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...

Cystic fibrosis is caused by mutations in CFTR, the cystic fibrosis transmembrane conductance regulator gene. Disruption of CFTR-mediated anion conductance results in defective fluid and electrolyte movement in the epithelial cells of organs such as the pancreas, airways and sweat glands, but the function of CFTR in salivary glands is unclear. Salivary gland acinar cells produce an isotonic, plasma-like fluid, which is subsequently modified by the ducts to produce a hypotonic, NaCl-depleted final saliva. In the present study we investigated whether submandibular salivary glands (SMGs) in F508 mice (Cftr F/ F ) display ion transport defects characteristic of cystic fibrosis in other tissues. Immunolocalization and whole-cell recordings demonstrated that Cftr and the epithelial Na + (ENaC) channels are co-expressed in the apical membrane of submandibular duct cells, consistent with the significantly higher saliva [NaCl] observed in vivo in Cftr F/ F mice. In contrast, Cftr and ENaC channels were not detected in acinar cells, nor was saliva production affected in Cftr F/ F mice, implying that Cftr contributes little to the fluid secretion process in the mouse SMG. To identify the source of the NaCl absorption defect in Cftr F/ F mice, saliva was collected from ex vivo perfused SMGs. Cftr F/ F glands secreted saliva with significantly increased [NaCl]. Moreover, pharmacological inhibition of either Cftr or ENaC in the ex vivo SMGs mimicked the Cftr F/ F phenotype. In summary, our results demonstrate that NaCl absorption requires and is likely to be mediated by functionally dependent Cftr and ENaC channels localized to the apical membranes of mouse salivary gland duct cells.

The heterologous expression and activation of the human P2Y2 nucleotide receptor (P2Y2R) in human 1321N1 astrocytoma cells stimulates α‐secretase‐dependent cleavage of the amyloid precursor protein (APP), causing extracellular release of the non‐amyloidogenic protein secreted amyloid precursor protein (sAPPα). To determine whether a similar response occurs in a neuronal cell, we analyzed whether P2Y2R‐mediated production of sAPPα occurs in rat primary cortical neurons (rPCNs). In rPCNs, P2Y2R mRNA and receptor activity were virtually absent in quiescent cells, whereas overnight treatment with the pro‐inflammatory cytokine interleukin‐1β (IL‐1β) up‐regulated both P2Y2R mRNA expression and receptor activity by four‐fold. The up‐regulation of the P2Y2R was abrogated by pre‐incubation with Bay 11‐7085, an IκB‐α phosphorylation inhibitor, which suggests that P2Y2R mRNA transcript levels are regulated through nuclear factor‐κ‐B (NFκB) signaling. Furthermore, the P2Y2R agonist Uridine‐5′‐triphosphate (UTP) enhanced the release of sAPPα in rPCNs treated with IL‐1β or transfected with P2Y2R cDNA. UTP‐induced release of sAPPα from rPCNs was completely inhibited by pre‐treatment of the cells with the metalloproteinase inhibitor TACE inhibitor (TAPI‐2) or the phosphatidylinositol 3‐kinase (PI3K) inhibitor LY294002, and was partially inhibited by the MAPK/extracellular signal‐regulated kinase inhibitor U0126 and the protein kinase C inhibitor GF109203. These data suggest that P2Y2R‐mediated release of sAPPα from cortical neurons is directly dependent on a disintegrin and metalloproteinase (ADAM) 10/17 and PI3K activity, whereas extracellular signal‐regulated kinase 1/2 and PI3K activity may indirectly regulate APP processing. These results demonstrate that elevated levels of pro‐inflammatory cytokines associated with neurodegenerative diseases, such as IL‐1β, can enhance non‐amyloidogenic APP processing through up‐regulation of the P2Y2R in neurons.

Baker OJ, Camden JM, Redman RS, Jones JE, Seye CI, Erb L, Weisman GA. Proinflammatory cytokines tumor necrosis factor-␣ and interferon-␥ alter tight junction structure and function in the rat parotid gland Par-C10 cell line.

The amyloid precursor protein (APP) is proteolytically processed by ␤-and ␥-secretases to release amyloid ␤, the main component in senile plaques found in the brains of patients with Alzheimer disease. Alternatively, APP can be cleaved within the amyloid ␤ domain by ␣-secretase releasing the non-amyloidogenic product sAPP␣, which has been shown to have neuroprotective properties. Several G protein-coupled receptors are known to activate ␣-secretase-dependent processing of APP; however, the role of G protein-coupled nucleotide receptors in APP processing has not been investigated. Here it is demonstrated that activation of the G proteincoupled P2Y 2 receptor (P2Y 2 R) subtype expressed in human 1321N1 astrocytoma cells enhanced the release of sAPP␣ in a time-and dose-dependent manner. P2Y 2 Rmediated sAPP␣ release was dependent on extracellular calcium but was not affected by 1,2-bis(2-aminophenoxy)ethane-N,N,N,-trimethylammonium salt, an intracellular calcium chelator, indicating that P2Y 2 R-stimulated intracellular calcium mobilization was not involved. Inhibition of protein kinase C (PKC) with GF109203 or by PKC down-regulation with phorbol ester pre-treatment had no effect on UTP-stimulated sAPP␣ release, indicating a PKC-independent mechanism. U0126, an inhibitor of the mitogen-activated protein kinase pathway, partially inhibited sAPP␣ release by UTP, whereas inhibitors of Src-dependent epidermal growth factor receptor transactivation by P2Y 2 Rs had no effect. The metalloprotease inhibitors phenanthroline and TAPI-2 and the furin inhibitor decanoyl-ArgVal-Lys-Arg-chloromethylketone also diminished UTPinduced sAPP␣ release. Furthermore, small interfering RNA silencing of an endogenous adamalysin, ADAM10 or ADAM17/TACE, partially suppressed P2Y 2 R-activated sAPP␣ release, whereas treatment of cells with both ADAM10 and ADAM17/TACE small interfering RNAs completely abolished UTP-activated sAPP␣ release. These results may contribute to an understanding of the non-amyloidogenic processing of APP.P2 nucleotide receptors modulate a wide range of physiological responses following their activation by extracellular nucleotides (1, 2). The G protein-coupled P2Y 2 receptor (P2Y 2 R) 1 subtype is fully activated by equivalent concentrations of ATP or UTP (3-5) and is up-regulated in salivary gland models of stress and disease (6 -8) as well as in blood vessels after balloon angioplasty and in collared carotid arteries, where it induces intimal hyperplasia and inflammation by increasing smooth muscle cell proliferation and leukocyte infiltration (9, 10). Moreover, nucleotides are released from damaged cells of all tissues and from excited neurons, aggregating platelets, and contracting smooth muscle under physiological conditions (2, 11).The diversity of cellular responses mediated by P2Y 2 Rs is due in part to unique structural features that enable these receptors to stimulate a variety of signal transduction pathways. In addition to the classical stimulation of G␣ q -dependent phospholipase C (12, 13), the P2Y 2 R c...