(Received for publication, July 14, 1995; and in revised form, September 21, 1995)
From the
Functional domains of the mouse aryl hydrocarbon receptor (Ahr) were investigated by deletion analysis. Ligand binding was localized to a region encompassing the PAS B repeat. The ligand-mediated dissociation of Ahr from the 90-kDa heat shock protein (HSP90) does not require the aryl hydrocarbon receptor nuclear translocator (Arnt), but it is slightly enhanced by this protein. One HSP90 molecule appears to bind within the PAS region. The other molecule of HSP90 appears to require interaction at two sites: one over the basic helix-loop-helix region, and the other located within the PAS region. Each mutant was analyzed for dimerization with full-length mouse Arnt and subsequent binding of the dimer to the xenobiotic responsive element (XRE). In order to minimize any artificial steric hindrances to dimerization and XRE binding, each Ahr mutant was also tested with an equivalently deleted Arnt mutant. The basic region of Ahr is required for XRE binding but not for dimerization. Both the first and second helices of the basic helix-loop-helix motif and the PAS region are required for dimerization. These last results are analogous to those previously obtained for Arnt (Reisz-Porszasz, S., Probst, M.R., Fukunaga, B. N., and Hankinson, O.(1994) Mol. Cell. Biol. 14, 6075-6086) compatible with the notion that equivalent regions of Ahr and Arnt associate with each other. Deletion of the carboxyl-terminal half of Ahr does not affect dimerization or XRE binding but, in contrast to an equivalent deletion of Arnt, eliminates biological activity as assessed by an in vivo transcriptional activation assay, suggesting that this region of Ahr plays a more prominent role in transcriptional activation of the cyp1a1 gene than the corresponding region of Arnt.
The aryl hydrocarbon receptor (Ahr) ()binds a variety
of environmentally important carcinogens, including polycyclic aromatic
hydrocarbons, and certain halogenated aromatic hydrocarbons, such as
TCDD. Following ligand treatment, induction of CYP1A1 and several other
enzymes involved in xenobiotic metabolism occurs in most tissues.
Certain of these enzymes (including CYP1A1) are involved in the
metabolism of polycyclic aromatic hydrocarbons to active genotoxic
metabolites, and Ahr therefore plays an important role in
carcinogenesis by these compounds. Ahr also mediates most, if not all,
of the carcinogenic and toxic effects of the halogenated aromatic
hydrocarbons, although metabolism of these compounds does not appear to
be involved (reviewed in (1) ).
The unliganded Ahr is located in the cytoplasm of the mouse hepatoma cell line, Hepa-1, as part of a complex that also contains two molecules of the 90-kDa heat shock protein (HSP90) and perhaps another protein of approximately 43 kDa(2) . It is not known whether both, one, or neither of the HSP90 molecules bind Ahr directly. HSP90 appears to be required for ligand binding by Ahr(3) . Binding of ligand leads to dissociation of Ahr from HSP90. In ligand-treated cells, Ahr is found in the nuclear fraction, from which it can be extracted in the form of a complex with the aryl hydrocarbon receptor nuclear translocator protein (Arnt)(4, 5) . This complex is probably a heterodimer of the two proteins, although the presence of additional small protein(s) has not been rigorously excluded. Arnt appears to be a nuclear protein in Hepa-1 cells(5, 6) . Some evidence indicates that dissociation of Ahr from HSP90 occurs in the nucleus and that HSP90 may play a direct role in translocating Ahr into this organelle(7, 8) . It has been proposed that Arnt promotes the dissociation of Ahr from HSP90(9) . Transcriptional activation of the cyp1a1 gene results from the binding of the Ahr/Arnt heterodimer to short DNA sequences, termed xenobiotic responsive elements (XREs), located in the 5`-flanking region of the gene(4, 10, 11, 12) . Both Ahr and Arnt bind directly to the XRE sequence(13) .
