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INTRODUCTION |
In contrast to mammals, fish can undergo gonadal sex inversion in
either direction by treatment with exogenous sex steroid if it is
applied early enough during development (1, 2). These observations led
to the postulate that androgens and estrogens are the substances
responsible for sex differentiation of male and female fish,
respectively (3), and has resulted in the development of protocols for
the masculinization and feminization of large numbers of fish for
experimental or economic purposes (1, 4, 5). The androgen receptor
(AR)1 is a critical mediator
of male sexual differentiation and development in both fish and
mammals. Structurally and functionally, the AR belongs to the
superfamily of ligand-responsive transcription modifiers, which
encompasses the receptors for the steroid and thyroid hormones. Like
other nuclear receptors, steroid receptors are composed of three major
functional domains: an NH2-terminal hypervariable
transcriptional activation domain (TAD), a central highly conserved DNA
binding domain (DBD) consisting of two Cys-Cys zinc finger motifs, and
a COOH-terminal ligand binding domain (LBD) (6-9). It has been
reported that the AR regulates androgen target genes by binding to a
specific DNA sequence, the androgen-responsive element (ARE;
consensus = 5'-GGTACANNNTGTTCT), which is similar to the
glucocorticoid response element (12-14). The AR can either up- or
down-regulate the expression of androgen target genes, the outcome
probably depending on interactions with specific adapters or
co-activators (10, 11).
At present, complete AR cDNAs have been cloned only from mammalian
species (human, rat, and mouse) (6, 15). We have undertaken the
isolation of the rainbow trout homologue of the androgen receptor in
order to study its potential role in salmonid fish sexual
differentiation, development, and general physiology and to further
explore structure-function relationships through sequence comparisons.
We report that there are two isoforms of AR mRNA in rainbow trout.
These two isoforms, which were present in all tissues examined, encode
96.1- and 95.8-kDa proteins that are highly homologous to mammalian AR.
Surprisingly, when the biological activities of the two isoforms were
examined in a fish cell line using a transient expression assay, the
96.1-kDa protein was highly active, while the 95.8-kDa AR was inactive.
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EXPERIMENTAL PROCEDURES |
Isolation and Characterization of Rainbow Trout Androgen Receptor
cDNAs--
In order to isolate the rainbow trout androgen receptor
cDNA, a probe was first generated by PCR. Oligonucleotide primers were designed based on the amino acid sequence of the highly central conserved region of the AR and then used to amplify rainbow trout testis cDNA as described previously (16). Products of the expected size were subcloned into the pSK vector (Stratagene) and sequenced. The
primers, which yielded a 569-bp fragment highly homologous to human
were as follows: primer 1, TA(T/C)TG(T/C)AA(A/G)AA(T/C)GG(A/G/C/T)GG (A/G/C/T)TT(T/C)TT; primer 2, CAT(A/G/C/T)GG(A/G/C/T)A(A/G)(A/G)AA(A/G/C/T)A(A/G)(A/G/T)AT(A/G/C/T)GC(T/C)TT(T/C)TG. A rainbow trout testis random hexanucleotide primed cDNA library in
ZapII (Stratagene) was screened by hybridization with a probe made
from the cloned PCR fragment. Positive clones were rescued as
pBluescript plasmids by in vivo excision and sequenced. Two independent clones, pAR-
and pAR-
, were isolated.
Reverse Transcriptase PCR--
Reverse transcriptase PCR was
