Comparison of the nuclear and cytosolic forms of the Ah receptor from Hepa 1c1c7 cells: Charge heterogeneity and ATP binding properties

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Abstract

2-[125I]iodo-7,8-dibromo-p-dioxin ([125I]Br2DpD) and 2-[125I]iodo-3-azido-7,8-dibromo-p-dioxin([125I]N3Br2-DpD) are both capable of binding to the Ah receptor (AhR) with a high degree of specificity in cultured Hepa 1c1c7 cells. After incubation with either [125I]N3Br2DpD or [125I]Br2DpD Hepa 1c1c7 cytosolic and high salt nuclear extracts were analyzed by sucrose density gradient analysis with the following results: (i) With both radio-ligands an ~9 S form of the AhR was observed in cytosolic extracts, (ii) Nuclear extracts labeled with [125I]N3Br2DpD revealed both ~6 S and ~9 S forms of the AhR. (iii) In contrast, analysis of nuclear extracts labeled with [125I]Br2DpD revealed only an ~6 S form of the AhR. The ~9 S [125I]N3Br2DpD-labeled AhR was preferentially extracted with 100 mm KCl from a nuclear fraction and mixed with monoclonal antibody 8D3, an anti-90-kDa heat shock protein antibody. Monoclonal antibody 8D3 was able to bind to the ~9 S nuclear form of the AhR and caused the receptor to sediment as a heavier complex on sucrose density gradients. This would indicate that the AhR can reside in the nucleus bound to 90-kDa heat shock protein. The [125I]N3Br2DpD-labeled ~6 S peak fractions were collected and subjected to denaturing two-dimensional gel electrophoresis. A comparison of [125I]N3Br2DpD-labeled cytosolic (9 S) AhR preparations with the nuclear (6 S) AhR by 2-D gel electrophoresis was performed. The cytosolic form of the AhR was present in the apparent pI range of 5.2–5.7; the nuclear form focused between 5.5 and 6.2. The [125I]N3Br2DpD-labeled nuclear extracts were incubated with ATP-agarose and 43% of the photoaffinity-labeled AhR bound to the affinity gel. In contrast, ~threefold lower binding of [125I]N3Br2DpD-labeled receptor was obtained when GTP-, AMP-, or ADP-agarose was used. Only 2% of the [125I]N3Br2DpD-labeled cytosolic AhR was able to bind to ATP-agarose. These results suggest that after the AhR translocates into the nucleus the following biochemical changes occur: (i) The sedimentation value for the AhR changes from an ~9 S to an ~6 S species. (ii) The AhR attains the ability to bind with specificity to ATP. (iii) The AhR undergoes a shift to a more basic pI.

References (47)

  • J.M. Fisher et al.

    J. Biol. Chem

    (1990)
  • M.S. Denison et al.

    J. Biol. Chem

    (1989)
  • C. Legraverend et al.

    J. Biol. Chem

    (1982)
  • A.G. Miller et al.

    J. Biol. Chem

    (1983)
  • S.O. Karenlampi et al.

    J. Biol. Chem

    (1988)
  • S. Cuthill et al.

    J. Biol. Chem

    (1987)
  • R.D. Prokipcak et al.

    Arch. Biochem. Biophys

    (1988)
  • C.J. Elferink et al.

    J. Biol. Chem

    (1990)
  • A. Poland et al.

    J. Biol. Chem

    (1986)
  • A.C. Smith et al.

    J. Biol. Chem

    (1986)
  • M. Denis et al.

    Biochem. Biophys. Res. Commun

    (1988)
  • D.B. Mendel et al.

    J. Biol. Chem

    (1988)
  • G.H. Perdew

    J. Biol. Chem

    (1988)
  • J.-M. Renoir et al.

    J. Biol. Chem

    (1990)
  • R.D. Prokipcak et al.

    Arch. Biochem. Biophys

    (1989)
  • M. Rexin et al.

    FEBS Lett

    (1988)
  • J.C. Knutson et al.

    Cell

    (1982)
  • R.D. Prokipcak et al.

    Biochem. Biophys. Res. Commun

    (1990)
  • A. Poland et al.

    Biochem. Biophys. Res. Commun

    (1987)
  • E.A. Roberts et al.

    Arch. Biochem. Biophys

    (1990)
  • M.S. Denison et al.

    Arch. Biochem. Biophys

    (1990)
  • N.J. Holbrook et al.

    J. Biol. Chem

    (1983)
  • E. Orti et al.

    J. Steroid Biochem

    (1989)
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    This work was supported in part by National Institute of Environmental Health Science Grant ES-04869. This is technical paper No. 12,910 Indiana Agricultural Experiment Station.

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