Abstract. Vibrio cholerae and Escherichia coli heat labile toxins (CT and LT) elicit a secretory response from intestinal epithelia by binding apical receptors (ganglioside GM1 ) and subsequently activating basolateral effectors (adenylate cyclase). We have recently proposed that signal transduction in polarized cells may require transcytosis of toxin-containing membranes (Lencer, W. I., G. Strohmeier, S. Moe, S. L. Carlson, C. T. Constable, and J. L. Madara. 1995. Proc. Natl. Acad. Sci. USA. 92:10094-10098). Targeting of CT into this pathway depends initially on binding of toxin B subunits to GMI at the cell surface. The anatomical compartments in which subsequent steps of CT processing occur are less clearly defined. However, the enzymatically active A subunit of CT contains the ER retention signal KDEL (RDEL in LT). Thus if the KDEL motif were required for normal CT trafficking, movement of CT from the Golgi to ER would be implied. To test this idea, recombinant wild-type (wt) and mutant CT and LT were prepared. The COOH-terminal KDEL sequence in CT was replaced by seven unrelated amino acids: LEDERAS. In LT, a single point mutation replacing leucine with valine in RDEL was made. Wt and mutant toxins displayed similar enzymatic activities and binding affinities to GM1 immobilized on plastic. Biologic activity of recombinant toxins was assessed as a C1-secretory response elicited from the polarized human epithelial cell line T84 using standard electrophysiologic techniques. Mutations in K(R)DEL of both CT and LT delayed the time course of toxin-induced C1-secretion. At T1/2, dose dependencies for K(R)DELmutant toxins were increased ~>10-fold. KDELmutants displayed differentially greater temperature sensitivity. In direct concordance with a slower rate of signal transduction, KDEL-mutants were trafficked to the basolateral membrane more slowly than wt CT (assessed by selective cell surface biotinylation as transcytosis of B subunit). Mutation in K(R)DEL had no effect on the rate of toxin endocytosis. These data provide evidence that CT and LT interact directly with endogenous KDEL-receptors and imply that both toxins may require retrograde movement through Golgi cisternae and ER for efficient and maximal biologic activity.ETROGRADE transport through Golgi cisternae has been shown to occur for soluble and membrane proteins of the ER (49) and for certain protein toxins (6, 55). Targeting of soluble ER and some type II membrane proteins in this pathway depends on the COOH-terminal sorting signal Lys-Asp-Glu-Leu (KDEL or HDEL

Cholera and Escherichia coli heat-labile toxins (CT and LT) require proteolysis of a peptide loop connecting two major domains of their enzymatic A subunits for maximal activity (termed "nicking"). To test whether host intestinal epithelial cells may supply the necessary protease, recombinant rCT and rLT and a protease-resistant mutant CTR192H were prepared. Toxin action was assessed as a Cl ؊ secretory response (Isc) elicited from monolayers of polarized human epithelial T84 cells. When applied to apical cell surfaces, wild type toxins elicited a brisk increase in Isc (80 A/cm 2 ). Isc was reduced 2-fold, however, when toxins were applied to basolateral membranes. Pretreatment of wild type toxins with trypsin in vitro restored the "basolateral" secretory responses to "apical" levels. Toxin entry into T84 cells via apical but not basolateral membranes led to nicking of the A subunit by a serine-type protease. T84 cells, however, did not nick CTR192H, and the secretory response elicited by CTR192H remained attenuated even when applied to apical membranes. Thus, T84 cells express a serine-type protease(s) fully sufficient for activating the A subunits of CT and LT. The protease, however, is only accessible for activation when the toxin enters the cell via the apical membrane.

In the polarized human intestinal epithelial cell line T84, signal transduction by cholera toxin (CT) follows a complex series of events in which CT enters the apical endosome and moves through multiple vesicular compartments before it activates adenylate cyclase. As with processing of many other surface ligands, it has been suggested that CT must enter acidic vesicles to exert its downstream effects. To determine if intravesicular pH may regulate signal transduction by CT, we examined the cAMP-dependent Cl- secretory response [short-circuit current (Isc)] to CT in T84 cell monolayers treated with chloroquine (500 microM), methylamine (50 mM), NH4Cl (10 mM), nigericin (4 microM), or bafilomycin A1 (1 microM). Each of these reagents collapsed intravesicular pH gradients as confirmed by accumulation of acridine orange within subcellular compartments of living T84 cells imaged by confocal epifluorescence microscopy. Both acidotropic amines and nigericin inhibited the cAMP-dependent Cl secretory response in T84 cells. However, none of these reagents specifically affected adenylate cyclase itself or coupling of adenylate cyclase with the heterotrimeric guanosinetriphosphatase Gs as judged by the secretory response to the adenosine 3',5'-cyclic monophosphate (cAMP) agonists vasoactive intestinal peptide (VIP), forskolin, or 8-bromo-cAMP. In vitro enzyme-linked immunosorbent assay showed that CT binding to ganglioside GM1 was not dependent on pH between 5.0 and 10. Maximal Isc elicited by apical CT relative to maximal Isc elicited by VIP was not affected by pretreatment with chloroquine, methylamine, NH4Cl, or bafilomycin AI. Nigericin was the only reagent to inhibit CT-induced Isc (5 +/- 2% maximal response to VIP). The data indicate that low intravesicular pH will have little or no effect on CT association with its membrane receptor GM1, or on subsequent processing/signal transduction events.