Chemico-Biological Interactions 141 (2002) 3Á/24 www.elsevier.com/locate/chembiont Ligand binding and activation of the Ah receptor Michael S. Denison a,*, Alessandro Pandini c, Scott R. Nagy a, Enoch P. Baldwin b, Laura Bonati c a Department of Environmental Toxicology, Meyer Hall, One Shields Avenue, University of California, Davis, CA 95616-8588, USA b Section of Molecular and Cellular Biology, Briggs Hall, University of California, Davis, CA 95616, USA c Dipartimento di Scienze dell’Ambiente e del Territorio, Universita` degli Studi di Milano-Bicocca, Piazza della Scienza, 1-20126 Milano, Italy Abstract The Ah receptor (AhR) is a ligand-dependent transcription factor that can be activated by structurally diverse synthetic and naturally-occurring chemicals. Although a significant amount of information is available with respect to the planar aromatic hydrocarbon AhR ligands, the actual spectrum of chemicals that can bind to and activate the AhR is only now being elucidated. In addition, the lack of information regarding the actual three-dimensional structure of the AhR ligand binding domain (LBD) has hindered detailed analysis of the molecular mechanisms by which these ligands bind to an active AhR signal transduction. In this review we describe the current state of knowledge with respect to naturally occurring AhR ligands and present and discuss the first theoretical model of the AhR LBD based on crystal structures of homologous PAS family members. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Ah receptor; 2,3,7,8-Tetrachlorodibenzo-p-dioxin; TCDD; Dioxin; Homology model; Ligand binding domain Abbreviations: AA, arachidonic acid; AhR, aryl hydrocarbon receptor; ARNT, AhR nuclear translocator; bHLH, basic helixÁ/loopÁ/helix; BNF, b-naphthoflavone; CYP1A1, cytochrome P4501A1; HAH, halogenated aromatic hydrocarbon; ICZ, indolo-[3,2,-b]-carbazole; LBD, ligand binding domain; NLS, nuclear localization sequence; PAH, polycyclic aromatic hydrocarbon; PAS, PerÁ/ArntÁ/Sim; PCDD, polychlorinated dibenzo-p-dioxin; PGG2, prostaglandin G2 PYP, photoactive yellow protein; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; TRP, tryptophan; XAP2, X-associated protein 2. * Corresponding author. Tel.: /1-530-752-3879; fax: /1-530-752-3394 E-mail address: [email protected] (M.S. Denison). 0009-2797/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 0 9 - 2 7 9 7 ( 0 2 ) 0 0 0 6 3 - 7 4 M.S. Denison et al. / Chemico-Biological Interactions 141 (2002) 3Á/24 1. Introduction The Ah receptor (AhR) is a basic helixÁ/loopÁ/helix (bHLH)- and PerÁ/ArntÁ/Sim (PAS)-containing transcription factor that regulates the expression of genes in a ligand-dependent manner [1Á/4]. Although recent studies have demonstrated that the AhR can bind and be activated by a structurally diverse range of chemicals [5,6], the best characterized high affinity ligands for the AhR include a wide variety of ubiquitous and hydrophobic environmental contaminants [7,8] such as the haloge- nated aromatic hydrocarbons (HAHs) and the non-halogenated polycyclic aromatic hydrocarbons (PAHs). Exposure to numerous HAHs, including 2,3,7,8-tetrachlor- odibenzo-p-dioxin (TCDD, dioxin), the most potent member of this class of chemicals, produces a wide variety of species- and tissue-specific toxic and biological effects [7Á/11]. The induction of gene expression is one response observed in all species exposed to TCDD and related chemicals. Induction of expression of cytochrome P4501A1 (CYP1A1) has been used as a model system to define the mechanism of action of HAHs. Biochemical and genetic studies over the past 20 years has revealed that induction of CYP1A1 and other HAH/PAH-responsive genes, as well as the toxicity of TCDD and related HAHs, is mediated by the AhR, a soluble intracellular receptor to which these chemicals bind with high affinity [3,7,8,12]. Mechanistically, the inducing chemical diffuses across the plasma membrane and binds to the AhR which is present in the cytosolic compartment as a multiprotein complex containing two molecules of hsp90 (a heat shock protein of 90 kDa), the X-associated protein 2 (XAP2 [13]) (also referred to as AIP or ara9 [14,15]) and p23 (a co-chaperone protein of 23 kDa [16]). Following ligand binding, the cytosolic ligand:AhR complex is presumed to undergo a conformation change exposing a nuclear localization sequence(s) (NLS(s)). The complex then translocates into the nucleus [17,18], dissociates from the protein complex and binds to a closely related nuclear bHLHÁ/PAS protein called Arnt (AhR nuclear translocator [1]). Formation of the AhR:Arnt heterodimer converts the complex into its high affinity DNA binding form [1,19] and binding of the complex to its specific DNA recognition site, the dioxin