Src Homology 3 Binding Sites in the P2Y2 Nucleotide Receptor Interact with Src and Regulate Activities of Src, Proline-rich Tyrosine Kinase 2, and Growth Factor Receptors

Abstract

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...

Disordered Pancreatic Inflammatory Responses and Inhibition of Fibrosis in CD39-Null Mice

“…We propose desensitization of P2Y receptors on CD39-null PSC perturbs proliferative responses to growth factor agonists by precluding transactivation processes. 29 Further compelling evidence that CD39 and purinergic elements are involved in the complex process of fibrogenesis in comparable cellular systems has been provided by Dranoff et al 7 Activation of hepatic stellate cells (HSC) leads to heightened expression of another closely related ecto-nucleotidase, CD39L1, on HSC. Furthermore, addition of extracellular UDP to HSC can triple mRNA levels of procollagen-α1 that encodes a major constituent of the fibrotic ECM.…”

Neuroprotective roles of the P2Y2 receptor

“…Deletion of the SH3-binding domains in the P2Y 2 R prevented nucleotides from activating VEGFR-2-dependent VCAM-1 upregulation [22,89]. VCAM-1 expression in endothelial cells also was found to be dependent on increases in [Ca 2+ ] i and p38 and Rho kinase activation but was independent of ERK1/2 activity [92].…”

“…Although P2Y 2 R-mediated activation of mitogen-activated protein (MAP) kinases is stimulated by G q -dependent increases in [Ca 2+ ] i , P2Y 2 R activation also stimulates epidermal growth factor receptor (EGFR) phosphorylation and significantly enhances the activities of the MAP kinases ERK1/2 and related adhesion focal tyrosine kinase (RAFTK) via a mechanism involving Src and Shc/Grb2 [21,88]. Other studies have shown that the P2Y 2 R contains 2 Src-homology-3 (SH3) binding domains in the intracellular C terminus that facilitate the binding of Src and the association of the P2Y 2 R with the EGFR, thereby enabling nucleotides to induce Src-dependent phosphorylation of the EFGR [22,89]. This P2Y 2 R-mediated transactivation of growth factor receptors has implications in the regulation of cell growth, motility, differentiation, and cytoskeleton-associated morphological changes [22,89].…”

“…Other studies have shown that the P2Y 2 R contains 2 Src-homology-3 (SH3) binding domains in the intracellular C terminus that facilitate the binding of Src and the association of the P2Y 2 R with the EGFR, thereby enabling nucleotides to induce Src-dependent phosphorylation of the EFGR [22,89]. This P2Y 2 R-mediated transactivation of growth factor receptors has implications in the regulation of cell growth, motility, differentiation, and cytoskeleton-associated morphological changes [22,89]. Studies also have shown that P2Y 2 Rs in the presence of nerve growth factor co-localize with tyrosine receptor kinase A via a Src-dependent event that promotes neurite outgrowth and cell division [27].…”

“…Another approach is to analyze the effects of uridine nucleotides (i.e., UTP/UDP) that only activate G qcoupled P2Y 2 , P2Y 4 , and P2Y 6 receptors among the known P2 receptor subtypes [2,4,19,20]. Among these three receptors, the P2Y 2 receptor (P2Y 2 R) has unique motifs that promote interactions with integrins and growth factor receptors, thereby enabling activation of signaling pathways beyond G q -dependent phospholipase C (PLC) [21][22][23][24]. Furthermore, the contribution of the P2Y 2 R subtype to CNS functions is becoming better understood [14,15,25] and appears to be most important under pathophysiological conditions, such as inflammation and bacterial infection [15,26].…”