| The P2Y2 receptor is a G protein-coupled receptor activated equally well by UTP or ATP. The receptor regulates diverse physiological responses such as ion secretion from epithelial tissue and modulation of vascular tone. Up-regulation of P2Y2 receptors has been associated with pathological conditions such as atherosclerosis and restenosis. Thus, it is important to study the signaling pathways whereby P2Y2 receptors mediate these physiological and pathological responses. Using human 1321N1 astrocytoma cells expressing P2Y2 receptors, we studied two structural domains of the receptor, SH3 binding sites and an integrin binding site.; Consensus SH3 binding sites were identified in the carboxy-terminal tail of the receptor and found to directly associate with Src in protein binding assays. A mutant P2Y2 receptor lacking the consensus SH3 binding sites was found to stimulate calcium mobilization and ERK1/2 phosphorylation like the wild type receptor, but was defective in its ability to stimulate tyrosine phosphorylation of Src and Src-dependent phosphorylations of Pyk2, EGFR, and PDGFR. Dual immunofluorescence labeling of the P2Y2 receptor and EGFR indicated that they co-localize in the plasma membrane upon P2Y 2 receptor activation. These data suggest that agonist-induced co-localization of the P2Y2 receptor with the EGFR allows Src, bound to the SH3 binding sites in the P2Y2 receptor, to efficiently phosphorylate the EGFR.; We also found that the consensus SH3 binding sites directly associate with the p85 subunit of PI3Kα in protein binding assays. Furthermore, phosphorylation of PDGFR and ERK1/2 by the P2Y2 receptor was inhibited by LY294002, an inhibitor of PI3K, suggesting that PI3K may also interact directly with the SH3 binding sites to promote downstream signaling.; The P2Y2 receptor also contains an integrin-binding sequence (RGD) in its first extracellular loop, raising the possibility that the receptor may interact with integrins. A mutant receptor whose RGD sequence was mutated to RGE required 1000-fold higher agonist concentrations to induce phosphorylation of focal adhesion kinase (FAK) and ERK1/2, as compared to the wild type P2Y 2 receptor. A soluble RGDS peptide inhibited phosphorylation of FAK and ERK1/2 by the wild type P2Y2 receptor. Together with previous findings that the αVβ3 integrins co-localization with P2Y2 receptor and mobilization of intracellular Ca 2+ are dependent on RGD sequence, we conclude the RGD sequence mediates the interaction of the P2Y2 receptor with αVβ 3 integrins and controls Ca2+, FAK and ERK1/2 signaling.; Taken together, these results indicate that the SH3 and integrin binding sites in the P2Y2 receptor mediate formation of a multi-protein complex involving EGFR, integrins, PI3K and Src, which allows downstream divergent signaling to mobilize intracellular Ca2+, and activate FAK, Pyk2, EGFR, and ERK1/2. |
