Intoxication of cultured cells by cholera toxin: evidence for different pathways when bound to ganglioside GM1 or neoganglioproteins.
We previously reported that when the oligosaccharide of ganglioside GM1 is covalently attached to cell surface proteins of GM1-deficient rat glioma C6 cells, the cells bind large amounts of cholera toxin (CT) but their cAMP response to CT is not enhanced [Pacuszka, T., & Fishman, P. H. (1990) J. Biol. Chem. 265, 7673-7668]. We now report that when such cells were exposed to CT in the presence of chloroquine, an acidotropic agent, they accumulated cAMP. This raised the possibility that CT bound to cell surface "neoganglioproteins" may be entering the cells through a different pathway from that of CT-bound GM1. To further explore this phenomenon, we covalently attached GM1 oligosaccharide to human transferrin (Tf). The modified protein (GM1OS-Tf) bound with high affinity to Tf receptors on HeLa cells and increased the binding of CT to the cells. The bound CT, however, was unable to activate adenylyl cyclase as measured by cyclic AMP accumulation. By contrast, treatment of HeLa cells with GM1 increased both CT binding and stimulation of cyclic AMP accumulation. Control cells and cells treated with either GM1 or GM1OS-Tf were exposed to CT in the presence of chloroquine. Whereas chloroquine had little or no effect on the response of control or GM1-treated cells to CT, it made the cells treated with GM1OS-Tf responsive to the toxin. Our results indicate that CT bound to its natural receptor GM1 enters the cells through a pathway different from that of toxin bound to neoganglioproteins.
Pacuszka
T
T
Membrane Biochemistry Section, National Institute of Neurological Disorders and Stroke, Bethesda, Maryland 20892.
Fishman
P H
PH
D000818:Animals; D002738:Chloroquine / Q000494:pharmacology; D002772:Cholera Toxin / Q000633:toxicity*; D000242:Cyclic AMP / Q000378:metabolism; D004705:Endocytosis; D005677:G(M1) Ganglioside / Q000378:metabolism*; D005910:Glioma / Q000378:metabolism*; D006367:HeLa Cells / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D051381:Rats; D011990:Receptors, Transferrin / Q000032:analysis / Q000187:drug effects; D014168:Transferrin / Q000737:chemistry / Q000187:drug effects; D014407:Tumor Cells, Cultured
D011990:Receptors, Transferrin; D014168:Transferrin; D005677:G(M1) Ganglioside; D002738:Chloroquine; D002772:Cholera Toxin; D000242:Cyclic AMP