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. 2003 Aug 15;374(Pt 1):51–61. doi: 10.1042/BJ20030585

P2X7 receptor activates extracellular signal-regulated kinases ERK1 and ERK2 independently of Ca2+ influx.

Jan Amstrup 1, Ivana Novak 1
PMCID: PMC1223572  PMID: 12747800

Abstract

P2X7 nucleotide receptors modulate a spectrum of cellular events in various cells including epithelia, such as exocrine pancreas. Although the pharmacology and channel properties of the P2X7 receptors have been studied intensively, signal transduction pathways are relatively unknown. In this study we applied a heterologous expression system of rat P2X7 receptors in HEK-293 cells. We followed the receptor expression and function using the enhanced green fluorescent protein (EGFP) tag, activation of intracellular proteins and increases in cellular Ca2+. EGFP-P2X7 receptors localized to the plasma membrane, clusters within the membrane and intracellularly. Stimulation of P2X7 receptors in HEK-293 cells led to an activation of extracellular signal-regulated kinases ERK1 and ERK2 and this activation was seen after just 1 min of stimulation with ATP. Using C- and N-terminal P2X7-receptor mutants we show that the N-terminus is important in activation of ERKs, whereas deletion of the last 230 amino acids in the C-terminus did not effect ERK activation. On the other hand, Ca2+ entry was impaired in C-terminal but not in N-terminal mutants. In cell suspensions prepared from rat pancreas we show that P2X7 receptors also activate ERK1 and ERK2, indicating that these signalling pathways are also turned on in native epithelium.

