Characterization of multiple forms of the Ah receptor: recognition of a dioxin-responsive enhancer involves heteromer formation.
We have employed a combination of gel retardation, protein-DNA cross-linking, and protein-protein cross-linking techniques to further examine the 2,3,7,8-tetrachlorodibenzo-p- dioxin-(TCDD-) dependent changes in the Ah receptor that result in a DNA-binding conformation. Gel retardation analysis of DNA-Sepharose chromatographic fractions of rat hepatic cytosol indicated that TCDD-dependent and sequence-specific DNA binding coeluted with a 200-kDa form of the Ah receptor (peak 2) previously characterized as being multimeric and having high affinity for calf thymus DNA. The TCDD-bound, 100-kDa form of the receptor (peak 1) bound weakly to the DNA recognition motif. These results indicated that the DNA-binding form of the Ah receptor is a multimer. SDS-polyacrylamide gel electrophoresis of peak 2 cross-linked to a bromodeoxyuridine-substituted DNA recognition motif indicated that this form of the receptor present in rat hepatic cytosol is composed of at least two DNA-binding proteins of approximately 100 and 110 kDa. Using the chemical cross-linking agent dimethyl pimelimidate, we further established that the 100-kDa form of the receptor (peak 1) associates with a different protein to generate the receptor form (peak 2) that binds to the dioxin-responsive enhancer. Photoaffinity-labeling studies indicated that only the 100-kDa protein (peak 1), and not the 110-kDa protein, binds ligand. Together, these observations imply that the DNA-binding form of the Ah receptor exists as a heteromer.
Gasiewicz
T A
TA
Department of Biophysics, University of Rochester School of Medicine, New York 14642.
Elferink
C J
CJ
Henry
E C
EC
D000345:Affinity Labels; D000818:Animals; D001483:Base Sequence; D002846:Chromatography, Affinity; D006224:Cricetinae; D003600:Cytosol / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004147:Dioxins / Q000378:metabolism; D004742:Enhancer Elements, Genetic; D006168:Guinea Pigs; D008099:Liver / Q000378:metabolism*; D008297:Male; D008647:Mesocricetus; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis; D000072317:Polychlorinated Dibenzodioxins / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D018336:Receptors, Aryl Hydrocarbon; D011955:Receptors, Drug / Q000302:isolation & purification / Q000378:metabolism*
D000345:Affinity Labels; D004268:DNA-Binding Proteins; D004147:Dioxins; D009838:Oligodeoxyribonucleotides; D000072317:Polychlorinated Dibenzodioxins; D018336:Receptors, Aryl Hydrocarbon; D011955:Receptors, Drug; C056902:2-iodo-7,8-dibromodibenzo-1,4-dioxin; D004247:DNA