Mouse Ahr and Arnt are 20% identical in amino acid sequence. They also show a striking resemblance in overall structure(14, 15, 16) . Both proteins contain bHLH motifs toward their amino termini. However, Ahr and Arnt represent a novel subclass of bHLH-containing transcription factors because they differ from most or all other such proteins in that (i) activation of the Ahr complex requires ligand, (ii) the XRE sequence differs from the E-box sequence, which is the recognition sequence for nearly all other bHLH-containing transcription factors (reviewed in (17) ), and (iii) both proteins contain an approximately 300-amino acid segment of sequence similarity, called the PAS domain. The PAS domains of each protein contain two copies of an approximately 50-amino acid degenerate direct repeat, referred to as the PAS A and PAS B repeats. The Drosophila proteins single-minded (Sim) and period (Per) also contain PAS domains, and this domain has been shown to mediate homodimerization of Per and heterodimerization of Per with Sim(18) .
Deletion analysis indicates that both
-helices of the bHLH region of Arnt are required for dimerization
with Ahr and that the basic region is required for XRE binding but not
for dimerization(16) . The XRE sequence is asymmetrical. Arnt
binds to the side of the XRE that is identical in sequence to a
half-site of an E-box, while Ahr binds to the side of the XRE not
resembling an E-box half site(19) . This is consistent with the
observation that the basic region of Arnt conforms well to the
consensus sequence for other bHLH proteins, while the basic region of
Ahr conforms only poorly. Deletion of either the A or B segments of the
PAS region of Arnt slightly reduces dimerization with Ahr, while
deletion of the complete PAS region severely affects
dimerization(16) . Thus Arnt possesses multiple domains
required for maximal heterodimerization with Ahr.
We describe here a mutational analysis of Ahr that complements our previous mutational analysis of Arnt. We have used Ahr and Arnt proteins that are derived from the same species (mouse); have designed the deletion mutants such that individual functional domains are deleted precisely, so that we could accurately assess their putative roles in Ahr function; and have performed a comprehensive in vitro and in vivo phenotypic analysis of the mutants.
The C mutant of Ahr was generated by polymerase chain reaction
using pSportAhr as template. The 5` primer for
C was the same 5`
polymerase chain reaction primer used to generate the Ahr expression
construct pcDNAI/Neo/Ahr(16) , while the 3` primer contained a
four-nucleotide random sequence, a XhoI restriction site, a
stop codon, and the complement of Ahr bases 1181-1191. After
digestion with the appropriate restriction enzymes, the polymerase
chain reaction product was ligated into the similarly digested
pcDNAI/Neo vector (Invitrogen, San Diego, CA), for in vitro and in vivo expression. Internal deletion mutants of Ahr
were generated by the oligonucleotide directed mutagenesis system of
Nakamaye and Eckstein (20) using the Oligonucleotide-Directed In Vitro Mutagenesis System, version 2.1. (Amersham Corp.).
Mutagenesis of the Ahr cDNA was performed on a 1.2-kilobase SalI/BamHI fragment (1-1254 bp) from pSportAhr
cloned into M13 mp18. After confirmation of each mutation by
sequencing, the mutagenized fragment was transferred to pcDNAI/Neo for
expression. This was accomplished by a three-way ligation of a
1.039-kilobase SacII/NarI fragment of pcDNAI/Neo/Ahr
(containing pcDNAI/Neo sequence 1183-2196 bp and Ahr cDNA
sequence -15 to 11 bp), a NarI/BamHI-mutagenized fragment of the Ahr cDNA (bp
11-1254 less the deletion), and a 8.715-kilobase SacII/BamHI fragment of pcDNAI/Neo/Ahr (containing
pcDNAI/Neo sequences 0-1182 bp and 2258-6969 bp and Ahr
cDNA sequence 1255-3077 bp). This ligation altered the 5` region
of the pSportAhr fragment to that of pcDNAI/Neo/Ahr, which resulted in
a 4-fold increase in in vitro expression(16) .
Plasmids were prepared by the Qiagen maxiprep procedure according to
the supplier's protocols (Qiagen, Chatsworth, CA).
