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J. Cell Biol.,
Volume 141, Number 4, May 18, 1998 905-915
Membrane Biochemistry Section, Laboratory of Molecular and Cellular Neurobiology, National Institute of Neurological
Disorders and Stroke, The National Institutes of Health, Bethesda, Maryland 20892-4440
The mechanism by which cholera toxin (CT)
is internalized from the plasma membrane before its intracellular reduction and subsequent activation of adenylyl cyclase is not well understood. Ganglioside GM1,
the receptor for CT, is predominantly clustered in detergent-insoluble glycolipid rafts and in caveolae, noncoated, cholesterol-rich invaginations on the plasma
membrane. In this study, we used filipin, a sterol-binding agent that disrupts caveolae and caveolae-like
structures, to explore their role in the internalization and activation of CT in CaCo-2 human intestinal epithelial cells. When toxin internalization was quantified,
only 33% of surface-bound toxin was internalized by
filipin-treated cells within 1 h compared with 79% in
untreated cells. However, CT activation as determined by its reduction to form the A1 peptide and CT activity
as measured by cyclic AMP accumulation were inhibited in filipin-treated cells. Another sterol-binding
agent, 2-hydroxy-
-cyclodextrin, gave comparable results. The cationic amphiphilic drug chlorpromazine, an
inhibitor of clathrin-dependent, receptor-mediated endocytosis, however, affected neither CT internalization,
activation, nor activity in contrast to its inhibitory effects on diphtheria toxin cytotoxicity. As filipin did not
inhibit the latter, the two drugs appeared to distinguish
between caveolae- and coated pit-mediated processes. In addition to its effects in CaCo-2 cells that express
low levels of caveolin, filipin also inhibited CT activity
in human epidermoid carcinoma A431 and Jurkat T
lymphoma cells that are, respectively, rich in or lack caveolin. Thus, filipin inhibition correlated more closely
with alterations in the biochemical characteristics of
CT-bound membranes due to the interactions of filipin
with cholesterol rather than with the expressed levels of
caveolin and caveolar structure. Our results indicated
that the internalization and activation of CT was dependent on and mediated through cholesterol- and glycolipid-rich microdomains at the plasma membrane
rather than through a specific morphological structure
and that these glycolipid microdomains have the necessary components required to mediate endocytosis.
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