Agonist but Not Antagonist Ligands Induce Conformational Change in the Mouse Aryl Hydrocarbon Receptor as Detected by Partial Proteolysis

Abstract

The cytosolic transcription factor known as the aryl hydrocarbon receptor (AhR) undergoes transformation to a DNA-binding form by a series of processes initiated by binding of ligand. Subsequent steps include dissociation of several proteins that are complexed with the inactive receptor, nuclear translocation, and dimerization with Arnt. We have used limited proteolysis of the in vitro-translated mouse AhR to determine whether this technique can detect conformational change(s) associated with AhR transformation and whether the effect of agonist and antagonist ligands can be distinguished by this assay. Limited digestion of [35 S]AhR/AhR nuclear translocator (Arnt) by trypsin produced a peptide of approximately 40 kDa that was more resistant to proteolysis in the presence of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) than vehicle and was also Arnt-dependent. This trypsin-resistant peptide was also elicited in the presence of other agonist ligands, but not with antagonist ligands that do not form the DNA-binding AhR/Arnt complex. Immunoblot of trypsin-treated AhR/Arnt Ϯ TCDD indicated that the trypsin-resistant peptide did not include the N-terminal portion of the AhR against which the antibody was made. Truncated AhRs were also subjected to limited trypsinization. From AhR(1-399), a TCDD-dependent peptide of approximately 35 kDa was observed; from the constitutively active AhR(1-348), a band of approximately 30 kDa was produced from vehicle-and TCDD-treated protein. From these observations, we hypothesize that the trypsin-resistant peptide from full-length AhR spans approximately from amino acid 80 to 440. We conclude that agonist ligands initiate structural alteration in AhR that is Arnt-dependent and at least partially involves the ligand-binding/Per-Arnt-Sim domain.

Covalent binding of quinones activates the Ah receptor in Hepa1c1c7 cells

“…Collectively, these findings suggest that quinone-mediated induction of Cyp1a1 is not cell-specific. It is generally considered that efficient AhR ligands, such as TCDD, MC, and benzo[a]pyrene exhibit a high affinity for this transcription factor and thus cause a conformational change in AhR, leading to facilitation of its translocation into the nucleus in cells (Henry and Gasiewicz, 2003;Harper et al, 1991). In the present study, 1,4-BQ, TBQ, 1,2-NQ, and 1,4-NQ also translocated AhR into the nucleus and induced Cyp1a1 in Hepa1c1c7 cells, whereas benzene and naphthalene without electrophilic properties did not translocate AhR in the cells because of their poor ligand characteristics (Badham and Winn, 2007;Genter et al, 2006).…”

An Aryl Hydrocarbon Receptor Agonist Amplifies the Mitogenic Actions of Estradiol in Granulosa Cells: Evidence of Involvement of the Cognate Receptors1

“…The effects of two aromatic hydrocarbons (bnaphthoflavone and a-naphthoflavone) that have been described as agonist and antagonist of this receptor in a variety of systems [22][23][24][25][26][27][28][29][30] were tested in a defined culture system of rat granulosa cells that has been extensively characterized in terms of hormonally regulated cell proliferation [31].…”

The Aryl Hydrocarbon ReceptorsansXenobiotics: Endogenous Function in Genetic Model Systems

“…In mammalian cells, unliganded AHR is primarily found in a cytoplasmic complex, which includes an Hsp90 homodimer and the immunophilinlike Ara9, also known as XAP2 or AIP (the AHR:chaperone complex is reviewed in Petrulis and Perdew, 2002). Ligand binding increases nuclear shuttling of the receptor via a process presumed to involve structural transformation and/or nuclear localization sequence (NLS) presentation (Ikuta et al, 1998;Henry and Gasiewicz, 2003;Petrulis et al, 2003).…”