conducted as described previously (17) with some modifications.
Briefly, poly(A)-selected RNA was prepared from various rainbow trout
tissues. The cDNAs were synthesized from 100 ng of poly(A) selected
RNA using a first strand synthesis kit (Stratagene). An aliquot of the
first strand reaction was then subjected to PCR analysis using rtAR-
specific primers
-1 (GGACATCCATGCACAGATA) and
-2
(CATGTGCTGGGGGTT) (which generated a 225-bp product) or
rtAR-
-specific primers
-1 (ACAATATGGACCGAGGCA) and
-2
(ATGGGCAGTTCTTCCTTCTC) (which generated a 451-bp product).
Plasmid Construction--
The pARE3TK-CAT reporter plasmid,
which carries three androgen-responsive elements in front of a TK
(thymidine kinase) promoter, was a kind gift of Dr. J. Trapman (18).
The pMSG-CAT reporter plasmid, which carries the MMTV (mouse mammary
tumor virus) promoter, was purchased from Amersham Pharmacia Biotech.
To construct the expression vectors, plasmids pAR-
and pAR-
were
amplified by PCR using a sense primer homologous to the NH2-terminal end of the coding sequence and an antisense
primer homologous to the COOH-terminal end. In addition, the coding
sequences in each primer were flanked by EcoRI or
HindIII sites for the rtAR-
and rtAR-
sequences,
respectively. The resultant products were cleaved with EcoRI
or HindIII to yield fragments of 2577 bp (rtAR-
) or 2574 bp (rtAR-
) and then subcloned into EcoRI- or
HindIII-digested pBluescript SK+ (Stratagene) to yield
pSK-rtAR-
and pSK-rtAR-
, respectively. Following
verification of their sequences, the EcoRI and
HindIII fragments were excised and subcloned into
EcoRI or HindIII digested pcDNA3
(Invitrogen) to yield pCMV-rtAR-
and pCMV-rtAR-
, respectively.
Chimeric Plasmid Construction--
To facilitate construction of
chimeric receptors, plasmids were first made in which the TAD, DBD, or
LBD could be separately excised by flanking the DBD with
ApaI and XbaI sites using site-directed mutagenesis as described previously (19, 20). Single-stranded DNA was
prepared from pSK-rtAR-
, pSK-rtAR-
, and pSK-rtGR-I (21) for
mutagenesis, and mutations were confirmed by DNA sequencing. The
positions at which these sites were introduced and the resultant plasmids were as follows: at amino acids 495 and 562 for rtAR-
, yielding pSK-AX/rtAR-
; at amino acids 494 and 561 for rtAR-
, yielding pSK-AX/rtAR-
; and at amino acids 420 and 485 for rtGR-I (21), yielding pSK-AX/rtGR-I. Chimeras were then constructed by
subcloning combinations of fragments from these three plasmids into
appropriately digested pcDNA3 as follows: pCMV-A
A
G contains the 1683-bp EcoRI/XbaI fragment from
pSK-AX/rtAR-
and the 813-bp XbaI/EcoRI
fragment from pSK-AX/rtGR-I; pCMV-A
GG contains the 1482-bp
EcoRI/ApaI fragment from pSK-AX/rtAR-
and the
1008-bp ApaI/EcoRI fragment from pSK-AX/rtGR-I;
pCMV-GA
A
contains the 1077-bp ApaI/EcoRI
fragment from pSK-AX/rtAR-
and and the 1260-bp EcoRI/ApaI fragment from pSK-AX/rtGR-I;
pCMV-GGA
contains the 876-bp XbaI/EcoRI:
fragment from pSK-AX/rtAR-
and the 1455-bp EcoRI/XbaI fragment from pSK-AX/rtGR-I; and
pCMV-A
A
A
contains the 1683-bp
EcoRI/XbaI: fragment from pSK-AX/rtAR-
and the
813-bp XbaI/HindIII fragment from
pSK-AX/rtAR-
.
Constructs chimeric for subregions of the LBD were created by using PCR
methodology, and all chimeric constructs were verified by DNA
sequencing. The LBD of rtAR-
was divided into four segments, corresponding to amino acid positions 561-630, 631-712, 713-784, and
785-853, carrying 7, 5, 4, and 5 amino acid substitutions, respectively, when compared with rtAR-
. Plasmids
pCMV-AR-