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Selected References

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  1. Armstrong John N., Brust Tyson B., Lewis Randall G., MacVicar Brian A. Activation of presynaptic P2X7-like receptors depresses mossy fiber-CA3 synaptic transmission through p38 mitogen-activated protein kinase. J Neurosci. 2002 Jul 15;22(14):5938–5945. doi: 10.1523/JNEUROSCI.22-14-05938.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Baricordi O. R., Melchiorri L., Adinolfi E., Falzoni S., Chiozzi P., Buell G., Di Virgilio F. Increased proliferation rate of lymphoid cells transfected with the P2X(7) ATP receptor. J Biol Chem. 1999 Nov 19;274(47):33206–33208. doi: 10.1074/jbc.274.47.33206. [DOI] [PubMed] [Google Scholar]
  3. Bradford Michelle D., Soltoff Stephen P. P2X7 receptors activate protein kinase D and p42/p44 mitogen-activated protein kinase (MAPK) downstream of protein kinase C. Biochem J. 2002 Sep 15;366(Pt 3):745–755. doi: 10.1042/BJ20020358. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Budagian Vadim, Bulanova Elena, Brovko Luba, Orinska Zane, Fayad Raja, Paus Ralf, Bulfone-Paus Silvia. Signaling through P2X7 receptor in human T cells involves p56lck, MAP kinases, and transcription factors AP-1 and NF-kappa B. J Biol Chem. 2002 Nov 6;278(3):1549–1560. doi: 10.1074/jbc.M206383200. [DOI] [PubMed] [Google Scholar]
  5. Buell G., Collo G., Rassendren F. P2X receptors: an emerging channel family. Eur J Neurosci. 1996 Oct;8(10):2221–2228. doi: 10.1111/j.1460-9568.1996.tb00745.x. [DOI] [PubMed] [Google Scholar]
  6. Christoffersen B. C., Hug M. J., Novak I. Different purinergic receptors lead to intracellular calcium increases in pancreatic ducts. Pflugers Arch. 1998 Jun;436(1):33–39. doi: 10.1007/s004240050601. [DOI] [PubMed] [Google Scholar]
  7. Dutton J. L., Poronnik P., Li G. H., Holding C. A., Worthington R. A., Vandenberg R. J., Cook D. I., Barden J. A., Bennett M. R. P2X(1) receptor membrane redistribution and down-regulation visualized by using receptor-coupled green fluorescent protein chimeras. Neuropharmacology. 2000 Aug 23;39(11):2054–2066. doi: 10.1016/s0028-3908(00)00058-7. [DOI] [PubMed] [Google Scholar]
  8. Dwivedi Prem P., Hii Charles S. T., Ferrante Antonio, Tan Joseph, Der Channing J., Omdahl John L., Morris Howard A., May Brian K. Role of MAP kinases in the 1,25-dihydroxyvitamin D3-induced transactivation of the rat cytochrome P450C24 (CYP24) promoter. Specific functions for ERK1/ERK2 and ERK5. J Biol Chem. 2002 Jun 4;277(33):29643–29653. doi: 10.1074/jbc.M204561200. [DOI] [PubMed] [Google Scholar]
  9. Fernando K. C., Gargett C. E., Wiley J. S. Activation of the P2Z/P2X7 receptor in human lymphocytes produces a delayed permeability lesion: involvement of phospholipase D. Arch Biochem Biophys. 1999 Feb 15;362(2):197–202. doi: 10.1006/abbi.1998.1045. [DOI] [PubMed] [Google Scholar]
  10. Gendron Fernand-Pierre, Neary Joseph T., Theiss Patty M., Sun Grace Y., Gonzalez Fernando A., Weisman Gary A. Mechanisms of P2X7 receptor-mediated ERK1/2 phosphorylation in human astrocytoma cells. Am J Physiol Cell Physiol. 2003 Feb;284(2):C571–C581. doi: 10.1152/ajpcell.00286.2002. [DOI] [PubMed] [Google Scholar]
  11. Good D. W., Di Mari J. F., Watts B. A., 3rd Hyposmolality stimulates Na(+)/H(+) exchange and HCO(3)(-) absorption in thick ascending limb via PI 3-kinase. Am J Physiol Cell Physiol. 2000 Nov;279(5):C1443–C1454. doi: 10.1152/ajpcell.2000.279.5.C1443. [DOI] [PubMed] [Google Scholar]
  12. Gu B. J., Zhang W. Y., Bendall L. J., Chessell I. P., Buell G. N., Wiley J. S. Expression of P2X(7) purinoceptors on human lymphocytes and monocytes: evidence for nonfunctional P2X(7) receptors. Am J Physiol Cell Physiol. 2000 Oct;279(4):C1189–C1197. doi: 10.1152/ajpcell.2000.279.4.C1189. [DOI] [PubMed] [Google Scholar]
  13. Gudipaty L., Humphreys B. D., Buell G., Dubyak G. R. Regulation of P2X(7) nucleotide receptor function in human monocytes by extracellular ions and receptor density. Am J Physiol Cell Physiol. 2001 Apr;280(4):C943–C953. doi: 10.1152/ajpcell.2001.280.4.C943. [DOI] [PubMed] [Google Scholar]
  14. Hede S. E., Amstrup J., Christoffersen B. C., Novak I. Purinoceptors evoke different electrophysiological responses in pancreatic ducts. P2Y inhibits K(+) conductance, and P2X stimulates cation conductance. J Biol Chem. 1999 Nov 5;274(45):31784–31791. doi: 10.1074/jbc.274.45.31784. [DOI] [PubMed] [Google Scholar]
