Skip to main content
SpringerLink
Log in

Agonist-induced phosphorylation and desensitization of the P2Y2 nucleotide receptor

  • Published:
Molecular and Cellular Biochemistry Aims and scope Submit manuscript

Abstract

Purification of HA-tagged P2Y2 receptors from transfected human 1321N1 astrocytoma cells yielded a protein with a molecular size determined by SDS-PAGE to be in the range of 57–76 kDa, which is typical of membrane glycoproteins with heterogeneous complex glycosylation. The protein phosphatase inhibitor, okadaic acid, attenuated the recovery of receptor activity from the agonist-induced desensitized state, suggesting a role for P2Y2 receptor phosphorylation in desensitization. Isolation of HA-tagged P2Y2 nucleotide receptors from metabolically [32P]-labelled cells indicated a (3.8 ± 0.2)-fold increase in the [32P]-content of the receptor after 15 min of treatment with 100 μM UTP, as compared to immunoprecipitated receptors from untreated control cells. Receptor sequestration studies indicated that ∼40% of the surface receptors were internalized after a 15-min stimulation with 100 μM UTP. Point mutation of three potential GRK and PKC phosphorylation sites in the third intracellular loop and C-terminal tail of the P2Y2 receptor (namely, S243A, T344A, and S356A) extinguished agonist-induced receptor phosphorylation, caused a marked reduction in the efficacy of UTP to desensitize P2Y2 receptor signalling to intracellular calcium mobilization, and impaired agonist-induced receptor internalization. Activation of PKC isoforms with phorbol 12-myristate 13-acetate that caused heterologous receptor desensitization did not increase the level of P2Y2 receptor phosphorylation. Our results indicate a role for receptor phosphorylation by phorbol-insensitive protein kinases in agonist-induced desensitization of the P2Y2 nucleotide receptor. (Mol Cell Biochem xxx: 35–45, 2005)

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

AAA-P2Y2, S243A, T344A, and S356A:

triple mutant of P2Y2

BSA:

bovine serum albumin

[Ca2+] i :

intracellular free calcium ion concentration

DMEM:

Dulbecco's modified Eagle's medium

EC50 :

agonist concentration that produces 50% of a response

FITC:

fluorescein isothiocyanate

GPCR:

G protein-coupled receptor

GRK:

G protein-coupled receptor kinase

HA:

hemagglutinin

Hepes:

N-[2-Hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid]

IC50 :

agonist concentration that inhibits 50% of the response

MAPK:

mitogen-activated protein kinase

PKC:

protein kinase C

PLC-β:

phospholipase C-β

P2Y2-1321N1:

HA-tagged P2Y2 transfected 1321N1 cells

PMA:

phorbol 12-myristate 13-acetate

PMSF:

phenylmethylsulfonyl fluoride

S.E.M.:

standard error of the mean

WT-P2Y2 :

wild type P2Y2

References

  1. Hanley PJ, Musset B, Renigunta V, Limberg SH, Dalpke AH, Sus R, Heeg KM, Preisig-Muller R, Daut J: Extracellular ATP induces oscillations of intracellular Ca2+ and membrane potential and promotes transcription of IL-6 in macrophages. Proc Natl Acad Sci USA 101: 9479–9484, 2004

    Article  CAS  PubMed  Google Scholar 

  2. Kannan S: Neutrophil chemotaxis: potential role of chemokine receptors in extracellular nucleotide induced Mac-1 expression. Med Hypotheses 61: 577–579, 2003

    CAS  PubMed  Google Scholar 

  3. Neary JT, Baker L, Jorgensen SL, Norenberg MD: Extracellular ATP induces stellation and increases glial fibrillary acidic protein content and DNA synthesis in primary astrocyte cultures. Acta Neuropathol (Berl) 87: 8–13, 1994

    CAS  Google Scholar 

  4. Chorna NE, Santiago-Perez LI, Erb L, Seye CI, Neary JT, Sun GY, Weisman GA, Gonzalez FA: P2Y receptors activate neuroprotective mechanisms in astrocytic cells. J Neurochem 91: 119–132, 2004

    Article  CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  6. Nour M, Quiambao AB, Peterson WM, Al-Ubaidi MR, Naash MI: P2Y(2) receptor agonist INS37217 enhances functional recovery after detachment caused by subretinal injection in normal and rds mice. Invest Ophthalmol Vis Sci 44: 4505–4514, 2003

    Article  PubMed  Google Scholar 

  7. Kellerman DJ: P2Y(2) receptor agonists: a new class of medication targeted at improved mucociliary clearance. Chest 121: 201S–205S, 2002

    Article  CAS  PubMed  Google Scholar 

  8. Garrad RC, Otero MA, 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  CAS  PubMed  Google Scholar 

  9. Otero M, Garrad RC, Velazquez B, Hernandez-Perez MG, Camden JM, Erb L, Clarke LL, Turner JT, Weisman GA, Gonzalez FA: Mechanisms of agonist-dependent and -independent desensitization of a recombinant P2Y2 nucleotide receptor. Mol Cell Biochem 205: 115–123, 2000

