Structural determinants of P2Y(2) receptor functions .

摘要

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 α_Vβ₃ 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 α_Vβ 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.