  15. Henriksen Katerine L., Novak Ivana. Effect of ATP on intracellular pH in pancreatic ducts involves P2X7 receptors. Cell Physiol Biochem. 2003;13(2):93–102. doi: 10.1159/000070253. [DOI] [PubMed] [Google Scholar]
  16. Humphreys B. D., Rice J., Kertesy S. B., Dubyak G. R. Stress-activated protein kinase/JNK activation and apoptotic induction by the macrophage P2X7 nucleotide receptor. J Biol Chem. 2000 Sep 1;275(35):26792–26798. doi: 10.1074/jbc.M002770200. [DOI] [PubMed] [Google Scholar]
  17. Hung Amos C., Sun Synthia H. The P2X(7) receptor-mediated phospholipase D activation is regulated by both PKC-dependent and PKC-independent pathways in a rat brain-derived Type-2 astrocyte cell line, RBA-2. Cell Signal. 2002 Jan;14(1):83–92. doi: 10.1016/s0898-6568(01)00230-3. [DOI] [PubMed] [Google Scholar]
  18. Hurd Toby W., Culbert Ainsley A., Webster Kenneth J., Tavaré Jeremy M. Dual role for mitogen-activated protein kinase (Erk) in insulin-dependent regulation of Fra-1 (fos-related antigen-1) transcription and phosphorylation. Biochem J. 2002 Dec 1;368(Pt 2):573–580. doi: 10.1042/BJ20020579. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Ishiguro H., Naruse S., Kitagawa M., Hayakawa T., Case R. M., Steward M. C. Luminal ATP stimulates fluid and HCO3- secretion in guinea-pig pancreatic duct. J Physiol. 1999 Sep 1;519(Pt 2):551–558. doi: 10.1111/j.1469-7793.1999.0551m.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Khakh B. S., Smith W. B., Chiu C. S., Ju D., Davidson N., Lester H. A. Activation-dependent changes in receptor distribution and dendritic morphology in hippocampal neurons expressing P2X2-green fluorescent protein receptors. Proc Natl Acad Sci U S A. 2001 Apr 10;98(9):5288–5293. doi: 10.1073/pnas.081089198. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kim M., Jiang L. H., Wilson H. L., North R. A., Surprenant A. Proteomic and functional evidence for a P2X7 receptor signalling complex. EMBO J. 2001 Nov 15;20(22):6347–6358. doi: 10.1093/emboj/20.22.6347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kim M., Spelta V., Sim J., North R. A., Surprenant A. Differential assembly of rat purinergic P2X7 receptor in immune cells of the brain and periphery. J Biol Chem. 2001 Apr 19;276(26):23262–23267. doi: 10.1074/jbc.M102253200. [DOI] [PubMed] [Google Scholar]
  23. Lee M. G., Zeng W., Muallem S. Characterization and localization of P2 receptors in rat submandibular gland acinar and duct cells. J Biol Chem. 1997 Dec 26;272(52):32951–32955. doi: 10.1074/jbc.272.52.32951. [DOI] [PubMed] [Google Scholar]
  24. Nicke A., Bäumert H. G., Rettinger J., Eichele A., Lambrecht G., Mutschler E., Schmalzing G. P2X1 and P2X3 receptors form stable trimers: a novel structural motif of ligand-gated ion channels. EMBO J. 1998 Jun 1;17(11):3016–3028. doi: 10.1093/emboj/17.11.3016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Novak Ivana. ATP as a signaling molecule: the exocrine focus. News Physiol Sci. 2003 Feb;18:12–17. doi: 10.1152/nips.01409.2002. [DOI] [PubMed] [Google Scholar]
  26. Novak Ivana, Nitschke Roland, Amstrup Jan. Purinergic receptors have different effects in rat exocrine pancreas. Calcium signals monitored by fura-2 using confocal microscopy. Cell Physiol Biochem. 2002;12(2-3):83–92. doi: 10.1159/000063784. [DOI] [PubMed] [Google Scholar]
  27. Panenka W., Jijon H., Herx L. M., Armstrong J. N., Feighan D., Wei T., Yong V. W., Ransohoff R. M., MacVicar B. A. P2X7-like receptor activation in astrocytes increases chemokine monocyte chemoattractant protein-1 expression via mitogen-activated protein kinase. J Neurosci. 2001 Sep 15;21(18):7135–7142. doi: 10.1523/JNEUROSCI.21-18-07135.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Ralevic V., Burnstock G. Receptors for purines and pyrimidines. Pharmacol Rev. 1998 Sep;50(3):413–492. [PubMed] [Google Scholar]
  29. Rubio M. E., Soto F. Distinct Localization of P2X receptors at excitatory postsynaptic specializations. J Neurosci. 2001 Jan 15;21(2):641–653. doi: 10.1523/JNEUROSCI.21-02-00641.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Saxena M., Mustelin T. Extracellular signals and scores of phosphatases: all roads lead to MAP kinase. Semin Immunol. 2000 Aug;12(4):387–396. doi: 10.1006/smim.2000.0219. [DOI] [PubMed] [Google Scholar]
