Download Production of Recombinant Androgen Receptor in a Heterologous

CLIN. CHEM. 39/2, 346-352
(1993)
Production of Recombinant Androgen Receptor in a Heterologous Expression
System
0111 A. Jflnne,”2
Jorma J. Palvimo,’
Pekka
Kallio,’
Merja Mehto,’
To facilitate detailed studies of androgen receptor, we
have produced a full-length receptor protein and some of
its deletion mutants in Spodopterafrugiperda(Sf9) insect
cells, using the baculovirus expression system. Recombinant baculovirus DNA-infected Sf9 cells expressed
these proteins in very high quantities, which represented
as much as 30-40% of total insect cell proteinat 72 h after
infection. Only <1% of the recombinant protein was
soluble in low-salt buffers; the majority formed electrondense cytoplasmic aggregates 30-40 nm in diameter.
These aggregates could be solubilized in 6 mol/L guanidine HCI, and biologically active receptor was generated
by diluting the guanidine HCI preparation 20- to 50-fold.
The full-length receptor, expressed either in a soluble or
aggregated form, had characteristics typical of a native
receptor: it bound steroids with high affinity and specificity, interacted with DNA in a sequence-specific fashion,
and was recognized by domain-specific receptor antibodies. Androgen-receptor protein purifiedto homogeneity in
guanidine HCI required the presence of Zn2 ions during
the refolding to reconstitute its DNA-binding form; ZnCl2
was not, however, needed to restore the receptor’s steroid-binding activity.
IndexingTerms:steroid hormones
Ic virus
receptorprotein .
‘
gene expression
recombinant
bacu-
Yan-Bo
induced phenotype.
Xie,2 and
Ya-Ping
Acquisition
Sui2
of this phenotype
nonreproductive
organs (1).
Availability
of purified androgen-receptor
protein is
essential
for many structural
and functional
studies;
therefore,
the protein should be available in large quantities. However, this receptor is present in androgentarget tissues in very low concentrations (7), which,
along with its poor stability,
has made itimpossible to
purify
the receptor in amounts
needed for detailed
structure-function
experiments.Truncated forms ofthe
androgen-receptor protein have been produced in both
prokaryotic and eukaryotic expression vectors (8-12).
Our laboratory has adopted the baculovirus expression
system (13, 14) to generate a full-length form of this
protein in large quantities (15). Here, we will review
our experience
with the baculovirus
expression system
in a large-scale production of the full-length androgen
receptor and some of its variant forms, and compare our
data with those obtained in other laboratories, who have
used a similar approach for a heterologous production of
other members ofthe steroid/thyroid
hormone receptor
superfamily.
DNA
Structural Features of the Androgen-Receptor
Androgens play an essential role in the differentiation, development, and maintenance
of normal male
reproductive
functions.
In addition, many sexually dimorphic responses in nongenital
tissues are controlled
by androgens through mechanisms
that appear to be
identical to those in the organs of the reproductive tract;
therefore, classification
of biological responses to male
sex steroids
as either androgenic or anabolic is not very
informative
(1). Physiological
androgens
such as testosterone and 5a-dthydrotestosterone
elicit their actions by
binding to intracellular
receptor proteins from a family
of ligand-responsive
transcription
factors. This large
protein family includes receptors for different classes of
steroid hormones, multiple receptor species for thyroid
hormones and retinoic acids, vitamin D receptor, and
several so-called orphan receptors, whose physiological
ligands are currently unknown (2-6). Receptor-androgen complexes interact with regulatory regions of specific genes and thereby modulate their activity, which
eventually results in the expression of an androgen1 Department
tavuorenpenger
correspondence).
of Physiology, University of Helsinki, Sil20 J, SF-00170 Helsinki, Finland (address for
2The Population Council, New York, NY 10021.
Received August 6, 1992; accepted October 19, 1992.
346
occurs
in a cell- and tissue-specific fashion; it involves both
hyperplastic and hypertrophic
growth responses in the
accessory
sex organs but only a hypertrophic
response
in
CLINICAL CHEMISTRY, Vol. 39, No. 2, 1993
Protein
Typical structural features of a protein belonging to
the steroid/thyroid
hormone receptor superfamily
are
depicted in Figure 1. The androgen receptor is one of the
largestproteinsin this family; its deduced amino acid
sequence contains 917-919 residues,from which one
can predict a molecular size of ---98 000 Da for the
human receptor (16-19). The reported sequence for the
rat androgen receptor is slightly shorter, 902 amino
acids (16, 18). The overall molecular size of an individual member in the receptor superfamily is largely determined by the length of its modulator (or transactivation) domain, also termed the A/B region, which extends
from the N-terminus to the beginning
of the DNAbinding domain (Figure 1). The length of this sequence
ranges from ‘-25 amino acid residues (vitamin
D receptor) to >600 residues (mineralocorticoid
receptor). The
function of the A/B domain has not been delineated in
sufficient detail for any one of the members, but this
region contains sequence
information
for at least two
key features:(a)optimizationof transactivation
capability of the receptor, and (b) gene recognition specificity
for the transcriptional
regulation.