Figure 1: Structures of the Ahr mutants. The solid boxes represent the PAS A and PAS B direct repeats. The hatched areas flanking the repeats represent segments that show sequence similarity to Per, Sim, and Arnt.
Figure 2:
Ligand
binding analysis. Equimolar amounts of each protein were assayed for
binding to the photoaffinity ligand
2-azido-3-[I]iodo-7,8-dibromodibenzo-p-dioxin.
The results of a representative experiment are shown. The arrow in the
C lane points to the receptor-ligand complex, while
the uppermost band corresponds to material trapped between the
stacking gel and the separation gel.
Figure 3:
Interaction of Ahr with HSP90. A,
[S]-methionine labeled Ahr was incubated with 10
nM TCDD (+) or solvent alone(-), in the presence of an
equimolar amount of unlabeled, in vitro synthesized Arnt
protein and/or 7.5 nM double-stranded synthetic
oligonucleotide containing mouse XRE-1. The mixtures were incubated
with monoclonal antibody 3G3, immunoprecipitated with
anti-IgM-Sepharose, and subjected to SDS-PAGE and
-scanning. The
position of Ahr is indicated by an arrow. B, aliquots
of each protein were incubated with monoclonal antibody 3G3 (I) or a corresponding amount of control IgM (C),
immunoprecipitated with anti-IgM-Sepharose, and subjected to SDS-PAGE
and
-scanning. The molecular mass markers (in kDa) are indicated
on the left.
We also analyzed the binding of certain
Ahr mutants with HSP90. The results of a representative experiment are
presented in Fig. 3B. The degree of HSP90 binding to
each Ahr derivative was determined by quantitative radioanalytic
imaging (referred to as -scanning). The value for each mutant
relative to that for full-length Ahr in the same experiment was
calculated, and the mean values for four experiments are presented in Table 1.
A bound HSP90 at undiminished efficiency.
bHLH
and
B each bound HSP90 at approximately 50% efficiency.
AB
did not bind HSP90. HSP90 was not precipitated by the control
monoclonal antibody from any incubation.
Figure 4:
Dimerization analysis of the Ahr mutants.
The results of a representative experiment are shown. The mixtures
contained equimolar amounts of the indicated Ahr derivatives and 3-fold
greater molar amounts of radiolabeled Arnt. The Ahr antibody was used
throughout, except in the fifth and sixth lanes,
where preimmune IgG was used. C was subjected to SDS-PAGE using
15% polyacrylamide; 7.5% polyacrylamide was used for all the other Ahr
mutants. -, no TCDD treatment; +, TCDD treatment; p, immunoprecipitate; s, supernatant. The positions
of the molecular mass markers are indicated on the left.
The first six lanes of Fig. 4represent the controls for the co-immunoprecipitation assay and utilized full-length Ahr and Arnt incubated in the absence or presence of TCDD, and treated with preimmune IgG or Ahr antibodies, as indicated. The data demonstrate that TCDD treatment increased the amount of Arnt co-immunoprecipitated with Ahr and that very little Arnt was precipitated from the co-incubation mixture upon treatment with the preimmune IgG preparation.
Deletion of the complete helix region or individual
helices (bHLH,
HLH,
H1, and
H2) resulted in
proteins with greatly reduced or nondetectable ability to
co-immunoprecipitate with Arnt, demonstrating a requirement for both H1
and H2 for dimerization. Deletion of the complete PAS domain (
AB)
or either PAS A (
A) or PAS B (
B), also resulted in proteins
lacking the ability to dimerize with Arnt. The dimerization activity of
A was much lower than its ligand binding activity, suggesting that
the PAS A region may contribute toward dimerization. However, an
alternative explanation is that the PAS A region is not directly
involved in dimerization and that
A cannot dimerize because the
HLH and PAS B regions of the
A derivative of Ahr and full-length
Arnt cannot properly align (as discussed below). The basic domain
deletion mutant (
b) retained full ability to co-immunoprecipitate
with Arnt, demonstrating that this region is not involved in
dimerization. In addition, deletion of amino acids 398-805
(
C) also produced a protein with undiminished capacity to
associate with Arnt, indicating that no domains required for
dimerization exist in this region.
