AH RECEPTOR NUCLEAR TRANSLOCATOR PROTEIN HETEROGENEITY IS ALTERED AFTER HETERODIMERIZATION WITH THE AH RECEPTOR

The Ah receptor (AhR) and the Ah receptor nuclear translocator (ARNT) are capable of forming a transcriptionally active heterodimeric comple x. The biochemical events that are required for dimerization and trans activation are not fully understood. The purpose of this study was to determine whether covalent modifications of ARNT occur between ARNT ex isting in the monomeric form and after heterodimerization with the AhR and subsequent binding to DNA. Mouse hepatoma cell line lclc7 (Heps 1 ) cytosol and ARNT immunoprecipitations were subjected to two-dimensio nal gel electrophoresis. ARNT was visualized with two antibodies, with distinct epitope specificity, and each detected a considerable level of charge heterogeneity. The pi range observed was 5.7-6.4, with the p redominant form at a pl of 6.2. The AhR/ARNT heterodimer was immunopre cipitated from high-salt nuclear extract obtained from Hepa 1 cells tr eated with beta-naphthoflavone using an anti-AhR polyclonal antibody. This immunoprecipitate was subjected to two-dimensional gel electropho resis, and coimmunoprecipitated ARNT was visualized. The results indic ated that ARNT complexed with the AhR in the nucleus has an isoform pa ttern shifted toward the basic end, with the predominant isoform havin g a pl of 6.8. Thus, a significant shift in pl occurs during the dimer ization and/or after binding to DNA. In vitro transformation of the Ah R with 2,3,7,8-tetrachlorodibenzo-p-dioxin in cytosol leads to heterod imerization with ARNT. Two-dimensional gel electrophoresis of ARNT coi mmunoprecipitated with the AhR revealed the same isoform pattern as se en in cytosol. This would indicate that each isoform of ARNT is capabl e of heterodimerizing with the AhR in vitro. ARNT is a phosphoprotein, and the more acidic isoforms appear to have a higher level of phospho rylation.