Regulation of aryl hydrocarbon receptor nuclear translocator (ARNT) and AH receptor function
Abstract
Aryl hydrocarbon receptor nuclear translocator (Arnt) and Ah receptor (AhR) belong to the family of basic helix-loop-helix (bHLH)-PAS transcription factors. Upon exposure to TCDD, AhR and Arnt form the transcriptionally active heterodimer and bind to xenobiotic response elements (XREs) in DNA. Arnt is also considered the central dimerization partner for bHLH-PAS proteins, and regulates various cellular processes. The aim of my thesis research is to study the regulation of Arnt. Two-dimensional gel electrophoresis (IEF-SDS-PAGE) was used to examine the posttranslational modifications (e.g. phosphorylation) of Arnt. Considerable charge heterogeneity of Arnt in Hepa 1 cell with multiple isoforms was determined by visualization with two distinct anti-Arnt antibodies. Arnt complexed with AhR in the nucleus after $\beta$-naphthoflavone ($\beta$NF) treatment had an isoform pattern shifted toward the basic end, implying a dephosphorylation event occurred after heterodimerization and/or DNA-binding. However, each isoform of Arnt was capable of heterodimerizing with AhR in vitro. Arnt is demonstrated for the first time to be a phosphoprotein in vivo, by transient transfection and ($\sp{32}$P) orthophosphate labeling. Two C-terminal truncated Arnt mutants were generated, and the region of Arnt phosphorylation was determined between amino acids 241 and 474. Arnt is phosphorylated exclusively on threonine residues, and phosphorylation is conserved among monkey, human, and mouse species. Effects of regulatory pathways that impinge on AhR/Arnt functions were also investigated by generating HepG2 stable transfectants (e.g., HG40/6 cells) that carry an integrated luciferase reporter gene driven solely by four XREs. Simultaneous treatment of HG40/6 cells with PMA and TCDD resulted in a synergistic enhancement on XRE/luciferase gene transcription, which was antagonized by two PKC inhibitors. These results indicate that a PKC-dependent pathway is involved in the transactivation function of AhR/Arnt. The epidermal growth factor (EGF) and the MAPK pathway were also shown to modulate TCDD-mediated AhR/Arnt transactivation function, demonstrating the importance of multiple signaling pathways in modulating AhR/Arnt function.
Degree
Ph.D.
Advisors
Perdew, Purdue University.
Subject Area
Molecular biology
Off-Campus Purdue Users:
To access this dissertation, please log in to our
proxy server.