An example of the
latter feature is the presence of two forms of progester-
one receptor (A and B forms), which differ in the length
of their A/B domains and which have been shown to
A/B
[25-600]
H2N-
C
D
[66-68] [50-70]
E
[220-250]
MODULATOR
#{149}
Transactivatlon
Highly variable
#{149}
Gene-specificresponses
Immuneresponse
SpecIlic DNA
binding
#{149}
Nuclear
localization
Dim.rizatIon
#{149}
Specificilgand binding
#{149}
Transactivation
#{149}
NuclearlocalIzatIon
#{149}
Dlmerlzatibn
hspeObinding
#{149}
hsp9ObInding(7)
Fig. 1. Schematicrepresentationof the structuraldomainsof a proteinbelongingto the steroid/thyroidhormonereceptorsuperfamily
The numbers in brackets depict the approximatenumberof aminoacidresiduesin each of the four regions.
Some of the putativefunctionalcharacteiistlcsof the
domainsare also listed. Adapted fromreferences2-6
exhibit gene-specific
regulatory
functions (20). The A/B
domains among the different family members have the
least-conserved
amino
acid sequences; consequently,
they represent most immunogenic
parts of the proteins.
It is also probable that this lack of identity of amino acid
sequences will enable interaction of a given A/B domain
with other cellular proteins in a receptor-specific fashion.
The human
androgen
receptor contains
long homopolymeric
amino acid segments that comprise motifs
of 21 glutamine
residues, 8 proline residues, and 24
glycines in the transactivation domain (6). The lengths
of these segments vary among healthy individuals,
indicating that the androgen-receptor
protein is polymorphic. The rat receptor contains similar poly(amino
acid) motifs, although their positions in the A/B domain
differ from those in the human
receptor (16,18).
Interestingly, X-linked spinal and bulbar muscular atrophy
(Kennedy disease), an adult-onset form of motor neuron
disease, has recently been found to contain an increased
size of the polyglutamine region in the transactivation
domain (CAG repeats in the receptor gene) (21,22). The
amplified CAG repeats were absolutely associated
with
the disease and were about twice as long in the affected
patients as in healthy control subjects (21, 22). The
biological function of the polyglutamine region or the
other poly(amino acid) motifs of the A/B domain is not
known;
however, similar repeats have been identified,
not only in other steroid receptors, but also in several
transcription-regulating
proteins,
particularly
in the
homeotic
gene products (23). Enlargement
of the polyglutamine
repeat appears to impair the function of the
androgen
receptor, at least to some extent, because the
patients with Kennedy disease show late-onset signs of
defective androgen action. The affected patients suffer
from degeneration of motor neurons, the growth
of
which may be controlled in a cell-specific fashion by the
transactivation
domain
of the androgen
receptor.
The DNA-binding domain of steroid receptors consists
of 66-68 amino acid residues. This region is cysteinerich and appears to fold into two so-called zinc finger
structures, with one zinc atom being tetrahedrically
coordinated
with four cysteines in each case (2-6).
Human
and rat androgen receptors have identical
amino acid sequences in this domain. The similarity
between the androgen receptor and other steroid hormone receptors is also greatest
in the DNA-binding
domain. For example, this region has an 82% amino acid
sequence identity with the human progesterone
receptor, 79% with human glucocorticoid and mineralocorticoid receptors, 59% with the human estrogen receptor,
and --40-50% with other members of the steroid/thyroid
hormone receptor superfamily
(16-19). The DNA-binding domain interacts
with cia-acting
elements of the
regulated
genes, known as the hormone-responsive
elements, which are usually located
in the 5’-fianking
region of the gene. A unique androgen-responsive
element (ARE) has not yet been defined; in some instances,
such as the mouse mammary
tumor virus long terminal
repeat, the same element that mediates induction by
glucocorticoids
and progestins also confers androgenresponsiveness on a heterologous promoter (24, 25).
Plausibly, there are indeed no distinct AREs, or if there
are, they are very similar to the responsive elements for
glucocorticoids
and progestins. As mentioned above, the
DNA-binding
domain
of the androgen receptor also
displays a remarkable
amino acid sequence
identity
with the glucocorticoid, progesterone, and mineralocorticoid receptors.
