Skip to main content
SpringerLink
Log in

Stable expression of a functional rat angiotensin II (AT1A) receptor in CHO-K1 cells: Rapid desensitization by angiotensin II

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

Abstract

The octapeptide angiotensin II mediates the physiological actions of the renin-angiotensin system through activation of several angiotensin II receptor subtypes; in particular the AT1. In many tissues, the presence of multiple angiotensin II receptor subtypes, together with a low number of receptors, makes it difficult to study biological responses to physiological concentrations (10−11–10−9 M) of angiotensin II. Also, cultured cells show diminished angiotensin II receptor binding with respect to time in culture and passage number. To address these problems, we expressed the recombinant AT1A receptor in CHO-K1 cells. The stably transfected receptor was characterized using radioligand binding studies and functional coupling to cytosolic free calcium. Radioligand binding of [125I] angiotensin II to the angiotensin II receptor was specific, saturable, reversible and modulated by guanine nucleotides. Like the endogenous AT1A receptor, reported in a variety of tissues, the specific, noncompetitive, nonpeptide AII receptor antagonist, EXP3174, blocked binding of [125I] angiotensin II to the transfected receptor. Scatchard analysis demonstrated that the transfected receptor had a dissociation constant of 1.9 nM with a density of 3.4 pmol/mg protein.

An important feature of many of the responses to angiotensin II is the rapid desensitization that occurs following agonist occupancy and the development of tachyphylaxis. In AT1A receptor transfected CHO-K1 cells, angiotensin II (10−9 M) stimulated a rapid increase in cytosolic free calcium that was completely desensitized within 50 sec following receptor occupancy. Agonist induced desensitization was unaffected when receptor internalization was blocked by pretreatment with concanavalin A or incubation at 4°C, and no changes in AT1A receptor affinity or number were observed. Receptor desensitization was also unaffected by inhibition or activation of protein kinase C. Thus, we have established a permanent, high-level transfectant of the AT1A receptor in CHO-K1 cells and have shown that these receptors rapidly desensitize following exposure to physiological concentrations of agonist. The mechanism of rapid desensitization is not related to receptor sequestration, internalization or controlled by PKC phosphorylation. This provides an excellent model for studying AII actions mediated through a specific receptor subtype, at subnanomolar concentrations.

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

References

  1. Raizada MK, Phillips MI, Summers C: Cellular and molecular biology of the renin-angiotensin system. CRC press, Inc. USA. 1993

    Google Scholar 

  2. Catt KJ: Angiotensin II receptors. The Renin-Angiotensin System. J.I.S. Robertson and M.J. Nicholls (eds). Gower Medical Publishing vol.1: 12.1–12.14, 1993

  3. Dostal DE, Baker KM, peach MJ: Growth promoting effects of angiotensin II in the cardiovascular system. In Horizons in Endocrinology. Vol II, M. Maggi, V. Greenen, (eds). Raven Press, 76: 265–272, 1991

  4. Murphy TJ, Alexander RW, Griendling KK, Runge MS, Bernstein KE: Isolation of a cDNA encoding the vascular type-1 angiotensin II receptor. Nature 351: 233–236, 1991

    Google Scholar 

  5. Iwai N, Inagami T: Identification of two subtypes in the rat type I angiotensin II receptor. FEBS 298: 257–260, 1992

    Google Scholar 

  6. Mukoyama M, Nakajima M, Horiuchi M, Sasamura H. Pratt RE, Dzau VJ: Expression cloning of type 2 angiotensin II receptor reveals a unique class of seven-transmembrane receptors. J Biol Chem 268: 24539–24542, 1993

    Google Scholar 

  7. Kambayashi Y, Bardhan S, Takahashi K, Tsuzuki S, Inui H, Hamakubo T, Inagami T: Molecular cloning of a novel angiotensin II receptor isoform involved in phosphotyrosine phosphatase inhibition. J Bio Chem 268: 24543–24546, 1993

    Google Scholar 

  8. Dostal DE, Baker KM: Angiotensin II stimulation of left ventricular hypertrophy in adult rat heart: Mediation by the AT1A receptor. Am J Hypertens 5: 276–280, 1992

    Google Scholar 

  9. Dostal DE, Murahashi T, Peach MJ: Regulation of cytosolic calcium by angiotensins in vascular smooth muscle. Hypertension 15: 815–822, 1990

    Google Scholar 

  10. Sibley DR, Lefkowitz RJ: Molecular mechanisms of receptor desensitization using the B-adrenergic receptor-coupled adenylate cyclase system as a model. Nature 317: 124–129, 1985

    Google Scholar 

  11. Lohse JM, Benovic JL, Caron MG, Lefkowitz RJ: Multiple pathways of rapid B2-adrenergic receptor desensitization. delineation with specific inhibitors. J Biol Chem 265: 3202–3209, 1990

    Google Scholar 

  12. Lefkowitz RJ, Inglese J, Koch WJ, Pitcher J, Attramadal H, Caron MG: G-protein-coupled receptors: Regulatory role of receptor kinases and arrestin proteins. The cell Surface: Cold Spring Harbor Symposia on Quantitative Biology LVII: 127–133, 1992

