PHYSICOCHEMICAL AND IMMUNOCYTOCHEMICAL ANALYSIS OF THE ARYL-HYDROCARBON RECEPTOR NUCLEAR TRANSLOCATOR - CHARACTERIZATION OF 2 MONOCLONAL-ANTIBODIES TO THE ARYL-HYDROCARBON RECEPTOR NUCLEAR TRANSLOCATOR

The aryl hydrocarbon receptor nuclear translocator (Arnt) is a basic h elix-loop-helix transcription factor that heterodimerizes with the ary l hydrocarbon receptor to mediate signal transduction pathways inducib le by 2,3,7,8-tetrachlorodibenzo-p-dioxin and other planar aromatic hy drocarbons. Monoclonal antibodies (MAbs) have been raised against a ca rboxyl-terminal 19-amino acid peptide hapten (MAb 2B10) and against a carboxyl-terminal 378-amino acid polypeptide-staphylococcal Protein A fusion protein (MAb 4G9) of Amt and their characterization is describe d. Western blot experiments show that both MAbs specifically cross-rea ct with an similar to 85-kDa band in cytosol prepared from COS-7 cells transfected with the full length human Arnt cDNA pBMSNeo-D24-1 and in Hepa 1c1c7 cytosol but not in Arnt-deficient Hepa 1-C4 mutant cytosol . Velocity sedimentation of Hepa 1c1c7 cytosol on sucrose gradients an d Superose 6 gel permeation chromatography were used to estimate the s edimentation coefficient, Stokes radius, and relative molecular mass o f Amt as similar to 3.6-4.1 S, 6.8 nm, and 101-115 kDa, respectively. These results indicate that Arnt probably exists in monomeric form in Hepa 1c1c7 cytosolic extracts. Laser scanning confocal microscopy and indirect immunofluorescence microscopy revealed Amt to be distributed throughout the non-nucleolar portion of the nucleus of Hepa 1c1c7, VT{ 2} (Hepa 1-C4T mutant cell line deficient in Amt function and stably t ransfected with pBMSNeo D24-1, expressing the full length human Arnt c DNA), and HeLa cells. The establishment of the nuclear localization of Arnt in human and murine cell lines shown here indicates that its nuc lear localization may be conserved across species. Immunofluorescence analysis of Arnt in three cell lines using two MAbs (to distinct epito pes) provides evidence that suggests that the aryl hydrocarbon recepto r heterodimerizes with Amt in the nucleus.