Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

Regulation of intracellular calcium by a signalling complex of IRAG, IP3 receptor and cGMP kinase Iβ

Nature volume 404, pages 197–201 (2000)Cite this article

Abstract

Calcium release from the endoplasmic reticulum controls a number of cellular processes, including proliferation and contraction of smooth muscle and other cells1,2. Calcium release from inositol 1,4,5-trisphosphate (IP3)-sensitive stores is negatively regulated by binding of calmodulin to the IP3 receptor (IP3R)3,4 and the NO/cGMP/cGMP kinase I (cGKI) signalling pathway5,6. Activation of cGKI decreases IP3-stimulated elevations in intracellular calcium7, induces smooth muscle relaxation8 and contributes to the antiproliferative9 and pro-apoptotic effects of NO/cGMP10. Here we show that, in microsomal smooth muscle membranes, cGKIβ phosphorylated the IP3R and cGKIβ, and a protein of relative molecular mass 125,000 which we now identify as the IP3R-associated cGMP kinase substrate (IRAG). These proteins were co-immunoprecipitated by antibodies directed against cGKI, IP3R or IRAG. IRAG was found in many tissues including aorta, trachea and uterus, and was localized perinuclearly after heterologous expression in COS-7 cells. Bradykinin-stimulated calcium release was not affected by the expression of either IRAG or cGKIβ, which we tested in the absence and presence of cGMP. However, calcium release was inhibited after co-expression of IRAG and cGKIβ in the presence of cGMP. These results identify IRAG as an essential NO/cGKI-dependent regulator of IP3-induced calcium release.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Macromolecular complex of IP3R, IRAG and cGKIβ.
Figure 2: Identification of IRAG.
Figure 3: Expression and localization of IRAG.
Figure 4: IRAG attenuates bradykinin-induced Ca2+ release.
Figure 5: IRAG inhibits IP3-evoked Ca2+ release.

Similar content being viewed by others

References

  1. Berridge, M. J., Bootman, M. D. & Lipp, P. Calcium—a life and death signal. Nature 395, 645–648 ( 1998).

    Article  ADS  CAS  Google Scholar 

  2. Somlyo, A. P. & Somlyo, A. V. Signal transduction and regulation in smooth muscle. Nature 372, 231– 236 (1994).

    Article  ADS  CAS  Google Scholar 

  3. Missiaen, L. et al. The bell-shaped Ca2+ dependence of the inositol 1,4,5-trisphosphate-induced Ca2+ release is modulated by Ca2+/calmodulin. J. Biol. Chem. 274, 13748–13751 (1999).

    Article  CAS  Google Scholar 

  4. Michikawea, T. et al. Calmodulin mediates calcium-dependent inactivation of the cerebellar type I inositol 1,4,5-trisphosphate receptor. Neuron 23, 799–808 ( 1999).

    Article  Google Scholar 

  5. Eigenthaler, M., Lohmann, S. M., Walter, U. & Pilz, R. B. Signal transduction by cGMP-dependent protein kinases and their emerging roles in the regulation of cell adhesion and gene expression. Rev. Physiol. Biochem. Pharmacol. 135, 173–209 (1999).

    Article  CAS  Google Scholar 

  6. Pfeifer, A. et al. Structure and function of cGMP-dependent protein kinases. Rev. Physiol. Biochem. Pharmacol. 135, 105–149 (1999).

    Article  CAS  Google Scholar 

  7. Ruth, P. et al. Transfected cGMP-dependent protein kinase suppresses calcium transients by inhibition of inositol 1,4,5-triphosphate production. Proc. Natl Acad. Sci. USA 90, 2623–2627 (1993).

    Article  ADS  CAS  Google Scholar 

  8. Pfeifer, A. et al. Defective smooth muscle regulation in cGMP kinase I-deficient mice. EMBO J. 17, 3045– 3051 (1998).

    Article  CAS  Google Scholar 

  9. von der Leyen, H. et al. Gene therapy inhibiting neointimal vascular lesion: In vivo transfer of endothelial cell nitric oxide synthase gene. Proc. Natl Acad. Sci. USA 92, 1137–1141 (1994).