This amino acid sequence similarity
in
the DNA-binding domains and the nucleotide sequence
identity of hormone-responsive elements in genes regulated by four different steroids poses an intriguing
biological enigma: how are the unique actions of these
four steroids actually specified under physiological conditions? In some cases, this may be exerted through the
presence or absence of a given receptor; however, a
simple abundance of receptor may not suffice in most
cases. It is, therefore, logical to assume that an important additional determinant for the hormone specificity
lies in the interaction of the A/B domain with cell- and
tissue-specific proteins, along with general transcription factors.
The C-terminal domain of the receptor proteins, which
encompasses -250 amino acid residues, is involved in the
ligand-binding
and in the interaction
with heat-shock
proteins such as hsp90 (Figure 1). The similarity of this
sequence between the androgen
receptor and other steroid receptors agrees with previous data from in vitro
steroid-binding
studies and biological
experiments
in
vivo (26), in that androgens, progestins, and glucocorticoids can, at least to some extent, interact with more than
3Nonstandard
abbreviations: ARE, androgen-responsive
element; nt, nucleotides; Sf, Spodoptera frugiperda; and Gdn, gunnidine.
CLINICALCHEMISTRY, Vol. 39, No. 2, 1993
347
one receptor type. This is particularly
true for synthetic
steroids, which may have higher affinities for multiple
receptors than does the corresponding
physiological hormone for its cognate receptor (27). Although both of the
two physiological
androgens,
testosterone
and 5a-dihydrotestosterone,
interact directly with the androgen receptor and mediate hormonal responses, in certain tissues conversion
of testosterone
to the more potent agonist
5a-dihydrotestosterone
is required for the steroid action
to occur. This requirement
is particularly clear during
male sexual development, when formation
of 5a-dihydrotestosterone
is mandatory
for virilization
of external
genitalia and development of the prostate gland. Virilization of the Wolifian duct structures
is, however,
thought to be mediated by testosterone
(28). Because the
mstmmnlian
genome
contains only one androgen-receptor gene, these effects are most probably mediated by a
single receptor protein. Furthermore,
immunoblotting
studies with receptor antibodies have failed to uncover
the presence of multiple, tissue-specific
androgen-receptor species (29, 30).
Production of Androgen Receptors In a
System
Construction
of the Recombinant
Heterologous
Baculovirus Strains
A full-length rat androgen receptor cDNA was ininto the baculovirus transfer plasmid pVLl393
by use of the EcoRI and Pst I sites on the multiple
cloning sequence as described in detail previously (15).
The transfer plasmid, termed pVXrAR, contained the
entire protein-coding
region and 32 nucleotides (nt) and
87 nt of the 5’- and 3’-untranslated
regions of the
mRNA, respectively.
Because the initiation codon for
the polyhedrin protein translation
is mutated
in
pVL1393, translation of the RNA encoded by the recombinant baculovirus initiates at the authentic AUG of the
coding sequence for androgen receptor mRNA. Recombinant baculovirus
DNA (designated AcrAR) was generated in Spodoptera frugiperda (Sf9) cells by homologous recombination with wild-type viral (AcNPV) DNA
and pVXrAR DNA by standard techniques
(13-15).
Sf9 insect cells infected with AcrAR DNA for 3-4 days
produced extremely high quantities of androgen-receptor protein that migrated on polyacrylamide
gel electrophoresis at Mr = 110 000, indicating that a full-length
protein was indeed formed in the recipient cells. At the
time of peak protein production, the amount of receptor
in infected Sf9 cells ranged from 30% to 50% of total
insect cell protein (15). However, only a fraction of the
recombinant
receptor was soluble upon homogenization
of infected Sf9 cells in buffers containing
nonionic detergents or moderate salt concentrations, with the soluble fraction representing usually <1% of the total recombinant receptor content (15). The rest of the receptor
population remained
as aggregated
cytoplasmic particles, 30-40 nm in diameter, and exhibited a relatively
regular
shape in transmission
electron microscopy.
These two receptor populations had identical characteristics in all physical-chemical
and functional
studies
performed so far, including molecular size, steroid-bindserted
348 CLINICAL CHEMISTRY, Vol. 39, No. 2, 1993
ing affInity and specificity, antibody recognition,
and
binding to different AREs (15, and below). The reason
for this very high level of androgen-receptor
expression
is currently
unknown
to us, but it may relate to the
structure
or the length of the 5’-untranslated
region in
the construct used to generate the AcrAR baculovirus
strain. In this context, we note that the length of the
5’-untranslated
region reportedly
influences the expression of recombinant
l,25-dihydroxyvitamin
D3 receptor
in insect cells (31).
In addition to the full-length
receptor,
we also produced several deletion mutants for the rat androgen
receptor cDNA, and generated
respective
recombinant
baculovirus strains for expression of mutated proteins
in insect cells. Schematic representation
of some of the
mutated proteins is shown in Figure 2. In these experiments, the basic transfer plasmid construct
was the
same, i.e., the pVLl393 series, and the 5’-untranslated
sequence
of androgen
receptor
znRNA was kept unchanged.
