Characterization of the intracellular localization of the aryl hydrocarbon receptor, aryl hydrocarbon receptor nuclear translocator and HSP90 and the hydrodynamic properties of Arnt
Abstract
The aryl hydrocarbon receptor (AhR) and aryl hydrocarbon receptor nuclear translocator (Arnt) are basic helix-loop-helix transcription factors which heterodimerize and bind dioxin responsive regulatory elements in DNA. It is possible that differential intracellular localization of AhR and Arnt, cytoplasmic sequestration of AhR by hsp90 and AhR/Arnt levels may exert regulatory influences on AhR transformation and activation. Two monoclonal antibodies to different carboxy-terminal epitopes in Arnt were generated. MAb 2B10 was used to detect Arnt on Western blots; MAb 4G9 was used to detect Arnt in fixed/permeabilized cells. Using anti-AhR, anti-Arnt and anti-hsp86/84 antibodies, the intracellular localization of AhR and Arnt was determined in Hepa 1c1c7 cells by immunofluorescence using indirect and laser scanning confocal microscopy. Hsp90 was found to be localized primarily in the cytoplasm of Hepa 1c1c7 cells. AhR was localized both in the cytoplasm and nucleus while Arnt was found exclusively in the non-nucleolar nucleus of the three cell lines studied. Treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) caused a time-dependent uptake of AhR into the nucleus; Arnt localization was not affected by TCDD treatment. Quantitative Western blot analysis of relative levels of AhR and Arnt in Hepa 1c1c7 and VT$\{2\}$ cells indicated that Hepa 1c1c7 cells had two fold higher levels of AhR, while VT$\{2\}$ cells had nine fold higher levels of Arnt than Hepa 1c1c7 cells. TCDD-treated Hepa 1c1c7 and VT$\{2\}$ cell 0.5 M NaCl nuclear extracts had 6- and 5-fold more AhR associated with the nuclear matrix than untreated cells, respectively. TCDD caused a 2-fold increase in Arnt associated with the nuclear matrix in both cell lines. Arnt levels did not influence the amount of AhR associated with the nuclear matrix of TCDD-treated VT$\{2\}$ cells. Hydrodynamic analyses of Arnt from Hepa 1c1c7 cytosol revealed a sedimentation coefficient of $\sim$4S, a Stokes radius $\rm (R\sb{s})$ of 6.8 nm and a relative molecular weight estimate of 115 kDa. Arnt's nuclear localization, considered with the detection of nuclear matrix-associated 9S AhR in TCDD-treated Hepa 1c1c7 cells, implicate the nucleus as the site of AhR transformation.
Degree
Ph.D.
Advisors
Perdew, Purdue University.
Subject Area
Biology|Pharmacology
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