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Mechanisms of agonist-dependent and -independent desensitization of a recombinant P2Y2 nucleotide receptor

Molecular and Cellular Biochemistry volume 205, pages 115–123 (2000)Cite this article

Abstract

UTP activates P2Y2 receptors in both 1321N1 cell transfectants expressing the P2Y2 receptor and human HT-29 epithelial cells expressing endogenous P2Y2 receptors with an EC50 of 0.2- 1.0 μM. Pretreatment of these cells with UTP diminished the effectiveness of a second dose of UTP (the IC50 for UTP-induced receptor desensitization was 0.3 - 1.0 μM for both systems). Desensitization and down-regulation of the P2Y2 nucleotide receptor may limit the effectiveness of UTP as a therapeutic agent. The present studies investigated the phenomenon of P2Y2 receptor desensitization in human 1321N1 astrocytoma cells expressing recombinant wild type and C-terminal truncation mutants of the P2Y,2 receptor. In these cells, potent P2Y2 receptor desensitization was observed after a 5 min exposure to UTP. Full receptor responsiveness returned 5-10 min after removal of UTP. Thapsigargin, an inhibitor of Ca2+-ATPase in the endoplasmic reticulum, induced an increase in the intracellular free calcium concentration, [Ca2+]i, after addition of desensitizing concentrations of UTP, indicating that P2Y2 receptor desensitization is not due to depletion of calcium from intracellular stores. Single cell measurements of increases in [Ca2+]i induced by UTP in 1321N1 cell transfectants expressing the P2Y2 receptor indicate that time- and UTP concentration-dependent desensitization occurred uniformly across a cell population. Other results suggest that P2Y2 receptor phosphorylation/dephosphorylation regulate receptor desensitization/resensitization. A 5 min preincubation of 1321N1 cell transfectants with the protein kinase C activator, phorbol 12-myristate 13-acetate (PMA), reduced the subsequent response to UTP by about 50% whereas co-incubation of PMA with UTP caused a greater inhibition in the response. The protein phosphatases - 1 and -2A inhibitor, okadaic acid, partially blocked resensitization of the receptor. Furthermore, C-terminal truncation mutants of the P2Y2 receptor that eliminated several potential phosphorylation sites including two for PKC were resistant to UTP-, but not phorbol ester-induced desensitization. Down regulation of protein kinase C isoforms prevented phorbol ester-induced desensitization but had no effect on agonist-induced desensitization of wild type or truncation mutant receptors. These results suggest that phosphorylation of the C-terminus of the P2Y2 receptor by protein kinases other than protein kinase C mediates agonist-induced receptor desensitization. A better understanding of the molecular mechanisms of P2Y2 nucleotide receptor desensitization may help optimize a promising cystic fibrosis pharmacotherapy based on the activation of anion secretion in airway epithelial cells by P2Y2 receptor agonists.

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References

  1. Weisman GA, Gonzalez FA, Erb L, Garrad RC, Turner JT: The cloning and expression of G protein-coupled P2Y nucleotide receptors. In: J.T. Turner, GA Weisman, JS Fedan, (eds). The P2 Nucleotide Receptors, Humana Press, Totowa, NJ, 1998, pp. 63–79

    Google Scholar 

  2. Lustig KD, Shiau AK, Brake AJ, Julius D: Expression cloning of an ATP receptor from mouse neuroblastoma cells. Proc Natl Acad Sci USA 90: 5113–5117, 1993

    PubMed  Google Scholar 

  3. Parr CE, Sullivan DM, Paradiso AM, Lazarowski ER, Burch LH, Olsen JC, Erb L, Weisman GA, Boucher RC, Turner JT: Cloning and expression of a human P2U nucleotide receptor: A target for cystic fibrosis pharmacotherapy. Proc Natl Acad Sci USA 91: 3275-3279, 1994

    Google Scholar 

  4. Froldi G, Pandolfo L, Chinellato A, Ragazzi E, Caparrotta L, Fassina G: Dual effect of ATP and UTP on rat atria: which types of receptors are involved? Naunyn-Schmiedeberg's Arch Pharmacol 349: 381–386, 1994

    Google Scholar 

  5. Chang K, Hanaoka K, Kumada M, Takuwa Y: Molecular cloning and functional analysis of a novel P2 nucleotide receptor. J Biol Chem 270: 26152–26158, 1995

