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A more recent version of this article appeared on July 1, 2004
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Submitted on March 8, 2004
Revised on April 15, 2004
Accepted on April 16, 2004
1 Department of Biochemistry and Molecular Biology, Mayo Clinic and Foundation, 200 First Street, SW, Rochester, Minnesota 55905
2 Cardiovascular Diseases, Mayo Clinic and Foundation, 200 First Street, SW, Rochester, Minnesota 55905
3 Department of Biochemistry and Molecular Biology; Cardiovascular Diseases, Mayo Clinic and Foundation, 200 First Street, SW, Rochester, Minnesota 55905
4 Institute for Molecular Bioscience, Center for Microscopy and Microanalysis, and School of Biomedical Sciences, University of Queensland, St. Lucia, Brisbane QLD 4072, Australia
* Corresponding author. E-mail address: pagano.richard{at}mayo.edu.
Internalization of some plasma membrane constituents, bacterial toxins and viruses occurs via caveolae, however the factors that regulate caveolar internalization are still unclear. Here, we demonstrate that a brief treatment of cultured cells with natural or synthetic glycosphingolipids (GSLs) or elevation of cholesterol (either by acute treatment with m
-cyclodextrin/cholesterol or by alteration of growth conditions) dramatically stimulates caveolar endocytosis with little or no effect on other endocytic mechanisms. These treatments also stimulated the movement of GFP-labeled vesicles in cells transfected with caveolin-1-GFP, and reduced the number of surface connected caveolae seen by electron microscopy. In contrast, overexpression of caveolin-1 decreased caveolar uptake, but treatment with GSLs reversed this effect and stimulated caveolar endocytosis. Stimulation of caveolar endocytosis did not occur using ceramide or phosphatidylcholine, and was not due to GSL degradation since similar results were obtained using a nonhydrolyzable GSL analog. Stimulated caveolar endocytosis required src kinase and PKC-
activity as shown by (i) use of pharmacological inhibitors, (ii) expression of kinase inactive src or dominant negative PKC, and (iii) stimulation of src kinase activity upon addition of GSLs or cholesterol. These results suggest that caveolar endocytosis is regulated by a balance of caveolin-1, cholesterol, and GSLs at the plasma membrane.
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