P2Y(2) receptors regulate multiple signal transduction pathways in monocytic cells

Activation of P2Y(2) receptors by extracellular nucleotides induces cellula r responses required for differentiation of human promonocytic U937 cells. Multiple signal transduction pathways are independently coupled to P2Y2 rec eptors in U937 cells, including stimulation of phospholipase C (PLC)-depend ent 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 (P13-K) and c-Src. ERK1/2 ph osphorylation induced by UTP was inhibited by the P13-K inhibitors wortmann in and LY294002, the c-Src inhibitors radicicol and PP2, and the inhibitor of actin polymerization, cytochalasin D, but not by inhibitors of calcium-d ependent protein kinase C isoforms. The phosphorylation of ERK1/2 induced b y UTP was independent of calcium mobilization, since chelation of intracell ular calcium with BAPTA had no effect. The importance of multiprotein compl exes in mediating G-protein-coupled receptor signaling has recently been re cognized. Studies with an epitope-tagged P2Y(2) receptor expressed in human 1321 N1 astrocytoma cells indicated that activation of the P2Y(2) receptor with UTP causes it to colocalize with the EGF receptor. The P2Y(2) recepto r also contains the consensus integrin-binding motif arginine-glycine-aspar tic acid (RGD) in its first extracellular loop that was hypothesized to pro mote 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 alpha (v)beta (3)/beta (5 ) integrins and the integrin-associated thrombospondin receptor (CD47). Com pared to wild-type P2Y2 receptors, RGE mutant receptors required 1,000-fold higher concentrations of ATP or UTP to activate PLC-dependent calcium mobi lization and the phosphorylation of focal adhesion kinase (FAK) and ERK1/2. Competition studies indicated that an RGDS peptide inhibited the P2Y(2) re ceptor-mediated phosphorylation of FAK and ERK1/2. These findings suggest t hat the RGD domain of the P2Y(2) receptor is required to promote efficient coupling to intracellular signaling pathways. Thus, P2Y(2) receptors regula te divergent signal transduction pathways that are dependent on the formati on of multiprotein complexes including EGF receptors and integrins. Our stu dies encourage further attempts to develop strategies to independently regu late these pathways at steps proximal to P2Y(2) receptors. (C) 2001 Wiley-L iss, Inc.