P2Y2 Nucleotide Receptors Mediate Metalloprotease-dependent Phosphorylation of Epidermal Growth Factor Receptor and ErbB3 in Human Salivary Gland Cells

Abstract

The G protein-coupled receptor P2Y 2 nucleotide receptor (P2Y 2 R) has been shown to be up-regulated in a variety of tissues in response to stress or injury. Recent studies have suggested that P2Y 2 Rs may play a role in immune responses, wound healing, and tissue regeneration via their ability to activate multiple signaling pathways, including activation of growth factor receptors. Here, we demonstrate that in human salivary gland (HSG) cells, activation of the P2Y 2 R by its agonist induces phosphorylation of ERK1/2 via two distinct mechanisms, a rapid, protein kinase C-dependent pathway and a slower and prolonged, epidermal growth factor receptor (EGFR)-dependent pathway. The EGFR-dependent stimulation of UTP-induced ERK1/2 phosphorylation in HSG cells is inhibited by the adamalysin inhibitor tumor necrosis factor-␣ protease inhibitor or by small interfering RNA that selectively silences ADAM10 and ADAM17 expression, suggesting that ADAM metalloproteases are required for P2Y 2 R-mediated activation of the EGFR. G proteincoupled receptors have been shown to promote proteolytic release of EGFR ligands; however, neutralizing antibodies to known ligands of the EGFR did not inhibit UTP-induced EGFR phosphorylation. Immunoprecipitation experiments indicated that UTP causes association of the EGFR with another member of the EGF receptor family, ErbB3. Furthermore, stimulation of HSG cells with UTP induced phosphorylation of ErbB3, and silencing of ErbB3 expression inhibited UTP-induced phosphorylation of both ErbB3 and EGFR. UTP-induced phosphorylation of ErbB3 and EGFR was also inhibited by silencing the expression of the ErbB3 ligand neuregulin 1 (NRG1). These results suggest that P2Y 2 R activation in salivary gland cells promotes the formation of EGFR/ErbB3 heterodimers and metalloprotease-dependent neuregulin 1 release, resulting in the activation of both EGFR and ErbB3.G protein-coupled receptors (GPCRs), 3 the largest group of cell surface receptors, function not only as short term modulators of cell metabolism but also as regulators of cellular growth and differentiation via activation of the ERK/MAPK signaling cascade (1-5). The mechanisms whereby GPCRs activate the ERK/MAPK signaling pathway are complex and vary according to the type of GPCR and the tissue in which the receptor is expressed (1, 6, 7). During the past decade, numerous studies have demonstrated that GPCRs can couple to the ERK/MAPK signaling cascade directly through a G protein-dependent pathway or indirectly by activation of the epidermal growth factor receptor (EGFR, also known as ErbB1) (1, 8 -10). Mediators of GPCR-induced ERK/MAPK activation include Src tyrosine kinase (6,(11)(12)(13)(14)(15), protein kinase C (PKC) (6, 11, 12), prolinerich tyrosine kinase 2 (13, 14), increases in the intracellular calcium concentration ([Ca 2ϩ ] i ) (15, 16), reactive oxygen species (17-19), and metalloprotease-dependent shedding of an EGFR ligand (8, 20 -22). EGFR ligands promote homodimerization and autophosphorylation of the EGFR (23), resulting i...

P2Y2 receptor inhibits EGF-induced MAPK pathway to stabilise keratinocyte hemidesmosomes.

“…3A, UTP did not induce EGFR phosphorylation on any tyrosine residue tested in these experiments in HaCaT cells. Thus, in sharp contrast with data reported in other cell types (Ratchford et al, 2010;Liu et al, 2004;Morris et al, 2004;Soltoff, 1998;Boucher et al, 2011), P2Y2R was not able to transactivate EGFR in keratinocytes. When added simultaneously with EGF, UTP had no significant impact on the phosphorylation level of Tyr845, Tyr992, Tyr1045 and Tyr1068 upon EGF stimulation (Fig.…”

“…Journal of Cell Science Giltaire et al, 2011) and other cell types (Ratchford et al, 2010;Erb et al, 2006;Grimm et al, 2010;Liu et al, 2004;Seye et al, 2004) showing that P2Y2R classically activates ERK1/2, like most of the GPCR. Interestingly, similarly to our own observations in HaCaT cells, P2Y receptors activate MAPK pathway in serum-starved astrocytes and exhibit some ability to inhibit this pathway in the presence of growth factors (Lenz et al, 2001).…”

Neuroprotective roles of the P2Y2 receptor

“…Similarly, lymphocyte binding to epithelium is stimulated by P2Y 2 R activation in epithelial cells via EGFR-dependent VCAM-1 upregulation [93]. However, this pathway was found to be Src-independent and required the release of growth factors by P2Y 2 R-dependent activation of matrix metalloproteases (MMPs) [94].…”

“…The C terminus of the P2Y 2 R also has been shown to interact with the actin-binding protein filamin A (FLNa). P2Y 2 R activation can stimulate the activity of matrix metalloproteases (e.g., ADAM10 and ADAM17) leading to the α-secretase-dependent processing of APP to the non-amyloidogenic peptide sAPPα [38,48] and release of growth factors (e.g., NRG1) [94] cells as a result of caspase 3/7 activation, also have been shown to induce cell migration of phagocytic cells [143]. In an in vivo model of cell migration, supernatants from apoptotic cells produced a three-fold greater recruitment of monocytes and macrophages than supernatants from control cells and depletion of nucleotides in the apoptotic cell supernatants diminished cell migration [143].…”

Interaction of purinergic receptors with GPCRs, ion channels, tyrosine kinase and steroid hormone receptors orchestrates cell function

“…Thus, P2X 7 receptors are also likely to be important in the control of angiogenesis and wound repair [106]. P2Y 2 receptor activation in human salivary gland cells promotes the formation of EGFR/ ErbB3 heterodimers and metalloprotease-dependent neuregulin 1 release, resulting in the activation of both EGFR and ErbB3 [107]. P2X 7 receptors were also implicated in VEGF release in rat C6 glioma cells.…”