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Identification of a Critical Amino Acid in the Aryl Hydrocarbon Receptor∗

  • Eric A. Andreasen
    Affiliations
    Molecular and Environmental Toxicology Program, University of Wisconsin, Madison, Wisconsin 53706
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  • Robert L. Tanguay
    Affiliations
    Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Health Sciences Center, Denver, Colorado 80262
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  • Richard E. Peterson
    Affiliations
    Molecular and Environmental Toxicology Program, University of Wisconsin, Madison, Wisconsin 53706

    School of Pharmacy, University of Wisconsin, Madison, Wisconsin 53706
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  • Warren Heideman
    Correspondence
    To whom correspondence should be addressed: School of Pharmacy, 777 Highland Ave., Madison, WI 53705. Tel.: 608-262-1795; Fax: 608-262-3397
    Affiliations
    Molecular and Environmental Toxicology Program, University of Wisconsin, Madison, Wisconsin 53706

    School of Pharmacy, University of Wisconsin, Madison, Wisconsin 53706
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  • Author Footnotes
    ∗ This work was supported by the University of Wisconsin Sea Grant Institute under grants from the National Sea Grant College Program, National Oceanic and Atmospheric Administration, U. S. Department of Commerce, Sea Grant Project Numbers R/BT12 and R/BT14 (to W. H. and R. E. P.), by NIEHS, National Institutes of Health Grant ES10820 (to R. L. T.), and by NIEHS, NIH Developmental and Molecular Toxicology Center Grant P30ES09090 (to W. H. and R. E. P.). 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.
Open AccessPublished:January 31, 2002DOI:https://doi.org/10.1074/jbc.M200073200
      Two aryl hydrocarbon receptors (rtAHR2α and rtAHR2β) have been identified in the rainbow trout (Oncorhynchus mykiss). These receptors share 98% amino acid identity, yet their functional properties differ. Both rtAHR2α and rtAHR2β bind 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), dimerize with rainbow trout ARNTb (rtARNTb), and recognize dioxin response elements in vitro. However, in a transient transfection assay the two proteins show differential ability to recognize enhancers, produce transactivation, and respond to TCDD. To identify the sequence differences that confer the functional differences between rtAHR2α and rtAHR2β, we constructed chimeric rtAHRs, in which segments of one receptor form was replaced with the corresponding part from the other isoform. This approach progressively narrowed the region being examined to a single residue, corresponding to position 111 in rtAHR2β. Altering this residue in rtAHR2β from the lysine to glutamate found in rtAHR2α produced an rtAHR2β with the properties of rtAHR2α. All other known AHRs resemble rtAHR2α and carry glutamate at this position, located at the N terminus of the PAS-A domain. We tested the effect of altering this glutamate in the human and zebrafish AHRs to lysine. This lysine substitution produced AHRs with transactivation properties that were similar to rtAHR2β. These results identify a critical residue in AHR proteins that has an important impact on transactivation, enhancer site recognition, and regulation by ligand.
      AHR
      aryl hydrocarbon receptor
      hAHR
      human AHR
      rtAHR
      rainbow trout AHR
      ARNT
      Aryl Hydrocarbon Receptor NuclearTransporter
      bHLH
      basic helix-loop-helix
      PAS
      Per, ARNT, and Sim protein family
      TCDD
      2,3,7,8-tetrachlorodibenzo-p-dioxin
      HSP90
      heat-shock protein 90
      DRE
      dioxin response element
      CYP450
      cytochrome P450
      NLS
      nuclear localization signal
      NES
      nuclear export signal
      CMV
      cytomegalovirus
      ORF
      open reading frame
      MOPS
      4-morpholinepropanesulfonic acid
      PYP
      photoactive yellow protein
      The aryl hydrocarbon receptor (AHR)1 and its associated dimerization partner ARNT are members of the basic helix-loop-helix (bHLH) PAS family of proteins. These proteins transduce signals generated by environmental stresses into transcriptional responses. These stresses range from hypoxia to xenobiotic compounds (
      • Gu Y.Z.
      • Hogenesch J.B.
      • Bradfield C.A.
      ,
      • Rowlands J.C.
      • Gustafsson J.A.
      ). The AHR is activated by a structurally broad range of ligands. Among these, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is one of the most potent and well-studied agonists (
      • Denison M.S.
      • Heath-Pagliuso S.
      ). A broad spectrum of environmental contaminants, including TCDD, can produce toxic responses through activation of the AHR. Two hybrid and coprecipitation studies have revealed the presence of proteins that interact with AHR, including HSP90 and ARA9/AIP/XAP2 (
      • Carver L.A.
      • Jackiw V.
      • Bradfield C.A.
      ,
      • Carver L.A.
      • Bradfield C.A.
      ,
      • Chen H.S.
      • Perdew G.H.
      ,
      • Ma Q.
      • Whitlock Jr., J.P.
      ). These chaperone proteins stabilize and hold AHR in a conformation that is better able to bind ligand (
      • Meyer B.K.
