Identification of a novel C-terminal domain involved in the negative function of the rainbow trout Ah receptor nuclear translocator protein isoform a(rtARNTa) in Ah receptor-mediated signaling

Citation
B. Necela et Rs. Pollenz, Identification of a novel C-terminal domain involved in the negative function of the rainbow trout Ah receptor nuclear translocator protein isoform a(rtARNTa) in Ah receptor-mediated signaling, BIOCH PHARM, 62(3), 2001, pp. 307-318
Citations number
38
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Pharmacology & Toxicology
Journal title
BIOCHEMICAL PHARMACOLOGY
ISSN journal
0006-2952 → ACNP
Volume
62
Issue
3
Year of publication
2001
Pages
307 - 318
Database
ISI
SICI code
0006-2952(20010801)62:3<307:IOANCD>2.0.ZU;2-Q
Abstract
Rainbow trout aryl hydrocarbon receptor (AHR) nuclear translocator isoform a (rtARNTa) has a negative function in AHR-mediated signal transduction. Pr evious analyses suggest that the negative function is at the level of DNA b inding and may be due to the presence of 57 C-terminal amino acids that are strongly hydrophobic. To assess the negative activity of rtARNTa at the mo lecular level, hydrophobic-rich domains corresponding to amino acids 601-63 7, 601-631, and 616-631 were analyzed for the ability to affect the functio n of truncated rtARNT proteins in complementation and gel shift assays. Add ition of the hydrophobic-rich domains to these proteins reduced their abili ty to complement AHR-tnediated signal transduction in mouse hepatoma cells by 65-95%. The decrease in function was related to a reduced ability of the AHR.rtARNT complex to bind DNA and not. due to a lack of dimerization with AHR. Expression of the hydrophobic-rich domains on Gal4 proteins showed th at the C-terminal domain of rtARNTa was unlikely to contain transactivation function; however, the hydrophobic domains reduced the ability of the Gal4 proteins to bind DNA. Immunoprecipitation and mutational experiments indic ate that the hydrophobic-rich domains do not interact with the bHLH motif o f AHR. interestingly, immunoprecipitation experiments also revealed that th e C-terminal hydrophobic-rich region of rtARNTa could oligomerize in vitro in a chimera with the Gal4 DNA binding domain. These findings indicate that the C-terminal hydrophobic amino acids are critical for the negative funct ion of rtARNTa in AHR-mediated signaling and suggest that multiple mechanis ms may be involved in the repression of DNA binding. (C) 2001 Elsevier Scie nce Inc. All rights reserved.