Cationic Amphiphilic Drugs Inhibit the Internalization of Cholera Toxin to the Golgi Apparatus and the Subsequent Elevation of Cyclic AMP

doi:10.1074/jbc.270.20.12117

Cationic Amphiphilic Drugs Inhibit the Internalization of Cholera Toxin to the Golgi Apparatus and the Subsequent Elevation of Cyclic AMP (*)

Anat Sofer and
Anthony H. Futerman(§)

From the (1) Department of Membrane Research and Biophysics, Weizmann Institute of Science, Rehovot 76100, Israel

^§Incumbent of the Recanati Career Development Chair in Cancer Research. To whom correspondence should be addressed. Tel.: 972-8-342704; Fax: 972-8-344112; E mail: BMFUTER{at}WEIZMANN.WEIZMANN.AC.IL.

Abstract

Cholera toxin (CT) consists of a pentameric B subunit which binds with high affinity to ganglioside GM₁, and an A subunit which stimulates adenylate cyclase, resulting in the elevation of cAMP. We now examine the effect of cationic amphiphilic drugs (CADs) on the internalization of rhodamine (Rh)-CT in cultured hippocampal neurons. CADs have recently been shown to inhibit receptor recycling by disrupting the assembly-disassembly of clathrin at the plasma membrane and on endosomes (Wang, L.-H., Rothberg, K. G., and Anderson, R. G. W.(1993) J. Cell Biol. 123, 1107-1117). Rh-CT was internalized by an energy- and temperature-dependent (presumably vesicular) mechanism to the Golgi apparatus. Internalization to the Golgi apparatus was completely but reversibly blocked by CADs, and the ability of CT to stimulate the elevation of cAMP was significantly reduced. In control cells, cAMP levels were elevated 2.3-fold after 20 min of incubation with CT, but in CAD-treated cells cAMP levels were only elevated 1.3-fold. The effect of CADs on CT internalization was not due to a direct effect of CADs on the Golgi apparatus. Our data demonstrate that CADs inhibit vesicular transport of CT to the Golgi apparatus and imply that the sorting of CT to the Golgi apparatus occurs in the same endosomal compartment involved in sorting recycling receptors to the plasma membrane, since both pathways are inhibited by CADs.

Footnotes

↵* This work was supported by a grant from the Basic Research Foundation of the Israel Academy of Sciences and Humanities, and by Grant 91-00278 from the United States-Israel Binational Science Foundation, Jerusalem, Israel. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

PM

plasma membrane

C₅-Bodipy-ceramide

N-[5-(5,7-dimethylBodipy)-1-pentanoyl]-D-erythro-sphingosine

CAD

cationic amphiphilic drug

C₆-NBD-ceramide

N-{6-[(7-nitrobenzo-2-oxa-1,3-diazol-4-yl)amino]caproyl}-D-erythro-sphingosine

CT

cholera toxin

CT-B

B-subunit of cholera toxin

Rh

rhodamine.
↵²A. Sofer and A. H. Futerman, unpublished observations.