Purinergic Signalling

, Volume 5, Issue 3, pp 289–298

Differential role of the carboxy-terminus of the A2B adenosine receptor in stimulation of adenylate cyclase, phospholipase Cβ, and interleukin-8

  • Sergey Ryzhov
  • Rinat Zaynagetdinov
  • Anna E. Goldstein
  • Anton Matafonov
  • Italo Biaggioni
  • Igor Feoktistov
Original Article


In human mast cells and microvascular endothelial cells, the A2B adenosine receptor controls at least three independent signaling pathways, i.e., Gs-mediated stimulation of adenylate cyclase, Gq-mediated stimulation of phospholipase Cβ, and Gs/Gq-independent upregulation of IL-8. Functional analysis of cells transfected with full-length and truncated receptor constructs revealed that the A2B receptor C-terminus is important for coupling to Gs and Gq proteins. Removal of the entire cytoplasmic portion in the A2B receptor C-terminus rendered it incapable of stimulating adenylate cyclase and phospholipase Cβ. Conversely, removal of the distal 16 amino acids facilitated signal transduction from the receptor to the downstream Gs but not Gq proteins. However, the A2B receptor C-terminus is not essential for upregulation of IL-8. Analysis of chimeric A2A/A2B receptors demonstrated that only chimeras containing the third intracellular loop of the A2B receptor mediated agonist-dependent IL-8 reporter stimulation, suggesting that this domain is important for upregulation of IL-8.


Adenosine Purinergic receptors P1 Interleukin-8 Adenylate cyclase Type C phospholipases GTP-binding proteins 