responsive element (DRE), upstream of the CYP1A1 gene leads to chromatin and nucleosome disruption, increased promoter accessibility and an increase in transcription of the CYP1A1 gene [12,20Á/22]. DREs have also been identified in the upstream region of most other TCDD-inducible genes [3] and they also appear to be responsible for conferring TCDD- and AhR-responsiveness upon these genes. The presence of the AhR and AhR signal transduction pathway in a diverse range of species, tissues and cell types [23Á/27] and its ability to act as a ligand-dependent transcription factor suggests that many of the toxic and biological effects of AhR ligands result from differential alteration of gene expression in susceptible cells. In addition, since the majority of the toxic effects of TCDD/HAHs are not observed until weeks following chemical exposure [7,11], the adverse effects of these chemicals likely result from the continuous and inappropriate expression of specific genes in target cells which ultimately results in the delayed toxic responses. Although significant advances in the field over the past 10 years have clearly defined the role of AhR in the toxic and biological effects of AhR ligands, the exact M.S. Denison et al. / Chemico-Biological Interactions 141 (2002) 3Á/24 5 biochemical events which lead to the spectrum of species- and tissue-specific toxic responses to these chemicals still remain elusive. All of the high affinity AhR ligands identified to date (HAHs and PAHs) are planar hydrophobic molecules and are able to induce gene expression in an AhR- dependent manner. In previous reviews [5,6], we detailed the spectrum of chemicals that have been documented in the literature to bind to and active the AhR- and/or induce AhR-dependent gene expression. Accordingly, rather than reiterating what we have described previously, this review will highlight more recent developments in our knowledge about AhR ligands with an emphasis on naturally occurring ligands that activate the AhR and AhR signaling pathway. In addition, we will describe reported differences in AhR ligand binding specificity between species and describe recent structural modeling studies of the AhR ligand binding domain (LBD) itself. For a more in depth description of AhR signal transduction and the effects of AhR ligands, the reader is referred both to additional reviews contained within this special issue of Chemico-Biological Interactions and to the many excellent published reviews [1Á/3,5,7,11,12,28]. 2. AhR ligands HAHs (such as the polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans and biphenyls and related chemicals) and the PAHs (such as benzo(a)pyrene, 3- methylcholanthrene, benzoflavones, rutacarpine alkaloids, aromatic amines and related chemicals) are the most extensively studied classes of AhR ligands [5,7,8,29Á/ 32]. HAHs have a relatively high binding affinity for the AhR (in the pM to nM range) whereas the PAHs have a significantly lower affinity (in the high nM to mM range). StructureÁ/activity relationship studies with a large number of HAH and PAH AhR ligands suggest that the AhR binding pocket can accept planar ligands with maximal dimensions of 14/12/5 A˚ . However, high affinity binding appears to be critically dependent upon key thermodynamic and electronic properties of the ligands [29Á/37]. The overall picture emerging from these studies highlights the role of electrostatic and dispersion-type interactions in ligandÁ/AhR binding. Although the results of these modeling studies have some predictive applications for identification of new high affinity AhR ligands, the constraints of these models are too restrictive, especially given that a large number of chemicals that are reported to bind the AhR have physicochemical and structural properties that deviate significantly from the currently defined structural requirements for AhR ligands (reviewed in Refs. [5,6]). Although the majority of these chemicals are relatively weak inducers or AhR ligands when compared with TCDD, their striking structural diversity is clearly evidenced by comparison of the chemicals for which direct AhR binding has been demonstrated (Fig. 1). While binding remains to be demonstrated for many of these structurally diverse AhR activators [5], their ability to induce CYP1A1 and/or activate the AhR- and AhR-dependent gene expression is consistent with their ability to interact with the AhR. Because most of these chemicals do not fit 6 M.S. Denison et al. / Chemico-Biological Interactions 141 (2002) 3Á/24 Fig. 1. Structures of selected AhR ligands. See text and Denison et al. [5] for more details. the established characteristics for known AhR ligands, their identification as ligands supports a reevaluation of the currently accepted