Identification of a Domain Responsible for the Negative Function of rtARNTa in Ah Receptor SignallingEPA Grant Number: U915218
Title: Identification of a Domain Responsible for the Negative Function of rtARNTa in Ah Receptor Signalling
Investigators: Necela, Brian M.
Institution: Medical University of South Carolina
EPA Project Officer: Michaud, Jayne
Project Period: January 1, 1997 through January 1, 2000
Project Amount: $102,000
RFA: STAR Graduate Fellowships (1997) RFA Text | Recipients Lists
Research Category: Fellowship - Toxicology , Academic Fellowships , Health Effects
The objective of this research project was to determine whether specific domains exist within the COOH-terminal end of rtARNTa, which are responsible for the negative function of rtARNTa in aromatic hydrocarbon receptor (AHR)-mediated signal transduction.
The secondary structure of the PST-rich COOH-end of rtARNTa was evaluated by computer analysis (Motif database). Computer analysis revealed that the sequence was dominated by ß-sheets, numerous turn regions, and a lack of helices. In addition, the the last 20 amino acids contained a region that appeared be strongly hydrophobic. To assess the impact of these regions within the PST-rich COOH-end, the rtARNTa protein was truncated and function evaluated by electromobility shift assay and the complementation of P4501A1 induction in ARNT deficient Hepa-1 cells. Complementation studies revealed that truncation of the hydrophobic domain that spanned the final 20-40 amino acids of rtARNTa restored the protein's ability to induce P4501A1. Gel -shift analysis confirmed that these results were related to the recovery of DNA binding of the rtARNT/AHR heterodimer. Addition of the hydrophobic domain to mARNT or rtARNT resulted in proteins with reduced DNA binding and the inability to complement AHR-mediated signal transduction. These findings indicate that the hydrophobic domain is primarily responsible for the negative function of rtARNTa, suggesting that repressor function is related to protein misfolding or masking of the DNA binding domain. In addition, the Gal4 fusion approach was employed to identify possible transactivation domains within the truncated rtARNTa proteins. Gal4 analysis revealed the lack of transactivation function in the truncated COOH terminal domains of rtARNTa, in contrast to the QN-rich COOH-end of rtARNTb. The lack of transcriptional activity was not related to DNA binding, as the Gal4 fusion proteins bound DNA. Collectively, these studies narrow the negative function of rtARNTa to the presence of a C-terminal hydrophobic domain, and illustrates how changes in the C-terminal domain can affect protein function.