An RGD sequence in the P2Y2 receptor interacts with αVβ3 integrins and is required for G0-mediated signal transduction

The P2Y(2) nucleotide receptor (P2Y(2)R) contains the integrin-binding domain arginine-glycine-aspartic acid (RGD) in its first extracellular loop, raising the possibility that this G protein-coupled receptor interacts directly with an integrin, Binding of a peptide corresponding to the first extracellular loop of the P2Y(2)R to K562 erythroleukemia cells was inhibited by antibodies against alpha (V)beta (3)/beta (5) integrins and the integrin-associated thrombospondin receptor, CD47. Immunofluorescence of cells transfected with epitope-tagged P2Y(2)Rs indicated that cry integrins colocalized 10-fold better with the wild-type P2Y(2)R than with a mutant P2Y(2)R in which the RGD sequence was replaced with RGE. Compared with the wild-type P2Y(2)R, the RGE mutant required 1,000-fold higher agonist concentra tions to phosphorylate focal adhesion kinase, activate extracellular signal-regulated kinases, and initiate the PLC-dependent mobilization of intracellular Ca(2+). Furthermore, an anti-cry integrin antibody partially inhibited these signaling events mediated by the wild-type P2Y(2)R. Pertussis toxin, an inhibitor of G(i/o) proteins, partially inhibited Ca(2+) mobilization mediated by the wild-type P2Y(2)R, but not by the RGE mutant, suggesting that the RGD sequence is required for P2Y(2)R-mediated activation of G(o), but not G(q). Since CD47 has been shown to associate directly with GU, family proteins, these results suggest that interactions between P2Y(2)Rs, integrins, and CD47 may be important for coupling the P2Y(2)R to G(o).