  1. 1.
    Londos C, Wolff J (1977) Two distinct adenosine-sensitive sites on adenylate cyclase. Proc Natl Acad Sci U S A 74:5482–5486 doi:10.1073/pnas.74.12.5482 PubMedCrossRefGoogle Scholar
  2. 2.
    Londos C, Cooper DMF, Wolff J (1980) Subclasses of external adenosine receptors. Proc Natl Acad Sci U S A 77:2551–2554 doi:10.1073/pnas.77.5.2551 PubMedCrossRefGoogle Scholar
  3. 3.
    Fredholm BB, Ijzerman AP, Jacobson KA, Klotz KN, Linden J (2001) International Union of Pharmacology. XXV. Nomenclature and classification of adenosine receptors. Pharmacol Rev 53:527–552PubMedGoogle Scholar
  4. 4.
    Bruns RF, Lu GH, Pugsley TA (1987) Adenosine receptor subtypes: binding studies. In: Gerlach E, Becker BF (eds) Topics and perspectives in adenosine research. Springer, Berlin, pp 59–73Google Scholar
  5. 5.
    Feoktistov I, Biaggioni I (1993) Characterization of adenosine receptors in human erythroleukemia cells. Further evidence for heterogeneity of adenosine A2 receptors. Mol Pharmacol 43:909–914PubMedGoogle Scholar
  6. 6.
    Feoktistov I, Biaggioni I (1995) Adenosine A2B receptors evoke interleukin-8 secretion in human mast cells. An enprofylline-sensitive mechanism with implications for asthma. J Clin Invest 96:1979–1986 doi:10.1172/JCI118245 PubMedCrossRefGoogle Scholar
  7. 7.
    Linden J, Thai T, Figler H, Jin X, Robeva AS (1999) Characterization of human A2B adenosine receptors: radioligand binding, western blotting, and coupling to Gq in human embryonic kidney 293 cells and HMC-1 mast cells. Mol Pharmacol 56:705–713PubMedGoogle Scholar
  8. 8.
    Ryzhov S, Zaynagetdinov R, Goldstein AE, Novitskiy SV, Dikov MM, Blackburn MR, Biaggioni I, Feoktistov I (2008) Effect of A2B adenosine receptor gene ablation on proinflammatory adenosine signaling in mast cells. J Immunol 180:7212–7220PubMedGoogle Scholar
  9. 9.
    Zhong H, Belardinelli L, Maa T, Feoktistov I, Biaggioni I, Zeng D (2004) A2B adenosine receptors increase cytokine release by bronchial smooth muscle cells. Am J Respir Cell Mol Biol 30:118–125 doi:10.1165/rcmb.2003-0118OC PubMedCrossRefGoogle Scholar
  10. 10.
    Zhong H, Belardinelli L, Maa T, Zeng D (2005) Synergy between A2B adenosine receptors and hypoxia in activating human lung fibroblasts. Am J Respir Cell Mol Biol 32:2–8 doi:10.1165/rcmb.2004-0103OC PubMedCrossRefGoogle Scholar
  11. 11.
    Evans BA, Elford C, Pexa A, Francis K, Hughes AC, Deussen A, Ham J (2006) Human osteoblast precursors produce extracellular adenosine, which modulates their secretion of IL-6 and osteoprotegerin. J Bone Miner Res 21:228–236 doi:10.1359/JBMR.051021 PubMedCrossRefGoogle Scholar
  12. 12.
    Sitaraman SV, Merlin D, Wang L, Wong M, Gewirtz AT, Si-Tahar M, Madara JL (2001) Neutrophil-epithelial crosstalk at the intestinal lumenal surface mediated by reciprocal secretion of adenosine and IL-6. J Clin Invest 107:861–869 doi:10.1172/JCI11783 PubMedCrossRefGoogle Scholar
  13. 13.
    Rees DA, Lewis BM, Lewis MD, Francis K, Scanlon MF, Ham J (2003) Adenosine-induced IL-6 expression in pituitary folliculostellate cells is mediated via A2b adenosine receptors coupled to PKC and p38 MAPK. Br J Pharmacol 140:764–772 doi:10.1038/sj.bjp.0705488 PubMedCrossRefGoogle Scholar
  14. 14.
    Schwaninger M, Neher M, Viegas E, Schneider A, Spranger M (1997) Stimulation of interleukin-6 secretion and gene transcription in primary astrocytes by adenosine. J Neurochem 69:1145–1150PubMedCrossRefGoogle Scholar
  15. 15.
    Fiebich BL, Akundi RS, Biber K, Hamke M, Schmidt C, Butcher RD, van Calker D, Willmroth F (2005) IL-6 expression induced by adenosine A2b receptor stimulation in U373 MG cells depends on p38 mitogen activated kinase and protein kinase C. Neurochem Int 46:501–512 doi:10.1016/j.neuint.2004.11.009 PubMedCrossRefGoogle Scholar