    Article  ADS  Google Scholar 

  10. Chiche, J. D. et al. Adenovirus-mediated gene transfer of cGMP-dependent protein kinase increases the sensitivity of cultured vascular smooth muscle cells to the antiproliferative and pro-apoptotic effects of nitric oxide/cGMP. J. Biol. Chem. 273, 34263–34267 (1998).

    Article  CAS  Google Scholar 

  11. Casnelli, J. E. & Greengard, P. Guanosine 3′:5′-cyclic monophosphate-dependent phosphorylation of endogenous substrate proteins in membranes of mammalian smooth muscle. Proc. Natl Acad. Sci. USA 71, 1891–1895 ( 1974).

    Article  ADS  Google Scholar 

  12. Koga, T., Yoshida, Y., Cai, J. Q., Islam, M. O. & Imai, S. Purification and characterization of 240-kDa cGMP-dependent protein kinase substrate of vascular smooth muscle. Close resemblance to inositol 1,4,5-trisphosphate receptor. J. Biol. Chem. 269, 11640–11647 (1994).

    CAS  PubMed  Google Scholar 

  13. Komalavilas, P. & Lincoln, T. M. Phosphorylation of the inositol 1,4,5-trisphosphate receptor by cyclic GMP-dependent protein kinase. J. Biol. Chem. 269, 8701– 8707 (1994).

    CAS  PubMed  Google Scholar 

  14. Haug, L. S., Jensen, V., Hvalby, O., Walaas, S. I. & Ostvold, A. C. Phosphorylation of the inositol 1,4,5-trisphosphate receptor by cyclic nucleotide-dependent kinases in vitro and in rat cerebellar slices in situ. J. Biol. Chem. 274, 7467–7473 (1999).

    Article  CAS  Google Scholar 

  15. Rooney, T. A., Joseph, S. K., Queen, C. & Thomas, A. P. Cyclic GMP induces oscillatory calcium signals in rat hepatocytes. J. Biol. Chem. 271, 19817–19825 ( 1996).

    Article  CAS  Google Scholar 

  16. Tertyshnikova, S., Yan, X. & Fein, A. cGMP inhibits IP3-induced Ca2+ release in intact rat megakaryocytes via cGMP- and cAMP-dependent protein kinases. J. Physiol. (Lond.) 512, 89–96 (1998).

    Article  CAS  Google Scholar 

  17. Baltensperger, K., Carafoli, E. & Chiesi, M. The Ca2+-pumping ATPase and the major substrates of the cGMP-dependent protein kinase in smooth muscle sarcolemma are distinct entities. Eur. J. Biochem. 172, 7–16 (1988).

    Article  CAS  Google Scholar 

  18. Fujita, S. et al. Characterization of major phosphoproteins in the cGMP-mediated protein phosphorylation system of vascular smooth muscle membranes. J. Vasc. Res. 36, 299–310 (1999).

    Article  CAS  Google Scholar 

  19. Surks, H. K. et al. Regulation of myosin phosphatase by a specific interaction with cGMP-dependent protein kinase Iα. Science 286, 1583–1587 (1999).

    Article  CAS  Google Scholar 

  20. Behrens, T. W. et al. Jaw1, a lymphoid-restricted membrane protein localized to the endoplasmic reticulum. J. Immunol. 153, 682–690 (1994).

    CAS  PubMed  Google Scholar 

  21. Brenman, J. E., Xia, H., Chao, D. S., Black, S. M. & Bredt, D. S. Regulation of neuronal nitric oxide synthase through alternative transcripts. Dev. Neurosci. 19, 224–231 (1997).

    Article  CAS  Google Scholar 

  22. Newton, C. L., Mignery, G. A. & Südhof, T. C. Co-expression in vertebrate tissues and cell lines of multiple inositol 1,4,5-trisphosphate (InsP3) receptors with distinct affinities for InsP3. J. Biol. Chem. 269, 28613– 28619 (1994).