      • Petrulis J.R.
      • Perdew G.H.
      ). TCDD binding causes the AHR protein to dissociate from cytosolic HSP90 and move into the nucleus where it forms a functional dimer with ARNT. This dimer then binds DNA to regulate the transcription of target genes. The AHR·ARNT dimer binds to specific enhancer elements that are often referred to asDioxin Response Elements, or DREs. The best-characterized DREs lie upstream of genes encoding cytochrome P450s (CYP450s) (
      • Schmidt J.V.
      • Bradfield C.A.
      ,
      • Whitlock Jr., J.P.
      ). Following nuclear localization and DNA binding, AHR exits the nucleus and is then degraded by the proteasome pathway (
      • Ma Q.
      • Renzelli A.J.
      • Baldwin K.T.
      • Antonini J.M.
      ,
      • Ma Q.
      • Baldwin K.T.
      ,
      • Pollenz R.S.
      ,
      • Pollenz R.S.
      • Barbour E.R.
      ).
      The AHR protein is composed of several functional domains. The N terminus contains a domain rich in basic amino acids followed by a helix-loop-helix domain that is conserved among a variety of DNA binding proteins. The basic domain is required for DNA binding, whereas the helix-loop-helix domain is involved in dimer formation with ARNT. The N terminus also contains nuclear localization (NLS) and export (NES) domains (
      • Berg P.
      • Pongratz I.
      ,
      • Ikuta T.
      • Eguchi H.
      • Tachibana T.
      • Yoneda Y.
      • Kawajiri K.
      ). C-terminal to the bHLH domain is a pair of PAS domains, PAS-A and PAS-B, that are conserved among a family of proteins. The PAS domains are named for several founding members of this protein family, Per, ARNT, andSim (
      • Gu Y.Z.
      • Hogenesch J.B.
      • Bradfield C.A.
      ). PAS domains act as regulated protein interaction surfaces and are involved in a wide variety of sensory/signaling processes in both eukaryotes and prokaryotes. These domains are involved in ligand binding to AHR, and the subsequent change in protein associations, subcellular location, and activity. The ligand-binding domain encompasses the PAS-B domain whereas HSP90 is thought to interact with the bHLH and PAS domains (
      • Fukunaga B.N.
      • Probst M.R.
      • Reisz-Porszasz S.
      • Hankinson O.
      ,
      • Coumailleau P.
      • Poellinger L.
      • Gustafsson J.A.
      • Whitelaw M.L.
      ). ARA9/AIP interacts with the PAS-B/ligand-binding domains (
      • Bell D.R.
      • Poland A.
      ,
      • Meyer B.K.
      • Perdew G.H.
      ). Potential retinoblastoma protein binding sites have also been identified (
      • Puga A.
      • Barnes S.J.
      • Dalton T.P.
      • Chang C.
      • Knudsen E.S.
      • Maier M.A.
      ). The C-terminal domain is necessary for transcriptional activation and is the least conserved among AHR proteins (
      • Fukunaga B.N.
      • Probst M.R.
      • Reisz-Porszasz S.
      • Hankinson O.
      ).
      Developing fish are especially sensitive to the toxic effects of TCDD (
      • Elonen G.E.
      • Sphear R.L.
      • Holcombe G.W.
      • Johnson R.D.
      ). AHR and ARNT proteins have been identified in a variety of fish species and presumably mediate these effects. The ability of ligands to activate the AHR pathway is similar to their ability to cause TCDD-like toxicity (
      • Walker M.K.
      • Peterson R.E.
      ,
      • Zabel E.W.
      • Cook P.M.
      • Peterson R.E.
      ,
      • Zabel E.W.
      • Pollenz R.
      • Peterson R.E.
      ). In contrast to mammals, most fish species appear to have at least two AHR genes. Generally, one AHR (AHR1) is more similar to the mammalian AHR and a second (AHR2) is fish-specific (
      • Hahn M.E.
      • Karchner S.I.
      • Shapiro M.A.
      • Perera S.A.
      ). Full-length AHRs have been cloned in tomcod and two each in rainbow trout, Fundulus heteroclitus, and zebrafish (
      • Abnet C.C.
      • Tanguay R.L.
      • Hahn M.E.
      • Heideman W.
      • Peterson R.E.
      ,
      • Karchner S.I.
      • Powell W.H.
      • Hahn M.E.
      ,
      • Roy N.K.
      • Wirgin I.
      ,
      • Tanguay R.L.
      • Abnet C.C.
      • Heideman W.
      • Peterson R.E.
      ). In addition, partial AHR sequences have been cloned from several fish (
      • Hahn M.E.
      • Karchner S.I.
      • Shapiro M.A.
      • Perera S.A.
      ). ARNT isoforms have been cloned from Fundulus (ARNT2) rainbow trout (rtARNTa and b) and zebrafish (zfARNT2a, b, and c) (
      • Pollenz R.S.
      • Sullivan H.R.
      • Holmes J.
      • Necela B.
      • Peterson R.E.
