EVIDENCE FOR 2 FUNCTIONALLY DISTINCT FORMS OF THE HUMAN AH RECEPTOR

The Ah receptor (AhR) was visualized using monoclonal antibody Rpt 1 o n protein blots of HeLa cell cytosol; two bands were detected at 104 a nd 106 kDa. The photoaffinity ligand, 2-azido-3-[I-125]iodo-7,8-dibrom odibenzo-p-dioxin, was added to HeLa cells in culture, and after 1 hou r the cells were UV irradiated. Cytosolic and high salt nuclear prepar ations were isolated and subjected to sodium dodecyl sulfate-polyacryl amide gel electrophoresis (SDS-PAGE), followed by transfer of the prot ein 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 f raction contained only the photoaffinity-labeled 106 kDa form of the A hR. 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 a nalysis of HeLa cell cytosol indicated that both forms cosedimented at 9 S. Both the 106 and 104 kDa AhR bands were detected in four differe nt human cell lines. Together, these results would indicate that the A hR in human cell lines exists in two distinct forms.