Figure 5:
XRE binding analysis of the Ahr mutants.
Equimolar amounts of Ahr and its mutant derivatives were mixed with
equimolar amounts of Arnt, incubated with or without 10 nM TCDD, as indicated, and subjected to gel mobility shift analysis.
C/bHLHAB is a mixture of Ahr
C and Arnt bHLHAB. The solid
arrow indicates the Ahr
Arnt
XRE complex. The open
arrow indicates free probe. The Ahr + TCDD lane is
from the same gel as the other samples.
Figure 6:
Dimerization between equivalent Ahr and
Arnt mutants. The results of a representative experiment are shown.
Mixtures contained equimolar amounts of Ahr or one of its mutant
derivatives (as indicated in the lower row) and 3-fold greater
molar amounts of radiolabeled Arnt or one of its mutant derivates (as
indicated in the upper row). The Ahr antibody was used
throughout. The Arnt bHLHAB plus Ahr C mixture was subjected to
SDS-PAGE using 15% polyacrylamide; 7.5% polyacrylamide was used for the
other mixtures. -, no TCDD treatment; +, TCDD treatment; p, immunoprecipitate; s,
supernatant.
Figure 7:
XRE binding of mixtures of mutant
derivatives of Ahr and Arnt. In the heading for each lane, the name of the Arnt derivative is indicated first, and
the name of the Ahr derivative is indicated second. The solid arrow indicates the AhrArnt
XRE complex. The open arrow indicates the free probe.
In the presence of TCDD, Ahr A dimerized with Arnt
A at 35% of the efficiency with which full-length Ahr and
full-length Arnt dimerized with each other. This is approximately the
same efficiency with which Ahr
A binds ligand and suggests that,
like the PAS A region of Arnt(16) , the PAS A region of Ahr
plays little if any role in dimerization. The poor dimerization
efficiency of Ahr
A with full-length Arnt is therefore probably
due to inappropriate association of the two proteins. In contrast to
their partial ability to dimerize, the two
A constructs, when
mixed together, were completely incapable of binding the XRE. Thus
either PAS A is directly involved in binding to the XRE (which would
appear to be unlikely), or the conformation of the heterodimer of Ahr
A and Arnt
A is still aberrant to such a degree that DNA
binding is precluded.
Certain Ahr mutant constructs were contransfected
with pMC6.3k, with and without Arnt into CV-1 cells. The cells were
treated with TCDD and then assayed for CAT activity. The CAT activity
of each construct (with or without Arnt) is presented as a percentage
of the activity obtained in the same experiment with cells
contransfected with full-length Ahr and Arnt and treated with TCDD. As
expected, bHLH,
b, and
A, which were shown not to bind
the XRE in the presence of full-length Arnt, did not produce
appreciable levels of CAT activity.
C, although possessing nearly
full in vitro dimerization and XRE-binding activities,
produced no significant CAT activity, even with Arnt cotransfection.
In no previous investigations have the roles of the individual subdomains of the bHLH motif or of the individual PAS regions of Ahr been reported, and in addition, in no previous reports have either the bHLH or PAS regions been excised precisely, which is necessary if the roles of these regions are to be assessed accurately. We have previously reported on analogous mutational studies of Arnt(16) . The various functional domains of Ahr deduced, as discussed below, from the current work and that of other investigators (29, 30, 31, 32, 33) are illustrated in Fig. 8.
Figure 8: Functional domains of mouse Ahr.