      ,
      • Powell W.H.
      • Karchner S.I.
      • Bright R.
      • Hahn M.E.
      ,
      • Tanguay R.L.
      • Andreasen E.
      • Heideman W.
      • Peterson R.E.
      ). No ARNT1 has been identified in fish.
      To date, salmonids are the group of fish species that are most sensitive to the effects of TCDD. Two AHR genes encoding rtAHR2α and rtAHR2β have been identified in rainbow trout. These two AHR isoforms are ∼98% identical in primary sequence. Despite this similarity in structure, the two proteins have distinct properties. In general, rtAHR2α has stronger transactivation properties than rtAHR2β. This is somewhat surprising in light of the fact that these two proteins are identical in sequence in the C-terminal domain that is thought to mediate transcriptional activation. In addition, rtAHR2α and rtAHR2β have different enhancer sequence requirements. rtAHR2β appears to be active with a more limited set of enhancer sequences than rtAHR2α. To explore the structural nature of these differences, we constructed a set of chimeric proteins in which segments of rtAHR2β were exchanged with the cognate sequence from rtAHR2α. These experiments indicate that the functional differences between rtAHR2α and rtAHR2β are conferred by a single amino acid difference corresponding to position 111 in rtAHR2β.

      RESULTS

      The rtAHR2α and rtAHR2β proteins are almost 98% identical in primary sequence yet have distinctly different properties. We used transient transfection assays in COS-7 cells to measure transactivation properties of these proteins, because these cells are devoid of endogenous AHR and express only a low amount of ARNT (
      • Ema M.
      • Ohe N.
      • Suzuki M.
      • Mimura J.
      • Sogawa K.
      • Ikawa S.
      • Fujii-Kuriyama Y.
      ). When assayed for reporter activation in a transient transfection assay using COS-7 cells and DRE-containing luciferase reporters, these proteins gave different results. When we used the prt1Aluc reporter construct, in which sequences from the rainbow trout CYP1A promoter are used to drive the luciferase reporter (
      • Abnet C.C.
      • Tanguay R.L.
      • Heideman W.
      • Peterson R.E.
      ), rtAHR2α produced a luciferase signal that was readily detected and sensitive to TCDD induction. In contrast, rtAHR2β produced only a low level of reporter activity, which was not increased by the addition of TCDD (Fig. 1). However, when we used pGudluc1.1, a reporter driven by mammalian sequences derived from the mouse cyp1A1 gene (
      • Garrison P.M.
      • Tullis K.
      • Aarts J.M.
      • Brouwer A.
      • Giesy J.P.
      • Denison M.S.
      ), we observed a different response. In this case, both AHR proteins were able to produce a readily detected reporter signal that was induced by TCDD. Although rtAHR2α produced stronger transactivation with the pGudluc1.1 reporter than rtAHR2β, rtAHR2β was more responsive to TCDD, as indicated in the bottom right panel of Fig. 1. In this assay, rtAHR2α was induced ∼10-fold by TCDD, whereas the activity of rtAHR2β was induced by more than 50-fold, owing to the very low activity in the absence of TCDD. This rtAHR2β basal activity is close to the limit of detection, making calculated values for -fold TCDD induction of rtAHR2β somewhat variable. However, both the low basal activity and the high -fold induction by TCDD were consistently observed with this receptor. These results demonstrate several different properties of these receptor molecules: First, rtAHR2α appears to have stronger transactivation properties than rtAHR2β, producing more luciferase expression with either reporter construct. Second, when assayed with the pGudluc1.1 reporter, rtAHR2β is more tightly regulated by TCDD, owing to the very low basal activity. Finally, the two AHR proteins appear to have different requirements for DNA target sequences.
      To identify the domain that confers these differences in activity, we constructed chimeric rtAHR2 proteins by exchanging similar domains between rtAHR2α and rtAHR2β and measured the activities of these chimeras with prt1Aluc and pGudluc1.1. We then attempted to correlate the presence of a region from the α or β receptor isoforms with the different receptor characteristics. The C-terminal portions of the rtAHR2s are entirely conserved (Fig. 2), so our chimeras concentrated on the N-terminal half of the AHR proteins. The first set of chimeric proteins were made by taking advantage of conserved SphI and BglII restriction sites found in both rtAHR2α and rtAHR2β. These were used to transfer domains from rtAHR2α to rtAHR2β and vice versa (Fig. 3, and see Fig. 2). In chimeras A through F, the ability to produce a robust transactivation signal with the prt1Aluc reporter, a characteristic of rtAHR2α, was observed only in chimeras in which the N-terminal 250 amino acids were from rtAHR2α. Chimeras carrying rtAHR2β sequence in this region were relatively inactive with the prt1Aluc reporter. Similarly, the tight regulation by TCDD observed in rtAHR2β, characterized by very low basal expression and resulting high -fold induction by TCDD with the pGudluc1.1 reporter, correlated with the presence of the rtAHR2β sequence in this N-terminal SphI portion of the protein. In addition, the marked preference for pGudluc1.1, which characterizes rtAHR2β, was also conferred by this part of the protein.
