[Dioxin-l] molecular mechanism of dioxin action

[Jon Campbell]

Dr. Bell,

     Excuse the last question; I didn't fully understand the import of what
you had written.

     It appears that the Ah receptor, but not the ARNT, is degraded, that
the transcription initiated by the presence of dioxin in the nucleus
continues, and that the mechanism you are describing appears to merely
curtail further transmission of other dioxin molecules into the nucleus.

     Is this a correct interpretation?

Thanks
Jon

-----Original Message-----
From: david bell [mailto:burnt_paper@hotmail.com]
Sent: Tuesday, February 15, 2000 1:59 AM
To: Jon.Campbell@MetraTech.com
Subject: Re: [Dioxin-l] molecular mechanism of dioxin action


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

Roberts, B. J.
Whitelaw, M. L.
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 
translocator and initiates transcription. Concurrently, ligand binding 
down-regulates receptor levels via an unknown mechanism. In this study we 
show that receptor levels are dependent upon cellular compartmentalization, 
with entry into the nucleus leading to the rapid destruction of the DR. 
Ligand-induced DR translocation was bypassed by adding a heterologous 
nuclear localization signal to the DR, creating a constitutively nuclear 
form of the dioxin receptor (DRNLS). The DRNLS protein was shown to be 
unstable with a half-life of less than or equal to 1 h whether partnering 
ARNT or HSP90. Thus, the structural changes induced by ligand binding have 
no inherent effect on DR stability 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 MG132 or co-expression of DRNLS with the 
ubiquitin mutant UbK48R. Incubation of cells expressing DRNLS with the 
phosphatase inhibitor calyculin resulted in the rapid phosphorylation and 
ubiquitination of DRNLS, suggesting that a nuclear kinase is required to 
trigger receptor proteolysis. Overall, this study demonstrates a novel 
mechanism of proteolysis whereby the simple relocation of a transcription 
factor from cytoplasm to nucleus initiates its rapid destruction.

The aryl-hydrocarbon receptor, but not the aryl-hydrocarbon receptor nuclear

translocator protein, is rapidly depleted in hepatic and nonhepatic culture 
cells exposed to 2,3,7,8-tetrachlorodibenzo-p- dioxin

Molecular Pharmacology49:391-398
Western blot analysis and indirect immunofluorescence microscopy were used 
to evaluate the fate of the aryl-hydrocarbon receptor (AhR) and 
aryl-hydrocarbon receptor nuclear translocator (Arnt) protein in culture 
cell models exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In 
wild-type (WT) murine Hepa-1c1c7 cells, AhR protein was depleted by 85% 
after 4 hr of TCDD treatment as measured in total cell lysates. In contrast,

the concentration of Arnt protein was unaffected by TCDD treatment in WT 
cells. Analysis of the AhR with immunofluorescence microscopy revealed that 
nuclear translocation of the liganded AI IR preceded its depletion from 
cells. AhR protein was depleted from Hepa-l type I variants (that contain a 
concentration of AhR that is 10% of WT) with a similar time course and to 
the same maximal level observed in WT cells (85%). The EC(50) for AhR 
depletion in Hepa-l cells was 39 pM TCDD and corresponded to the EC(50) for 
induction of P4501A1 protein. Murine embryonic fibroblasts (NIH-3T3), rat 
aortic smooth muscle cells (A7), and murine skeletal muscle cells (C2C12) 
all exhibited >90% depletion of the AhR after 2- 4 hr of TCDD treatment. 
Arnt concentration was not affected by TCDD in these cell lines. These 
results indicate that the liganded AhR is rapidly depleted within the 
nuclear compartment of hepatic and nonhepatic cells in a manner independent 
of the Arnt protein.

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