An RGD sequence in the P2Y(2) receptor interacts with alpha(V)beta(3) integrins and is required for G(0)-mediated signal transduction

The P2Y(2) nucleotide receptor (P2Y(2)R) contains the integrin-binding doma in arginine-glycine-aspartic acid (RGD) in its first extracellular loop, ra ising the possibility that this G protein-coupled receptor interacts direct ly with an integrin, Binding of a peptide corresponding to the first extrac ellular 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-as sociated thrombospondin receptor, CD47. Immunofluorescence of cells transfe cted 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 whi ch 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 p hosphorylate focal adhesion kinase, activate extracellular signal-regulated kinases, and initiate the PLC-dependent mobilization of intracellular Ca2. Furthermore, an anti-cry integrin antibody partially inhibited these sign aling events mediated by the wild-type P2Y(2)R. Pertussis toxin, an inhibit or of G(i/o) proteins, partially inhibited Ca2+ mobilization mediated by th e wild-type P2Y(2)R, but not by the RGE mutant, suggesting that the RGD seq uence is required for P2Y(2)R-mediated activation of G(o), but not G(q). Si nce CD47 has been shown to associate directly with GU, family proteins, the se results suggest that interactions between P2Y(2)Rs, integrins, and CD47 may be important for coupling the P2Y(2)R to G(o).