cAMP-Independent Activation of the Unfolded Protein Response by Cholera Toxin

Cholera toxin (CT) is an AB₅ protein toxin that activates the stimulatory alpha subunit of the heterotrimeric G protein (Gsα) through ADP-ribosylation. Activation of Gsα produces a cytopathic effect by stimulating adenylate cyclase and the production of cAMP.

ABSTRACT

Cholera toxin (CT) is an AB₅ protein toxin that activates the stimulatory alpha subunit of the heterotrimeric G protein (Gsα) through ADP-ribosylation. Activation of Gsα produces a cytopathic effect by stimulating adenylate cyclase and the production of cAMP. To reach its cytosolic Gsα target, CT binds to the plasma membrane of a host cell and travels by vesicle carriers to the endoplasmic reticulum (ER). The catalytic CTA1 subunit then exploits the quality control mechanism of ER-associated degradation to move from the ER to the cytosol. ER-associated degradation is functionally linked to another quality control system, the unfolded protein response (UPR). However, the role of the UPR in cholera intoxication is unclear. We report here that CT triggers the UPR after 4 h of toxin exposure. A functional toxin was required to induce the UPR, but, surprisingly, activation of the adenylate cyclase signaling pathway was not sufficient to trigger the process. Toxin-induced activation of the UPR coincided with increased toxin accumulation in the cytosol. Chemical activation of the heterotrimeric G protein or the UPR also enhanced the onset of CTA1 delivery to the cytosol, thus producing a toxin-sensitive phenotype. These results indicate there is a cAMP-independent response to CT that activates the UPR and thereby enhances the efficiency of intoxication.

INTRODUCTION

Cholera toxin (CT) is the main virulence factor secreted by Vibrio cholerae (1, 2). The toxin is an ADP-ribosyltransferase that modifies and activates the stimulatory alpha subunit of the heterotrimeric G protein (Gsα). Activated Gsα stimulates adenylate cyclase (AC) and the production of intracellular cAMP. The elevated levels of cAMP initiate a signaling pathway that results in the opening of chloride channels at the apical membrane of intestinal epithelial cells. The resulting osmotic movement of water which follows chloride efflux into the intestinal lumen produces the profuse watery diarrhea of cholera.

CT is an AB-type protein toxin that consists of a catalytic A subunit with a cell-binding B subunit (3). The A subunit is a disulfide-linked A1/A2 heterodimer, while the B subunit is a homopentameric ring-like structure. The intact holotoxin is secreted into the extracellular space by V. cholerae, but Gsα is found at the cytosolic face of the host plasma membrane. CT must therefore cross a membrane barrier in order to act on its Gsα target. This event only occurs after CT is delivered from the cell surface to the endoplasmic reticulum (ER) through a series of vesicle-mediated events that are collectively termed retrograde transport (4).

Redox conditions in the ER promote the chaperone-assisted dissociation of CTA1 from CTA2/CTB₅ (5–9). As the isolated CTA1 subunit is a thermally unstable protein (10), holotoxin disassembly at physiological temperature leads to the spontaneous unfolding of CTA1. The loss of structure that occurs during this unfolding event identifies the toxin as a substrate for ER-associated degradation (ERAD) (11–13), a quality control mechanism that recognizes misfolded or misassembled proteins in the ER and exports them to the cytosol for degradation by the ubiquitin-proteasome system (14). ERAD transfers CTA1 to the cytosol through protein-conducting channels in the ER membrane (15–18), but CTA1 avoids the standard degradative fate of ERAD substrates because a strong arginine-over-lysine bias in the amino acid content of CTA1 limits the number of potential sites for ubiquitination (19, 20). Thus, CTA1 exploits ERAD for translocation to the cytosol and then evades ERAD processing by the ubiquitin-proteasome system. Other AB toxins such as ricin and Shiga toxin also move from the cell surface to the ER and use ERAD for A chain translocation to the cytosol (21, 22).

