Role of the carboxy-terminal transactivation domain and active transcription in the ligand-induced and ligand-independent degradation of the mouse Ahb−1 receptor

Pollenz, Richard S.; Popat, Jesal; Dougherty, Edward J.

doi:10.1016/j.bcp.2005.09.006

Cited by 17 publications

(12 citation statements)

References 43 publications

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“…It is also important to note that if the latent 95kDa Ah b-1 AHR required calpain cleavage for a nuclear translocation event, this likely have a substantial impact on the transactivation potential of the AHR. For example, truncation of 165 amino acids from the C-terminus of the Ah b-1 receptor (approximately 21kDa) has no affect on the cytoplasmic localization of the AHR, but reduces the ability of the AHR to induce expression of endogenous CYP1A1 by 50% [ 24 ]. In addition, removal of 305 amino acids from the C-terminus of the AHR (approximately 40kDa) causes the latent AHR to become nuclear but completely abolishes the ability of the AHR to induce endogenous CYP1A1 [ 24 ].…”

Section: Discussionmentioning

confidence: 99%

“…For example, truncation of 165 amino acids from the C-terminus of the Ah b-1 receptor (approximately 21kDa) has no affect on the cytoplasmic localization of the AHR, but reduces the ability of the AHR to induce expression of endogenous CYP1A1 by 50% [ 24 ]. In addition, removal of 305 amino acids from the C-terminus of the AHR (approximately 40kDa) causes the latent AHR to become nuclear but completely abolishes the ability of the AHR to induce endogenous CYP1A1 [ 24 ]. A cleavage from 95kDa to 70kDa (as suggested by Poland and Glover [ 44 ]) would be a loss of approximately 270 amino acids.…”

Section: Discussionmentioning

confidence: 99%

“…For these studies, Hepa-1 cells were evaluated since they exhibit a predominant cytoplasmic AHR in the absence of ligand exposure and this allows the nuclear translocation event to be prominently observed [ 21,22,24,[28][29][30]35 ]. Hepa-1 cells were pre-incubated with 15μM of each inhibitor for an effective total concentration of 45μM and then exposed to TCDD for an additional hour.…”

Section: Effect Of Calpain Proteases Inhibitors On Tcdd-induced Degramentioning

confidence: 99%

“…For these studies, Hepa-1 cells were exposed to MG-132 (15μM), lactacystin (10μM), PSI-1 (15μM), or epoxomicin (2.5μM) for 2 hrs and the cells fixed and stained for the AHR as detailed in Methods. Hepa-1 cells were used since the endogenous AHR is predominantly cytoplasmic in the absence of ligand exposure in this line [ 21,24,25,28,30 ]. Fields of cells from a representative experiment and the relative nuclear fluorescence intensities are presented in Figure 7.…”

Section: Effect Of Proteasome Inhibitors On Tcdd-induced Degradation mentioning

confidence: 99%

“…Presently, the ubiquitin-proteasome pathway has been implicated in the ligand-induced degradation of the AHR due to the ability of the proteasome inhibitors, MG-132 and lactacystin to inhibit AHR degradation [ 21,23 ], as well as studies showing that AHR degradation is blocked in cells that contain a temperature sensitive mutation in the E1 ubiquitin conjugating enzyme [ 23 ]. More recently it has been shown that the ligand-bound AHR may be a target for the ubiquitin-proteasome pathway only after ligand binding, association with ARNT and DNA binding [ 23,24 ]. Interestingly, these studies also indicate that C-terminal truncations of the AHR are targeted for degradation via the proteasome, but sugges that they may be targeted there by a mechanism distinct from the full-length AHR.…”

Section: Introductionmentioning

confidence: 99%

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Specific blockage of ligand-induced degradation of the Ah receptor by proteasome but not calpain inhibitors in cell culture lines from different species

Pollenz

2007

Biochemical Pharmacology

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To firmly establish the pathway involved in ligand-induced degradation of the AHR, cell lines derived from mouse rat or human tissues were exposed to inhibitors specific to the proteasome or calpain proteases and exposed to TCDD. The level of endogenous AHR and CYP1A1 protein was then evaluated by quantitative Western blotting. Treatment of cells with the calpain inhibitors: calpeptin, calpain inhibitor III, or PD150606 either individually or in combinations up to 75μM did not reduce TCDD-induced degradation of the AHR, the induction of endogenous CYP1A1 or the nuclear accumulation of the AHR. The activity of the inhibitors was verified with an in vivo calpain assay. In contrast, exposure of cells to the specific proteasome inhibitors: epoxomicin (1-5μM), proteasome inhibitor I (5-10μM) or lactacystin (5-15μM) completely inhibited TCDD-induced degradation of the AHR. Inhibition of AHR degradation with these compounds did not reduce the induction of endogenous CYP1A1. In addition, exposure of the Hepa-1 line to the various proteasome inhibitors caused an accumulation of the AHR in the nucleus in the absence of TCDD exposure. Finally, Western blot analysis of the DNA bound AHR showed that its molecular mass was unchanged in comparison to the unliganded (cytoplasmic) AHR. Thus, these studies conclusively implicate the proteasome and not calpain proteases in the ligand-induced degradation of the mouse, rat and human AHR and suggest that the pharmacological use of proteasome inhibitors may impact the time course and magnitude of gene regulatory events mediated through the AHR.

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Section: Discussionmentioning

confidence: 99%