  16. 16.
    Fiebich BL, Biber K, Guyfko K, Berger M, Bauer J, van Calker D (1996) Adenosine A2b receptors mediate an increase in interleukin (IL)-6 mRNA and IL-6 protein synthesis in human astroglioma cells. J Neurochem 66:1426–1431PubMedGoogle Scholar
  17. 17.
    Feoktistov I, Goldstein AE, Ryzhov S, Zeng D, Belardinelli L, Voyno-Yasenetskaya T, Biaggioni I (2002) Differential expression of adenosine receptors in human endothelial cells: role of A2B receptors in angiogenic factor regulation. Circ Res 90:531–538 doi:10.1161/01.RES.0000012203.21416.14 PubMedCrossRefGoogle Scholar
  18. 18.
    Zeng D, Maa T, Wang U, Feoktistov I, Biaggioni I, Belardinelli L (2003) Expression and function of A2B adenosine receptors in the U87MG tumor cells. Drug Dev Res 58:405–411 doi:10.1002/ddr.10212 CrossRefGoogle Scholar
  19. 19.
    Merighi S, Benini A, Mirandola P, Gessi S, Varani K, Simioni C, Leung E, Maclennan S, Baraldi PG, Borea PA (2007) Caffeine inhibits adenosine-induced accumulation of hypoxia-inducible factor-1alpha, vascular endothelial growth factor, and interleukin-8 expression in hypoxic human colon cancer cells. Mol Pharmacol 72:395–406 doi:10.1124/mol.106.032920 PubMedCrossRefGoogle Scholar
  20. 20.
    Ryzhov S, Goldstein AE, Matafonov A, Zeng D, Biaggioni I, Feoktistov I (2004) Adenosine-activated mast cells induce IgE synthesis by B lymphocytes: an A2B-mediated process involving Th2 cytokines IL-4 and IL-13 with implications for asthma. J Immunol 172:7726–7733PubMedGoogle Scholar
  21. 21.
    Ryzhov S, Goldstein AE, Biaggioni I, Feoktistov I (2006) Cross-talk between Gs- and Gq-coupled pathways in regulation of interleukin-4 by A2B adenosine receptors in human mast cells. Mol Pharmacol 70:727–735 doi:10.1124/mol.106.022780 PubMedCrossRefGoogle Scholar
  22. 22.
    Olah ME (1997) Identification of A2a adenosine receptor domain involved in selective coupling to Gs. Analysis of chimeric A1/A2a adenosine receptors. J Biol Chem 272:337–344PubMedGoogle Scholar
  23. 23.
    Tucker AL, Jia LG, Holeton D, Taylor AJ, Linden J (2000) Dominance of Gs in doubly Gs/Gi-coupled chimaeric A1/A2A adenosine receptors in HEK-293 cells. Biochem J 352:203–210 doi:10.1042/0264-6021:3520203 PubMedCrossRefGoogle Scholar
  24. 24.
    Klinger M, Kuhn M, Just H, Stefan E, Palmer T, Freissmuth M, Nanoff C (2002) Removal of the carboxy terminus of the A2A-adenosine receptor blunts constitutive activity: differential effect on cAMP accumulation and MAP kinase stimulation. Naunyn Schmiedebergs Arch Pharmacol 366:287–298 doi:10.1007/s00210-002-0617-z PubMedCrossRefGoogle Scholar
  25. 25.
    Pankevych H, Korkhov V, Freissmuth M, Nanoff C, Pankevych H, Freissmuth M, Nanoff C (2003) Truncation of the A1 adenosine receptor reveals distinct roles of the membrane-proximal carboxyl terminus in receptor folding and G protein coupling. J Biol Chem 278:30283–30293 doi:10.1074/jbc.M212918200 PubMedCrossRefGoogle Scholar
  26. 26.
    Feoktistov I, Biaggioni I (1997) Adenosine A2B receptors. Pharmacol Rev 49:381–402PubMedGoogle Scholar
  27. 27.
    Daunt DA, Hurt C, Hein L, Kallio J, Feng F, Kobilka BK (1997) Subtype-specific intracellular trafficking of alpha2-adrenergic receptors. Mol Pharmacol 51:711–720PubMedGoogle Scholar
  28. 28.
    Seuwen K, Lagarde A, Pouyssegur J (1988) Deregulation of hamster fibroblast proliferation by mutated ras oncogenes is not mediated by constitutive activation of phosphoinositide-specific phospholipase C. EMBO J 7:161–168PubMedGoogle Scholar
  29. 29.
