A point mutation responsible for defective function of the aryl-hydrocarbon-receptor nuclear translocator in mutant Hepa-1c1c7 cells.
A 3,4-benzopyrene-resistant mutant clone (c4) of the mouse hepatoma Hepa-1c1c7 cell line was examined for the mutation that causes the defective function of aryl-hydrocarbon receptor (AHR) nuclear translocator (Arnt). Arnt dimerizes with AHR and mediates the induction signal of aryl-hydrocarbon hydroxylase activity. The Arnt cDNAs of c4 cells were cloned by reverse-transcription/PCR to compare the sequences with that of wild-type Arnt cDNA. The Arnt cDNA of c4 cells was found to have a single point mutation, leading to replacement of Gly326 with Asp between two internal repeats in the highly conserved Per-Arnt-Sim (PAS) domain, PAS A and PAS B. The inability of [Asp326]Arnt/AHR heterodimers to enhance reporter gene transcription under the control of the CYP1A1 gene promoter and enhancer confirmed that the G326-->D substitution was a causative mutation. While fluorescence microscopy and coimmunoprecipitation experiments showed that this mutant form of Arnt was not changed from wild-type Arnt in terms of nuclear localization or heterodimer formation with AHR, the binding activity of the [Asp326]Arnt x AHR heterodimer to the xenobiotic-responsive element was reduced markedly. Determination of the turnover rate in COS-7 cells transfected with expression plasmids for mutant Arnt or normal Arnt showed that the mutant protein turned over with an accelerated rate compared with that of the normal. Moreover, the mutant protein displayed increased proteolytic digestibility in vitro with various proteases.
Numayama-Tsuruta
K
K
Department of Chemistry, Graduate School of Science, Tohoku University, Sendai, Japan.
Kobayashi
A
A
Sogawa
K
K
Fujii-Kuriyama
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D019556:COS Cells; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D006868:Hydrolysis; D051379:Mice; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011233:Precipitin Tests; D018336:Receptors, Aryl Hydrocarbon / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
C497397:Arnt protein, mouse; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D018336:Receptors, Aryl Hydrocarbon; D014157:Transcription Factors; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator