P2Y2 receptors regulate multiple signal transduction pathways in monocytic cells

Activation of P2Y2 receptors by extracellular nucleotides induces cellular responses required for differentiation of human promonocytic U937 cells. Multiple signal transduction pathways are independently coupled to P2Y2 receptors in U937 cells, including stimulation of phospholipase C (PLC)‐dependent calcium mobilization and phosphorylation of mitogen‐activated protein (MAP) kinases. In U937 cells, P2Y2 receptors couple to the MAP kinases MEK1/2 and ERK1/2 via phosphatidylinositol 3‐kinase (PI3‐K) and c‐Src. ERK1/2 phosphorylation induced by UTP was inhibited by the PI3‐K inhibitors wortmannin and LY294002, the c‐Src inhibitors radicicol and PP2, and the inhibitor of actin polymerization, cytochalasin D, but not by inhibitors of calcium‐dependent protein kinase C isoforms. The phosphorylation of ERK1/2 induced by UTP was independent of calcium mobilization, since chelation of intracellular calcium with BAPTA had no effect. The importance of multiprotein complexes in mediating G‐protein‐coupled receptor signaling has recently been recognized. Studies with an epitope‐tagged P2Y2 receptor expressed in human 1321N1 astrocytoma cells indicated that activation of the P2Y2 receptor with UTP causes it to colocalize with the EGF receptor. The P2Y2 receptor also contains the consensus integrin‐binding motif arginine‐glycine‐aspartic acid (RGD) in its first extracellular loop that was hypothesized to promote interactions with integrins. Results of immunofluorescence and peptide binding experiments suggest that wild‐type, RGD‐containing receptors, but not RGE mutant receptors, can form complexes with αVβ3/β5 integrins and the integrin‐associated thrombospondin receptor (CD47). Compared to wild‐type P2Y2 receptors, RGE mutant receptors required 1,000‐fold higher concentrations of ATP or UTP to activate PLC‐dependent calcium mobilization and the phosphorylation of focal adhesion kinase (FAK) and ERK1/2. Competition studies indicated that an RGDS peptide inhibited the P2Y2 receptor‐mediated phosphorylation of FAK and ERK1/2. These findings suggest that the RGD domain of the P2Y2 receptor is required to promote efficient coupling to intracellular signaling pathways. Thus, P2Y2 receptors regulate divergent signal transduction pathways that are dependent on the formation of multiprotein complexes including EGF receptors and integrins. Our studies encourage further attempts to develop strategies to independently regulate these pathways at steps proximal to P2Y2 receptors. Drug Dev. Res. 53:186–192, 2001. © 2001 Wiley‐Liss, Inc.