Abstract
Cholera toxin (CT) consists of a pentameric B subunit which binds to ganglioside GM1 on the cell surface and an A subunit which activates adenylylcyclase. The latter process involves the reduction of A to the A1 peptide which ADP-ribosylates the stimulatory G protein, Gs of adenylylcyclase. There is a distinct lag phase between toxin binding and activation of adenylylcyclase. Little is known about the events during this lag including where A1 is generated and how it gains access to Gs on the cytoplasmic side of the plasma membrane. We explored the effects of several inhibitors of intracellular trafficking on the response of human SK-N-MC neurotumor and Caco-2 intestinal tumor cells to CT. Whereas chloroquine or monensin had little or no effect on CT stimulation of cyclic AMP accumulation, brefeldin A (BFA) totally inhibited the response to CT in a time- and dose-dependent and reversible manner. BFA was effective when added at the same time as CT and had an IC50 of 30 ng/ml. BFA did not alter cell surface GM1 as cells treated with BFA for 30 min bound as much 125I-CT as control cells. Furthermore, BFA inhibited CT stimulation of GM1-treated rat glioma C6 cells. BFA treatment did not affect beta-adrenergic agonist stimulation of cyclic AMP. In addition, adenylylcyclase was activated by A1 peptide and NAD+ to the same extent in membranes from control and BFA-treated cells, or when BFA was added directly to the assay. Whereas control cells generated small amounts of A1 from bound CT with time, no A1 was detected in BFA-treated cells. BFA treatment did not prevent the internalization of CT but did inhibit its degradation. BFA is known to disrupt the organization of the Golgi complex, resulting in inhibition of protein transport from the endoplasmic reticulum and redistribution of Golgi enzymes to the endoplasmic reticulum. BFA also prevents the formation of non-clathrin-coated vesicles from Golgi membranes and thus vesicular transport between Golgi cisternae. We confirmed that BFA caused the morphological disruption of the Golgi apparatus in Caco-2 cells. The data support a role for a functional Golgi apparatus with its associated vesicular routing in CT action.
Highlights
Cholera toxin (CT)consists of a pentameric B subunit disease cholera, and mediates its effects by increasing CAMP which binds to ganglioside G Mo~n the cell surface and (Finkelstein, 1973)
The A subunit, which consists of two peptides, A, and Az linked by a disulfide bond, activates adenylylcyclase.The A, peptide is an ADP-ribosyltransferase which catalyzes the transfer of ADP-ribose from NAD’ to the a subunit of the stimulatory G protein, G., resulting in thepersistent activation of adenysurface Gml as cells treated withBFA for 30 min bound lylcyclase (Moss and Vaughan, 1979)
We explored the effects of chloroquine, monensin, and brefeldin A on the response of several cultured
Summary
Cholera toxin (CT)consists of a pentameric B subunit disease cholera, and mediates its effects by increasing CAMP which binds to ganglioside G Mo~n the cell surface and (Finkelstein, 1973). The human small intestinal an A subunit which activates adenylylcyclase. The latter process involves the reductionof A to thAe1peptide which ADP-ribosylates the stimulatory G protein, G. mucosal cell is the normal target of the toxin, CT is a ubiquitious activator of adenylylcyclase in most vertebrate cells (van Heyningen, 1983;Fishman, 1990).The structure of CT is well defined, especially as the crystal structure of the Little isknown about the events during this inlacglud- homologous Escherichia coli heat-labile enterotoxin has reing where A1 is generated and how it gains access to cently been published (Sixma et al, 1991).CT is composed of. Adenylylcyclase was activated by AI peptide and NAD+ to the same extentin membranes fromcontroland BFA-
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