The unfolded protein response (UPR) is a quality control mechanism linked to ERAD (23, 24). When the basal level of ERAD activity cannot cope with the accumulation of misfolded proteins in the ER, the UPR is activated. The following three separate but interrelated pathways respond to ER stress conditions in mammalian cells: the Perk pathway downregulates overall protein synthesis in order to reduce the load of misfolded proteins in the ER, while both the ATF6 and the Ire1 pathways upregulate the production of ER chaperones and other ERAD components in order to compensate for the elevated levels of misfolded proteins in the ER. Proteins related to vesicle trafficking in the biosynthetic secretory pathway are also upregulated by the UPR (25). Transcriptional activators for the ATF6 and Ire1 pathways are generated by distinct mechanisms and bind to distinct promoter elements. Collectively, the three branches of the mammalian UPR act to reestablish homeostasis after the cell encounters ER stress conditions.

Since CTA1 follows an ERAD-dependent translocation route (26, 27), the UPR could also be involved with CT intoxication. To examine this possibility, we used an ATF6-dependent UPR reporter assay to determine whether CT intoxication triggers the UPR. Cells exposed to wild-type CT but not to nontoxic CT mutants exhibited an UPR. The timing of toxin-induced UPR activation coincided with markedly elevated levels of cytosolic CTA1. In addition, chemical activation of the UPR stimulated the onset of CTA1 delivery to the cytosol and sensitized cells to CT. Cells with a ligand-activated G protein likewise exhibited a UPR and enhanced CTA1 accumulation in the cytosol. Surprisingly, however, UPR activation and enhanced CTA1 accumulation in the cytosol were not observed in cells with an activated AC. Our collective data suggest there is a physiological target for CT-activated G protein, other than AC, which activates the UPR and thereby enhances the efficiency of intoxication.

RESULTS

CT activity is required for AC-independent activation of the UPR.

To determine whether CT could activate the UPR, we employed an established reporter assay (28) in which the expression of firefly luciferase is controlled by a UPR-responsive ATF6 promoter element (Fig. 1). A 2.8-fold increase in luciferase activity was recorded after treating cells with thapsigargin, a Ca²⁺-ATPase inhibitor that activates the UPR by depleting ER Ca²⁺ stores and consequently disrupting the function of Ca²⁺-dependent ER chaperones. Cells exposed to 100 ng CT/ml produced a comparable 2.2-fold increase in luciferase activity. No UPR was detected until 4 h after toxin challenge, even though elevated levels of cAMP could be detected after 2 h of intoxication. In contrast, a thapsigargin-induced UPR could be detected with the luciferase assay following just 2 h of drug treatment (29). CT-induced activation of the UPR was a dose-dependent event; exposure to 1 ng CT/ml did not stimulate luciferase activity above the baseline value compared to unintoxicated control cells. Incubation with 100 ng/ml of the inactive CT Y149A or CT Y149S mutants did not trigger the UPR. These mutants do not interact with host ADP-ribosylation factors, which are allosteric activators of CTA1 required for cytosolic toxin activity (30). Dose-dependent activation of the UPR by CT therefore required a functional toxin. However, cells treated with the AC agonist forskolin did not exhibit elevated levels of luciferase activity. Control experiments confirmed that cAMP levels rose substantially in forskolin-treated cells (data not shown). Thus, while CT activity was required to trigger the UPR, elevated cAMP levels were not sufficient for UPR activation.

FIG 1.

CT activity triggers the UPR through a cAMP-independent mechanism. A luciferase-based reporter assay was used to monitor UPR activation in CHO cells exposed for 4 h to thapsigargin (+Tg), forskolin, CT, or inactive CT mutants. To calculate the extent of UPR induction, values from the experimental conditions were divided by the control value from untreated cells. The mean ± standard error of the mean (SEM) values for at least 4 independent experiments per condition are shown. Asterisks denote a statistically significant difference (P ≤ 0.002; Student’s t test) from the untreated control. A statistically significant difference between groups was confirmed by one-way analysis of variance (ANOVA) (P = 0.005).

UPR activation sensitizes cells to CT.

