Expression and characterization of mouse angiotensin II type 1a receptor tagging hemagglutinin epitope in cultured cells.

  • Authors:
    • J Ishida
    • S Asada
    • H Daitoku
    • K Fujiwara
    • Y Kon
    • T Sugaya
    • K Murakami
    • T Nakajima
    • Y Kasuya
    • A Fukamizu
  • View Affiliations

  • Published online on: March 1, 1999     https://doi.org/10.3892/ijmm.3.3.263
  • Pages: 263-333
Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )
Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )


Abstract

The octapeptide angiotensin II mediates the physiological actions of the renin-angiotensin system through activation of several angiotensin II receptor (AT) subtypes, in particular AT1 (AT1a and AT1b in the case of rodents). Although we and others have generated mutant mice in which the AT1a gene was disrupted, the function of mouse AT1 remains to be fully elucidated, due to the lack of effective tools involving antibodies against AT1 for detecting biological responses in cellular conditions. To avoid these problems, we constructed the hemagglutinin (HA)-tagged mouse AT1a, and stably introduced this recombinant receptor into human embryonic kidney 293-T cells. Radioligand binding of [(125)I] angiotensin II to AT1a was specific, saturable, and reversible. Scatchard analysis demonstrated that the transfected receptor had a dissociation constant of 1.7 nM with a density of 1.2 x 10(5) sites/cells. Angiotensin II stimulated a rapid increase in cytosolic free calcium, and angiotensin II-induced phosphorylation of extracellular signal-regulated kinases (Erk) was found in a dose-dependent manner. After solubilization, Western blot analysis showed specific interactions between an anti-HA antibody and HA-tagged mouse AT1a. Furthermore, a significant proportion of HA-tagged mouse AT1a was specifically immunoprecipitated with this antibody. In the immunocytochemical and electronmicroscopic studies, treatment of this cell line with angiotensin II resulted in decrease in signals of the surface receptors. Based on these results, the cell line established here provides an excellent tool for studying angiotensin II actions mediated through mouse AT1a, at sub-nanomolar concentrations.

Related Articles

Journal Cover

Mar 1999
Volume 3 Issue 3

Print ISSN: 1107-3756
Online ISSN:1791-244X

Sign up for eToc alerts

Recommend to Library

Copy and paste a formatted citation
x
Spandidos Publications style
Ishida J, Asada S, Daitoku H, Fujiwara K, Kon Y, Sugaya T, Murakami K, Nakajima T, Kasuya Y, Fukamizu A, Fukamizu A, et al: Expression and characterization of mouse angiotensin II type 1a receptor tagging hemagglutinin epitope in cultured cells.. Int J Mol Med 3: 263-333, 1999
APA
Ishida, J., Asada, S., Daitoku, H., Fujiwara, K., Kon, Y., Sugaya, T. ... Fukamizu, A. (1999). Expression and characterization of mouse angiotensin II type 1a receptor tagging hemagglutinin epitope in cultured cells.. International Journal of Molecular Medicine, 3, 263-333. https://doi.org/10.3892/ijmm.3.3.263
MLA
Ishida, J., Asada, S., Daitoku, H., Fujiwara, K., Kon, Y., Sugaya, T., Murakami, K., Nakajima, T., Kasuya, Y., Fukamizu, A."Expression and characterization of mouse angiotensin II type 1a receptor tagging hemagglutinin epitope in cultured cells.". International Journal of Molecular Medicine 3.3 (1999): 263-333.
Chicago
Ishida, J., Asada, S., Daitoku, H., Fujiwara, K., Kon, Y., Sugaya, T., Murakami, K., Nakajima, T., Kasuya, Y., Fukamizu, A."Expression and characterization of mouse angiotensin II type 1a receptor tagging hemagglutinin epitope in cultured cells.". International Journal of Molecular Medicine 3, no. 3 (1999): 263-333. https://doi.org/10.3892/ijmm.3.3.263