Whitelaw and co-workers (30) showed that a fusion protein containing only amino acids
230-421 of Ahr possessed nearly full ligand binding activity,
while Poland and co-workers (34) demonstrated that an Ahr
mutant containing amino acids 1-403 bound ligand with normal
efficiency. Since deletion of amino acids 398-805 in C had
no effect on ligand binding, our data allow us to narrow the boundaries
of a domain absolutely required for ligand binding to amino acids
230-397. Deletion of the PAS A region (amino acids 121-182)
reduced ligand binding by 70%. This is consistent with previous results
obtained with amino-terminal deletion mutants of
Ahr(29, 34) . Since HSP90 may be required for binding
of ligand to Ahr(3) , ligand binding assays probably define a
minimal region required for binding both HSP90 and ligand rather than a
region that contacts ligand alone.
Our Ahr-HSP90
co-immunoprecipitation experiments were carried out under conditions in
which increasing the amount of the antibodies to HSP90 led to a
proportionate increase in the amount of HSP90 precipitated. Therefore
the amount of each Ahr derivative coprecipitated with HSP90 should
reflect the degree to which the derivative binds HSP90. Since bHLH
binds only 50% of the amount of HSP90 as the full-length Ahr protein,
the bHLH region appears to contain a necessary binding site for one of
the HSP90 molecules. Interestingly, HSP90 has been shown to transiently
interact with several bHLH proteins, and a binding site on MyoD has
been mapped to a small region encompassing the bHLH region of this
protein(35, 36) . The results from
B suggest that
a necessary binding site for the other HSP90 molecule is contained
within amino acids 259-374. Since
AB is totally deficient in
HSP90 binding, but
A is unaffected, the segment from amino acid
182 to 374 appears to contain a region (or regions) required for
binding both HSP90 molecules. The HSP90 molecule binding in the region
between amino acids 259 and 374 may bind to an additional site
contained between amino acids 182 and 259. The results of Poellinger
and co-workers (30, 31, 37) also suggest that
HSP90 binds over the bHLH region and over a region encompassing the PAS
B repeat. However, these workers did not quantitate their results, and
they could not deduce that different HSP90 molecules bind over the two
regions. Furthermore many of their results were obtained with chimeric
proteins containing Ahr fragments fused with a portion of the
glucocorticoid receptor, and interpretation of these results is
potentially confounded by the fact that the glucocorticoid receptor
also binds HSP90, since it is possible that the segment of the
glucocorticoid receptor contained in the chimeras contributes toward
HSP90 binding.
In contrast to the results of McGuire and co-workers(9) , we observed that TCDD treatment reduced the binding of the in vitro translated Ahr to HSP90, thus reflecting the in vivo situation and contradicting the proposal of McGuire and co-workers (9) that Arnt is required for dissociation of Ahr from HSP90. It should be noted that a requirement for Arnt in the latter regard is difficult to reconcile with the observation that the free Ahr monomer appears to be an intermediate in the dissociation process (38) and with evidence that TCDD can trigger dissociation of HSP90 from Ahr in Arnt-defective mutants of Hepa-1 cells(30) . We did, nevertheless, obtain evidence that Arnt can enhance the dissociation of Ahr from HSP90.
As with Arnt, deletion of either -helix of the bHLH domain of
Ahr eliminated dimerization and XRE binding, while deletion of the
basic region eliminated XRE binding but not dimerization. Thus, like
Arnt, the basic region of Ahr is required for DNA binding but not for
dimerization. The observation that equivalent helix 1 deletion mutants
of Arnt and Ahr failed to dimerize and that equivalent helix 2 deletion
mutants also failed to dimerize reinforces the conclusion that both
helices are required for dimerization. Although the PAS A-deleted
mutant (
A) of Ahr was unable to dimerize with Arnt, this mutant
dimerized with Arnt
A at the same efficiency as it bound ligand.
This suggests that the inability of Ahr
A to dimerize with
full-length Arnt is due to steric hindrance to dimer formation between
these two proteins, and suggests that PAS A of Ahr is dispensable for
dimerization. Deletion of PAS A of Arnt was previously shown to have
little effect on its dimerization with Ahr(16) .