To examine the impact of UPR activation on the extent of CT intoxication, we performed toxicity assays on CHO cells that had been incubated with the N-glycosylation inhibitor tunicamycin (Fig. 2). Improper glycosylation of nascent secretory proteins triggers the UPR in tunicamycin-treated cells, with preliminary experiments confirming there was a 6- or 13-fold induction of the UPR after an 8-h exposure to tunicamycin (n = 2). Untreated and tunicamycin-treated cells were challenged with various concentrations of CT for 90 min before cAMP levels were quantified. In comparison to the untreated cells, tunicamycin-treated cells displayed a dramatic sensitization to CT. This observation was consistent with previous reports documenting the sensitizing effect of either tunicamycin or thapsigargin on cholera intoxication (15, 31). Thus, chemical activation of the UPR greatly increases the potency of CT. A similar sensitizing effect was recorded for tunicamycin-treated HeLa cells as well (data not shown).

FIG 2.

UPR activation sensitizes cells to CT. Untreated and tunicamycin-treated CHO cells were challenged with the stated concentrations of CT. Cell extracts generated after 90 min of continual toxin exposure were then screened for cAMP content. Data are presented as the mean ± SEM values of 5 independent experiments with triplicate samples.

UPR activation enhances the onset of CTA1 delivery to the cytosol.

An elevated level of cytosolic toxin could be responsible for UPR-induced sensitization to CT. To examine this possibility, we monitored the time-dependent accumulation of cytosolic CTA1 in control cells and cells with a chemically activated UPR (Fig. 3). Untreated or tunicamycin-treated HeLa cells were exposed to 1 μg CT/ml at 4°C, a temperature that allows toxin binding to the cell surface but prevents endocytosis of the bound toxin. After removal of unbound toxin, the cells were warmed to 37°C in order to permit toxin endocytosis, retrograde transport to the ER, and translocation to the cytosol. Cell extracts collected after 15, 30, 45, or 60 min of chase at 37°C were partitioned into separate organelle and cytosol fractions. The translocated, cytosolic pools of CTA1 were then detected by perfusing the cytosolic fractions over a surface plasmon resonance (SPR) sensor coated with an anti-CTA1 antibody. The fidelity of our fractionation procedure was confirmed with a Western blot which demonstrated that protein disulfide isomerase (PDI; a soluble ER protein) was only found in the pellet fraction containing intact organelles, while the vast majority of cytosolic chaperone Hsp90 was found in the supernatant fraction containing the cytosol (Fig. 3A, inset). The detection of a minor pool of membrane-associated Hsp90 was consistent with previous studies that have used similar fractionation protocols (32–35).

FIG 3.

Chemical activation of the UPR enhances CTA1 delivery to the cytosol. Untreated (A) and tunicamycin-treated (B) cells were placed on ice and exposed to 1 μg/ml of CT for 30 min. Unbound toxin was removed, and the cells were warmed to 37°C for 15, 30, 45, or 60 min before selective permeabilization of the plasma membrane with digitonin. The Western blot inset of panel A confirms the fidelity of the permeabilization procedure by locating protein disulfide isomerase (PDI), a soluble ER protein, in the organelle-containing pellet fraction (P) and the cytosolic protein Hsp90 in the cytosol-containing supernatant fraction (S). The cytosolic samples were perfused over an SPR sensor coated with an anti-CTA1 antibody. All samples from both panels were perfused over the same sensor, but data are shown in separate panels for clarity. The sensor was regenerated at the end of each perfusion by stripping bound sample from the slide. CTA standards were perfused over the sensor as positive controls. The cytosol from unintoxicated cells and cells intoxicated in the presence of BfA were used as negative controls. One of two representative experiments is shown. MicroRIU, micro-refractive index units.

