https://mcardle.wisc.edu/bradfield/publications/Dunham%20Kelch%20paper.pdf
The Aryl Hydrocarbon Receptor Signaling Pathway Is Modified through Interactions with a Kelch Protein
Elizabeth E. Dunham, Emily A. Stevens, Edward Glover, and Christopher A. Bradfield
McArdle Laboratory for Cancer Research, University of Wisconsin Medical School, Madison, WisconsinReceived March 16, 2006; accepted March 31, 2006ABSTRACTThe aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor with important roles in metabolic adaptation,dioxin toxicology, and vascular development. To understand the details of this signal transduction pathway, we have used the yeast two-hybrid system to identify proteins that physically interact with the AHR in a ligand-dependent manner. Using this strategy, we identified a novel modifier of the AHR signaling pathway that we named Ah-receptor associated protein 3(ARA3). Coexpression of ARA3 with an AHR chimera in yeast and mammalian cells enhances signaling in response to agonists. The human full-length cDNA previously was described as influenza virus nonstructural protein-1 binding protein (NS1BP).This protein contains four apparent domains a “broad complex/tramtrack/bric-a-brac” (BTB) domain, a “kelch” domain, a“BTB and C-terminal kelch” (BACK) domain, and an intervening region (IVR). The carboxyl terminus of the AHR “Per-ARNT-Sim” (periodicity/AHR nuclear translocator/simple-minded) do-main and the BACK/IVR domains of ARA3 mediate the AHR-ARA3 interaction. The BACK/IVR domains of ARA3 also are sufficient to modify AHR signaling in yeast and mammalian cells. In an effort to provide a preliminary model of NS1BP activity in AHR signaling, we demonstrate that NS1BP regulates the concentration of functional AHR in mammalian cell.
The aryl hydrocarbon receptor (AHR) is a member of the basic helix-loop-helix Per-Arnt-Sim(bHLH-PAS) superfamily. Upon binding ligands, the AHR mediates an adaptive metabolic response by up-regulating the transcription of a battery of xenobiotic metabolizing enzymes, including the cytochromes P450, CYP1A1, CYP1A2, and CYP1B1 (Schmidt and Bradfield, 1996). When stimulated by high-potency agonists, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD),the AHR mediates an additional toxic response that includes hepatocellular damage, thymic involution, teratogenesis, chloracne, and cancer (Pohjanvirta and Tuomisto, 1994;Fernandez-Salguero et al., 1996). It has been shown that theAHR also plays an important role in vascular development.
In this regard, patent ductus venosusis observed in 100 % of mice with a null allele at the Ahr locus (Lahvis et al., 2005).The presentation of vascular aberrations in AHR-mutant mice is consistent with the idea that the AHR is activated by an un known endogenous ligand.The AHR signaling pathway is understood at a basic level. In the absence of ligand, the AHR has a higher affinity for the cytosol, where it exists in a complex with the 90-kDa heat shock protein and the co chaperones ARA9 (also known as AIP1 or XAP2) and p23 (Wilhelmsson et al., 1990; Ma and Whitlock, 1997; Carver et al., 1998; Meyer et al., 1998; Ka-zlauskas et al., 1999). Upon ligand binding, the AHR attains a higher affinity for the nuclear compartment, where it dimerizes with another bHLH-PAS protein known as the AHR nuclear translocator (ARNT) (Reyes et al., 1992). The transcriptional activity of the AHR-ARNT heterodimer is modulated through interactions with cofactors such as SRC-1and RIP140 (Kumar et al., 1999; Beischlag et al., 2002).
The AHR pathway can be attenuated by two mechanisms, proteasome-dependent degradation of the AHR and the AHR-mediated transcription of a dominant-negative bHLH-PAS protein known as the Ahreceptor repressor (Davarinos andPollenz, 1999; Mimura et al., 1999). In previous studies, we have demonstrated that the yeast Saccharomyces cerevisiaeis an excellent model of AHR signaltransduction (Carver et al., 1994; Yao et al., 2004). In an effort to identify unknown components of the AHR signalingpathway, we have been employing a yeast-two-hybrid strategy to screen for proteins that interact with the AHR in a ligand-dependent manner. This approach has led to the identification of ARA9, now a proven AHR co chaperone (Ma andWhitlock, 1997; Bell and Poland, 2000; LaPres et al., 2000;Petrulis et al., 2000). As a result of this earlier success, we began characterizing the second clone identified in our yeast-two-hybrid assay [i.e.,Ah receptor associated 3(ARA3)](Carver and Bradfield, 1997). Herein, we characterize the AHR-ARA3 interaction and its effect on AHR signaling inyeast and mammalian cells.
We previously reported a modified yeast two-hybrid assayto screen for proteins that interact with the AHR in a ligand-
Fig. 1.Analysis of the ARA3 cDNA revealed three characterized proteindomains. The ARA3 yeast two hybrid clones originally identified in our screen encoded a 531-AA protein. There was no Kozak consensus initiation ATG present. Therefore, the human full-length ARA3 (FLARA3) was cloned from a human heart cDNA library and sequenced. FLARA3 is 642AA long, whereas the original ARA3 clone was missing the first 111 AA.Similarity searches identified FLARA3 as a protein originally called NS1BP. The NS1BP/FLARA3 protein contains a BTB domain from AA 22 to 129, a BACK domain from AA 134 to 233, and a kelch domain consisting of six imperfect 50-AA kelch repeats from AA 357 to 635 (http://www.sanger.ac.uk). A low homology region exists between the BACK and kelch domains that we refer to as the IVR
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