Characterization of multiple forms of the Ah receptor: comparison of species and tissues

Biochemical and toxic responses to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) appear to be mediated via the Ah receptor, a gene-regulatory protein that, like steroid hormone receptors, undergoes a ligand-dependent acquisition of affinity for nuclei and DNA. Since responses to TCDD are highly species- and tissue-specific, we compared DNA-binding properties of Ah receptor from several tissues of rat, C57BL/6 mouse, hamster, and guinea pig, using DNA-Sepharose chromatography. Hepatic cytosol from all species contained TCDD.receptor complexes that eluted at approximately 0.15 (peak 1) and approximately 0.33 M NaCl (peak 2). The relative proportions of these forms as well as of TCDD-receptor that did not bind to DNA (i.e., was present in flowthrough fractions) varied among species. In each case, the yield of the higher affinity form (peak 2) increased with time or temperature of incubation. Cytosol from lung, thymus, kidney, and testis contained the same two forms; peak 2 was the major DNA-binding form only in thymus. In KCl extracts of hepatic nuclei from animals treated with [3H]TCDD, only the higher affinity form (peak 2) was found. Peak 1 isolated from cytosol by DNA-Sepharose and incubated with hepatic cytosol from D2 mouse (which contains no detectable receptor) transformed into peak 2, suggesting that these two forms are different conformations of the same protein. Sucrose density gradient and gel filtration analyses of peaks 1 and 2 isolated from DNA-Sepharose indicated that (i) the untransformed form (peak 1) was smaller than the unoccupied and the transformed forms, (ii) 0.4 M KCl in the density gradients had little effect on these isolated forms, and (iii) nuclear receptor sedimented like peak 2. On the basis of these results, we hypothesize that the Ah receptor exists in several forms: When occupied, it has no affinity for DNA. Ligand binding initially yields a smaller form with low DNA affinity (i.e., peak 1), as well as, in some cases, a form with no DNA affinity (flowthrough fractions); further incubation in the presence of cytosolic factor(s) induces a change conferring higher DNA affinity and faster sedimentation (i.e., peak 2). The latter form is likely the transcriptionally active form in vivo. Species and tissue differences in this scheme are quantitative rather than qualitative.