    Article  PubMed  Google Scholar 

  6. Gobran LI, Xu Z-X, Lu Z, Rooney SA: P2u purinoceptor stimulation of surfactant secretion coupled to phosphatidylcholine hydrolysis in type II cells. Am J Physiol 267: L625–L633, 1994

    PubMed  Google Scholar 

  7. Rice WR, Burton FM, Fiedeldey DT: Cloning and expression of the alveolar type II cell P2u-purinergic receptor. Am J Respir Cell Mol Biol 12: 27–32, 1995

    PubMed  Google Scholar 

  8. Kaplan AD, Reimer WJ, Feldman RD, Dixon SJ: Extracellular nucleotides potentiate the cytosolic Ca2+, but not cyclic adenosine 3′,5′-monophosphate response to parathyroid hormone in rat osteoblastic cells. Endocrinol 136: 1674-1685, 1995

    Google Scholar 

  9. Lustig KD, Sportiello MG, Erb L, Weisman GA: A nucleotide receptor in vascular endothelial cells is specifically activated by the fully ionized forms of ATP and UTP. Biochem J 284: 733–739, 1992

    PubMed  Google Scholar 

  10. Communi D, Raspe E, Pirotton S, Boeynaems J-M: Coexpression of P2Y and P2U receptors on aortic endothelial cells: comparison of cell localization and signaling pathways. Circ Res 76: 191–198, 1995

    PubMed  Google Scholar 

  11. Bowden A, Patel V, Brown C, Boarder MR: Evidence for requirement of tyrosine phosphorylation in endothelial P2Y-and P2U-purinoceptor stimulation of prostacyclin release. Br J Pharmacol 116: 2563–2568, 1995

    PubMed  Google Scholar 

  12. Motte S, Communi D, Pirotton S, Boeynaems J-M: Involvement of multiple receptors in the actions of extracellular ATP: the example of vascular endothelial cells. Internatl. J Biochem Cell Biol 27: 1–7, 1995

    Article  Google Scholar 

  13. Mason SJ, Paradiso AM, Boucher RC: Regulation of transepithelial ion transport and intracellular calcium by extracellular ATP in human normal and cystic fibrosis airway epithelium. Br J Pharmacol 103: 1649–1656, 1991

    PubMed  Google Scholar 

  14. Stutts MJ, Chinet TC, Mason SJ, Fulton JM, Clarke LL, Boucher RC: Regulation of chloride channels in normal and cystic fibrosis airway epithelial cells by extracellular ATP. Proc Natl Acad Sci USA 89: 1621–1625, 1992

    PubMed  Google Scholar 

  15. Knowles MR, Clarke LL, Boucher RC: Activation by extracellular nucleotides of chloride secretion in the airway epithelia of patients with cystic fibrosis. N Engl J Med 325: 533–538, 1991

    PubMed  Google Scholar 

  16. Clarke LL, Boucher RC: Chloride secretory response to extracellular ATP in human normal and cystic fibrosis nasal epithelia. Am J Physiol 263: C348–C356, 1992

    PubMed  Google Scholar 

  17. Clarke LL, Harline MC, Otero MA, Glover GG, Garrad RC, Krugh B, Walker NM, Gonzá lez FA, Turner JT, Weisman GA: Desensitization of P2Y2 receptor-activated transepithelial anion secretion. Am J Physiol 276: C777–C787, 1999

    PubMed  Google Scholar 

  18. Garrad RC, Otero M, Erb L, Theiss PM, Clarke LL, Gonzalez FA, Turner JT, Weisman GA: Structural basis of agonist-induced desensitization and sequestration of the P2Y2 nucleotide receptor: Consequences of truncation of the C-terminus. J Biol Chem 273: 29437–29444, 1998

    Article  PubMed  Google Scholar 

  19. Erb L, Garrad R, Wang Y, Quinn T, Turner JT, Weisman GA: Sitedirected mutagenesis of P2Y2 purinoceptors: Positively charged amino acids in transmembrane helices 6 and 7 affect agonist potency and specificity. J Biol Chem 270: 4185–4188, 1995

    Article  PubMed  Google Scholar 

  20. Gonzalez FA, Alfonzo RG, Toro JR, Heppel LA: A receptor specific for certain nucleotides stimulates inositol phosphate metabolism and Ca2+ fluxes in A431 cells. J Cell Physiol 141: 606–617, 1989

