Evidence for two functionally distinct forms of the human Ah receptor.
The Ah receptor (AhR) was visualized using monoclonal antibody Rpt 1 on protein blots of HeLa cell cytosol; two bands were detected at 104 and 106 kDa. The photoaffinity ligand, 2-azido-3-[125I]iodo-7,8-dibromodibenzo-p-dioxin, was added to HeLa cells in culture, and after 1 hour the cells were UV irradiated. Cytosolic and high salt nuclear preparations were isolated and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by transfer of the protein to membrane. The AhR was visualized on the membrane, revealing two bands. Alignment of an autoradiogram with the membrane revealed that only the 106 kDa (upper) band was photoaffinity labeled. The nuclear fraction contained only the photoaffinity-labeled 106 kDa form of the AhR. The 104 kDa AhR does not appear to be a proteolytic product of the 106 kDa form. Cyanogen bromide fragmentation revealed that both forms contain the same size N-terminal fragment. Sucrose density gradient analysis of HeLa cell cytosol indicated that both forms cosedimented at 9 S. Both the 106 and 104 kDa AhR bands were detected in four different human cell lines. Together, these results would indicate that the AhR in human cell lines exists in two distinct forms.
Perdew
G H
GH
Department of Foods and Nutrition, Purdue University, West Lafayette, IN 47907, USA.
Hollenback
C E
CE
D000345:Affinity Labels; D000911:Antibodies, Monoclonal; D001943:Breast Neoplasms; D002294:Carcinoma, Squamous Cell; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D003488:Cyanogen Bromide; D003600:Cytosol / Q000378:metabolism; D004147:Dioxins / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006801:Humans; D007457:Iodine Radioisotopes; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010449:Peptide Mapping; D018336:Receptors, Aryl Hydrocarbon / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D000345:Affinity Labels; D000911:Antibodies, Monoclonal; D004147:Dioxins; D007457:Iodine Radioisotopes; D010446:Peptide Fragments; D018336:Receptors, Aryl Hydrocarbon; C054916:2-azido-3-iodo-7,8-dibromodibenzo-1,4-dioxin; D003488:Cyanogen Bromide