In control cells, CTA1 could not be detected in the cytosolic fraction after 15 min of chase (Fig. 3A). Increasing quantities of CTA1 appeared in the cytosol of control cells after 30, 45, and 60 min of chase (Fig. 3A). A time-dependent increase in cytosolic toxin was also recorded for the UPR-activated cells (Fig. 3B). However, the cytosolic fractions from UPR-activated cells contained larger quantities of CTA1 than the cytosolic fractions from control cells collected at the same time point. A minor but detectable pool of CTA1 was even present in the cytosol of UPR-activated cells after just 15 min of chase. No CTA1 was detected in the cytosol of unintoxicated cells or CT-treated cells exposed to brefeldin A (BfA), a drug that prevents toxin transport to the ER translocation site (36, 37) (Fig. 3B). Thus, the positive signals from control and UPR-activated cells could be attributed to the presence of cytosolic CTA1. These collective observations indicated that UPR activation increases the accumulation of toxin in the cytosol.

Toxin-induced activation of the UPR stimulates CTA1 delivery to the cytosol.

Induction of the UPR occurs approximately 4 h after toxin challenge. Interestingly, we previously observed a remarkable jump in the concentration of cytosolic CTA1 after 4 h of intoxication (12). We accordingly hypothesized that the jump in cytosolic CTA1 results from toxin-induced activation of the UPR. To test this prediction, we monitored the time-dependent accumulation of cytosolic CTA1 from either wild-type CT or the CT Y149S mutant (Fig. 4A). HeLa cells labeled with toxin at 4°C were chased at 37°C in the absence of additional toxin as described above. Both wild-type and mutant toxins displayed similar patterns of CTA1 delivery to the cytosol during the first 4 h of chase, but an obvious difference could be seen after 5 h, namely, a dramatic increase in the level of cytosolic CTA1 that occurred between 4 and 5 h of chase for wild-type CT but not for CT Y149S. The enzymatically inactive CT E110D/E112D mutant (38) also failed to exhibit the dramatic jump in cytosolic toxin from 4 to 5 h of chase (Fig. 4B). Thus, the lack of cellular enzymatic function for both CT Y149S and CT E110D/E112D appeared to be responsible for the diminished accumulation of cytosolic toxin at 5 h of chase.

FIG 4.

CT activity enhances CTA1 delivery to the cytosol. (A) HeLa cells were placed on ice for 30 min in the presence of 1 μg/ml of either wild-type CT (wt, solid line) or mutant CT Y149S (mt, dashed line). Unbound toxin was removed, and the cells were warmed to 37°C for 1, 4, or 5 h before selective permeabilization of the plasma membrane with digitonin. The collected cytosolic fractions were then perfused over an SPR sensor coated with an anti-CTA1 antibody. The cytosol from unintoxicated cells and BfA-treated cells intoxicated with wt CT for 1 h (+BfA) were used as negative controls. (B) The experiment was repeated using the CT E110D/E112D mutant (mt, dashed line). One of three representative experiments is shown for each panel.

Chemical activation of the heterotrimeric G protein stimulates toxin delivery to the cytosol and induces the UPR.

Enhanced toxin delivery to the cytosol apparently occurs when the catalytic activity of CTA1 triggers the UPR through a cAMP-independent pathway. To determine whether this pathway involved a heterotrimeric G protein signaling mechanism, we followed the accumulation of cytosolic CTA1 in isoproterenol-treated CHO cells with constitutive expression of a recombinant β₂-adrenergic receptor (β₂AR) (Fig. 5). Isoproterenol binds to the β₂AR, which in turn activates Gsα (39, 40). This provided us with a means to activate the heterotrimeric G protein independently of toxin activity.

FIG 5.