    Article  CAS  PubMed  Google Scholar 

  10. Bouvier M, Hausdorff WP, De Blasi A, O'Dowd BF, Kobilka BK, Caron MG, Lefkowitz RJ: Removal of phosphorylation sites from the β 2-adrenergic receptor delays onset of agonist-promoted desensitization. Nature 333: 370–373, 1988

    Article  CAS  PubMed  Google Scholar 

  11. Hausdorff WP, Bouvier M, O'Dowd BF, Irons GP, Caron MG, Lefkowitz RJ: Phosphorylation sites on two domains of the β 2-adrenergic receptor are involved in distinct pathways of receptor desensitization. J Biol Chem 264: 12657–12665, 1989

    CAS  PubMed  Google Scholar 

  12. Premont RT, Inglese J, Lefkowitz RJ: Protein kinases that phosphorylate activated G protein-coupled receptors. Faseb J 9: 175–182, 1995

    CAS  PubMed  Google Scholar 

  13. Gurevich VV, Pals-Rylaarsdam R, Benovic JL, Hosey MM, Onorato JJ: Agonist-receptor-arrestin, an alternative ternary complex with high agonist affinity. J Biol Chem 272: 28849–28852, 1997

    Article  CAS  PubMed  Google Scholar 

  14. Ferguson SS, Barak LS, Zhang J, Caron MG: G-protein-coupled receptor regulation: role of G-protein-coupled receptor kinases and arrestins. Can J Physiol Pharmacol 74: 1095–1110, 1996

    Article  CAS  PubMed  Google Scholar 

  15. Sterne-Marr R, Benovic JL: Regulation of G protein-coupled receptors by receptor kinases and arrestins. Vitam Horm 51: 193–234, 1995

    CAS  PubMed  Google Scholar 

  16. Koenig JA, Edwardson JM: Endocytosis and recycling of G protein-coupled receptors. Trends Pharmacol Sci 18: 276–287, 1997

    CAS  PubMed  Google Scholar 

  17. Zuker CS, Ranganathan R: The path to specificity. Science 283: 650–651, 1999

    Article  CAS  PubMed  Google Scholar 

  18. Erb L, Lustig KD, Sullivan DM, Turner JT, Weisman GA: Functional expression and photoaffinity labeling of a cloned P2U purinergic receptor. Proc Natl Acad Sci USA 90: 10449–10453, 1993

    CAS  PubMed  Google Scholar 

  19. Boarder MR, Weisman GA, Turner JT, Wilkinson GF: G protein-coupled P2 purinoceptors: from molecular biology to functional responses. Trends Pharmacol Sci 16: 133–139, 1995

    Article  CAS  PubMed  Google Scholar 

  20. Santiago-Perez LI, Flores RV, Santos-Berrios C, Chorna NE, Krugh B, Garrad RC, Erb L, Weisman GA, Gonzalez FA: P2Y(2) nucleotide receptor signaling in human monocytic cells: activation, desensitization and coupling to mitogen-activated protein kinases. J Cell Physiol 187: 196–208, 2001

    CAS  PubMed  Google Scholar 

  21. 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

    CAS  PubMed  Google Scholar 

  22. Nguyen T, Erb L, Weisman GA, Marchese A, Heng HH, Garrad RC, George SR, Turner JT, O'Dowd BF: Cloning, expression, and chromosomal localization of the human uridine nucleotide receptor gene. J Biol Chem 270: 30845–30848, 1995

    CAS  PubMed  Google Scholar 

  23. Grynkiewicz G, Poenie M, Tsien RY: A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260: 3440–3450, 1985

    CAS  PubMed  Google Scholar 

  24. Freedman NJ, Liggett SB, Drachman DE, Pei G, Caron MG, Lefkowitz RJ: Phosphorylation and desensitization of the human β 1-adrenergic receptor. Involvement of G protein-coupled receptor kinases and cAMP-dependent protein kinase. J Biol Chem 270: 17953–17961, 1995

    Article  CAS  PubMed  Google Scholar 

  25. Holloway AC, Qian H, Pipolo L, Ziogas J, Miura S, Karnik S, Southwell BR, Lew MJ, Thomas WG: Side-chain substitutions within angiotensin II reveal different requirements for signaling, internalization, and phosphorylation of type 1A angiotensin receptors. Mol Pharmacol 61: 768–777, 2002

    Article  CAS  PubMed  Google Scholar 

  26. Seibold A, January BG, Friedman J, Hipkin RW, Clark RB: Desensitization of β2-adrenergic receptors with mutations of the proposed G protein-coupled receptor kinase phosphorylation sites. J Biol Chem 273: 7637–7642, 1998

