Ligand-dependent Interaction of the Aryl Hydrocarbon Receptor with a Novel Immunophilin Homolog in Vivo

doi:10.1074/jbc.272.17.11452

Ligand-dependent Interaction of the Aryl Hydrocarbon Receptor with a Novel Immunophilin Homolog *in Vivo**

From the McArdle Laboratory for Cancer Research, University of Wisconsin Medical School, Madison, Wisconsin 53706-1599

^‡ To whom correspondence should be addressed:
McArdle Laboratory for Cancer Research, 1400 University Ave., Madison, WI 53706-1599.
Tel.: 608-262-2024; Fax: 608-262-2824; E-mail: bradfield{at}oncology.wisc.edu

Abstract

In an effort to identify regulators of aryl hydrocarbon receptor (AHR) signaling, we have employed the yeast two-hybrid system to screen for human proteins that interact in a ligand-dependent manner with the AHR. After screening 1.4 × 10⁶ clones from a human B cell library, two distinct clones were identified that associated specifically with the liganded receptor. No clones were identified that interacted preferentially with the unliganded AHR. One of the ligand-dependent clones, ARA9, encodes a novel 330-amino acid protein with regions of amino acid sequence similarity to the 52-kDa FK506-binding protein known to be associated with the glucocorticoid receptor. Yeast two-hybrid experiments with ARA9 demonstrated a strong interaction with the AHR that is enhanced 11-fold in the presence of the ligand β-naphthoflavone. In vitro experiments using proteins generated in reticulocyte lysates confirmed this interaction and indicated that ARA9 can be co-immunoprecipitated with the AHR using antisera raised specifically for either the AHR or the 90-kDa heat shock protein. The observation that ARA9 has a high affinity for both the 90-kDa heat shock protein-associated and ligand-activated forms of the AHR suggests that ARA9 is a component of the AHR-signaling pathway in vivo.

Footnotes

↵* This work was supported by National Institutes of Health Grants P30-CA07175 and ES05703 and The Burroughs Wellcome Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) U78521[GenBank].
↵¹ The abbreviations used are:

AHR

aryl hydrocarbon receptor

PAS

Per-ARNT-Sim homology domain

ARNT

AHR nuclear translocator

GR

glucocorticoid receptor

hsp90

90-kDa heat shock protein

hsp70

70-kDa heat shock protein

FKBP52

52-kDa FK506-binding protein

FKBP12

12-kDa FK506-binding protein

β NF

β-naphthoflavone

TAD

transcriptional activation domain

TPR

tetratricopeptide repeat

hsp56

56-kDa heat shock protein.
- Received February 19, 1997.