      Figure thumbnail gr2
      Figure 2Amino acid sequence alignment of rtAHR2α, rtAHR2β, and the human AHR. Sequence alignment of rtAHR2α (GenBankTM accession number AF065137), rtAHR2β (AF065138), and human AHR (L19872) was done using ClustalW1.8.Asterisks indicate perfect identity to rtAHR2α, and dashes indicate gaps in sequence alignment. The basic, HLH, and PAS domains are indicated by lines above the sequence. The ligand-binding domain is indicated by underlining (
      • Fukunaga B.N.
      • Probst M.R.
      • Reisz-Porszasz S.
      • Hankinson O.
      ,
      • Coumailleau P.
      • Poellinger L.
      • Gustafsson J.A.
      • Whitelaw M.L.
      ). The nuclear localization signal and nuclear export signal (
      • Ikuta T.
      • Eguchi H.
      • Tachibana T.
      • Yoneda Y.
      • Kawajiri K.
      ) are shaded.
      Figure thumbnail gr3
      Figure 3TCDD responsiveness of rtAHR2 chimeras in a transient transfection assay. COS-7 cells were transiently transfected with expression vectors for the indicated rtAHR2 chimeras as described for . Maps of the AHR open reading frames indicate the positions of the chimera junctions. Dark bars indicate rtAHR2α sequence, and the light bars represent rtAHR2β sequence. Data are expressed in the upper panels as β-galactosidase normalized relative light units: light bars, TCDD-exposed; dark bars, vehicle control. Results are expressed as -fold induction by TCDD in the lower panels. The results are expressed as the means of three independent replicates ± S.E.
      This N-terminal SphI fragment encoding the first 250 amino acids contains the majority of the differences between the rtAHR2α and rtAHR2β sequences. To narrow down the residues responsible for the functional differences between rtAHR2α and rtAHR2β and to possibly dissociate these functional differences, we made an additional set of chimeras, G and H. In these chimeras the regions between position 86 and the SphI site, containing the PAS-A domain, are swapped. The designation of the junction at position 86 is arbitrary, because this junction occurs in a region of sequence identity; the actual point of the junction can be considered anywhere between positions 84 and 93 on rtAHR2α. The ability to produce a robust transactivation signal with the prt1Aluc reporter was observed in chimera H, which carried rtAHR2α sequence between positions 86 and 250, but not in the chimera containing rtAHR2β sequence in this region (Fig. 3). As observed with the previous set of chimeras, the tight regulation by TCDD observed with rtAHR2β and the pGudluc1.1 reporter was also conferred by this region of the protein. The chimera G, containing rtAHR2β sequence between positions 86 and 250, showed low activity with prt1Aluc and high -fold induction with pGudluc1.1. Thus, this portion of the protein confers both the rtAHR2α- and rtAHR2β-specific characteristics.
      The domain from residues 86 to 250 found to be responsible for the differential transactivation properties of the two rtAHR2s is differentiated by 8 residues. Seven of these differences lie near the N terminus of the PAS-A domain (Fig. 2). To identify specific residues that confer the differential transactivation properties, we made a chimera in which the residues in rtAHR2β from position 105 through 111 were converted to those found in the rtAHR2α sequence. This involves three alterations: glutamate 105 was converted to asparagine, threonine 107 was converted to proline, and lysine 111 was changed to glutamate. This produced a chimera that behaved like rtAHR2α (Fig. 4). Activity with prt1Aluc was markedly increased, while the -fold stimulation by TCDD measured with the pGudluc1.1 reporter was decreased. Single substitutions demonstrated that a single residue conveyed much of the differential properties of the rtARH2s: conversion of the rtAHR2β lysine at position 111 to the corresponding glutamate from rtAHR2α changed the properties of rtAHR2β to those of rtAHR2α. The other single residue changes had no effect nor did altering rtAHR2β isoleucine 101 to methionine or alanine 135 to serine (not shown). This single-residue corresponding to position 111 in rtAHR2β therefore has an effect on transactivation, -fold regulation by ligand, and DNA sequence preference.
      Figure thumbnail gr4
      Figure 4TCDD responsiveness of rtAHR2 chimeras created by point mutations in rtAHR2β.COS-7 cells were transiently transfected with expression vectors for the indicated rtAHR2 chimeras as described for . The change in amino acid altered in rtAHR2β to that found in rtAHR2α is labeled as the rtAHR2β residue followed by the position number and corresponding altered amino acid. A, data are expressed in the upper panels as β-galactosidase normalized relative light units. Light bars, TCDD-exposed; dark bars, vehicle control. B, data are expressed as -fold induction by TCDD. The results in both A and B are expressed as the means of three independent replicates ± S.E. C, Western blot of the transfected FLAG epitope-tagged rtAHR2α, rtAHR2β, and the rtAHR2β K111E substitution chimera proteins.
