The role of the P2Y2 nucleotide receptor in inflammation: The mechanisms of P2Y2 receptor-mediated activation of G proteins.

摘要

The extracellular ATP/UTP receptor, i.e., the P2Y 2 receptor (P2Y2R), mediates mitogenic and pro-inflammatory responses in the vasculature, including increased smooth muscle cell proliferation and migration, and the endothelium-dependent infiltration of monocytes and their transmigration into sites of infection, injury, or stress, responses associated with the in vivo up-regulation of the P2Y 2 receptor in vascular cells (i.e., smooth muscle and endothelial cells).;This dissertation concerns the mechanisms whereby the P2Y2R mediates chemotaxis as well as the modulation of endothelial intercellular junctions. Since G proteins, such as heterotrimeric G12 and G i/o and the monomeric Rho family of GTPases, are responsible for regulating cellular actin dynamics and cytoskeletal changes that are central to chemotaxis and the stability of intercellular junctions, this dissertation focuses on the mechanisms underlying the P2Y2R-mediated activation of G proteins.;The P2Y2R is a G protein-coupled receptor with an extracellular integrin binding domain (Arg-Gly-Asp or RGD) that enables this receptor to directly interact with alphavbeta3/beta5 integrins, cell adhesion molecules known to regulate chemotaxis. Mutation of the RGD sequence to Arg-Gly-Glu (RGE) prevented association between the P2Y2R and alphav integrins but did not prevent activation of Gq or Gq-mediated calcium mobilization by the P2Y 2R agonist UTP. On the contrary, UTP-induced activation of G12 and G12-mediated events, including RhoA activation, cofilin and myosin light chain-2 phosphorylation, stress fiber formation and chemotaxis were all inhibited by mutation of the RGD domain to RGE in the human P2Y 2R expressed in human 1312N1 astrocytoma cells that are devoid of endogenous P2 nucleotide receptors. Similarly, UTP-induced activation of Go and Go-mediated events, including activation of Rac and vav2, a guanine nucleotide exchange factor for Rac, as well as chemotaxis towards UTP, were inhibited by the RGD to RGE mutation in the P2Y2R. These responses were also inhibited by anti-alphavbeta5 integrin antibodies or alphav integrin-specific antisense oligonucleotides, further confirming that alphav integrin activity and expression are required for the P2Y2R to activate Go and G 12, and subsequently, chemotaxis. Involvement of Go and G 12 in these UTP-triggered events was confirmed using the Gi/o inhibitor, pertussis toxin or dominant negative Ga12. Thus, the interaction between the P2Y2R and alphav integrin is required for the P2Y2R to activate heterotrimeric Go and G12 proteins that control chemotaxis.;In migrating leukocytes, P2Y2Rs remain evenly distributed on the cell surface and are thought to provide directional sensing and amplification of chemotactic signals by sensing nucleotides released at the leading edge of the migrating cell. In chapter IV of this study, we analyzed the distribution of P2Y2Rs in quiescent endothelial cells. Activation of P2Y2Rs with the agonist UTP caused a rapid and transient clustering of GFP-tagged P2Y2Rs at the intercellular junctions of human coronary artery endothelial cells (HCAEC). Co-immunoprecipitation experiments indicate that UTP causes a rapid and transient association of the P2Y2R and the vascular endothelial growth factor receptor-2 (VEGFR-2) with VE-cadherin, a transmembrane component of endothelial adherens junctions. Given that UTP infusion around stressed vascular tissue has been found to increase monocyte infiltration through the endothelium in collared rabbit carotid arteries, this dissertation addresses the mechanisms underlying monocyte infiltration by investigating P2Y2R interaction with endothelial intercellular junction proteins. The Rho family of GTPases has been shown to regulate endothelial permeability and leukocyte transmigration by controlling cytoplasmic actin dynamics. However, it is not well understood how a G protein-coupled receptor (GPCR) and junctional proteins coordinately regulate Rho GTPase activity. This dissertation reports that siRNA-mediated down-regulation of VE-cadherin or p120 catenin, a protein known to associate with VE-cadherin in adherens junctions, inhibited Rac activation induced by UTP. In addition, UTP promoted a prolonged interaction between p120 catenin and vav2, a response that correlates with the tyrosine phosphorylation of VE-cadherin and p120 catenin. UTP-induced responses, including the association of the P2Y2R with VE-cadherin, the association of p120 catenin with vav2, tyrosine phosphorylation of VE-cadherin and p120 catenin, and the activation of Rac, were inhibited by the Src inhibitor PP2 and the VEGFR-2 inhibitor SU1498, indicating that Src and VEGFR-2 activity are required for controlling P2Y2R-initiated signaling to adherens junctions, consistent with the presence of SH3-binding motifs in the C-terminal domain of the P2Y2R known to regulate Src-dependent VEGFR-2 transactivation by the P2Y2R.;Taken together, these data suggest that the P2Y2R requires direct interactions with alphav integrin, growth factor receptors and VE-cadherin to activate G proteins involved in chemotaxis and modulate the activities of intercellular junction proteins. Although in different types of cells, the expression or distribution of P2Y2R-associated proteins may vary, this dissertation provides novel findings indicating that to be fully functional, the P2Y2Rs must couple to a large complex containing multiple receptors and signaling proteins, each of which could be a potential target for the prevention and treatment of inflammatory diseases. (Abstract shortened by UMI.).