Degradation of the basic helix-loop-helix/Per-ARNT-Sim homology domain dioxin receptor via the ubiquitin/proteasome pathway

The basic helix-loop-helix/Per-ARNT-Sim homology domain dioxin receptor (DR ) translocates to the nucleus upon binding of aromatic hydrocarbon ligands typified by dioxin, whereupon it partners the Ah receptor nuclear transloca tor and initiates transcription. Concurrently, ligand binding down-regulate s receptor levels via an unknown mechanism. In this study we show that rece ptor levels are dependent upon cellular compartmentalization, with entry in to the nucleus leading to the rapid destruction of the DR. Ligand-induced D R translocation was bypassed by adding a heterologous nuclear localization signal to the DR, creating a constitutively nuclear form of the dioxin rece ptor (DRNLS). The DRNLS protein was shown to be unstable with a half-life o f less than or equal to 1 h whether partnering ARNT or HSP90. Thus, the str uctural changes induced by ligand binding have no inherent effect on DR sta bility but are critical in transporting the receptor prior to degradation. The proteolytic pathway that degrades the nuclear receptor is suggested to involve ubiquitination as it was inhibited by the proteasome inhibitor MG13 2 or co-expression of DRNLS with the ubiquitin mutant UbK48R. Incubation of cells expressing DRNLS with the phosphatase inhibitor calyculin resulted i n the rapid phosphorylation and ubiquitination of DRNLS, suggesting that a nuclear kinase is required to trigger receptor proteolysis. Overall, this s tudy demonstrates a novel mechanism of proteolysis whereby the simple reloc ation of a transcription factor from cytoplasm to nucleus initiates its rap id destruction.