      With both the prt1Aluc and pGudluc1.1 reporters, rtAHR2α consistently produced a higher level of transactivation than rtAHR2β. This might have been caused by a difference in the level of expression between the two different receptor types. To test this we transfected epitope-tagged rtAHR2α and rtAHR2β into COS-7 cells and used Western blotting to determine the relative levels of the tagged proteins (Fig. 4, bottom). We observed that the two receptors were expressed at comparable levels. In addition, the rtAHR2β K111E mutant was also expressed at levels similar to rtAHR2α and rtAHR2β. This indicates that the stronger transactivation signal produced by rtAHRα and the rtAHR2β K111E mutant is not due to higher levels of expression.
      The glutamate at position 110 in rtAHR2α corresponding to lysine 111 in rtAHR2β is conserved among the known AHRs that bind TCDD. To be certain that the lysine identified at residue 111 in rtAHR2β was not an allelic variant or cloning artifact, RNA was isolated from four separate hatchery strains: Arlee, Eagle Lake, McConoughy, and Shasta (n = 3 for each strain). Amplified cDNAs for rtAHR2α and rtAHR2β were generated. In addition, clones were obtained from cDNA prepared from a rainbow trout gonadal cell line (RTG-2). All samples yielded cDNAs for both rtAHR2α and rtAHR2β, and all of the rtAHR2β cDNAs encoded lysine at position 111 (not shown).
      To determine whether the characteristics of rtAHR2β are determined by the presence of lysine at position 111 or the absence of the glutamate residue found at this position in all other AHRs, an rtAHR2β mutant was constructed in which the lysine at position 111 was replaced by alanine (rtAHR2βK111A). This produced a receptor with characteristics that were somewhat intermediate between rtAHR2α and rtAHR2β (Fig. 5). This protein was a stronger transactivator than rtAHR2β, but weaker than rtAHR2α. Similarly, when assayed with the pGudluc1.1 reporter, the rtAHRβK111A mutant had a higher -fold induction by TCDD than the K111E mutant, but lower -fold induction than the normal rtAHR2β. This suggests that the glutamate in rtAHR2α and the corresponding lysine in rtAHR2β both play important roles in determining the characteristics of their receptors.
      Figure thumbnail gr5
      Figure 5TCDD responsiveness of rainbow trout and zebrafish AHR2 chimeras created by site-directed point mutations.COS-7 cells were transiently transfected with expression vectors for the indicated chimeras as described for . A, data are expressed as β-galactosidase normalized relative light units and -fold induction by TCDD. Light bars, TCDD-exposed;dark bars, vehicle control. B, the results are expressed as the means of three independent replicates ± S.E.
      We also determined whether the presence of this lysine at the corresponding position in other AHR proteins would produce characteristics of rtAHR2β in these AHRs. When the zebrafish AHR2 was mutated from glutamate to lysine at position 114 (corresponding to position 111 in rtAHR2β) the mutated zfAHR2 displayed the characteristics of rtAHR2β. These included decreased transactivation, a requirement for prtGudluc1.1 as a reporter, and a low basal level of activity with a corresponding increase in -fold responsiveness to TCDD (Fig. 5).
      A similar mutation was made in the human AHR (hAHR) in which glutamate 118 corresponding to K111 in rtAHR2β was mutated to lysine (hAHRE118K) (Fig. 6). This also produced a decrease in transactivation and an increase in TCDD responsiveness characteristic of rtAHR2β. These results underscore the importance of this residue, which is consistently conserved among all known AHRs, other than rtAHR2β.
      Figure thumbnail gr6
      Figure 6TCDD responsiveness of human AHR carrying a E to K substitution at the position corresponding to rtAHR2β K111. Y-1 cells were transiently transfected with expression vectors for the indicated AHR as described in the legend. Data are expressed as (A) β-galactosidase normalized relative light units and (B) -fold stimulation by TCDD. Light bars, TCDD-exposed; dark bars, vehicle control. The results are expressed as the means of three independent triplicates ± S.E.

      DISCUSSION

      Our results indicate that the lysine at position 111 in rtAHR2β and the corresponding glutamate at position 110 in rtAHR2α play important roles in the functions of the proteins that they reside in. The residue at this position affects transactivation, the degree of activation by ligand, and enhancer specificity. In rtAHR2α the glutamate confers increased transactivation function at either of the two reporters used in our experiments. Substitution of glutamate for the lysine at position 111 on rtAHR2β produced a mutant rtAHR2β with increased transcriptional activity, similar to that of rtAHR2α. Although the rtAHR2β K111E mutant gained these rtAHR2α-like properties, it lost other properties, i.e. high -fold induction by TCDD with pGudluc1.1 and enhancer specificity, that are characteristic of rtAHR2β. These changes in receptor properties are apparently entirely the result of the lysine at position 111. The residues that differ at this position between rtAHR2α and rtAHR2β (glutamate in rtAHR2α and lysine in rtAHR2β) are substantially different in charge and could therefore be expected to produce different conformations in the respective proteins.