, pCMV-AR-


, pCMV-AR-


, and
pCMV-AR-


were then constructed by replacing segment I, II,
III, or IV of rtAR-
with the corresponding segment of rtAR-
,
respectively (Fig. 6). Details of plasmid constructions are available
on request.
Transient AR Expression in Cultured Cells--
EPC cells (22),
derived from carp, were cultured at 27 °C in Dulbecco's modified
Eagle's/Ham's F-12 medium (1:1) supplemented with 10% fetal calf
serum. A mixture of the indicated amounts of each receptor plasmid and
2 µg of reporter plasmid (pARE3TK-CAT or pMSG-CAT) was co-transfected
into subconfluent cultures of EPC cells in each well of a six-well
plate (Corning) using the calcium phosphate method (16). CAT assays
were performed as described previously (21).
Antibody Preparation--
The amino-terminal portion of AR-
(amino acids 1-200, Fig. 1A) was expressed as a histidine
fusion protein in Escherichia coli, purified by
nickel-agarose (23) and used to immunize rabbits. The specificity of
the antibody was examined by immunoblot analysis. Immunoblotting was
performed as described previously (17).
Ligand Binding Assays--
COS-1 cells were plated at 3 × 106 cells/150-cm2 flask and were transfected
with 24 µg of each expression plasmid. Forty-eight hours after
transfection, cells were harvested in phosphate-buffered saline,
centrifuged at 5000 rpm for 10 min at 4 °C, and the cell pellet was
resuspended in 1 ml of TEGM buffer (10 mM Tris-HCl, 1 mM EDTA, 10% (v/v) glycerol, 1 mM
mercaptoethanol, 10 mM sodium molybdate, pH 7.2). The
suspension was homogenized and then sonicated on ice and centrifuged at
55,000 rpm for 30 min. The supernatant was used to perform binding
assays using the method described by Tilley et al. (24),
except that we used [3H]mibolerone instead of
[3H]dihydrotestosterone. The Kd
(dissociation constant) and Bmax were calculated
by nonlinear regression of specific binding data (Graphpad Prism, San
Diego, CA).
Immunocytochemistry--
COS-1 cells were plated at 1 × 105 cells/well on two-well chamber slides and were
transfected with 2 µg of rtAR-
expression plasmid or rtAR-
expression plasmid. Twenty-four hours after transfection, MT was added
to 10
7 M, and after another 24 h the
cells were washed in phosphate-buffered saline and fixed with 4%
paraformaldhyde in phosphate-buffered saline. Immunocytochemical
staining was performed as described previously (25), except that
Cy3-conjugated sheep anti-rabbit IgG (Sigma) was used for visualization.
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RESULTS |
Isolation of Two Distinct cDNAs Encoding the Rainbow Trout
Androgen Receptors--
As a first step in isolating the rainbow trout
AR cDNA clone, we designed a set of degenerate oligonucleotide
primers homologous to an amino acid sequence that is highly conserved
between the human and mouse AR. These primers were then used to
generate PCR fragments from rainbow trout testis cDNA to isolate a
fragment that would serve as a screening probe. A PCR product of the
length predicted from the human AR gene was isolated, cloned, and
sequenced. The sequence encoded 190 amino acids that had 70% identity
with the corresponding region of the human AR. Using a probe generated from this fragment, approximately 1 × 106 plaques
from a rainbow trout testis cDNA library were screened, and several
strongly hybridizing clones were obtained and sequenced. The sequences
obtained revealed two different AR homologues, designated rtAR-
, and
rtAR-
(accession numbers for nucleotide sequences are AB012095 for
rtAR-
and AB012096 for rtAR-
). Both sequences contain an ATG
initiation codon followed by an extended open reading frame; the
rtAR-
cDNA encodes an 854 amino acid protein of a molecular mass
of 96.1 kDa, and the rtAR-
cDNA encodes an 853-amino acid
protein of a molecular mass of 95.8 kDa (Fig.
1, A and B, respectively). Both of the encoded proteins contain all of the domains
that characterize the steroid hormone receptor family, including a
striking canonical leucine zipper structure in the COOH-terminal
LBD.

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Fig. 1.
Deduced amino acid sequence of rainbow trout
androgen receptor AR- (A) and
AR- (B). DNA binding
domains are shown by black boxes. The heptad repeat of
leucines in the putative leucine zipper in the LBD is shown by
asterisks.
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Structure of Rainbow Trout AR-
and AR-
--
Comparison of
the predicted amino acid sequence of the rtAR-
with that of rtAR-
reveals 85% identity with continuity. Comparison of the three
different functional domains of the rtAR-
to those of rtAR-
shows
a homology of 79% for the TAD (amino acids 1-495), 97% for the DBA
(amino acids 496-562), and 93% for the LBD (amino acids 563-854)
(Fig. 2). The observation that
differences occur throughout the coding regions indicates that these
two predicted proteins arise from different genes rather than by
alternative splicing. However, since the testis cDNA library used
to isolate the clones was constructed from pooled RNA from several
fishes, it was also possible that they are encoded by two different
alleles in the population. To test this, PCR fragments spanning the
NH2-terminal portion of the two cDNAs were amplified
and sequenced from one rainbow trout testis and ovary. The presence of
the two isoforms were confirmed in each case (data not shown).

