-induced Rac1 activation, tyrosine phosphorylation of p120 catenin and VE-cadherin, and association of the P2Y2R with VE-cadherin and p120 catenin with vav2. These findings suggest that the P2Y2R uses Src and VEGFR-2 to mediate association of the P2Y2R with VE-cadherin complexes in endothelial adherens junctions to activate Rac1.
Highlights
The vascular endothelium separates circulating blood from the underlying tissue and provides a semipermeable barrier for normal fluid, nutrient and waste exchange
Thirty-six hours after transfection, most enhanced green fluorescent protein (eGFP)-Human P2Y2R (hP2Y2R) were localized to an intracellular compartment, possibly early endosomes, whereas 84 h after transfection, the GFP-hP2Y2R appeared uniformly distributed in the plasma membrane (Figure 1)
Interaction between P2Y2 nucleotide receptor (P2Y2R) and VE-cadherin induced by UTP was prevented by pretreatment of endothelial cells with PP2 or SU1498, compounds that inhibit Src and VEGF receptor-2 (VEGFR-2) kinase activity, respectively. This suggests that Src and VEGFR-2 kinases regulate assembly of a VE-cadherin-associated complex with the P2Y2R that is important for downstream signaling to Rac1. In support of this idea, both Src and VEGFR-2 have been found to associate with the activated P2Y2R [15] [16] and we show that PP2 and SU1498 inhibited UTP-induced tyrosine phosphorylation of VE-cadherin and p120 catenin, interaction between p120 catenin and vav2, and activation of Rac1
Summary
The vascular endothelium separates circulating blood from the underlying tissue and provides a semipermeable barrier for normal fluid, nutrient and waste exchange. Other studies demonstrated that the P2Y2R, by virtue of an arginine-glycine-aspartate (RGD) integrin-binding motif in its extracellular domain, mediates the activation of small Rho GTPases, Rac and RhoA [13] [14] and, by virtue of SH3-binding motifs in its intracellular domain, interacts with Src and promotes the Src-dependent activation of several growth factor receptors, including VEGFR2 that up-regulates the expression of vascular cell adhesion molecule-1 (VCAM-1), a leukocyte binding protein in endothelial cells [15] [16]. Since the P2Y2R regulates vascular integrity, leukocyte adhesion and extravasation, and Rho GTPase activities [5]-[11] [13]-[17], we speculated that the P2Y2R may modulate the permeability of endothelium by affecting the stability of adherens junctions
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