Role of the Per/Arnt/Sim Domains in Ligand-dependent Transformation of the Aryl Hydrocarbon Receptor

Michael S Denison,Anatoly Soshilov

doi:10.1074/jbc.m802414200

Abstract

The aryl hydrocarbon receptor (AhR) mediates the toxic and biological effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin and related compounds. In a process termed transformation, ligand binding converts the AhR into its high affinity DNA binding form that represents a dimer of the AhR and Arnt, a closely related nuclear protein. During transformation, protein chaperone Hsp90 is thought to be replaced by Arnt in overlapping binding sites in the basic helix loop helix and PASB domains of the AhR. Here, analysis of AhR variants containing a modified PASB domain and AhR PASA-PASB fragments of various lengths revealed (i) an inhibitory effect on transformation concomitant with Hsp90 binding in the PASB domain, (ii) an ability of the PASA-PASB fragment of the AhR to reproduce key steps in the transformation process, and (iii) a ligand-dependent conformational change in the PASA domain consistent with increased PASA exposure during AhR transformation. Based on these results, we propose a new mechanism of AhR transformation through initiation of Arnt dimerization and Hsp90 displacement in AhR PASA/B domains. This study provides insights into mechanisms of AhR transformation, dimerization of PAS domain proteins, and Hsp90 dissociation in activation of its client proteins.

Highlights

Absence of ligand, Hsp90 binding is thought to maintain the aryl hydrocarbon receptor (AhR) in an inactive state, possibly, through association in the regions that overlap AhR Arnt binding sites [5, 6]
These observations raise the question whether the Hsp90-mediated inhibitory effect on AhR transformation is binding site dependent
Because Hsp90 and Arnt have overlapping binding sites in the AhR and do not appear to bind to the AhR at the same time, it was hypothesized that Hsp90 is displaced by Arnt during transformation and that in the absence of ligand Hsp90 maintains the AhR in a dimerization-incompetent state by masking the Arnt dimerization interface(s) on the receptor [6, 13, 25, 27]

Summary

EXPERIMENTAL PROCEDURES

Antibodies—Monoclonal 3G3p90 anti-Hsp antibody [6, 7] was produced at Antibodies Incorporated (Davis, CA) from hydridoma cells kindly provided by Dr G. AhR/PASB-Arnt was generated by inserting an MfeI site at position ϩ782 in m␤AhR/pcDNA3 using the Stratagene QuikChange technique and substituting the MfeI-BglII fragment of the resulting construct for that amplified from mArnt/pBKCMV using primers S1 and S2 with subsequent digestion with MfeI and BamHI. In FLAG co-immunoprecipitations, the transformation mixtures were incubated with 1 ␮g of the anti-FLAG antibody or IgG control for 1 h at 4 °C and with 10 ␮l of Protein G Plus-agarose (Santa Cruz) (pre-washed with 2% (w/v) bovine serum albumin in PBS) for 1 h at 4 °C with shaking. [3H]TCDD specific binding to the in vitro synthesized proteins diluted in MEDG buffer to 8 mg/ml protein was conducted in the presence of 2 nM [3H]TCDD for the indicated periods of time and measured following the previously published protocol for cytosolic AhR [22]. The primary anti-AhR antibody SE-6 was added at 0.2 mg/ml and detection performed using the Amersham Biosciences ECL Western blotting analysis system (GE Healthcare)

RESULTS

Number of methionines

DISCUSSION

Role of PAS Domains in AhR Transformation a b c