Multiple roles of ligand in transforming the dioxin receptor to an active basic helix-loop-helix/PAS transcription factor complex with the nuclear protein Arnt

The dioxin receptor is a ligand-activated transcription factor belonging to an emerging class of basic helix-loop-helix/PAS proteins which show intera ction with the molecular chaperone hsp90 in their latent states and require heterodimerization with a general cofactor, Amt, to form active DNA bindin g complexes. Upon binding of polycyclic aromatic hydrocarbons typified by d ioxin, the dioxin receptor translocates from the cytoplasm to the nucleus t o allow interaction with Arnt. Here we have bypassed the nuclear translocat ion step by creating a cell line which expresses a constitutively nuclear d ioxin receptor, which we find remains in a latent form, demonstrating that ligand has functional roles beyond initiating nuclear import of the recepto r. Treatment of the nuclear receptor with dioxin induces dimerization with Arnt to form an active transcription factor complex, while in stark contras t, treatment with the hsp90 ligand geldanamycin results in rapid degradatio n of the receptor. Inhibition of degradation by a proteasome inhibitor allo wed geldanamycin to transform the nuclear dioxin receptor to a heterodimer with Arnt (DR-Amt). Our results indicate that unchaperoned dioxin receptor is extremely labile and is consistent with a concerted nuclear mechanism fo r receptor activation whereby hsp90 is released from the ligand-bound dioxi n receptor concomitant with Arnt dimerization. Strikingly, artificial trans formation of the receptor by geldanamycin provided a DR-Arnt complex capabl e of binding DNA but incapable of stimulating transcription. Limited proteo lysis of DR-Arnt heterodimers indicated different conformations for dioxin versus geldanamycin-transformed receptors. Our studies of intracellular dio xin receptor transformation indicate that ligand plays multiple mechanistic roles during receptor activation, being important for nuclear translocatio n, transformation to an Arnt heterodimer, and maintenance of a structural i ntegrity key for transcriptional activation.