      With the exception of rtAHR2β, all other known AHRs have a glutamate at the residue corresponding to rtAHR2β's lysine 111. This complete conservation in sequence implies a conserved function among these AHR proteins. Because rtAHR2β does not share this conserved residue, our results suggest that rtAHR2β diverges in function in some way from these other AHRs.
      Our results complement a body of work that has identified distinct functional domains in the AHR protein. A number of laboratories have identified residues that are required for transactivation, ligand binding, protein·protein interactions, and DNA binding. In addition to identifying the C terminus as a transactivation domain (
      • Fukunaga B.N.
      • Probst M.R.
      • Reisz-Porszasz S.
      • Hankinson O.
      ,
      • Whitelaw M.L.
      • Gustafsson J.A.
      • Poellinger L.
      ,
      • Jain S.
      • Dolwick K.M.
      • Schmidt J.V.
      • Bradfield C.A.
      ,
      • Ma Q.
      • Dong L.
      • Whitlock Jr., J.P.
      ) specific subdomains within the C terminus that are necessary for transactivation have been identified (
      • Jones L.C.
      • Whitlock Jr., J.P.
      ,
      • Kumar M.B.
      • Ramadoss P.
      • Reen R.K.
      • Vanden Heuvel J.P.
      • Perdew G.H.
      ). Mutation of residue 678 in hAHR blocks transactivation without affecting DNA binding (
      • Kumar M.B.
      • Ramadoss P.
      • Reen R.K.
      • Vanden Heuvel J.P.
      • Perdew G.H.
      ). The domain spanning residues 230–421 containing the PAS-B domain has been shown to bind ligand, and residue 381 in hAHR is important for ligand binding (
      • Ema M.
      • Ohe N.
      • Suzuki M.
      • Mimura J.
      • Sogawa K.
      • Ikawa S.
      • Fujii-Kuriyama Y.
      ). Alterations at positions 78 and 216 in the mouse AHR affect DNA binding (
      • Levine S.L.
      • Petrulis J.R.
      • Dubil A.
      • Perdew G.H.
      ,
      • Sun W.
      • Zhang J.
      • Hankinson O.
      ). Substitutions in the basic domain alter DNA binding activity (
      • Bacsi S.G.
      • Hankinson O.
      ). Differences in sensitivity to TCDD toxicity among rodent species have been attributed to differences in the C-terminal sequences of the respective AHRs (
      • Korkalainen M.
      • Tuomisto J.
      • Pohjanvirta R.
      ,
      • Korkalainen M.
      • Tuomisto J.
      • Pohjanvirta R.
      ). Our results are significant, because they identify a part of the AHR protein that appears to play a role in the ligand-regulated interactions between these previously identified domains. This suggests that the residue at this position in AHR is in communication with domains responsible for these different processes.
      Position 111 in rtAHR2β corresponds to the N-terminal border of the PAS-A domain that is strongly conserved in AHR proteins (Fig. 2). This position lies between the bHLH and PAS domains. Thus, it is close to regions involved in DNA binding, dimerization with ARNT and other proteins, as well as nuclear import and export signals. In addition, ligand binding in the PAS-B region must in some way transmit conformational changes to the PAS-A and helix-loop-helix domains. The residue at position 111 may be in a position to affect these processes. Clearly, a difference in residue polarity at such a central location could alter the function of the rtAHR2s.
      The crystal structure for the PAS domain of photoactive yellow protein (PYP) from Halorhodospira halophila has been determined (
      • Borgstahl G.E.
      • Williams D.R.
      • Getzoff E.D.
      ). This structure has been proposed as a model for PAS domains in eukaryotic proteins such as ARNT and AHR (
      • Pellequer J.L.
      • Wager-Smith K.A.
      • Kay S.A.
      • Getzoff E.D.
      ). Fig. 7 shows the structure of the PYP PAS domain with the position corresponding to the location of the rtAHR2β K111 highlighted in yellow on the polypeptide backbone (in PYP, this residue is methionine). Although lysine in position 111 in rtAHR2β is unique among AHRs, this region is not strongly conserved among all PAS proteins, and lysine is found at this position in some bacterial PAS family proteins. The degree of conservation between PAS domains from different proteins is indicated by color as well, with warmer colors indicating higher conservation (Fig. 7). It can be seen that the residue in question lies at the junction between the conserved PAS-A domain and the remainder of the N terminus of the protein. In PYP, this segment is referred to as the N-terminal cap, a part of the structure that helps enclose the hydrophobic portion of the PAS core (
      • Pellequer J.L.
      • Wager-Smith K.A.
      • Kay S.A.
      • Getzoff E.D.
      ). PAS domains are thought to function as protein interaction domains that can be regulated by signals. In the case of PYP, the signal is produced by isomerization of bound chromophore, a p-hydroxycinnamoyl anion. In the case of AHR, the signal is presumably generated by occupancy of the ligand-binding domain. Lysine 111 is situated at the edge of the PAS-A domain, in close proximity to the basic and helix-loop-helix domains. This residue is therefore in a position that might impact the interaction between these domains and PAS-A. Such a central position is consistent with the finding that this residue affects transactivation, choice of enhancer elements, and response to agonist.