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Fig. 2.
Comparison of the deduced amino acid
sequences for AR- and AR-
of rainbow trout, human, and mouse. Sequence identities are
shown byblack boxes. The individual leucines of the putative
leucine zipper structure in the COOH-terminal LBD are marked by
asterisks. Gaps are indicated by dashes.
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Comparison of the amino acid sequence of rtAR-
and rtAR-
with
that of the mouse and human ARs is shown in Fig. 2. The amino acid
sequence of the DBD of rtAR-
and rtAR-
shows 92 and 95% identity
with that of human AR (the DBD of human AR is identical to that of
mouse AR). Similarly, the amino acid sequence of the LBD of rtAR-
and rtAR-
shows 65 and 68% identity with that of human AR (the LBD
of human AR is 97% identical to that of mouse AR). Thus, both the DNA
binding and ligand binding domains are highly conserved. On the other
hand, the NH2-terminal TAD shows less sequence similarity
than does the COOH-terminal portion (only 19% sequence identity
between rtAR-
/rtAR-
and the human AR).
Tissue Distribution of Rainbow Trout AR-
and AR-
mRNA--
We used the highly sensitive RT-PCR technique to survey
the tissue distribution of rtAR-
and rtAR-
mRNA in rainbow
trout. The PCR primers used were targeted to non-homologous segments of
the two sequences so that the presence of each transcript could be
evaluated (Fig. 3, lanes 1 and
2). Both rtAR-
and rtAR-
mRNA expression could be
detected in all tissues tested (Fig. 3). No amplification was seen when
the AMV reverse transcriptase was omitted from the cDNA synthesis
reaction (data not shown).

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Fig. 3.
Expression of rainbow trout
AR- and AR-
mRNA. Poly(A)+ RNA from various tissues was
used to generate cDNA for PCR amplification using rainbow trout
AR- - or AR- -specific primers asdescribed under"Experimental
Procedures." The PCR products were size fractionated and visualized
by ethidium bromide staining. Primers specific for the rainbow trout
-actin were used as a control.
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Biological Activities of Rainbow Trout AR-
and AR-
--
To
determine the biological activities of rtAR-
and rtAR-
, cells
were co-transfected with an rtAR expression vector and an
androgen-responsive reporter gene. The expression vectors were made by
inserting the rtAR-
and rtAR-
cDNAs into a CMV expression vector to yield pCMV-rtAR-
and pCMV-rtAR-
. For the reporter gene
we used pMSG-CAT, which carries the MMTV promoter driving expression of
the bacterial CAT reporter gene (Fig.
4A). Although the expression
of many genes is known to be androgen dependent, only a few have been
shown to be directly regulated by androgen, and the MMTV promoter is
the best studied among these (14). The MMTV promoter is regulated by
glucocorticoids and progestins, but is also able to confer androgen
responsiveness to a reporter gene, although less efficiently.

