ARA9 modifies agonist signaling through an increase in cytosolic aryl hydrocarbon receptor.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates the effects of agonists like 2,3, 7,8-tetrachlorodibenzo-p-dioxin. In the current model for AHR signaling, the unliganded receptor is found in the cytosol as part of a complex with a dimer of the 90-kDa heat shock protein and an immunophilin-like molecule, ARA9. In yeast, expression of ARA9 results in an increase in the maximal agonist response and a leftward shift in the AHR dose-response curve. To better understand the mechanism by which ARA9 modifies AHR signal transduction, we performed a series of coexpression experiments in yeast and mammalian cells. Our results demonstrate that ARA9's influence on AHR signaling is not due to inhibition of a membrane pump or modification of the receptor's transactivation properties. Using receptor photoaffinity labeling experiments, we were able to show that ARA9 enhances AHR signal transduction by increasing the available AHR binding sites within the cytosolic compartment of the cell. Our evidence suggests that ARA9's effects are related to its role as a cellular chaperone; i.e. we observed that expression of ARA9 increases the fraction of AHR in the cytosol and also stabilized the receptor under heat stress.

Characterization of the Ah receptor-associated protein, ARA9.

The unliganded aryl hydrocarbon receptor (AHR) is found in a complex with other proteins including the 90-kDa heat shock protein (Hsp90) and a 37-kDa protein we refer to as ARA9. We found that the three tetratricopeptide repeats found in the COOH terminus of ARA9 are necessary and sufficient for interaction with the AHR complex. Conversely, the AHR's "repressor"/Hsp90 binding domain is required for interaction with ARA9. Because ARA9 closely resembles the 52-kDa FK506-binding protein (FKBP52), found in the unliganded glucocorticoid receptor (GR) complex, we compared the binding specificities of ARA9 and FKBP52 for AHR and GR. In co-immunoprecipitation experiments, ARA9 specifically associated with AHR-Hsp90 complex but not with GR-Hsp90 complexes. In addition, ARA9 showed a greater capacity than FKBP52 to associate with AHR-Hsp90 complexes. The biological importance of this interaction was suggested by the observation that in a yeast expression system ARA9 expression enhanced the response of AHR to the agonist beta-napthoflavone, decreasing the EC50 by greater than 5-fold and increasing the maximal response 2.5-fold. In contrast, co-expression of FKBP52 had no effect on AHR signaling. In addition, although ARA9 contains a domain similar to that found in other FK506-binding proteins, ARA9 binding to 3H-FK506 could not be detected. Finally, we have characterized the developmental and expression pattern of ARA9 in the developing mouse embryo and mapped the ARA9 locus to human chromosome 11q13.3.