Advertisement

Identification of GPCR Localization in Detergent Resistant Membranes

  • Ranju Kumari
  • Anna FrancesconiEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 746)

Abstract

Lipid domains of the plasma membrane were originally described as a cell matrix insoluble in cold ­nonionic detergents and enriched in glycosphingolipids. Because of these biochemical properties, these membrane domains were termed detergent-resistant membranes (DRMs) or detergent-insoluble ­glycolipid-enriched (DIG) membranes. Membrane rafts and caveolae are two types of lipid domains that share these properties, as well as structural/functional dependence on membrane cholesterol. Membrane rafts and caveolae are believed to act as signaling platforms for ligand-activated receptors, thereby contributing to the regulation of receptor function. Here we describe a simple method to assess the association of GPCRs with detergent resistant membranes in native brain tissue and cultured cells.

Key words

GPCR mGluR Membrane rafts Caveolae Lipid domains Detergent resistant membranes DRM DIG Density gradients Flotation 

Notes

Acknowledgments

Supported by the U.S. National Institute of Health/NIMH (MH082870 to A.F.)

References

  1. 1.
    Simons, K. and Ikonen, E. (1997) Functional rafts in cell membranes. Nature 387, 569–572.PubMedCrossRefGoogle Scholar
  2. 2.
    Parton, R.G. and Simons, K. (2007) The multiple faces of caveolae. Nat. Rev. Mol. Cell Biol. 8, 185–194.PubMedCrossRefGoogle Scholar
  3. 3.
    Simons, K. and Toomre, D. (2000) Lipid rafts and signal transduction. Nat. Rev. Mol. Cell Biol. 1, 31–39.PubMedCrossRefGoogle Scholar
  4. 4.
    Allen, J.A., Halverson-Tamboli, R.A., and Rasenick, M.M. (2007) Lipid raft microdomains and neurotransmitter signalling. Nat. Rev. Neurosci. 8, 128–140.PubMedCrossRefGoogle Scholar
  5. 5.
    Hanzal-Bayer, M.F. and Hancock, J.F. (2007) Lipid rafts and membrane traffic. FEBS Lett. 581, 2098–2104.PubMedCrossRefGoogle Scholar
  6. 6.
    Tsui-Pierchala, B.A., Encinas, M., Milbrandt, J., and Johnson, E.M., Jr. (2002) Lipid rafts in neuronal signaling and function. Trends Neurosci. 25, 412–417.PubMedCrossRefGoogle Scholar
  7. 7.
    Hering, H., Lin, C.C., and Sheng, M. (2003) Lipid rafts in the maintenance of synapses, dendritic spines, and surface AMPA receptor stability. J. Neurosci. 23, 3262–3271.PubMedGoogle Scholar
  8. 8.
    Kamiguchi, H. (2006) The region-specific activities of lipid rafts during axon growth and guidance. J. Neurochem. 98, 330–335.PubMedCrossRefGoogle Scholar
  9. 9.
    Munro, S. (2003) Lipid rafts: elusive or illusive? Cell 115, 377–388.PubMedCrossRefGoogle Scholar
  10. 10.
    Nichols, B. (2005) Cell biology: without a raft. Nature 436, 638–639.PubMedCrossRefGoogle Scholar
  11. 11.
    Hancock, J.F. (2006) Lipid rafts: contentious only from simplistic standpoints. Nat. Rev. Mol. Cell Biol. 7, 456–462.PubMedCrossRefGoogle Scholar
  12. 12.
    Eggeling, C., Ringemann, C., Medda, R., Schwarzmann, G., Sandhoff, K., Polyakova, S., Belov, V.N., Hein, B., von Middendorff, C., Schonle, A., and Hell, S.W. (2009) Direct observation of the nanoscale dynamics of membrane lipids in a living cell. Nature 457, 1159–1162.PubMedCrossRefGoogle Scholar
  13. 13.
    Ostrom, R.S., and Liu, X. (2007) Detergent and detergent-free methods to define lipid rafts and caveolae. Methods Mol. Biol. 400, 459–468.PubMedCrossRefGoogle Scholar
  14. 14.
    Macdonald, J.L., and Pike, L.J. (2005) A simplified method for the preparation of ­detergent-free lipid rafts. J. Lipid Res. 46, 1061–1067.PubMedCrossRefGoogle Scholar
  15. 15.
    Francesconi, A., Kumari, R., and Zukin, R.S. (2009) Regulation of group I metabotropic glutamate receptor trafficking and signaling by the caveolar/lipid raft pathway. J. Neurosci. 29, 3590–3602.PubMedCrossRefGoogle Scholar
  16. 16.
    Jiang, L., Fang, J., Moore, D.S., Gogichaeva, N.V., Galeva, N.A., Michaelis, M.L., and Zaidi, A. (2008) Age-associated changes in synaptic lipid raft proteins revealed by two-dimensional fluorescence difference gel electrophoresis. Neurobiol. Aging, doi: 10.1016/j.neurobiolaging.2008.11.005Google Scholar
  17. 17.
    Ostermeyer, A.G., Beckrich, B.T., Ivarson, K.A., Grove, K.E., and Brown, D.A. (1999) Glycosphingolipids are not essential for formation of detergent-resistant membrane rafts in melanoma cells. methyl-beta-cyclodextrin does not affect cell surface transport of a ­GPI-anchored protein. J. Biol. Chem. 274, 34459–34466.PubMedCrossRefGoogle Scholar
  18. 18.
    Kong, M.M., Hasbi, A., Mattocks, M., Fan, T., O’Dowd, B.F., and George, S.R. (2007) Regulation of D1 dopamine receptor ­trafficking and signaling by caveolin-1. Mol. Pharmacol. 72, 1157–1170.PubMedCrossRefGoogle Scholar
  19. 19.
    Francesconi, A., and Duvoisin, R.M. (1998) Role of the second and third intracellular loops of metabotropic glutamate receptors in mediating dual signal transduction activation. J. Biol. Chem. 273, 5615–5624.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Dominick P. Purpura Department of NeuroscienceAlbert Einstein College of MedicineNew YorkUSA

Personalised recommendations