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Imaging nanometer domains of β-adrenergic receptor complexes on the surface of cardiac myocytes

Nature Chemical Biology volume 1, pages 196–202 (2005)Cite this article

Abstract

The contraction of cardiac myocytes is initiated by ligand binding to adrenergic receptors1,2 contained in nanoscale multiprotein complexes called signalosomes3. The composition and number of functional signalosomes within cardiac myocytes defines the molecular basis of the response to adrenergic stimuli3,4,5,6. For the first time, we demonstrated the ability of near-field scanning optical microscopy to visualize β-adrenergic receptors at the nanoscale in situ. On H9C2 cells, mouse neonatal and mouse embryonic cardiac myocytes, we showed that functional receptors are organized into multiprotein domains of ∼140 nm average diameter. Colocalization experiments in primary cells at the nanometer scale showed that 15–20% of receptors were preassociated in caveolae. These nanoscale complexes were sufficient to effect changes in ligand-induced contraction rate without the requirement for substantial changes in receptor distribution in the cellular membrane. Using fluorescence intensities associated with these nanodomains, we estimated the receptor density within the observed nanometer features and established a lower limit for the number of receptors in the signalosome.

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Figure 1: Confocal fluorescence and NSOM images for fixed H9C2 cells immunostained for β2AR, β1AR or caveolin-3.
Figure 2: Confocal fluorescence and NSOM images for neonatal and embryonic cardiac myocytes immunostained for β2AR and caveolin-3.
Figure 3: Confocal fluorescence and NSOM images for the colocalization of β2AR with caveolae on neonatal cardiac myocytes.

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References

  1. Hall, R.A. & Lefkowitz, R.J. Regulation of G protein-coupled receptor signaling by scaffold proteins. Circ. Res. 91, 672–680 (2002).

    Article  CAS  Google Scholar 

  2. Rockman, H.A., Koch, W.J. & Lefkowitz, R.J. Seven-transmembrane-spanning receptors and heart function. Nature 415, 206–212 (2002).

    Article  CAS  Google Scholar 

  3. Razani, B., Woodman, S.E. & Lisanti, M.P. Caveolae: from cell biology to animal physiology. Pharmacol. Rev. 54, 431–467 (2002).

    Article  CAS  Google Scholar 

  4. Luttrell, L.M. et al. β-arrestin-dependent formation of β(2) adrenergic receptor Src protein kinase complexes. Science 283, 655–661 (1999).

    Article  CAS  Google Scholar 

  5. Rybin, V.O., Xu, X.H., Lisanti, M.P. & Steinberg, S.F. Differential targeting of β-adrenergic receptor subtypes and adenylyl cyclase to cardiomyocyte caveolae—a mechanism to functionally regulate the cAMP signaling pathway. J. Biol. Chem. 275, 41447–41457 (2000).

    Article  CAS  Google Scholar 

  6. Lisanti, M.P., Scherer, P.E., Tang, Z. & Sargiacomo, M. Caveolae, caveolin and caveolin-rich membrane domains: a signalling hypothesis. Trends Cell Biol. 4, 231–235 (1994).

    Article  CAS  Google Scholar 

  7. Davare, M.A. et al. A β(2) adrenergic receptor signaling complex assembled with the Ca2+ channel Ca(v)1.2. Science 293, 98–101 (2001).

    Article  CAS  Google Scholar 

  8. Bouvier, M. Oligomerization of G-protein-coupled transmitter receptors. Nat. Rev. Neurosci. 2, 274–286 (2001).

    Article  CAS  Google Scholar 

  9. Hebert, T.E. & Bouvier, M. Structural and functional aspects of G protein-coupled receptor oligomerization. Biochem. Cell Biol. 76, 1–11 (1998).

    Article  CAS  Google Scholar 

  10. Bouvier, M. et al. Removal of phosphorylation sites from the β-2-adrenergic receptor delays onset of agonist-promoted desensitization. Nature 333, 370–373 (1988).

    Article  CAS  Google Scholar 

  11. Fotiadis, D. et al. Atomic-force microscopy: rhodopsin dimers in native disc membranes. Nature 421, 127–128 (2003).

    Article  CAS  Google Scholar 

  12. Lewis, A. et al. Near-field optics: from subwavelength illumination to nanometric shadowing. Nat. Biotechnol. 21, 1378–1386 (2003).

    Article  CAS  Google Scholar 

  13. Koopman, M. et al. Near-field scanning optical microscopy in liquid for high resolution single molecule detection on dendritic cells. FEBS Lett. 573, 6–10 (2004).

    Article  CAS  Google Scholar 

  14. Ianoul, A. et al. Near-field scanning fluorescence microscopy study of ion channel clusters in cardiac myocyte membranes. Biophys. J. 87, 3525–3535 (2004).

    Article  CAS  Google Scholar 

  15. Hescheler, J. et al. Morphological, biochemical, and electrophysiological characterization of a clonal cell (H9C2) line from rat-heart. Circ. Res. 69, 1476–1486 (1991).

    Article  CAS  Google Scholar 

  16. Salahpour, A. et al. Homodimerization of the β-2-adrenergic receptor as a prerequisite for cell surface targeting. J. Biol. Chem. 279, 33390–33397 (2004).

    Article  CAS  Google Scholar 

  17. Flesch, M. et al. Differential effects of carvedilol and metoprolol on isoprenaline-induced changes in β-adrenoceptor density and systolic function in rat cardiac myocytes. Cardiovasc. Res. 49, 371–380 (2001).

    Article  CAS  Google Scholar 

  18. Hegener, O. et al. Dynamics of β (2)-adrenergic receptor - Ligand complexes on living cells. Biochemistry 43, 6190–6199 (2004).

