PHOTOAFFINITY-LABELING OF THE AH RECEPTOR WITH 3-[H-3]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 bin ds halogenated ligands such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TC DD) and nonhalogenated ligands such as 3-methylcholanthrene (MC) and b enzo[alpha]pyrene. The best characterized biological response mediated by the Ah receptor is induction of cytochrome P4501A1 (CYP1A1). Photo affinity labeling of the Ah receptor has been reported only with halog enated ligands such as TCDD and some of its iodinated derivatives. In this study, photolabeling of the Ah receptor was achieved with the non halogenated aromatic hydrocarbon [H-3]MC. Sources of Ah receptor were the mouse hepatoma cell line Hepa-1c1c9 and the human colon adenocarci noma line LS180. Cytosolic fractions either were used in a crude form or were enriched by glycerol density gradient centrifugation. These th en were incubated with [H-3]MC, irradiated with UV light (>300 nm), pr ecipitated with acetone, and analyzed by SDS-polyacrylamide gel electr ophoresis. The yield of photoadduct formation was lower with [H-3]MC ( similar to 1%) compared with [H-3]TCDD (3.5%) in Hepa-1c1c9 cells. The same was true in LS180 cells, i.e. the yield was 0.2% for [H-3]MC ver sus 5.48 +/- 0.26% for [H-3]TCDD. The relative molecular mass of the [ H-3]MC-labeled receptor estimated by SDS-polyacrylamide gel electropho resis was 94,600 +/- 2,400 (mean +/- S.E.) for Hepa-1c1c9 cells and 11 3,600 +/- 3,200 for LS180 cells; these are the same molecular masses a s determined by photolabeling with [H-3]TCDD. In velocity sedimentatio n assays of mouse cytosol, [H-3]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 ex periments. [H-3]MC photolabeling of the human Ah receptor from LS180 c ells was detected only in experiments using enriched cytosolic prepara tions. In addition to the 95-kDa ligand-binding subunit, a specificall y radiolabeled protein of 164,900 +/- 5,800 kDa was also detected in H epa-1c1c9 cytosol photolabeled with [H-3]MC, suggesting cross-linking, by MC, of another subunit of the multimeric Ah receptor complex to th e ligand-binding subunit. Immunochemical analysis showed that the liga nd-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 antibodi es 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.