DEFINITION OF A NOVEL LIGAND-BINDING DOMAIN OF A NUCLEAR BHLH RECEPTOR - COLOCALIZATION OF LIGAND AND HSP90 BINDING ACTIVITIES WITHIN THE REGULABLE INACTIVATION DOMAIN OF THE DIOXIN RECEPTOR

The dioxin receptor mediates signal transduction by dioxin (2,3,7,8-te trachlorodibenzo-p-dioxin) and binds to DNA target sequences as a hete rodimer of the approximately 100 kDa ligand binding receptor and the a pproximately 85 kDa auxiliary factor, Arnt. Both of these factors enco mpass an N-terminal basic helix - loop - helix (bHLH) motif required f or DNA binding and dimerization. In this study we describe the constru ction of glucocorticoid/dioxin receptor fusion proteins which allow th e regulation of glucocorticoid receptor activity by dioxin in transien t transfections of CHO and hepatoma celts. Thus, in the absence of dio xin, chimeric receptor constructs which contain large 500-720 amino ac id C-terminal dioxin receptor fragments, but lack the N-terminal bHLH motif, confer repression upon the transcriptional activity of a glucoc orticoid receptor derivative, tauDBD, containing its N-terminal strong transactivating signal (tau) and its DNA binding domain (DBD). In the presence of dioxin, this repression is reversed. Importantly, these c himeric receptors did not require the bHLH Arnt co-factor for function . A considerably smaller region of the dioxin receptor, located betwee n amino acids 230 and 421, showed specific dioxin binding activity in vitro. Moreover, dioxin binding in vitro correlated with the ability o f receptor fragments to form stable complexes in vitro with the molecu lar chaperone hsp90. These findings support the notion that hsp90 may be important for folding of a dioxin binding configuration of the rece ptor. Finally, tauDBD activity was constitutively repressed in a dioxi n non-responsive manner by dioxin receptor fragments which failed to b ind ligand but also failed to bind hsp90 in vitro, indicating that alt ernative mechanisms in addition to hsp90 binding may contribute to the inactivation function. In summary, the dioxin receptor system provide s a novel and complex . model of regulation of bHLH factors that may a lso give important insights into the mechanism of action of ligand-act ivated nuclear receptors.