    Article  PubMed  Google Scholar 

  21. Brown HA, Lazarowski ER, Boucher RC, Harden TK: Evidence that UTP and ATP regulate phospholipase C through a common extracellular 5′-nucleotide receptor in human airway epithelial cells. Mol Pharmacol 40: 648–655, 1991

    PubMed  Google Scholar 

  22. Thastrup O, Cullen PJ, Drobak BK, Hanley MR, Dawson AP: Thapsigargin, a tumor promoter, discharges intracellular Ca2+ stores by specific inhibition of the endoplasmic reticulum Ca2+-ATPase. Proc Natl Acad Sci U S A 87: 2466–2470, 1990

    PubMed  Google Scholar 

  23. Takemura H, Thastrup O, Putney JW Jr: Calcium efflux across the plasma membrane of rat parotid acinar cells is unaffected by receptor activation or by the microsomal calcium ATPase inhibitor, thapsigargin. Cell Calcium 11: 11–17, 1990

    Article  PubMed  Google Scholar 

  24. Schmidt R, Hecker E: Autoxidation of phorbol esters under normal storage conditions. Cancer Res 35: 1375–1377, 1975

    PubMed  Google Scholar 

  25. Bialojan C, Takai A: Inhibitory effect of a marine-sponge toxin, okadaic acid, on protein phosphatases. Biochem J 256: 283–290, 1988

    PubMed  Google Scholar 

  26. Newton AC: Protein kinase C: structure, function and regulation. J Biol Chem 270: 28495–28498, 1995

    Article  PubMed  Google Scholar 

  27. Collins FS: Cystic fibrosis: molecular biology and therapeutic implications. Science 256: 774–779, 1992

    PubMed  Google Scholar 

  28. Bennett WD, Olivier KN, Zeman KL, Hohneker KW, Boucher RC, Knowles MR: Effect of uridine 5′-triphosphate plus amiloride on mucociliary clearance in adult cystic fibrosis. Am J Resp Crit Care Med 153: 1796–1801, 1996

    PubMed  Google Scholar 

  29. Liggett SB, Freedman NJ, Schwinn DA, Lefkowitz RJ: Structural basis for receptor subtype-specific regulation revealed by a chimeric beta 3/ beta 2-adrenergic receptor. Proc Natl Acad Sci U S A 90: 3665–3669, 1993

    PubMed  Google Scholar 

  30. Lattion AL, Diviani D, Cotecchia S: Truncation of the receptor carboxyl terminus impairs agonist-dependent phosphorylation and desensitization of the a1B-adrenergic receptor. J Biol Chem 269: 22887–22893, 1994

    PubMed  Google Scholar 

  31. Yuan N, Friedman J, Whaley BS, Clark RB: cAMP-dependent protein kinase and protein kinase C consensus site mutations of the badrenergic receptor. Effect on desensitization and stimulation of adenylylcyclase. J Biol Chem 269: 23032–23038, 1994

    PubMed  Google Scholar 

  32. Freedman NJ, Lefkowitz RJ: Desensitization of G protein-coupled receptors. Recent Prog Horm Res 51: 319–351, 1996

    PubMed  Google Scholar 

  33. Gonzalez FA, Gross DJ, Heppel LA, Webb WW: Studies on the increase in cytosolic free calcium induced by epidermal growth factor, serum, and nucleotides in individual A431 cells. J Cell Physiol 135: 269–276, 1988

    Article  PubMed  Google Scholar 

  34. Lustig KD, Erb L, Landis DM, Hicker-Taylor CS, Zhang X, Sportiello MG, Weisman GA: Mechanisms by which extracellular ATP and UTP stimulate the release of prostacyclin from bovine pulmonary artery endothelial cells. Biochim Biophys Acta 1134: 61–72, 1992

    Article  PubMed  Google Scholar 

  35. Lin TA, Lustig KD, Sportiello MG, Weisman GA, Sun GY: Signal transduction pathways coupled to a P2U receptor in neuroblastoma x glioma (NG108-15) cells. J Neurochem 60: 1115–1125, 1993

    PubMed  Google Scholar 

  36. Zeitlin PL, Crawford I, Lu L, Woel S, Cohen ME, Donowitz M, Montrose MH, Hamosh A, Cutting GR, Gruenert D: CFTR protein expression in primary and cultured epithelia. Proc Natl Acad Sci U S A 89: 344–347, 1992