Chemical activation of the heterotrimeric G protein enhances CTA1 delivery to the cytosol and induces the UPR. (A) CHO β₂AR cells were incubated in the absence of treatment (N.T.; no treatment) or in the presence of thapsigargin (+Tg), forskolin (+For), isoproterenol (+Iso), or 100 ng/ml of CT. cAMP levels were quantified after a 90-min or 4-h treatment. Data are presented as the averages ± ranges (n = 2) of two independent experiments with triplicate samples. (B and C) CHO β₂AR cells were placed on ice after either 90 min (B) or 4 h (C) of drug treatment. Cells were exposed to 1 μg/ml of CT for 30 min at 4°C. Unbound toxin was removed, and the cells were warmed to 37°C for 30 min before selective permeabilization of the plasma membrane with digitonin. The collected cytosolic fractions were then perfused over an SPR sensor coated with an anti-CTA1 antibody. The cytosol from unintoxicated cells and cells intoxicated in the presence of BfA were used as negative controls. All samples were perfused over the same sensor, but results are presented in two graphs for clarity. The sensor was regenerated at the end of each perfusion by stripping bound sample from the slide. One of three representative experiments is shown. (D) A luciferase-based reporter assay was used to monitor UPR activation in CΗΟ β₂AR cells exposed to thapsigargin, forskolin, isoproterenol, or 100 ng/ml CT for 4 h. To calculate the extent of UPR induction, values from the experimental conditions were divided by the control value from untreated cells. The means ± SEM of at least 4 independent experiments per condition are shown. Asterisks denote a statistically significant difference from the untreated control (*, P = 0.002; **, P = 0.0001; Student’s t test). A statistically significant difference between groups was confirmed by one-way ANOVA (P = 0.003).

Control experiments documented greatly elevated cAMP levels in CHO β₂AR cells exposed to forskolin (Fig. 5A). Isoproterenol-treated cells produced cAMP levels similar to, or greater than, those obtained from cells challenged with 100 ng/ml of CT for an equivalent time frame. Treatment with the Ca²⁺-ATPase inhibitor thapsigargin generated a small increase in intracellular cAMP.

To examine the impact of G protein activation on CTA1 delivery to the cytosol, we used our SPR system to detect the presence of cytosolic CTA1 after 30 min of toxin exposure. Preliminary experiments found that under control conditions, CTA1 could be detected in the cytosol of CHO β₂AR cells after 45 but not 30 min of toxin exposure. CTA1 was not found in the cytosol of forskolin-treated cells after 30 min of toxin exposure but was present in the cytosol of cells with a thapsigargin-induced UPR (Fig. 5B and C). Thus, the elevated levels of cAMP resulting from forskolin treatment did not stimulate CTA1 passage into the cytosol. In contrast, CTA1 delivery to the cytosol was enhanced through G protein activation with a 90-min (Fig. 5B) or 4-h (Fig. 5C) exposure to isoproterenol. G protein activation thus appeared to stimulate CTA1 delivery to the cytosol in a cAMP-independent fashion. Isoproterenol treatment did not alter the cytosolic quantity of CTA1 in wild-type CHO cells (data not shown), thus demonstrating the isoproterenol-stimulated delivery of cytosolic toxin in CHO β₂AR cells was mediated through the G-coupled β₂AR.

Activation of the UPR through a G protein signaling pathway most likely accounted for the elevated levels of cytosolic CTA1 in isoproterenol-treated CHO β₂AR cells. This prediction was tested with the ATF6 reporter assay (Fig. 5D). An active UPR was detected in cells incubated with CT, thapsigargin, or isoproterenol for 4 h. Forskolin-treated cells only exhibited a minor, 1.2-fold induction of the UPR, despite the robust production of cAMP in forskolin-treated cells (Fig. 5A). The data shown in Fig. 5 thus confirmed that UPR activation was not linked to the extent of cAMP production; treatment with either isoproterenol or forskolin resulted in high levels of intracellular cAMP (Fig. 5A), but only isoproterenol treatment enhanced toxin delivery to the cytosol (Fig. 5B and C) and triggered a substantial UPR (Fig. 5D). CT activity likewise activated the UPR (Fig. 1 and 5D) and stimulated CTA1 passage into the cytosol (Fig. 4). These collective observations suggested CT triggers the UPR through a G protein signaling pathway that does not involve AC. The inability of cAMP-dependent signaling events to trigger a UPR is consistent with previous work that demonstrated that plasmid-based expression of the catalytic subunit from PKA does not induce a UPR (28).

