Chemical cross-linking of the cytosolic and nuclear forms of the Ah receptor in hepatoma cell line 1c1c7

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Abstract

Both cytosolic and high salt nuclear extracts were isolated from Hepa 1c1c7 cells incubated with 2-azido-3[1251]iodo-7, 8-dibromo-dibenzo-p-dioxin ([125I]N3Br2DpD). The [125I]N3Br2DpD-labeled cytosolic fraction was subjected to chemical cross-linking with dimethyl pimelimidate and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Chemical cross-linking of the cytosolic form of the AhR revealed monomeric (97 kDa), dimeric (185 kDa), trimeric (281 kDa), and tetrameric (327 kDa) complexes. In a time course of exposure to the cross-linking reagent, the largest form given above became the predominant AhR form observed in the cytosolic extracts. The 327 kDa cytosolic species apparently consists of a 97 kDa AhR, an ∼ 88 kDa protein, an ∼ 96 kDa protein, and an ∼ 46 kDa protein. Nuclear extracts from [125I]N3Br2DpD-labeled Hepa 1c1c7 cells were applied to sucrose density gradients. The 6 S nuclear receptor peak fractions were pooled and subjected to chemical cross-linking. Analysis by SDS-PAGE revealed a monomeric (97 kDa) ligand binding protein and a dimeric (182 kDa) complex. This would suggest that the nuclear 6 S AhR consists of a 97 kDa AhR and an ∼ 85 kDa protein. These findings would indicate that the AhR exists in cytosol as a tetrameric species, while in the nucleus the AhR exists as a heterodimer.

Original languageEnglish (US)
Pages (from-to)55-62
Number of pages8
JournalBiochemical and Biophysical Research Communications
Volume182
Issue number1
DOIs
StatePublished - Jan 15 1992

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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