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

Abstract

2-[ 125I]iodo-7,8-dibromo- p-dioxin ([ 125I]Br 2D pD) and 2-[ 125I]iodo-3-azido-7,8-dibromo- p-dioxin([ 125I]N 3Br 2-D pD) 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]N 3Br 2D pD or [ 125I]Br 2D pD 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]N 3Br 2D pD revealed both ~6 S and ~9 S forms of the AhR. (iii) In contrast, analysis of nuclear extracts labeled with [ 125I]Br 2D pD revealed only an ~6 S form of the AhR. The ~9 S [ 125I]N 3Br 2D pD-labeled AhR was preferentially extracted with 100 m m 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]N 3Br 2D pD-labeled ~6 S peak fractions were collected and subjected to denaturing two-dimensional gel electrophoresis. A comparison of [ 125I]N 3Br 2D pD-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 p I range of 5.2–5.7; the nuclear form focused between 5.5 and 6.2. The [ 125I]N 3Br 2D pD-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]N 3Br 2D pD-labeled receptor was obtained when GTP-, AMP-, or ADP-agarose was used. Only 2% of the [ 125I]N 3Br 2D pD-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 p I.