    Article  CAS  Google Scholar 

  19. Angers, S. et al. Detection of β (2)-adrenergic receptor dimerization in living cells using bioluminescence resonance energy transfer (BRET). Proc. Natl. Acad. Sci. USA 97, 3684–3689 (2000).

    CAS  PubMed  Google Scholar 

  20. Steinberg, S.F. The molecular basis for distinct β-adrenergic receptor subtype actions in cardiomyocytes. Circ. Res. 85, 1101–1111 (1999).

    Article  CAS  Google Scholar 

  21. Xiang, Y., Rybin, V.O., Steinberg, S.F. & Kobilka, B. Caveolar localization dictates physiologic signaling of β (2)-adrenoceptors in neonatal cardiac myocytes. J. Biol. Chem. 277, 34280–34286 (2002).

    Article  CAS  Google Scholar 

  22. Rohrer, D.K., Schauble, E.H., Desai, K.H., Kobilka, B.K. & Bernstein, D. Alterations in dynamic heart rate control in the beta(1)-adrenergic receptor knockout mouse. Am. J. Physiol. Heart Circ. Physiol. 43, H1184–H1193 (1998).

    Article  Google Scholar 

  23. Chruscinski, A.J. et al. Targeted disruption of the β-2 adrenergic receptor gene. J. Biol. Chem. 274, 16694–16700 (1999).

    Article  CAS  Google Scholar 

  24. Portbury, A.L. et al. Catecholamines act via a β-adrenergic receptor to maintain fetal heart rate and survival. Am. J. Physiol. Heart Circ. Physiol. 284, H2069–H2077 (2003).

    Article  CAS  Google Scholar 

  25. Ostrom, R.S. et al. Receptor number and caveolar co-localization determine receptor coupling efficiency to adenylyl cyclase. J. Biol. Chem. 276, 42063–42069 (2001).

    Article  CAS  Google Scholar 

  26. Manders, E.M.M., Verbeek, F.J. & Aten, J.A. Measurement of colocalization of objects in dual color confocal images. J. Microscopy-Oxford 169, 375–382 (1993).

    Article  Google Scholar 

  27. Burgos, P. et al. Near-field scanning optical microscopy probes: a comparison of pulled and double-etched bent NSOM probes for fluorescence imaging of biological samples. J. Microscopy-Oxford 211, 37–47 (2003).

    Article  CAS  Google Scholar 

  28. Rizzuto, R., Brini, M., Murgia, M. & Pozzan, T. Microdomains With High Ca2+ Close to Ip(3)-Sensitive Channels That Are Sensed By Neighboring Mitochondria. Science 262, 744–747 (1993).

    Article  CAS  Google Scholar 

  29. Zaccolo, M. & Pozzan, T. Discrete microdomains with high concentration of cAMP in stimulated rat neonatal cardiac myocytes. Science 295, 1711–1715 (2002).

    Article  CAS  Google Scholar 

  30. Ghanouni, P., Steenhuis, J.J., Farrens, D.L. & Kobilka, B.K. Agonist-induced conformational changes in the G-protein-coupling domain of the β(2) adrenergic receptor. Proc. Natl. Acad. Sci. USA 98, 5997–6002 (2001).

    Article  CAS  Google Scholar 

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Acknowledgements

L.J.J. and J.P.P. thank the Genomics and Health Initiative of the National Research Council Canada for partial financial support for this research. We thank R. Trembley for assistance in primary cell isolation, Z. Lu for NSOM imaging support, D. Moffatt for assistance with cluster analysis software, and R. Taylor and N.K. Goto (University of Ottawa) for useful discussions.

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  1. Anatoli Ianoul

    Present address: Department of Chemistry, Carleton University, 1125 Colonel By Drive, Ottawa, Canada, K1S 5B6

Authors and Affiliations

  1. The Steacie Institute for Molecular Sciences, National Research Council Canada, 100 Sussex Drive, Ottawa, K1A 0R6, Canada

    Anatoli Ianoul, Donna D Grant, Yanouchka Rouleau, Linda J Johnston & John Paul Pezacki

  2. The Institute for Biological Sciences, National Research Council Canada, 1200 Montreal Road, Bldg M-54, Ottawa, K1A 0R6, Canada

    Mahmud Bani-Yaghoub

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Correspondence to Linda J Johnston or John Paul Pezacki.

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Supplementary information

Supplementary Fig. 1

Examples of analyses of numbers of secondary antibodies (Ab) per cluster based on fluorescence intensities and an antibody dilution calibration. (PDF 65 kb)

Supplementary Fig. 2

Fluorescence microscopy images comparing wet vs. dry H9C2 cardiac myocytes stained with DAPI and with an anti-β2AR Ab. (PDF 31 kb)

Supplementary Fig. 3

Fluorescence microscopy images of β2AR-GFP fusion proteins overexpressed in HEK293 cells. (PDF 58 kb)

Supplementary Fig. 4

Analyses of two sets of colocalization data from NSOM imaging of neonatal mouse cardiac myocytes using Image J software with the Colocalization Threshold plug-in. (PDF 249 kb)

Supplementary Fig. 5

An example of cluster analysis performed for NSOM images. (PDF 183 kb)

Supplementary Table 1

Cluster densities for individual images for β2AR and β1AR in H9C2 cells and for β2AR in neonatal cardiac myocytes. (PDF 15 kb)

Supplementary Methods (PDF 25 kb)

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Ianoul, A., Grant, D., Rouleau, Y. et al. Imaging nanometer domains of β-adrenergic receptor complexes on the surface of cardiac myocytes. Nat Chem Biol 1, 196–202 (2005). https://doi.org/10.1038/nchembio726

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