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Fig. 4.
Transcriptional activity of rainbow trout
AR- and AR- in EPC
cells. A, schematic representation of reporter
plasmids. B, EPC cells were co-transfected with pMSG-CAT
reporter plasmid and either pCMV-rtAR- or pCMV-rtAR- expression
plasmid as described under "Experimental Procedures." C,
EPC cells were co-transfected with pARE3TK-CAT reporter plasmid and
either pCMV-rtAR- or pCMV-rtAR- expression plasmid. Averaged
values are presented from three independent-transfections. Note that
the scale of B is different from C.
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As it has been shown that the level of androgen inducibility in a
transient transfection assay is dependent on the cell type used, we
first screened several fish cell lines by co-transfection with the
pMSG-CAT reporter plasmid and either the pCMV-rtAR-
or pCMV-rtAR-
expression plasmid. As a result of this screening, we selected the EPC
cell line derived from carp (22). Transfection of the pMSG-CAT reporter
plasmid into the EPC cell line produced very low basal expression of
CAT, and it was not induced by MT (Fig. 4B). In contrast,
upon co-transfection of the pMSG-CAT reporter plasmid and the rtAR-
expression plasmid, MT stimulated the level of CAT protein expression
about 5-fold (Fig. 4B). Surprisingly, co-transfection of
pMSG-CAT and pCMV-rtAR-
yielded no significant transactivation (Fig.
4B).
To further test the activities of rtAR-
and rtAR-
, we used
another reporter plasmid, pARE3TK-CAT, which carries three androgen responsive elements derived from the prostate-specific antigen promoter
(15) just upstream of the TK basal promoter (Fig. 4A). The
results obtained were similar to those using the pMSG-CAT reporter
plasmid; the CAT expression levels produced by pARE3TK-CAT were low in
either the absence or presence of MT, but were significantly enhanced
(about 7-fold) in the presence of MT upon co-transfection with
pCMV-rtAR-
but not with pCMV-rtAR-
(Fig. 4C). These
results indicate that the rtAR-
is capable of producing a fully
functional receptor that mediates hormone-dependent
transcriptional activation, whereas the highly homologous rtAR-
is
inactive. When EPC cells were co-transfected with pCMV-rtAR-
and
pARE3TK-CAT in the presence of the other steroids (dexamethasone,
aldosterone, progesterone, and 17
-estradiol), CAT expression was not
induced (data not shown). When we also used the co-transfection assay
to examine the response of the rtAR-
to the same set of steroids
including MT, no induction was observed with any of them (data not shown).
Functional Domain Mapping of the rtAR-
--
To map the
region(s) of rtAR-
that renders it inactive, a series of chimeric
expression plasmids was constructed that carry various combinations of
the TAD, DBD, and LBD regions from rtAR-
, rtAR-
, or the rainbow
trout GR-I (21). To facilitate construction of the chimeric plasmids,
we flanked the DNA binding domains of each receptor with
ApaI and XbaI restriction sites. The chimeric constructs produced are illustrated in Fig.
5. These chimeric plasmids were
transiently co-transfected with pARE3TK-CAT into EPC cells in the
presence or absence of MT or dexamethasone. Co-transfection of
pCMV-rtGR-I into EPC cells results in strong
dexamethasone-dependent CAT expression from the pARE3TK-CAT
reporter plasmid (Fig. 5), indicating that the DBD of rtGR could
functionally recognize the androgen-responsive element. The results
from the various chimeric constructs suggest that the inactivity of the
rtAR-
is due to its LBD; for example, a chimera composed of the rtGR
TAD and DBD and the rtAR-
LBD is inactive, whereas one composed of
the rtAR-
TAD and DBD and a LBD from either the rtGR or rtAR-
is
highly active (Fig. 5). The activity of the latter chimera,
pCMV-A
A
A
, is in fact about 2-fold greater than that of
pCMV-rtAR-
.

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Fig. 5.
Mapping the inactive domain of
rtAR- using chimeric proteins. EPC cells
were co-transfected with pARE3TK-CAT reporter plasmid and theindicated
chimeric expression plasmid. Twenty-four hours after transfection, the
indicated steroids at 10 7 M were added and
48 h after transfection, the cells were harvested. The fold
induction of CAT expression levels was determined relative to basal
expression levels in the absence of steroids. Averaged values are
presented from three independent transfections. DEX,
dexamethasone.
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To further localize the region of rtAR-
responsible for its
inactivity, we subdivided its LBD into four segments and made constructs in which each was individually replaced with the
corresponding segment from rtAR-
(Fig.
6). The four segments extended from amino
acids 562-630, 631-712, 713-784, and 785-853 and contained 7, 5, 4, and 5 amino acid differences between the isoforms, respectively. The
results of transfections using the rtAR-
/
chimera constructs, expressed in fold induction by MT, are shown in Fig. 6. Surprisingly, three of the four chimeric constructs supported a dramatic MT-regulated response, similar to that seen with pCMV-rtAR-
. Thus the inability of rtAR-
to activate transcription depends on the simultaneous presence of sequences distributed throughout the LBD.

