Mechanism of action of cholera toxin: effect of receptor density and multivalent binding on activation of adenylate cyclase.
Choleragen (cholera toxin) activates adenylate cyclase in HeLa cells, which contain less than 15,000 toxin receptors per cell, in a time- and concentration-dependent manner. Activation is blocked by the addition of the oligosaccharide chain of the ganglioside GM1, the receptor for the toxin. When the cells are preincubated with choleragen at 4 degrees C and then incubated with oligosaccharide at 37 degrees C, adenylate cyclase is activated less than 10%. When the preincubation phase is above 18 degrees C, adenylate cyclase becomes activated and the amount of activation depends on the time of preincubation. This inhibitory effect of the oligosaccharide is also observed with human lymphocytes and rat glial C6 cells but not with Friend erythroleukemic and mouse neuroblastoma N18 cells. The latter two cell lines have large numbers ot toxin receptors, whereas the former two cell lines have few receptors. When the number of toxin receptors in HeLa and C6 cells is increased by treating the cells with GM1, activation of adenylate cyclase by choleragen is no longer blocked by the oligosaccharide. The oligosaccharide has a corresponding effect on the displacement of bound 125I-choleragen. When bound to cells at 4 degrees C, most of the radiotoxin is displaced from HeLa, C6, and lymphocytes but not from Friend, N18, or HeLa cells pretreated with GM1. In untreated HeLa cells, dissociation of toxin-receptor complexes by the oligosaccharide depends on the time and temperature of complex formation; above 18 degrees C, the toxin rapidly becomes stably bound to the cells. The inhibitory effect of GM1 oligosaccharide us reversible, as, once it is removed, the small amount of toxin that remains bound can activate adenylate cyclase. These results are consistent with a model in which choleragen, which is multivalent, must bind to several GM1 molecules on the cell surface in order to subsequently activate adenylate cyclase. Lateral mobility of toxin-receptor complexes may be required only to achieve multivalent binding in cells with few receptors.
D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D002772:Cholera Toxin / Q000494:pharmacology*; D004789:Enzyme Activation; D005677:G(M1) Ganglioside / Q000494:pharmacology; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D004915:Leukemia, Erythroblastic, Acute / Q000378:metabolism; D008214:Lymphocytes / Q000378:metabolism; D051379:Mice; D009380:Neoplasms, Nerve Tissue / Q000378:metabolism; D009447:Neuroblastoma / Q000378:metabolism; D051381:Rats; D011956:Receptors, Cell Surface; D011971:Receptors, Immunologic / Q000032:analysis / Q000378:metabolism*
D011956:Receptors, Cell Surface; D011971:Receptors, Immunologic; C023552:choleragen receptor; D005677:G(M1) Ganglioside; D002772:Cholera Toxin; D000262:Adenylyl Cyclases