  31. Schilling W. P., Sinkins W. G., Estacion M. Maitotoxin activates a nonselective cation channel and a P2Z/P2X(7)-like cytolytic pore in human skin fibroblasts. Am J Physiol. 1999 Oct;277(4 Pt 1):C755–C765. doi: 10.1152/ajpcell.1999.277.4.C755. [DOI] [PubMed] [Google Scholar]
  32. Schilling W. P., Wasylyna T., Dubyak G. R., Humphreys B. D., Sinkins W. G. Maitotoxin and P2Z/P2X(7) purinergic receptor stimulation activate a common cytolytic pore. Am J Physiol. 1999 Oct;277(4 Pt 1):C766–C776. doi: 10.1152/ajpcell.1999.277.4.C766. [DOI] [PubMed] [Google Scholar]
  33. Smart Megan L., Gu Ben, Panchal Rekha G., Wiley James, Cromer Brett, Williams David A., Petrou Steven. P2X7 receptor cell surface expression and cytolytic pore formation are regulated by a distal C-terminal region. J Biol Chem. 2002 Dec 20;278(10):8853–8860. doi: 10.1074/jbc.M211094200. [DOI] [PubMed] [Google Scholar]
  34. Smart Megan L., Panchal Rekha G., Bowser David N., Williams David A., Petrou Steven. Pore formation is not associated with macroscopic redistribution of P2X7 receptors. Am J Physiol Cell Physiol. 2002 Jul;283(1):C77–C84. doi: 10.1152/ajpcell.00456.2001. [DOI] [PubMed] [Google Scholar]
  35. Soltoff S. P., McMillian M. K., Cragoe E. J., Jr, Cantley L. C., Talamo B. R. Effects of extracellular ATP on ion transport systems and [Ca2+]i in rat parotid acinar cells. Comparison with the muscarinic agonist carbachol. J Gen Physiol. 1990 Feb;95(2):319–346. doi: 10.1085/jgp.95.2.319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Soltoff S. P., McMillian M. K., Talamo B. R. ATP activates a cation-permeable pathway in rat parotid acinar cells. Am J Physiol. 1992 Apr;262(4 Pt 1):C934–C940. doi: 10.1152/ajpcell.1992.262.4.C934. [DOI] [PubMed] [Google Scholar]
  37. Sorensen C. E., Novak I. Visualization of ATP release in pancreatic acini in response to cholinergic stimulus. Use of fluorescent probes and confocal microscopy. J Biol Chem. 2001 May 31;276(35):32925–32932. doi: 10.1074/jbc.M103313200. [DOI] [PubMed] [Google Scholar]
  38. Soto F., Garcia-Guzman M., Stühmer W. Cloned ligand-gated channels activated by extracellular ATP (P2X receptors). J Membr Biol. 1997 Nov 15;160(2):91–100. doi: 10.1007/s002329900298. [DOI] [PubMed] [Google Scholar]
  39. Surprenant A., Rassendren F., Kawashima E., North R. A., Buell G. The cytolytic P2Z receptor for extracellular ATP identified as a P2X receptor (P2X7). Science. 1996 May 3;272(5262):735–738. doi: 10.1126/science.272.5262.735. [DOI] [PubMed] [Google Scholar]
  40. Tombes R. M., Auer K. L., Mikkelsen R., Valerie K., Wymann M. P., Marshall C. J., McMahon M., Dent P. The mitogen-activated protein (MAP) kinase cascade can either stimulate or inhibit DNA synthesis in primary cultures of rat hepatocytes depending upon whether its activation is acute/phasic or chronic. Biochem J. 1998 Mar 15;330(Pt 3):1451–1460. doi: 10.1042/bj3301451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Torres G. E., Egan T. M., Voigt M. M. Hetero-oligomeric assembly of P2X receptor subunits. Specificities exist with regard to possible partners. J Biol Chem. 1999 Mar 5;274(10):6653–6659. doi: 10.1074/jbc.274.10.6653. [DOI] [PubMed] [Google Scholar]
  42. Williams J. A. Intracellular signaling mechanisms activated by cholecystokinin-regulating synthesis and secretion of digestive enzymes in pancreatic acinar cells. Annu Rev Physiol. 2001;63:77–97. doi: 10.1146/annurev.physiol.63.1.77. [DOI] [PubMed] [Google Scholar]
  43. Wilson Heather L., Wilson Stuart A., Surprenant Annmarie, North R. Alan. Epithelial membrane proteins induce membrane blebbing and interact with the P2X7 receptor C terminus. J Biol Chem. 2002 Jul 9;277(37):34017–34023. doi: 10.1074/jbc.M205120200. [DOI] [PubMed] [Google Scholar]
  44. Worthington R. A., Dutton J. L., Poronnik P., Bennett M. R., Barden J. A. Localisation of P2X receptors in human salivary gland epithelial cells and human embryonic kidney cells by sodium dodecyl sulfate-polyacrylamide gel electrophoresis/Western blotting and immunofluorescence. Electrophoresis. 1999 Jul;20(10):2065–2070. doi: 10.1002/(SICI)1522-2683(19990701)20:10<2065::AID-ELPS2065>3.0.CO;2-E. [DOI] [PubMed] [Google Scholar]
  45. Worthington R. A., Smart M. L., Gu B. J., Williams D. A., Petrou S., Wiley J. S., Barden J. A. Point mutations confer loss of ATP-induced human P2X(7) receptor function. FEBS Lett. 2002 Feb 13;512(1-3):43–46. doi: 10.1016/s0014-5793(01)03311-7. [DOI] [PubMed] [Google Scholar]

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