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Fig. 6.
Transactivation of
rtAR- /rtAR- chimeric
expression plasmid. EPC cells were co-transfected with pARE3TK-CAT
reporter plasmid and theindicated chimeric expression plasmid.
Twenty-four hours after transfection, the indicated steroids at
10 7 M were added, and 48 h after
transfection, the cells were harvested. The fold induction of CAT
expression levels was determined relative to basal expression levels in
the absence of MT. Averaged values are presented from three independent
transfections. a.a., amino acid.
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Ligand Binding Characteristics of the rtAR-
and
rtAR-
--
Since the inability of rtAR-
to activate
transcription resides in its LBD, we next examined the ligand binding
abilities of the rtAR-
, rtAR-
, and chimeric rtARs. A high level
of [3H]mibolerone binding was observed in extracts
prepared from cells transfected with pCMV-rtAR-
or the active
chimeric expression plasmids pCMV-AR-A
A
A
, -


,
-


, and -


(Fig.
7). However, when cells were transfected
with the pCMV vector as a negative control, or with the inactive
chimeras pCMV-rtAR-
and pCMV-AR-


, specific
[3H]mibolerone binding was absent (Fig. 7). The complete
correlation between mibolerone binding and transcriptional activation
strongly suggests that the inability of rtAR-
to transactivate is
due to its poor ligand binding affinity.

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Fig. 7.
Binding of [3H]mibolerone to
recombinant rtAR- ,
rtAR- , and
rtAR- /rtAR- chimeric
proteins expressed in COS-1 cells. Cell extracts were incubated
with 1 nM [3H]mibolerone in the absence total
binding) or presence nonspecific binding) of 1000-fold excess unlabeled
mibolerone. Specific binding was calculated as total minus nonspecific
binding.
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Next, we performed an analysis of [3H]mibolerone binding
in cytosol fractions derived from COS-1 cells transiently transfected with pCMV-rtAR-
. Saturation ligand binding analysis and Scatchard analysis revealed a Kd of 0.16 nM and
Bmax of 178 fmol/mg of protein (data not shown).
These values are similar to those of the human AR
(Kd = 0.5 nM and
Bmax = 357 fmol/mg protein) (24).
Nuclear Translocation Abilities of rtAR-
and rtAR-
--
If
rtAR-
binds androgens poorly in vivo, it might remain in
the cytoplasm in the presence of MT, whereas the rtAR-
would translocate to the nucleus. This possibility was tested by analyzing transfected COS-1 cells by fluorescent immunocytochemistry, using an
antibody raised against bacterially expressed rtAR-
protein (NH2-terminal 200 amino acids; see "Experimental
Procedures"). This antibody was shown by immunoblot analysis of
transfected cell lysates from COS-1 cells to specifically detect both
the rtAR-
and the rtAR-
proteins (data not shown). Only low
background staining was observed if cells were transfected with pCMV
lacking an AR sequence (Fig.
8A), and no staining was
observed following transfection of any of the expression plasmids when
preimmune antisera was used (data not shown). In the presence of MT,
the AR-
showed strong nuclear staining, whereas a striking
cytoplasmic distribution was observed for AR-
(Fig. 8, B
and C). The remaining chimeric expression plasmids (see Fig.
7) were also examined for their nuclear translocation ability. Only the
AR-


protein showed cytoplasmic staining in the presence of
MT (data not shown). Thus, the inability of rtAR-
to mediate
transactivation correlated with failure to be translocated to the
nucleus.