DISCUSSION

We have identified a cAMP-independent signaling pathway that activates the UPR and enhances the onset of CTA1 delivery to the host cytosol. Our data demonstrate that (i) CT activates the UPR and (ii) CT intoxication is enhanced by UPR activation. Taken together, these results suggest that CT intoxication is a biphasic process in which an initially inefficient intoxication is enhanced by the UPR activation that occurs after the initial pool of CTA1 reaches the cytosol. A catalytically active toxin was required to induce the UPR; however, surprisingly, the AC signaling pathway was not sufficient to trigger this process. Other investigators have documented cAMP-independent effects resulting from CT activity (41–43). However, this is the first demonstration of a functional role for a cAMP-independent signaling event in the CT intoxication process.

Dixit et al. (15) documented the upregulation of certain ER proteins in cells exposed to relatively high concentrations (0.6 μg/ml) of the CTB pentamer for as little as 15 min. The increased expression of BiP, Derlin-1, and Derlin-2 apparently occurred at the level of translation and generated a toxin-sensitive phenotype in the affected cells. In contrast, our data document a transcriptional induction of the UPR that results from the action of a functional toxin. Collectively, these observations indicate that CT can trigger multiple signaling pathways to alter host sensitivity to intoxication. Toxin sensitization results from either CT-induced activation of the UPR or CTB-induced changes to the translation of ER-localized proteins.

The UPR is usually activated through the accumulation of misfolded proteins in the ER. For CT-induced UPR activation, it is doubtful that the physical presence of the toxin in the ER is enough to trigger the UPR. Only a minor pool (∼5%) of surface-bound CT reaches the ER; the majority of internalized toxin is instead routed to the lysosomes for degradation (36, 37, 44, 45). Thus, CT is unlikely to accumulate at sufficiently high levels in the ER to activate the UPR. The absence of a UPR in cells treated with inactive CT mutants further supports our conclusion that the physical presence of CT in the ER is not sufficient to activate the UPR.

It is highly unlikely that CTA1 would modify a target in the ER lumen during the brief interval between its dissociation from the holotoxin and its transport to the cytosol. The latent activity of CT is typically manifested after (i) the CTA subunit is proteolytically nicked to generate separate CTA1 and CTA2 polypeptides; (ii) the disulfide bond linking CTA1 to CTA2 is reduced; (iii) a conformational change in CTA1 permits expression of catalytic activity at a basal level; and (iv) allosteric activation of cytosolic CTA1 by ARF permits expression of full catalytic activity (44, 46). Furthermore, the isolated CTA1 subunit is in a disordered state at physiological temperature and therefore requires an interaction with host cytosolic factors to attain an active conformation (30, 35, 47, 48). Finally, the NAD donor molecule for the ADP-ribosylation reaction is not present in the ER (49). These collective observations indicate that CTA1 activity will only be apparent in the host cytosol.

The cAMP-independent effects associated with CT could result from alternative Gα signaling pathways (41–43), but it is also possible that the dissociated βγ subunits of a CT-activated heterotrimeric G protein are responsible for triggering the UPR. Both Gα and Gβγ can contribute to downstream signaling events (43, 50). A pathway stimulated by Gβγ would explain why UPR activation occurred in response to the β₂AR agonist isoproterenol but not the AC agonist forskolin; both treatments elevate intracellular cAMP levels, but only isoproterenol activates the heterotrimeric G protein. The βγ subunits do not yet have an established link to the mammalian UPR, but Gβγ has been reported to regulate the UPR in Arabidopsis thaliana (51, 52). Furthermore, the βγ subunits have been shown to induce calcium release from the mammalian ER (53). This, like thapsigargin treatment, could lead to UPR activation. Further work will be required to elucidate the signaling pathway and potential role of Gβγ in CT-induced, cAMP-independent activation of the UPR.