    PubMed  Google Scholar 

  37. Fonseca MI, Button DC, Brown RD: Agonist regulation of a1Badrenergic receptor subcellular distribution and function. J Biol Chem 270: 8902–8909, 1995

    Article  PubMed  Google Scholar 

  38. Beeler JF, Cooper RH: Regulation of hepatocyte plasma membrane α1-adrenergic receptors by 4β-phorbol 12-myristate 13-acetate. Biochem J 305: 73–79, 1995

    PubMed  Google Scholar 

  39. Eason MG, Moreira SP, Liggett SB: Four consecutive serines in the third intracellular loop are the sites for beta-adrenergic receptor kinasemediated phosphorylation and desensitization of the α2A-adrenergic receptor. J Biol Chem 270: 4681–4688, 1995

    Article  PubMed  Google Scholar 

  40. Diviani D, Lattion AL, Larbi N, Kunapuli P, Pronin A, Benovic JL, Cotecchia S: Effect of different G protein-coupled receptor kinases on phosphorylation and desensitization of the α1B-adrenergic receptor. J Biol Chem 271: 5049–5058, 1996

    Article  PubMed  Google Scholar 

  41. Boarder MR, Weisman GA, Turner JT, Wilkinson GF:G proteincoupled P2 purinoceptors: From molecular biology to functional responses. Trends Pharmacol Sci 16: 133–139, 1995

    Article  PubMed  Google Scholar 

  42. Johnson JA, Clark RB, Friedman J, Dixon RA, Strader CD: Identification of a specific domain in the b-adrenergic receptor required for phorbol ester-induced inhibition of catecholamine-stimulated adenylyl cyclase. Mol Pharmacol 38: 289–293, 1990

    PubMed  Google Scholar 

  43. Wilkinson GF, Purkiss JR, Boarder MR: Differential heterologous and homologous desensitization of two receptors for ATP (P2Y purinoceptors and nucleotide receptors) coexisting on endothelial cells. Mol Pharmacol 45: 731–736, 1994

    PubMed  Google Scholar 

  44. Schossl C, Rossig L, von zur Muhlen A, Beil W, Jahue J, Manns MP, Wagner S: Extracellular nucleotides increase cytosolic free calcium by activating P2u receptors in single human gastric mucous cells. Biochem Biophys Res Commun 216: 637–641, 1995

    PubMed  Google Scholar 

  45. Czubayko U, Reiser G: Desensitization of P2U receptor in neuronal cell line. Different controls by the agonist ATP and UTP, as demonstrated by single-cell Ca2+ responses. Biochem J 320: 215–219, 1996

    PubMed  Google Scholar 

  46. Chen CC, Chen WC: ATP-evoked inositol phosphate formation through activation of P2U purinergic receptors in cultured astrocytes: regulation by PKC subtypes α, δ and θ. Glia 17: 63–71, 1996

    Article  PubMed  Google Scholar 

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Authors and Affiliations

  1. Department of Chemistry, University of Puerto Rico, Río Piedras Campus, San Juan, Puerto Rico, 00931, USA

    Miguel Otero, Betty Velázquez, Melvin G. Hernández-Pérez & Fernando A. González

  2. Department of Biochemistry, University of Missouri-Columbia, Columbia, MO, 65212, USA

    Richard C. Garrad, Laurie Erb & Gary A. Weisman

  3. Department of Pharmacology, University of Missouri-Columbia, Columbia, MO, 65212, USA

    Jean M. Camden & John T. Turner

  4. Veterinary Biomedical Sciences & Dalton Cardiovascular Research Center, University of Missouri-Columbia, Columbia, MO, 65212, USA

    Lane L. Clarke

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  1. Miguel Otero
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Otero, M., Garrad, R.C., Velázquez, B. et al. Mechanisms of agonist-dependent and -independent desensitization of a recombinant P2Y2 nucleotide receptor. Mol Cell Biochem 205, 115–123 (2000). https://doi.org/10.1023/A:1007018001735

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  • DOI: https://doi.org/10.1023/A:1007018001735

  • cystic fibrosis
  • uridine 5′-triphosphate
  • 1321N1 astrocytoma
  • HT-29 epithelial cells
  • protein kinase C isoforms