      Figure thumbnail gr7
      Figure 7Location of rtAHR2β residue K111 on a model of PAS domain structure. The amino acid sequence for the PAS-A domain from rtAHR2β was aligned by RPS-BLAST to the consensus PAS domain sequence in the NCBI data base. The structure model was produced by the Cn-3D 3.0 structure viewer (www.ncbi.nlm.nih.gov/Structure/CN3D/cn3d.shtml). Redindicates strongly conserved PAS-domain sequences. The position of the K111 residue is indicated in yellow.
      Whereas our experiments identify a critical residue in AHR proteins and suggest divergent functions for rtAHR2α and rtAHR2β, they do little to explain the functions of rtAHR2α and rtAHR2β in rainbow trout. Our results suggest that rtAHR2α is a more powerful transactivator than rtAHR2β and as such might be more suitable for the regulation of genes that are more highly expressed than those controlled by rtAHR2β. On the other hand, with some enhancers rtAHR2β appears to be more tightly regulated by TCDD than rtAHR2α. This tight control is produced primarily by virtue of extremely low basal activity in the absence of TCDD. This would suggest that rtAHR2β might regulate genes that must be very closely controlled. It is interesting that this high -fold induction by rtAHR2β is reporter-specific. This suggests that TCDD is regulating more than the process of translocation to the nucleus and dimerization with ARNT, processes that by themselves should be DNA-independent and unaffected by the enhancers.
      As noted previously by Whitelaw et al. (
      • Whitelaw M.L.
      • Gustafsson J.A.
      • Poellinger L.
      ) transcriptional activation and TCDD responsiveness are substantially influenced by the enhancer elements used in the experiments. rtAHR2α is more responsive to TCDD than rtAHR2β with prt1Aluc, but this is reversed with pGudluc1.1. Although rtAHR2α appears to be a stronger transactivator than rtAHR2β, we cannot rule out the possibility that there might be promoter sequences with which rtAHR2β is the stronger transactivator. Thus, using the in vitro activities of these proteins alone to draw conclusions about function is limited by our current uncertainty about the gene targets for these receptors. Identifying these targets will be an important step forward in understanding AHR function in fish.

      Acknowledgments

      Dr. Richard Pollenz provided the rainbow trout ARNTb construct. We are also grateful to Ennis National Fish Hatchery for providing rainbow trout. We thank Alicia Sato for assistance in making the chimeric AHRs and Dorothy Nesbit for excellent technical support.

      REFERENCES

        • Gu Y.Z.
        • Hogenesch J.B.
        • Bradfield C.A.
        Annu. Rev. Pharmacol. Toxicol. 2000; 40: 519-561
        • Rowlands J.C.
        • Gustafsson J.A.
        Crit. Rev. Toxicol. 1997; 27: 109-134
        • Denison M.S.
        • Heath-Pagliuso S.
        Bull. Environ. Contam. Toxicol. 1998; 61: 557-568
        • Carver L.A.
        • Jackiw V.
        • Bradfield C.A.
        J. Biol. Chem. 1994; 269: 30109-30112
        • Carver L.A.
        • Bradfield C.A.
        J. Biol. Chem. 1997; 272: 11452-11456
        • Chen H.S.
        • Perdew G.H.
        J. Biol. Chem. 1994; 269: 27554-27558
        • Ma Q.
        • Whitlock Jr., J.P.
        J. Biol. Chem. 1997; 272: 8878-8884
        • Meyer B.K.
        • Petrulis J.R.
        • Perdew G.H.
        Cell Stress Chaperones. 2000; 5: 243-254
        • Schmidt J.V.
        • Bradfield C.A.
        Annu. Rev. Cell Dev. Biol. 1996; 12: 55-89
        • Whitlock Jr., J.P.
        Annu. Rev. Pharmacol. Toxicol. 1999; 39: 103-125
        • Ma Q.
        • Renzelli A.J.
        • Baldwin K.T.
        • Antonini J.M.
        J. Biol. Chem. 2000; 275: 12676-12683
        • Ma Q.
        • Baldwin K.T.
        J. Biol. Chem. 2000; 275: 8432-8438
        • Pollenz R.S.
        Mol. Pharmacol. 1996; 49: 391-398
        • Pollenz R.S.
        • Barbour E.R.
        Mol. Cell. Biol. 2000; 20: 6095-6104
        • Berg P.
        • Pongratz I.
        J. Biol. Chem. 2001; 276: 43231-43238
        • Ikuta T.
        • Eguchi H.
        • Tachibana T.
        • Yoneda Y.
        • Kawajiri K.
        J. Biol. Chem. 1998; 273: 2895-2904
        • Fukunaga B.N.
        • Probst M.R.
        • Reisz-Porszasz S.
        • Hankinson O.
        J. Biol. Chem. 1995; 270: 29270-29278
        • Coumailleau P.