The UPR stimulates ERAD activity, but it also affects proteins involved with vesicle transport in the endomembrane system (25). Thus, enhanced delivery of CTA1 to the cytosol of UPR-activated cells may result from more efficient CT transport to the ER instead of, or in addition to, an increased rate of CTA1 translocation. In support of this interpretation, Sandvig et al. (31) have reported an increased rate of CT transport from the Golgi apparatus to the ER of thapsigargin-treated cells. This observation was made before the UPR was well defined, so the original interpretation of the Sandvig paper focused on the role of ER calcium stores in regulating the cycle of Golgi-to-ER/ER-to-Golgi toxin transport. Given our current knowledge of the UPR and the data presented in this report, we suggest that calcium depletion via thapsigargin treatment triggers a series of events which involve (i) UPR activation; (ii) enhanced vesicle-mediated transport of CT to the ER, which consequently results in faster onset of CTA1 delivery to the cytosol; and (iii) toxin sensitization resulting from increased toxin accumulation in the cytosol.

The UPR is also affected by ricin and Shiga toxin, two other ERAD-exploiting toxins. Ricin blocks the UPR to enhance its cytotoxic effect (54, 55). Shiga toxin activates the UPR, but this still contributes to its lethal effect (56). For both Shiga toxin and CT, activation of the ATF6 branch of the UPR requires a functional toxin. Ricin, which shares the same cellular target and catalytic action as Shiga toxin, also requires a functional toxin to affect the UPR (54). These observations suggest that ERAD-exploiting toxins can alter the UPR by a variety of mechanisms to facilitate the intoxication process. Here, we have provided new insights into UPR activation and CT intoxication by demonstrating that cAMP-independent activation of the UPR by CT increases the efficiency of CTA1 delivery to the host cytosol.

MATERIALS AND METHODS

Materials.

CT and CTA were purchased from List Biologicals (Campbell, CA); BfA, ganglioside GM1, forskolin, thapsigargin, tunicamycin, protease inhibitor cocktail, and isoproterenol were purchased from Sigma-Aldrich (St. Louis, MO); digitonin was purchased from Calbiochem (La Jolla, CA); rabbit anti-Hsp90 and anti-PDI antibodies were purchased from StressGen Bioreagents Corp. (Victoria, BC, Canada); and a horseradish peroxidase-conjugated goat anti-rabbit IgG antibody was purchased from Jackson Immunoresearch Laboratories, Inc. (West Grove, PA). Mutant toxins were provided by Randall K. Holmes (30). CHO β₂AR cells were provided by Scott E. Mills (39, 40). The p5xATF6-GL3 and pRLSV40P plasmids were provided by Ron Prywes (28).

cAMP assays.

Cells grown overnight to 80% confluence in 24-well plates were challenged with toxin for the stated intervals. When indicated, an 8-h (CHO cells) or 90-min (HeLa cells) preincubation with 10 μg/ml of tunicamycin was performed in order to activate the UPR before toxin treatment. A commercial kit (GE Healthcare, Piscataway, NJ) was used to quantify the cAMP levels from unintoxicated or toxin-treated cells. Basal cAMP levels from unintoxicated cells, which ranged from 50 to 200 pM per well for both untreated and tunicamycin-treated cells, were subtracted from all experimental results. Control experiments found that unintoxicated cells treated with both tunicamycin and forskolin, an AC agonist, only produced 57% of the cAMP levels recorded for cells treated with forskolin alone (n = 5). The raw cAMP data for CHO cells treated with tunicamycin and CT were corrected for this tunicamycin-induced inhibition of cAMP production. The maximal cAMP response from the experiment was set at 100%, and all other results were expressed as percentages of that value in order to determine the effect of UPR activation on the cellular response to CT.

UPR reporter assay.

