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 alpha(V) 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 concentrations to phosphorylate focal adhesion kinase, activate extracellular signal-regulated kinases, and initiate the PLC-dependent mobilization of intracellular Ca(2+). Furthermore, an anti-alpha(V) 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 G(i/o) 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).