    Fredholm BB, Irenius E, Kull B, Schulte G (2001) Comparison of the potency of adenosine as an agonist at human adenosine receptors expressed in Chinese hamster ovary cells. Biochem Pharmacol 61:443–448 doi:10.1016/S0006-2952(00)00570-0 PubMedCrossRefGoogle Scholar
  30. 30.
    Matharu AL, Mundell SJ, Benovic JL, Kelly E (2001) Rapid agonist-induced desensitization and internalization of the A2B adenosine receptor is mediated by a serine residue close to the COOH terminus. J Biol Chem 276:30199–30207 doi:10.1074/jbc.M010650200 PubMedCrossRefGoogle Scholar
  31. 31.
    Varani K, Gessi S, Merighi S, Vincenzi F, Cattabriga E, Benini A, Klotz KN, Baraldi PG, Tabrizi MA, Lennan SM, Leung E, Borea PA (2005) Pharmacological characterization of novel adenosine ligands in recombinant and native human A2B receptors. Biochem Pharmacol 70:1601–1612 doi:10.1016/j.bcp.2005.08.018 PubMedCrossRefGoogle Scholar
  32. 32.
    Cooper J, Hill SJ, Alexander SP (1997) An endogenous A2B adenosine receptor coupled to cyclic AMP generation in human embryonic kidney (HEK 293) cells. Br J Pharmacol 122:546–550 doi:10.1038/sj.bjp.0701401 PubMedCrossRefGoogle Scholar
  33. 33.
    Gao Z, Chen T, Weber MJ, Linden J (1999) A2B adenosine and P2Y2 receptors stimulate mitogen-activated protein kinase in human embryonic kidney-293 cells. cross-talk between cyclic AMP and protein kinase C pathways. J Biol Chem 274:5972–5980 doi:10.1074/jbc.274.9.5972 PubMedCrossRefGoogle Scholar
  34. 34.
    Alewijnse AE, Smit MJ, Rodriguez Pena MS, Verzijl D, Timmerman H, Leurs R (1997) Modulation of forskolin-mediated adenylyl cyclase activation by constitutively active GS-coupled receptors. FEBS Lett 419:171–174 doi:10.1016/S0014-5793(97)01440-3 PubMedCrossRefGoogle Scholar
  35. 35.
    Parker EM, Ross EM (1991) Truncation of the extended carboxyl-terminal domain increases the expression and regulatory activity of the avian beta-adrenergic receptor. J Biol Chem 266:9987–9996PubMedGoogle Scholar
  36. 36.
    Sitaraman SV, Wang L, Wong M, Bruewer M, Hobert M, Yun CH, Merlin D, Madara JL (2002) The adenosine 2b receptor is recruited to the plasma membrane and associates with E3KARP and ezrin upon agonist stimulation. J Biol Chem 277:33188–33195 doi:10.1074/jbc.M202522200 PubMedCrossRefGoogle Scholar
  37. 37.
    Hall RA, Ostedgaard LS, Premont RT, Blitzer JT, Rahman N, Welsh MJ, Lefkowitz RJ (1998) A C-terminal motif found in the beta2-adrenergic receptor, P2Y1 receptor and cystic fibrosis transmembrane conductance regulator determines binding to the Na+/H+ exchanger regulatory factor family of PDZ proteins. Proc Natl Acad Sci U S A 95:8496–8501 doi:10.1073/pnas.95.15.8496 PubMedCrossRefGoogle Scholar
  38. 38.
    Lau AG, Hall RA (2001) Oligomerization of NHERF-1 and NHERF-2 PDZ domains: differential regulation by association with receptor carboxyl-termini and by phosphorylation. Biochemistry 40:8572–8580 doi:10.1021/bi0103516 CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2008

Authors and Affiliations

  • Sergey Ryzhov
    • 1
  • Rinat Zaynagetdinov
    • 1
  • Anna E. Goldstein
    • 2
  • Anton Matafonov
    • 1
  • Italo Biaggioni
    • 3
  • Igor Feoktistov
    • 4
    • 5
  1. 1.Division of Cardiovascular Medicine, Department of MedicineVanderbilt UniversityNashvilleUSA
  2. 2.Division of Clinical Pharmacology, Department of PharmacologyVanderbilt UniversityNashvilleUSA
  3. 3.Division of Clinical Pharmacology, Departments of Medicine and PharmacologyVanderbilt UniversityNashvilleUSA
  4. 4.Division of Cardiovascular Medicine, Departments of Medicine and PharmacologyVanderbilt UniversityNashvilleUSA
  5. 5.360 PRBVanderbilt UniversityNashvilleUSA

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