To detect activation of the UPR, we used an established reporter assay (28) in which the transcription of firefly luciferase is controlled by a UPR-responsive ATF6 promoter element. CHO or CHO β₂AR cells grown to ∼75% confluence in 6-well plates were cotransfected with 0.5 μg of p5xATF6-GL3 and 0.1 μg of pRLSV40P using Lipofectamine (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. The UPR-responsive firefly luciferase is expressed from p5xATF6-GL3, while Renilla luciferase is constitutively expressed from pRLSV40P. After an overnight incubation, the transfected cells were left untreated or were challenged with 200 nM thapsigargin, 100 μM forskolin, 1 μM isoproterenol, 10 μg/ml tunicamycin, CT, and/or CT mutants for the stated intervals. Cells were washed twice with phosphate-buffered saline (pH 7.4) and solubilized by incubation at room temperature for 15 min with 0.5 ml of passive lysis buffer from Promega (Madison, WI). A 20-μl sample of the clarified lysate was placed in a clear-bottomed black-walled 96-well plate (Greiner Bio-One, Monroe, NC), and luciferase activity was detected using the Promega dual-luciferase reporter assay system with a Synergy 2 plate reader (Bio-Tek, Winooski, VT). Luminescence from the UPR-responsive firefly luciferase was detected using the Promega luciferase reagent II, while luminescence from the constitutive Renilla luciferase was subsequently detected using the Promega Stop and Glo reagent. Luminescence produced from firefly luciferase dissipates rapidly and does not interfere with the subsequent signal from Renilla luminescence, which serves as an internal control for transfection efficiency and overall levels of protein synthesis. The ratio of each sample’s Renilla signal to that of the untreated control was used to normalize signals from the UPR-responsive firefly luciferase. Normalized data for the signals from firefly luciferase generated under experimental conditions were expressed as a ratio of the signal obtained from firefly luciferase expression in the untreated control cells.

Detection of cytosolic toxin.

HeLa cells grown overnight to 80% confluence in 6-well plates were incubated for 1 h at 37°C with 100 ng/ml of GM1, the ganglioside receptor of CT. After washing, the cells were placed on ice for 30 min with 1 μg/ml of CT. After further washing, the cells were incubated at 37°C in toxin-free medium for the indicated intervals. The cells were then collected in 750 μl of phosphate-buffered saline (pH 7.4) containing 0.5 mM ethylenediaminetetraacetic acid, spun in a microcentrifuge tube at 5,000 × g for 5 min, and resuspended in 1 ml HCN buffer (50 mM HEPES [pH] 7.5, 150 mM NaCl, 2 mM CaCl₂, 10 mM N-ethylmaleimide, and a protease inhibitor cocktail) containing 0.04% digitonin. After 10 min at 4°C, the cells were partitioned into cytosolic (supernatant) and intact membrane (pellet) fractions with a 10-min spin at 16,000 × g. The cytosolic fractions were recovered, brought to a final volume of 1 ml in HCN buffer, and perfused over a SPR sensor slide coated with the anti-CTA1 monoclonal antibody 35C2 (57). Perfusions of a purified CTA1/CTA2 heterodimer (i.e., CTA) were used for a standard. The flow rate for all perfusions was 41 μl/min; a detailed step-by-step protocol for the procedure has been published (58). Detection of the cytosolic pool of CTA1 in the supernatant fractions, as well as Western blot analysis of the cellular fractions, was performed as previously described (13). Triplicate wells of cells were used for each condition. To activate the UPR, cells were treated with 10 μg/ml of tunicamycin for 90 min before initiating the experiment with GM1 pretreatment. When indicated, cells were treated with 5 μg/ml of BfA during the 4°C toxin labeling and the 37°C chase.

CHO β₂AR experiments.

Cells were challenged with 1 μM isoproterenol, 100 μM forskolin, 200 nM thapsigargin, or 100 ng/ml CT for 90 min or 4 h. Untreated, drug-treated, and toxin-treated cells were then processed for cAMP quantification as described above, with the exception that the data were presented as pM cAMP per well rather than as a percentage of the maximum response. In separate experiments, untreated and drug-treated cells were placed on ice with 1 μg/ml of CT for 30 min. The cells were then warmed to 37°C for 30 min before collecting cytosolic fractions for SPR detection using sensor slides coated with the anti-CTA1 monoclonal antibody 35C2. A third set of cells were cotransfected with plasmids encoding firefly and Renilla luciferases ∼24 h before conducting the UPR reporter assay with untreated, drug-treated, and toxin-treated cells.