    Google Scholar 

  13. Abdellatif MM, Neubauer FC, Lederer WJ, Rogers TB: Angiotensin-induced desensitization of the phosphoinositide pathway in cardiac cells occurs at the level of the receptor. Circulation Res 69: 800–809, 1991

    Google Scholar 

  14. Kumar RS, Thekkumkara TJ, Sen GC: The mRNAs encoding the two angiotensin-converting isozymes are transcribed from the same gene by a tissue-specific choice of alternative transcription initiation sites. J Bio Chem 266: 3854–3862, 1991

    Google Scholar 

  15. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K: Current Protocols in Molecular Biology, John Wiley and Sons, New York, 1987

    Google Scholar 

  16. Thekkumkara TJ, Livingston III W, Kumar RS, Sen GC: Use of alternative polyadenylation sites for tissue-specific transcription of two angiotensin-converting enzyme mRNAs. Nucleic Acids Res. 20: 683–687, 1992

    Google Scholar 

  17. Schorb W, Booz GW, Dostal DE, Conrad KM, Chang KC, Baker KM: Angiotensin II is mitogenic in neonatal rat cardiac fibroblasts. Circulation Res 72: 1245–1254, 1993

    Google Scholar 

  18. Bradford MM: Rapid and sensitive method for quantification of microgram quantities of protein utilizing the principal of protein-dye binding. Anal Biochem 72: 248–254, 1976

    Google Scholar 

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

    Google Scholar 

  20. Rogers TB, Gaa ST, Allen IS: Identification and characterization of functional angiotensin II receptors on cultured heart myoctes. J Pharmacol Exp Ther 236: 438–444, 1986

    Google Scholar 

  21. Baker KM, Singer HA, Aceto JF: Angiotensin II receptor mediated stimulation of cytosolic-free calcium and inositol phosphates in chick myocyte. J Pharmacol Exp Ther 251: 578–585, 1989

    Google Scholar 

  22. Anderson KM, Murahashi T, Dostal DE, Peach MJ: Morphological and biochemical analysis of angiotensin II internalization in cultured rat aortic smooth muscle cells. Am J Physiol 264: C179–C188, 1992

    Google Scholar 

  23. Catt KJ, Sanderg K, Balla T: Angiotensin II receptors and signal transduction mechanisms. In: M.K. Raizada, M.I. Phillips and C. Summers (eds). Cellular and molecular biology of the reninangiotensin system (CRC press, Inc.) 307–356, 1993

  24. Shimuta SI, Kanashiro CA, Ferreira AT, Oshiro MEM, Paiva TB, Paiva ACM: Role of Na+ and protein kinase C in angiotensin desensitization and tachyphylaxis in the guinea-pig ileum. Arch Pharmacol 347: 425–431, 1993

    Google Scholar 

  25. Sakuta H, Sekiguchi M, Okamoto K, Sakai Y: Desensitization of endogenous angiotensin II receptors in Xenopus oocytes: a role of protein kinase C. Euro J Pham 208: 41–47, 1991

    Google Scholar 

  26. Bergsma DJ, Ellis C, Nuthulaganti PR, Nambi P, Scaife K, Kumar C, Aiyar N: Isolation and expression of a noval angiotensin II receptor from Xenopus laevis heart. Mol Pharmacol 44: 277–284, 1993

    Google Scholar 

  27. Strader CD, Sigal IS, Dixon RA: Structural basis of β-adrenergic receptor function. FASEB J 3: 1825–1832, 1989

    Google Scholar 

  28. Liggett SB, Ostrowski J, Chesnut LC, Kurose H, Raymond JR, Caron MG, Lefkowitz RJ: Sites in the third intracellular loop of the 2A-adrenergic receptor confer short term agonist-promoted desensitization. J Biol Chem 267: 4740–4746, 1992

    Google Scholar 

  29. Sandberg K: Structural analysis and regulation of angiotensin II receptors. Trends Endocrinol Metab 5: 28–35, 1994

    Google Scholar 

  30. Onorato JJ, Palczewski K, Regan JW, Caron MC, Lefkowitz RJ, Benovic JL: Role of acidic amino acids in peptide substrates of the β-drenergic receptor kinase and rhodopsin kinase. Biochem 30: 5118–5125, 1991

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Geisinger Clinic, Weis Center for Research, 26-11, 100 North Academy Avenue, 17822, Danville, Pennsylvania, USA

    Thomas J. Thekkumkara, Jing Du, David E. Dostal, Thomas J. Motel, Walter G. Thomas & Kenneth M. Baker

Authors
  1. Thomas J. Thekkumkara
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jing Du
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. David E. Dostal
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Thomas J. Motel
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Walter G. Thomas
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Kenneth M. Baker
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Thekkumkara, T.J., Du, J., Dostal, D.E. et al. Stable expression of a functional rat angiotensin II (AT1A) receptor in CHO-K1 cells: Rapid desensitization by angiotensin II. Mol Cell Biochem 146, 79–89 (1995). https://doi.org/10.1007/BF00926885

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00926885

Key words

Search

Navigation