    Article  CAS  PubMed  Google Scholar 

  27. Mellor H, Parker PJ: The extended protein kinase C superfamily. Biochem J 332(Pt 2): 281–292, 1998

    Google Scholar 

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

  29. Turner JT, Weisman GA, Fedan JS: The P2 Nucleotide Receptors, 1998

  30. Lefkowitz RJ: G protein-coupled receptors. III. New roles for receptor kinases and β-arrestins in receptor signaling and desensitization. J Biol Chem 273: 18677–18680, 1998

    Article  CAS  PubMed  Google Scholar 

  31. von Zastrow M, Kobilka BK: Ligand-regulated internalization and recycling of human β 2-adrenergic receptors between the plasma membrane and endosomes containing transferrin receptors. J Biol Chem 267: 3530–3538, 1992

    CAS  PubMed  Google Scholar 

  32. Lattion AL, Diviani D, Cotecchia S: Truncation of the receptor carboxyl terminus impairs agonist-dependent phosphorylation and desensitization of the α 1B-adrenergic receptor. J Biol Chem 269: 22887–22893, 1994

    CAS  PubMed  Google Scholar 

  33. Huang Z, Chen Y, Nissenson RA: The cytoplasmic tail of the G-protein-coupled receptor for parathyroid hormone and parathyroid hormone-related protein contains positive and negative signals for endocytosis. J Biol Chem 270: 151–156, 1995

    CAS  PubMed  Google Scholar 

  34. Thomas WG, Thekkumkara TJ, Motel TJ, Baker KM: Stable expression of a truncated AT1A receptor in CHO-K1 cells. The carboxyl-terminal region directs agonist-induced internalization but not receptor signaling or desensitization. J Biol Chem 270: 207–213, 1995

    CAS  PubMed  Google Scholar 

  35. Chabry J, Botto JM, Nouel D, Beaudet A, Vincent JP, Mazella J: Thr-422 and Tyr-424 residues in the carboxyl terminus are critical for the internalization of the rat neurotensin receptor. J Biol Chem 270: 2439–2442, 1995

    CAS  PubMed  Google Scholar 

  36. Litosch I: Novel mechanisms for feedback regulation of phospholipase C-β activity. IUBMB Life 54: 253–260, 2002

    Article  CAS  PubMed  Google Scholar 

  37. Sato M, Moroi K, Nishiyama M, Zhou J, Usui H, Kasuya Y, Fukuda M, Kohara Y, Komuro I, Kimura S: Characterization of a novel C. elegans RGS protein with a C2 domain: evidence for direct association between C2 domain and Galphaq subunit. Life Sci 73: 917–932, 2003

    Article  CAS  PubMed  Google Scholar 

  38. Cunningham ML, Waldo GL, Hollinger S, Hepler JR, Harden TK: Protein kinase C phosphorylates RGS2 and modulates its capacity for negative regulation of Galpha 11 signaling. J Biol Chem 276: 5438–5444, 2001

    CAS  PubMed  Google Scholar 

  39. Krasel C, Dammeier S, Winstel R, Brockmann J, Mischak H, Lohse MJ: Phosphorylation of GRK2 by protein kinase C abolishes its inhibition by calmodulin. J Biol Chem 276: 1911–1915, 2001

    Article  CAS  PubMed  Google Scholar 

  40. Lorenz K, Lohse MJ, Quitterer U: Protein kinase C switches the Raf kinase inhibitor from Raf-1 to GRK-2. Nature 426: 574–579, 2003

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Author notes

  1. Edna Aquino

    Present address: Department of Microbiology and Medical Zoology, University of Puerto Rico, Medical Sciences Campus, San Juan, PR, USA

Authors and Affiliations

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

    Rosa V. Flores, Melvin G. Hernández-Pérez, Edna Aquino & Fernando A. Gonzalez

  2. Department of Biochemistry, University of Puerto Rico, Río Piedras Campus, San Juan, PR, USA

    Fernando A. Gonzalez

  3. Department of Biomedical Sciences, Southwest Missouri State University, Springfield, MO, USA

    Richard C. Garrad

  4. Department of Biochemistry, University of Missouri-Columbia, MO, USA

    Gary A. Weisman

  5. Department of Chemistry, University of Puerto Rico, Río Piedras Campus, P.O. Box 23346, San Juan, PR, 00931-3346, USA

    Fernando A. Gonzalez

Authors
  1. Rosa V. Flores
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Melvin G. Hernández-Pérez
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Edna Aquino
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Richard C. Garrad
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Gary A. Weisman
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Fernando A. Gonzalez
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Fernando A. Gonzalez.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Flores, R.V., Hernández-Pérez, M.G., Aquino, E. et al. Agonist-induced phosphorylation and desensitization of the P2Y2 nucleotide receptor. Mol Cell Biochem 280, 35–45 (2005). https://doi.org/10.1007/s11010-005-8050-5

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11010-005-8050-5

Keyword

Search

Navigation