Photoaffinity labeling of the Ah receptor with 3-[3H]methylcholanthrene and formation of a 165-kDa complex between the ligand-binding subunit and a novel cytosolic protein.
The aromatic hydrocarbon (Ah) receptor is a cytosolic protein that binds halogenated ligands such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and nonhalogenated ligands such as 3-methylcholanthrene (MC) and benzo[a]pyrene. The best characterized biological response mediated by the Ah receptor is induction of cytochrome P4501A1 (CYP1A1). Photoaffinity labeling of the Ah receptor has been reported only with halogenated ligands such as TCDD and some of its iodinated derivatives. In this study, photolabeling of the Ah receptor was achieved with the nonhalogenated aromatic hydrocarbon [3H]MC. Sources of Ah receptor were the mouse hepatoma cell line Hepa-1c1c9 and the human colon adenocarcinoma line LS180. Cytosolic fractions either were used in a crude form or were enriched by glycerol density gradient centrifugation. These then were incubated with [3H]MC, irradiated with UV light (> 300 nm), precipitated with acetone, and analyzed by SDS-polyacrylamide gel electrophoresis. The yield of photoadduct formation was lower with [3H]MC (approximately 1%) compared with [3H]TCDD (3.5%) in Hepa-1c1c9 cells. The same was true in LS180 cells, i.e. the yield was 0.2% for [3H]MC versus 5.48 +/- 0.26% for [3H]TCDD. The relative molecular mass of the [3H]MC-labeled receptor estimated by SDS-polyacrylamide gel electrophoresis was 94,600 +/- 2,400 (mean +/- S.E.) for Hepa-1c1c9 cells and 113,600 +/- 3,200 for LS180 cells; these are the same molecular masses as determined by photolabeling with [3H]TCDD. In velocity sedimentation assays of mouse cytosol, [3H]MC binds specifically to two cytosolic proteins: the 4 S carcinogen-binding protein and the Ah receptor (9 S). However, no photolabeling of the 4 S protein was detected in our experiments. [3H]MC photolabeling of the human Ah receptor from LS180 cells was detected only in experiments using enriched cytosolic preparations. In addition to the 95-kDa ligand-binding subunit, a specifically radiolabeled protein of 164,900 +/- 5,800 kDa was also detected in Hepa-1c1c9 cytosol photolabeled with [3H]MC, suggesting cross-linking, by MC, of another subunit of the multimeric Ah receptor complex to the ligand-binding subunit. Immunochemical analysis showed that the ligand-binding subunit of the Ah receptor is one component of the 165-kDa complex. The other protein in the complex could not be identified with antibodies to the heat shock proteins hsp90 or hsp70 or with antibodies to the p59 protein or Ah receptor nuclear translocator protein. The identity and function of the protein that becomes cross-linked to the ligand-binding subunit require further investigation.
de Morais
S M
SM
Department of Pharmacology, University of Toronto, Ontario, Canada.
Giannone
J V
JV
Okey
A B
AB
D000345:Affinity Labels / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis; D003600:Cytosol / Q000378:metabolism; D004790:Enzyme Induction; D007700:Kinetics; D008114:Liver Neoplasms, Experimental; D046911:Macromolecular Substances; D008748:Methylcholanthrene / Q000378:metabolism*; D051379:Mice; D000072317:Polychlorinated Dibenzodioxins / Q000378:metabolism*; D018336:Receptors, Aryl Hydrocarbon / Q000302:isolation & purification / Q000378:metabolism*; D014316:Tritium; D014407:Tumor Cells, Cultured
D000345:Affinity Labels; D046911:Macromolecular Substances; D000072317:Polychlorinated Dibenzodioxins; D018336:Receptors, Aryl Hydrocarbon; D014316:Tritium; D008748:Methylcholanthrene; D003577:Cytochrome P-450 Enzyme System