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Dominant-negative caveolin inhibits H-Ras function by disrupting cholesterol-rich plasma membrane domains

Nature Cell Biology volume 1, pages 98–105 (1999)Cite this article

Abstract

The plasma membrane pits known as caveolae have been implicated both in cholesterol homeostasis and in signal transduction. CavDGV and CavKSY, two dominant-negative amino-terminal truncation mutants of caveolin, the major structural protein of caveolae, significantly inhibited caveola-mediated SV40 infection, and were assayed for effects on Ras function. We find that CavDGV completely blocked Raf activation mediated by H-Ras, but not that mediated by K-Ras. Strikingly, the inhibitory effect of CavDGV on H-Ras signalling was completely reversed by replenishing cell membranes with cholesterol and was mimicked by cyclodextrin treatment, which depletes membrane cholesterol. These results provide a crucial link between the cholesterol-trafficking role of caveolin and its postulated role in signal transduction through cholesterol-rich surface domains. They also provide direct evidence that H-Ras and K-Ras, which are targeted to the plasma membrane by different carboxy-terminal anchors, operate in functionally distinct microdomains of the plasma membrane.

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Figure 2: Caveolin mutants inhibit H-Ras- but not K-Ras-mediated Raf activation.
Figure 3: CavDGV suppresses serum-dependent growth.
Figure 4: Immunolocalization of Cav DGV with respect to caveolin and markers of endocytic compartments.
Figure 5: Localization of Cav DGV with respect to internalized markers by immunoelectron microscopy and immunofluorescence.
Figure 6: CavDGV does not prevent plasma membrane recruitment of Raf by Ras.
Figure 7: Cav DGV decreases the buoyant density of membranes containing caveolin and H-Ras.
Figure 8: H-Ras but not K-Ras function is sensitive to perturbations in membrane cholesterol.
Figure 1: Caveolin mutants inhibit SV40 infection.

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Acknowledgements

We thank D. James, J. Gruenberg, R. McPherson, and G. van Meer for discussions. We also thank M. Way, J. Gruenberg, V. Gerke, B. Hock-Toh, and T. Nilsson for providing antibodies and constructs, and N. Zorzi, A. Lane, and C. Allen for excellent technical assistance. This research was supported by grants from the National Health and Medical Research Council of Australia to R.G.P. and J.F.H. J.F.H. is also supported by the Royal Children"s Hospital Foundation, Queensland. The Centre for Molecular and Cell Biology is a Special Research Centre of the Australian Research Council.

Correspondence should be addressed to J.F.H.

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  1. Espen Stang

    Present address: Institute of Pathology, The National Hospital, Oslo, 0027, Norway

Authors and Affiliations

  1. Queensland Cancer Fund Laboratory of Experimental Oncology Department of Pathology, University of Queensland Medical School, Herston Road, Brisbane, 4006, Australia

    Sandrine Roy, Angus Harding, Ann Apolloni, Maria Etheridge & John F. Hancock

  2. Department of Physiology and Pharmacology University of Queensland, Centre for Microscopy and Microanalysis, Centre for Molecular and Cellular Biology, Brisbane, 4072, Australia

    Robert Luetterforst, Espen Stang, Barbara Rolls & Robert G. Parton

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Roy, S., Luetterforst, R., Harding, A. et al. Dominant-negative caveolin inhibits H-Ras function by disrupting cholesterol-rich plasma membrane domains. Nat Cell Biol 1, 98–105 (1999). https://doi.org/10.1038/10067

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