    CAS  PubMed  Google Scholar 

  23. Lipp, P., Thomas, D., Berridge, M. J. & Bootman, M. D. Nuclear calcium signalling by individual cytoplasmic calcium puffs. EMBO J. 16, 7166–7173 ( 1997).

    Article  CAS  Google Scholar 

  24. Keilbach, A., Ruth, P. & Hofmann, F. Detection of cGMP-dependent protein kinase isozymes by specific antibodies. Eur. J. Biochem. 208, 467–473 (1992).

    Article  CAS  Google Scholar 

  25. Nguyen, T., Chin, W.-C. & Verdugo, P. Role of Ca2+/K+ ion exchange in intracellular storage and release of Ca2+. Nature 395, 908–912 ( 1998).

    Article  ADS  CAS  Google Scholar 

  26. O'Rourke, F. et al. Ca2+ release by inositol 1,4,5-trisphosphate is blocked by the K+-channel blockers apamin and tetrapentylammonium ion, and a monoclonal antibody to a 63 kDa membrane protein: reversal of blockade by K+ ionophores nigericin and valinomycin and purification of the 63 kDa antibody-binding protein. Biochem. J. 300, 673–683 ( 1994).

    Article  CAS  Google Scholar 

  27. Shaughnessy, J. D. Jr et al. Mrvi1, a common MRV integration site in BXH2 myeloid leukemias, encodes a protein with homology to a lymphoid-restricted membrane protein Jaw1. Oncogene 18, 2069 –2084 (1999).

    Article  CAS  Google Scholar 

  28. Wilm, M., Neubauer, G. & Mann, M. Parent ion scans of unseparated peptide mixtures. Anal. Chem. 68, 527–533 (1996).

    Article  CAS  Google Scholar 

  29. Broad, L. M., Armstrong, D. L. & Putney, J. W. Jr Role of inositol 1,4,5-trisphosphate receptor in Ca2+ feedback inhibition of calcium release-activated calcium current (I crac). J. Biol. Chem. 274, 32881–32888 ( 1999).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank S. Kamm and C. Wolf for technical assistance. This work was supported by Deutsche Forschungsgemeinschaft and Fond der Chemischen Industrie.

Author information

Author notes

  1. Hans-Dieter Allescher

    Present address: II. Medizinische Klinik und Poliklinik der TU München, Ismaninger Str. 22, D-81675, München, Germany

  2. Aldo Ammendola, Keith Ashman, Ge-Xin Wang and Michael Korth: These authors contributed equally to this work

Authors and Affiliations

  1. Institut für Pharmakologie und Toxikologie der Technischen Universität München, Biedersteiner Straße 29, München, 80802, Germany

    Jens Schlossmann, Aldo Ammendola, Xiangang Zong, Andrea Huber, Hans-Dieter Allescher, Franz Hofmann & Peter Ruth

  2. Protein and Peptide Group, EMBL, Meyerhofstraße 1, Heidelberg, 69117, Germany

    Keith Ashman, Gitte Neubauer, Ge-Xin Wang, Michael Korth & Matthias Wilm

  3. Abteilung Pharmakologie für Pharmazeuten, Universitäts-Krankenhaus Eppendorf, Martinistraße 52, Hamburg, 20246, Germany

    Ge-Xin Wang & Michael Korth

Authors
  1. Jens Schlossmann
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Aldo Ammendola
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Keith Ashman
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Xiangang Zong
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Andrea Huber
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Gitte Neubauer
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Ge-Xin Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Hans-Dieter Allescher
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Michael Korth
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Matthias Wilm
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Franz Hofmann
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Peter Ruth
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Franz Hofmann.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Schlossmann, J., Ammendola, A., Ashman, K. et al. Regulation of intracellular calcium by a signalling complex of IRAG, IP3 receptor and cGMP kinase Iβ. Nature 404, 197–201 (2000). https://doi.org/10.1038/35004606

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/35004606

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing