Fig 8. Selected AhR mutations demonstrate impaired ligand binding ability.

(A) Indicated AhR mutants were synthesized in vitro and analyzed for [³H]TCDD ligand binding. Specific binding of [³H]TCDD to each mutant AhR was calculated as a difference of [³H]TCDD bound to in vitro reticulocyte lysate expressed AhR mutant and to that of unprogrammed lysate (background value) reactions. Values are expressed as mean +/- standard deviation of three replicate reactions. Mean values for all mutants were significantly higher than the background value at p < 0.05 as determined by the Student’s t-test. The results are representative of three independent experiments. (B) Values for specific protein-DNA complex formation for indicated mutants (y axis) were plotted against the respective values for specific [³H]TCDD binding. All values are expressed as mean +/- standard deviation of three replicate reactions and were normalized to values obtained with wt AhR (set at 100%). The line represents values that have a direct 1:1 relationship between ligand binding and DNA binding. Results with mutant AhRs that fall in the lower-right half of the plot and circled in red (A119R, I324R) indicate a likely effect of the mutation on normal ligand-dependent AhR/ARNT dimerization and thus DNA binding, as described in the main text.