Identification of Functional Domains of the Aryl Hydrocarbon Receptor

Abstract

The aryl hydrocarbon receptor (Ahr) 1 binds a variety of environmentally important carcinogens, including polycyclic aromatic hydrocarbons, and certain halogenated aromatic hydrocarbons, such as TCDD. Following ligand treatment, induction of CYP1A1 and several other enzymes involved in xenobiotic metabolism occurs in most tissues. Certain of these enzymes (including CYP1A1) are involved in the metabolism of polycyclic aromatic hydrocarbons to active genotoxic metabolites, and Ahr therefore plays an important role in carcinogenesis by these compounds. Ahr also mediates most, if not all, of the carcinogenic and toxic effects of the halogenated aromatic hydrocarbons, although metabolism of these compounds does not appear to be involved (reviewed in Ref. 1).The unliganded Ahr is located in the cytoplasm of the mouse hepatoma cell line, Hepa-1, as part of a complex that also contains two molecules of the 90-kDa heat shock protein (HSP90) and perhaps another protein of approximately 43 kDa (2). It is not known whether both, one, or neither of the HSP90 molecules bind Ahr directly. HSP90 appears to be required for ligand binding by Ahr (3). Binding of ligand leads to dissociation of Ahr from HSP90. In ligand-treated cells, Ahr is found in the nuclear fraction, from which it can be extracted in the form of a complex with the aryl hydrocarbon receptor nuclear translocator protein (Arnt) (4, 5). This complex is probably a heterodimer of the two proteins, although the presence of additional small protein(s) has not been rigorously excluded. Arnt appears to be a nuclear protein in Hepa-1 cells (5, 6). Some evidence indicates that dissociation of Ahr from HSP90 occurs in the nucleus and that HSP90 may play a direct role in translocating Ahr into this organelle (7,8). It has been proposed that Arnt promotes the dissociation of Ahr from HSP90 (9). Transcriptional activation of the cyp1a1 gene results from the binding of the Ahr/Arnt heterodimer to short DNA sequences, termed xenobiotic responsive elements (XREs), located in the 5Ј-flanking region of the gene (4, 10 -12). Both Ahr and Arnt bind directly to the XRE sequence (13).Mouse Ahr and Arnt are 20% identical in amino acid sequence. They also show a striking resemblance in overall structure (14 -16). Both proteins contain bHLH motifs toward their amino termini. However, Ahr and Arnt represent a novel subclass of bHLH-containing transcription factors because they differ from most or all other such proteins in that (i) activation of the Ahr complex requires ligand, (ii) the XRE sequence differs from the E-box sequence, which is the recognition sequence for nearly all other bHLH-containing transcription factors (reviewed in Ref. 17), and (iii) both proteins contain an approximately 300-amino acid segment of sequence similarity, called the PAS domain. The PAS domains of each protein contain two copies of an approximately 50-amino acid degenerate direct repeat, referred to as the PAS A and PAS B repeats. The Drosophila proteins single-minded (Sim) and period * This work was sup...

The Aryl Hydrocarbon Receptor Complex and the Control of Gene Expression

“…74 As a result, a complementary analysis of the AhR domains was performed by Fukunaga and colleagues, 74 similar to previous domaindeletion studies with ARNT. 75 The ligand-binding region of the AhR was mapped to the PAS B domain.…”

“…HSP90 and ARNT both interact with the AhR via the receptors HLH and PAS domains, thereby making their association with the AhR mutually exclusive. 74,75,79,142 Similar to a number of other HSP90 substrates, the AhR interacts with HSP90 through the middle portion of the chaperone. 81 Upon ligand activation, the human and mouse AhR translocate into the nucleus still associated with HSP90, demonstrating that the cytoplasmic AhR complex does not have to shed its chaperone machinery before nuclear uptake.…”

“…Although ARNT could enhance the dissociation of the AhR from hsp90, ligand alone was sufficient to facilitate the dissociation of the AhR from hsp90 in vitro. 74 Whether the release of AhR from hsp90 in the presence of ligand happens in vivo has yet to be fully established, although evidence suggests that dissociation of the AhR from hsp90 may only be required for its heterodimerization with ARNT. 78 Indeed, the presence of monomeric AhR may not stably exist in the cell.…”

Structural and Functional Characterization of the Aryl Hydrocarbon Receptor Ligand Binding Domain by Homology Modeling and Mutational Analysis

“…Among the various domains of the AhR that exhibit distinct functional activities (1,(13)(14)(15), we have primarily focused our attention on that of PAS B, one of the two structural repeats (PAS A and PAS B) within the PAS domain, that is involved in both ligand and hsp90 binding. In contrast to the significant amount of information available regarding AhR ligands, essentially nothing is known about the AhR ligand binding domain (LBD) itself.…”

Development and characterization of monoclonal antibodies against human aryl hydrocarbon receptor

“…Given the homology of hAhR with murine AhR, functional domains of hAhR could be predicted based on those of mouse AhR (mAhR, Fukunaga et al, 1995;Whitelaw et al, 1994), including the PAS B domain (a.a. 230-397) of mAhR for exogenous ligand binding, the bHLH region (a.a. 27-79) and the region encompassing the PAS B repeat (a.a. 182-374) for Hsp 90 binding, a part of bHLH region (a.a. 27-39) for DNA binding, regions encompassing a.a. 40-79 and a.a. 121-289 for dimerization and the Q-rich region (a.a. 490 to 805) in the TAD domain for transcription activation. Thus, the epitopes of the commercial mAbs might specifically bind to the peptides located in only two function regions of hAhR, bHLH and Q-rich domains, which is insufficient to study the complex molecular events upon activation of hAhR involving different functional domains.…”