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Fig. 8.
Immunofluorescent localization of
rtAR- and rtAR-
expressed in COS-1 cells. COS-1 cells were transfected with
2 µg of the indicated expression plasmid. Twenty-four hours after
transfection, MT was added to 10 7 M, and
48 h after transfection, the cells were fixed for analysis. ARs
were visualized by indirect immunofluorescence with anti-rtAR-
antibody using Cy3-conjugate. Magnification was × 500. A, pCMV; B, pCMV-rtAR- ; C,
pCMV-rtAR- .
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 |
DISCUSSION |
The function of steroid hormones in the development and sexual
differentiation of fish has been well documented at the physiological and endocrinological levels. At the molecular level, both the estrogen
receptor and glucocorticoid receptor have been cloned and characterized
from various fish species, but sequence information for the AR has not
been available. In order to elucidate the molecular mechanism of
androgen action in the early stage of sexual differentiation and
spermatogenesis, we isolated AR cDNA clones from rainbow trout testis.
By using PCR to generate a homologous probe, we isolated two distinct
isoforms of rainbow trout AR cDNA clones that contain the entire
coding sequence for the protein. Both encoded protein sequences are
highly homologous to that of other species, especially in their
COOH-terminal halves. Because this region includes the DNA binding
domain, it is not unexpected that the DBD of both rtAR-
and rtAR-
could recognize the human AR response element (for rtAR-
based on
activity of LBD chimeras; e.g. pCMV-A
A
G in Fig. 6).
Homology between the human AR and rtAR-
is less strong at the
NH2-terminal half (19% identical) than at the
COOH-terminal half (DBD, 92% identical; LBD, 64% identical). The
lower homology in the NH2-terminal half could be
significant for regulation of gene activity, as this segment contains
the region involved in transcriptional activation.
Precedence for isoforms of a sex steroid receptor was set recently by
the discovery of a second estrogen receptor, ER-
(26, 27). ER-
shares many functional similarities with the classic estrogen receptor,
ER-
, including substrate and antagonist binding affinities. However,
there are differences in their tissue localization and in the
mechanisms regulating their transcriptional activities. In the case of
the AR, all data suggest that there is only a single gene in human
(28). In contrast, our results demonstrate the existence of two
distinct isoforms of ARs in rainbow trout. Interestingly, despite their
highly similar amino acid sequences, one is active in our transient
assay, and the other is not (Fig. 4).
Several lines of evidence demonstrate that the inactivity of rtAR-
in our assays is due to the sequence of its LBD. Interchanging the
rtAR-
or GR-I LBD for that of rtAR-
in chimeric proteins produced
an active receptor; and surprisingly, even replacement by the
corresponding rtAR-
segment of any one of three out of four
subregions of the LBD was sufficient to produce activity. Consistent
with this protein mapping data, ligand binding assays using cell
extracts from transfected COS-1 cells revealed that rtAR-
bound
[3H]mibolerone with an affinity comparable with that of
the human AR, whereas no binding could be detected with rtAR-
(Fig.
7). An absence of ligand binding by rtAR-
in vivo was
indicated by its failure to translocate to the nucleus when transfected
COS-1 cells were treated with MT (Fig. 8). Furthermore, tests of the various chimeric constructs show a perfect correlation between transcriptional activation, ligand binding, and nuclear translocation. Thus, all our data indicate that the rtAR-
is inactive in the transient assay due to a lack of ligand binding.
The rtAR-
that we have isolated presumably mediates the androgen
responses in the rainbow trout. The function of the rtAR-
, however,
is unclear. We consider four possibilities. One is that the rtAR-
gene is a duplication of the rtAR-
that has devolved to
nonfunctionality. This seems quite unlikely, however, as none of the
130 amino acid changes has resulted in introduction of a nonsense
codon, an unlikely probability (less than 0.5%) if rtAR-
has no
function. A second possibility is that rtAR-
is indeed inactive, but
serves to modulate the activity of rtAR-
by forming a heterodimer.
However, in preliminary experiments we have found no effect of
co-transfection of the rtAR-
expression vector on the activity
produced by rtAR-
. Generation of antibodies specific for either
rtAR-
or rtAR-
, now under way, will permit assay for heterodimers
in extracts of fish tissues. A third possibility is that the rtAR-
requires an accessory factor for hormone binding that is not present in
either the EPC or COS-1 cells that we have used. The alteration in
rtAR-
that prevents binding is apparently quite subtle, as
replacements of small subsections of the LBD with the corresponding
rtAR-
sequence restores activity. In fact, as the result of an
inadvertent PCR-induced mutation we found that activity was restored by
substitution of the glutamine at position 646 with an arginine (data
not shown). The tenuousness of the binding deficit suggests it might be
readily reversed by association of the rtAR-
with an accessory
factor. On the other hand, whereas there is ample precedent for
accessory proteins affecting the transcriptional potency of hormone
receptors (10, 11), there is none for affecting ligand binding.
Finally, it is possible that the ligand for rtAR-
differs from those
that we have tested (methyltestosterone, dexamethasone, aldosterone, progesterone, and 17
-estradiol), perhaps being unique to fish. The
antibody specific for rtAR-
that we are in the process of preparing
should also be useful for examining this question, by determining if
rtAR-
appears in the nucleus of fish cells.
In summary, the molecular cloning of the fish AR cDNAs described
here identifies AR proteins that will be important for determining the
mechanism of sex determination in fish. In addition, these proteins
will be useful for comparative, structure-function analyses of
mammalian ARs. Finally, the discovery of a highly homologous AR
isoform, rtAR-
, which is inactive in the conventional assays we have
used, raises the possibility of a novel function for a hormone receptor.