Characterization of a Subset of the Basic-Helix-Loop-Helix-PAS Superfamily That Interacts with Components of the Dioxin Signaling Pathway

doi:10.1074/jbc.272.13.8581

Characterization of a Subset of the Basic-Helix-Loop-Helix-PAS Superfamily That Interacts with Components of the Dioxin Signaling Pathway*

From the ^‡ Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Medical School, Chicago, Illinois 60611, the
^§ McArdle Laboratory for Cancer Research, University of Wisconsin Medical School, Madison, Wisconsin 53706, and the
^¶ Department of Veterinary Science, Pennsylvania State University, State College, Pennsylvania 16802

^∥To whom all correspondence should be addressed:
McArdle Laboratory for Cancer Research, 1400 University Ave., Madison, WI 53706
. Tel.: 608-262-2024; Fax: 608-262-2824; E-mail: bradfield{at}oncology.wisc.edu.

Abstract

In an effort to better understand the mechanism of toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin, we employed an iterative search of human expressed sequence tags to identify novel basic-helix-loop-helix-PAS (bHLH-PAS) proteins that interact with either the Ah receptor (AHR) or the Ah receptor nuclear translocator (ARNT). We characterized five new “embers f the AS superfamily,” or MOPs 1-5, that are similar in size and structural organization to the AHR and ARNT. MOPs 1-4 have N-terminal bHLH and PAS domains and C-terminal variable regions. MOP5 contained the characteristic PAS domain and a variable C terminus; it is possible that the cDNA contains a bHLH domain, but the entire open reading frame has yet to be completed. Coimmunoprecipitation studies, yeast two-hybrid analysis, and transient transfection experiments demonstrated that MOP1 and MOP2 dimerize with ARNT and that these complexes are transcriptionally active at defined DNA enhancer sequences in vivo. MOP3 was found to associate with the AHR in vitro but not in vivo. This observation, coupled with the fact that MOP3 formed tighter associations with the 90-kDa heat shock protein than the human AHR, suggests that MOP3 may be a conditionally active bHLH-PAS protein that requires activation by an unknown ligand. The expression profiles of the AHR, MOP1, and MOP2 mRNAs, coupled with the observation that they all share ARNT as a common dimeric partner, suggests that the cellular pathways mediated by MOP1 and MOP2 may influence or respond to the dioxin signaling pathway.

Footnotes

↵* This work was supported by the Burroughs Wellcome Foundation; by National Institutes of Health Grants P30-CA07175, ES05703, ES05660, ES05700, and GM08061; and by a postdoctoral fellowship sponsored by the Colgate-Palmolive Company. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) U29165[GenBank] and U51625[GenBank], U51626[GenBank], U51627[GenBank], U51628[GenBank].
↵¹ The abbreviations used are:

AHR

Ah receptor

ARNT

Ah receptor nuclear translocator

bHLH

basic-helix-loop-helix

PAS

PER/ARNT/SIM homology domain

TCDD

2,3,7,8-tetrachlorodibenzo-p-dioxin

PER

the protein product of the Drosophila period gene

SIM

the protein product of the Drosophila single-minded gene

HIF1α

hypoxia-inducible factor 1 α (also referred to as MOP1 in this paper)

HIF1β

hypoxia-inducible factor 1 β (also referred to as ARNT in this paper)

EST

expressed sequence tag

BLAST

basic local alignment search tool

PCR

polymerase chain reaction

ORF

open reading frame

MOP

members of PAS superfamily

IPTG

isopropyl-β-D-thiogalactopyranoside

βNF

β-napthoflavone

bp

base pair(s)

kb

kilobase pair(s)

MOPS

4-morpholinepropanesulfonic acid

PAGE

polyacrylamide gel electrophoresis.
↵² This estimate was provided by the IMAGE Consortium (http://www-bio.llnl.gov/bbrp/I.M.A.G.E./iachievements.htmlandftp://humpty.llnl.gov/pub/EST/IndexReport).
- Received April 1, 1996.
- Revision received December 3, 1996.