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 complex. The biochemical events that are required for dimerization and transactivation are not fully understood. The purpose of this study was to determine whether covalent modifications of ARNT occur between ARNT existing in the monomeric form and after heterodimerization with the AhR and subsequent binding to DNA. Mouse hepatoma cell line 1c1c7 (Hepa 1) cytosol and ARNT immunoprecipitations were subjected to two-dimensional 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 predominant form at a pI of 6.2. The AhR/ARNT heterodimer was immunoprecipitated from high-salt nuclear extract obtained from Hepa 1 cells treated with beta-naphthoflavone using an anti-AhR polyclonal antibody. This immunoprecipitate was subjected to two-dimensional gel electrophoresis, and coimmunoprecipitated ARNT was visualized. The results indicated that ARNT complexed with the AhR in the nucleus has an isoform pattern shifted toward the basic end, with the predominant isoform having a pI of 6.8. Thus, a significant shift in pI occurs during the dimerization and/or after binding to DNA. In vitro transformation of the AhR with 2,3,7,8-tetrachlorodibenzo-p-dioxin in cytosol leads to heterodimerization with ARNT. Two-dimensional gel electrophoresis of ARNT coimmunoprecipitated with the AhR revealed the same isoform pattern as seen in cytosol. This would indicate that each isoform of ARNT is capable of heterodimerizing with the AhRin vitro. ARNT is a phosphoprotein, and the more acidic isoforms appear to have a higher level of phosphorylation.
Tsai
J C
JC
Department of Veterinary Science, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Perdew
G H
GH
D000818:Animals; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D019556:COS Cells; D004268:DNA-Binding Proteins; D019281:Dimerization; D015180:Electrophoresis, Gel, Two-Dimensional; D018257:Helix-Loop-Helix Motifs; D007526:Isoelectric Point; D051379:Mice; D010766:Phosphorylation; D014176:Protein Biosynthesis; D011817:Rabbits; D018336:Receptors, Aryl Hydrocarbon / Q000737:chemistry / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D019324:beta-Naphthoflavone / Q000378:metabolism
C497397:Arnt protein, mouse; D004268:DNA-Binding Proteins; D018336:Receptors, Aryl Hydrocarbon; D014157:Transcription Factors; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D019324:beta-Naphthoflavone