It has
previously been shown that a mutant (C516), containing amino acids
1-289, and deleted for most of the PAS B repeat, binds the XRE at
normal efficiency, although it does not bind ligand(29) . Thus
the presence of ligand bound to Ahr per se, is not required
for dimerization and subsequent XRE binding. Instead, the role of
ligand in these processes appears to be to trigger release of HSP90
from Ahr, thereby allowing the latter to dimerize with Arnt. Ahr
B
is deficient in dimerization. However, we can make no conclusion as to
whether the segment missing from
B is required for dimerization,
because the inability of this mutant to dimerize could be fully
ascribable to the fact that it retains HSP90 binding. (The results
obtained with C
516 demonstrate that if the segment missing from
B is in fact required for dimerization, the relevant portion must
be between amino acids 259 and 289). Interestingly, the equivalent
mutant of Arnt (bHLHA) is only mildly deficient in dimerization.
AB does not bind ligand or HSP90 and does not dimerize with Arnt,
indicating that although the PAS A region (and also perhaps the PAS B
region) is dispensable for dimerization, the presence of either the PAS
A or the PAS B region is required and that both cannot be deleted
without eliminating dimerization potential; or alternatively, the
region that is deleted in
AB, but not in either
A or
B,
is necessary for dimerization. Like Ahr, deletion of the complete PAS
domain of Arnt also prevents heterodimerization(16) .
Poellinger (37) and co-workers reported that a mouse Ahr mutant
deleted for most of the PAS region (deleted for amino acids
84-340 and therefore not identical to our
AB mutant) could
dimerize with human Arnt but did not bind the XRE. However, they did
not quantitate their results, and importantly did not compare
dimerization of the mutant with that of full-length Ahr (37) .
Our results therefore indicate that helix 1, helix 2, and the PAS region of Ahr are all involved in dimerization with Arnt. The most plausible model for dimerization is that the various dimerization domains of Ahr associate with their corresponding counterparts in Arnt. All our results are consistent with this model.
Deletion of amino
acids 398-805 in C did not affect dimerization and reduced
XRE binding to 75% of that of full-length Ahr. This contrasts with
results of Dolwick and co-workers(29) . They found that a
mutant containing amino acids 1-492 only possessed 36% of
wild-type binding activity. However, they assessed binding using the
mouse Ahr mutant in conjunction with human Arnt, whereas our Ahr and
Arnt constructs (and the mutant derivatives) were both of mouse origin.
Our results are therefore of more biological relevance. The
carboxyl-terminal halves of both Ahr and Arnt have been shown to have
transcriptional activation potential when fused to heterologous DNA
binding domains. The transcriptional activation domains encompass the
glutamine-rich regions of the proteins and, at least in the case of
Ahr, includes flanking regions as well. The amino-terminal halves of
the proteins are devoid of transcriptional activation potential in
these assays.(31, 33, 39) . We found that Ahr
C was inactive in the in vivo CAT assay. However, we
previously found that an equivalently deleted mutant of Arnt (bHLHAB)
suffered only a 50% reduction of activity when analyzed in a similar
fashion(16) . Thus the transcriptional activation domain of Ahr
appears to play a more important role than that of Arnt with regard to
activation of the cyp1a1 gene. Whitelaw and co-workers (31) came to the completely opposite conclusion. Our results
are more likely to reflect the true in vivo situation,
however, because our reporter plasmid for the CAT assays contained the
normal architecture of the cyp1a1 enhancer-promoter, whereas
Whitelaw and co-workers' reporter plasmid was an artificial
construct, containing a single XRE element linked to the mouse mammary
tumor virus promoter. Transcriptional activation of the cyp1a1 gene results from cooperative interaction of Ahr
Arnt dimers
at several XREs and their coordinated interaction with proteins at the
promoter, including components of the general transcription
machinery(40) . In Whitelaw's construct, the proteins at
the heterologous promoter will probably not correspond to those at the cyp1a1 promoter, and transcriptional activation of this
construct may occur in an aberrant fashion relative to the normal cyp1a1 promoter.