        • Poellinger L.
        • Gustafsson J.A.
        • Whitelaw M.L.
        J. Biol. Chem. 1995; 270: 25291-25300
        • Bell D.R.
        • Poland A.
        J. Biol. Chem. 2000; 275: 36407-36414
        • Meyer B.K.
        • Perdew G.H.
        Biochemistry. 1999; 38: 8907-8917
        • Puga A.
        • Barnes S.J.
        • Dalton T.P.
        • Chang C.
        • Knudsen E.S.
        • Maier M.A.
        J. Biol. Chem. 2000; 275: 2943-2950
        • Elonen G.E.
        • Sphear R.L.
        • Holcombe G.W.
        • Johnson R.D.
        Environ. Toxicol. Chem. 1998; 17: 472-483
        • Walker M.K.
        • Peterson R.E.
        Aquat. Toxicol. 1991; 21: 219-238
        • Zabel E.W.
        • Cook P.M.
        • Peterson R.E.
        Aquat. Toxicol. 1995; 31: 315-328
        • Zabel E.W.
        • Pollenz R.
        • Peterson R.E.
        Environ. Toxicol. Chem. 1996; 15: 2310-2318
        • Hahn M.E.
        • Karchner S.I.
        • Shapiro M.A.
        • Perera S.A.
        Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13743-13748
        • Abnet C.C.
        • Tanguay R.L.
        • Hahn M.E.
        • Heideman W.
        • Peterson R.E.
        J. Biol. Chem. 1999; 274: 15159-15166
        • Karchner S.I.
        • Powell W.H.
        • Hahn M.E.
        J. Biol. Chem. 1999; 274: 33814-33824
        • Roy N.K.
        • Wirgin I.
        Arch. Biochem. Biophys. 1997; 344: 373-386
        • Tanguay R.L.
        • Abnet C.C.
        • Heideman W.
        • Peterson R.E.
        Biochim. Biophys. Acta. 1999; 1444: 35-48
        • Pollenz R.S.
        • Sullivan H.R.
        • Holmes J.
        • Necela B.
        • Peterson R.E.
        J. Biol. Chem. 1996; 271: 30886-30896
        • Powell W.H.
        • Karchner S.I.
        • Bright R.
        • Hahn M.E.
        Arch. Biochem. Biophys. 1999; 361: 156-163
        • Tanguay R.L.
        • Andreasen E.
        • Heideman W.
        • Peterson R.E.
        Biochim. Biophys. Acta. 2000; 1494: 117-128
        • Garrison P.M.
        • Tullis K.
        • Aarts J.M.
        • Brouwer A.
        • Giesy J.P.
        • Denison M.S.
        Fundam. Appl. Toxicol. 1996; 30: 194-203
        • Ema M.
        • Ohe N.
        • Suzuki M.
        • Mimura J.
        • Sogawa K.
        • Ikawa S.
        • Fujii-Kuriyama Y.
        J. Biol. Chem. 1994; 269: 27337-27343
        • Abnet C.C.
        • Tanguay R.L.
        • Heideman W.
        • Peterson R.E.
        Toxicol. Appl. Pharmacol. 1999; 159: 41-51
        • Whitelaw M.L.
        • Gustafsson J.A.
        • Poellinger L.
        Mol. Cell. Biol. 1994; 14: 8343-8355
        • Jain S.
        • Dolwick K.M.
        • Schmidt J.V.
        • Bradfield C.A.
        J. Biol. Chem. 1994; 269: 31518-31524
        • Ma Q.
        • Dong L.
        • Whitlock Jr., J.P.
        J. Biol. Chem. 1995; 270: 12697-12703
        • Jones L.C.
        • Whitlock Jr., J.P.
        J. Biol. Chem. 2001; 276: 25037-25042
        • Kumar M.B.
        • Ramadoss P.
        • Reen R.K.
        • Vanden Heuvel J.P.
        • Perdew G.H.
        J. Biol. Chem. 2001; 276: 42302-42310
        • Levine S.L.
        • Petrulis J.R.
        • Dubil A.
        • Perdew G.H.
        Mol. Pharmacol. 2000; 58: 1517-1524
        • Sun W.
        • Zhang J.
        • Hankinson O.
        J. Biol. Chem. 1997; 272: 31845-31854
        • Bacsi S.G.
        • Hankinson O.
        J. Biol. Chem. 1996; 271: 8843-8850
        • Korkalainen M.
        • Tuomisto J.
        • Pohjanvirta R.
        Biochem. Biophys. Res. Commun. 2000; 273: 272-281
        • Korkalainen M.
        • Tuomisto J.
        • Pohjanvirta R.
        Biochem. Biophys. Res. Commun. 2001; 285: 1121-1129
        • Borgstahl G.E.
        • Williams D.R.
        • Getzoff E.D.
        Biochemistry. 1995; 34: 6278-6287
        • Pellequer J.L.
        • Wager-Smith K.A.
        • Kay S.A.
        • Getzoff E.D.
        Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5884-5890