Abstract. The mitogen-activated protein (MAP) kinase signal transduction pathway represents an important mechanism by which growth factors regulate cell function. Targets of the MAP kinase pathway are located within several cellular compartments. Signal transduction therefore requires the localization of MAP kinase in each sub-cellular compartment that contains physiologically relevant substrates. Here, we show that serum treatment causes the translocation of two human MAP kinase isoforms, p40 "~k and p41 inapt, from the cytosol into the nucleus. In addition, we report that p41 m~k (but not p40 "~'k) is localized at the cell surface ruffling membrane in serum-treated cells.To investigate whether the protein kinase activity of MAP kinase is required for serum-induced redistribution within the cell, we constructed mutated kinasenegative forms of p40 ~pk and p41~. The kinasenegative MAP kinases were not observed to localize to the cell surface ruffling membrane. In contrast, the kinase-negative MAP kinases were observed to be translocated to the nucleus. Intrinsic MAP kinase activity is therefore required only for localization at the cell surface and is not required for transport into the nucleus.Together, these data demonstrate that the pattern of serum-induced redistribution of p40"~ is different from p41,,~k. Thus, in addition to common targets of signal transduction, it is possible that these MAP kinase isoforms may differentially regulate targets located in distinct sub-cellular compartments.

The P2Y2 nucleotide receptor (P2Y2R) 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 P2Y2R to K562 erythroleukemia cells was inhibited by antibodies against αVβ3/β5 integrins and the integrin-associated thrombospondin receptor, CD47. Immunofluorescence of cells transfected with epitope-tagged P2Y2Rs indicated that αV integrins colocalized 10-fold better with the wild-type P2Y2R than with a mutant P2Y2R in which the RGD sequence was replaced with RGE. Compared with the wild-type P2Y2R, 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 Ca2+. Furthermore, an anti-αV integrin antibody partially inhibited these signaling events mediated by the wild-type P2Y2R. Pertussis toxin, an inhibitor of Gi/o proteins, partially inhibited Ca2+ mobilization mediated by the wild-type P2Y2R, but not by the RGE mutant, suggesting that the RGD sequence is required for P2Y2R-mediated activation of Go, but not Gq. Since CD47 has been shown to associate directly with Gi/o family proteins, these results suggest that interactions between P2Y2Rs, integrins, and CD47 may be important for coupling the P2Y2R to Go.

Background-Extracellular uridine 5Ј-triphosphate (UTP) induces mitogenic activation of smooth muscle cells (SMCs) through binding to P2Y 2 nucleotide receptors. P2Y 2 receptor mRNA is upregulated in intimal lesions of rat aorta, but it is unclear how this G-protein-coupled receptor contributes to development of intimal hyperplasia. Methods and Results-This study used a silicone collar placed around rabbit carotid arteries to induce vascular injury and intimal thickening. Collar placement caused rapid upregulation of P2Y 2 receptor mRNA in medial SMCs before appearance of neointima. Fura-2 digital imaging of single SMCs was used to measure changes in myoplasmic calcium concentration (Ca m ) in response to P2Y receptor agonists. In contrast to UDP, activation by UTP or adenosine 5Ј-triphosphate (ATP) greatly increased Ca m , which indicates upregulation of functional P2Y 2 receptors at which UTP and ATP are equipotent agonists. The number of responsive cells was significantly greater for freshly dispersed SMCs from collared arteries than for controls. Perivascular infusion of UTP (100 mol/L) within the collar significantly enhanced neointimal development. Intimas that resulted from UTP exposure were infiltrated by macrophages. Moreover, increased expression of osteopontin occurred in response to in situ application of UTP. ATP or UTP also stimulated osteopontin expression in cultured SMCs in a dose-dependent manner. Furthermore, P2Y 2 antisense oligonucleotide inhibited osteopontin expression induced by UTP. Conclusions-These findings indicate for the first time a role for the UTP/ATP receptor, P2Y 2 , in development of intimal hyperplasia associated with atherosclerosis and restenosis.

Many G protein-coupled receptors activate growth factor receptors, although the mechanisms controlling this transactivation are unclear. We have identified two proline-rich, SH3 binding sites (PXXP) in the carboxyl-terminal tail of the human P2Y 2 nucleotide receptor that directly associate with the tyrosine kinase Src in protein binding assays. Furthermore, Src co-precipitated with the P2Y 2 receptor in 1321N1 astrocytoma cells stimulated with the P2Y 2 receptor agonist UTP. A mutant P2Y 2 receptor lacking the PXXP motifs was found to stimulate calcium mobilization and serine/threonine phosphorylation of the Erk1/2 mitogen-activated protein kinases, like the wild-type receptor, but was defective in its ability to stimulate tyrosine phosphorylation of Src and Srcdependent tyrosine phosphorylation of the proline-rich tyrosine kinase 2, epidermal growth factor receptor (EGFR), and platelet-derived growth factor receptor. Dual immunofluorescence labeling of the P2Y 2 receptor and the EGFR indicated that UTP caused an increase in the co-localization of these receptors in the plasma membrane that was prevented by the Src inhibitor PP2. Together, these data suggest that agonist-induced binding of Src to the SH3 binding sites in the P2Y 2 receptor facilitates Src activation, which recruits the EGFR into a protein complex with the P2Y 2 receptor and allows Src to efficiently phosphorylate the EGFR.Src and proline-rich tyrosine kinase 2 (Pyk2) 1 are non-receptor tyrosine kinases that have been implicated as intermediates in the signaling pathway by which some G protein-coupled receptors (GPCRs) transactivate growth factor receptors (1-3). Although Src and Pyk2 activities are thought to be necessary for the transactivation of growth factor receptors by GPCRs, there are differing opinions in the literature about the role these kinases play in the activation of downstream mitogenic signaling. For example, inhibition of Src activity by cellular expression of C-terminal Src kinase was found to impair lysophosphatidic acid (LPA) and ␤2-adrenergic receptor-mediated activation of MAP kinases in COS-7 cells (1, 4). In rat-1 fibroblasts, dominant-negative mutants of the epidermal growth factor receptor (EGFR) or Src were used to demonstrate that the EGFR and Src are important for linking GPCR activation with the activation of MAP kinases (5, 6). And in PC12 cells, a dominant-negative mutant of Pyk2 and the EGFR kinase inhibitor AG1478 inhibited GPCR-mediated MAP kinase activation (2, 7). In contrast, experiments performed with embryonic fibroblasts derived from Src Ϫ/Ϫ , Pyk2 Ϫ/Ϫ , or Src Ϫ/Ϫ Pyk2 Ϫ/Ϫ mice indicated that both Src and Pyk2 are essential for GPCRmediated transactivation of the EGFR but are dispensable for GPCR-mediated activation of MAP kinases (3).In the present study, we have expressed wild-type and mutant P2Y 2 nucleotide receptors in human 1321N1 astrocytoma cells to explore how this GPCR transactivates growth factor receptors and affects mitogenic signaling. The P2Y 2 receptor is a G o / q -coupled receptor tha...