In more recent studies, we used linearized
baculovirus DNA in lieu of the circular wild-type AcNPV DNA to produce recombinant
virus strains
by
homologous recombination and found that this improves
the yield of recombinant
plaques. As was the case with
the production of full-length
receptor protein, the mutated proteins were expressed to very high quantities
and represented
-30% of total insect cell protein 72 h
after infection
(Palvimo
et al., unpublished
results).
Distribution
of the expressed recombinant
protein between the soluble and aggregated
forms was not significantly influenced by the above mutations: only a minor
portion of each recombinant protein could be released
from Sf9 cells by homogenization in low-salt buffers.
NATIVE H,N
MODULATOR
1
46-408
542
907
690
902
H,N
-COOH
38-296
S40-147
787-902
H,N
n,N_Ilt\
OOH
r///Ao1
H,N
767
902
Fig. 2.Structures of thefull-length androgen receptor and Its mutants
produced ininsect cells by useof the baculowus expressionsystem
The numbers underneaththe full-length protein depict the beginnIngand the
endofthe venous domains in
the rat androgen
receptor.
Deletionmutants
were constructed as follows: M6-408, a fragment
betweentwoNhe I sites
was deleted;38-296, a fragment between two Sinai siteshas been deleted;
4O-147, the sequencebetweentwo Apa Isiteswas deleted; and 787-9O2,
a fragmentbetweenan EcoRl site and the end of the codingregionhas been
removed
Properties of the Recombinant Receptor Proteins
As mentioned previously, no significant functional
differences
were observed between the two forms in
which the recombinant
receptors were expressed; therefore, the experimental
data reviewed below for one form
can be extnded
to include the other form, too. The
aggregates receptor was routinely solubilized in 6 mol/L
guanidine
HCI (GdnHC1) at room temperature for 1 h,
after which it was either purified to virtual homogeneity
by gel-exclusion chromatography
(15) or used without
further
enrichment
for subsequent
studies. Recombinant receptor was, of course, biologically inactive in 6
mol/L GdnHC1, but it regained most of its functional
characteristics
upon dilution of the GdnHCl by a factor
of 20-50. This renaturation applied to all activities
tested, including sequence-specific
interaction with
DNA, steroid-specific hormone binding, and recognition
by domain-specific antisera (15).
The presence of Zn2 ions in the dilution buffer used
for reconstituting
the recombinant receptor from 6
moIIL GdnHC1 was required for the receptor’s acquisition of specific DNA binding. The optimal Zn2 ion
concentration was -50 imolIL, higher concentrations
being inhibitory. Of the several other divalent cations
tested, Cd2 ions were as active as Zn2 ions but at
-10-fold lower concentration; and, similar to ZnCl2,
higher CdCl2 concentrations prevented receptor binding
to ARE sequences. As mentioned previously, the DNAbinding
domain of all steroid receptors contains two
cysteine-rich zinc “fingers,” motifs that are required for
their binding to the hormone-responsive elements (2-6).
A recombinant glucocorticoid receptor fragment encompassing the DNA-binding domain (total length: 150
amino acids) purified to homogeneity has been previously shown to ligate reversibly two Zn2 ions (32).
Release of the zinc ions from the protein by chelating
agents yielded an apoprotein that failed to bind to its
target DNA element; the binding was restored by a
preincubation with Zn2 or Cd2 ions. Our studies with
the full-length
recombinant androgen receptor purified
to homogeneity in the presence of 6 mol/L GdnHC1 by
gel filtration thus agreed completely with the data on
the glucocorticoid receptor and indicated that Zn2 ions
are essential for folding the receptor from GdnHCl in
such a way that it acquires the specific DNA-binding
function. In our experiments
(15), the optimal ZnC12
concentration of -50 moI/L was one-fifth the optimum
(250 pmo1/L)
reported by Freedman et al. (32) for the
DNA-binding
domain of the recombinant glucocorticoid
receptor.
The reason for this difference is not known.
However, the optimal Cd2 ion concentration for the
full-length androgen receptor was the same as that for
the DNA-binding
domain of the glucocorticoid receptor
(5 moI/L in both cases) (15, 32).
The time course of appearance of androgen binding
(Id = 5 nmol/L for [3H}mibolerone, a synthetic
androgen) in the soluble cytosol of Sf9 cells revealed that, as
measured by the steroid-binding capacity, -150090
receptors/cell
were present by 72 h after infection (15).
Even though the solublereceptorfractionrepresented
<1% of the total expressed receptor population in Sf9
cells, its concentration was at least fivefold greater than
that reported for rat prostate, which is supposed to be
the richest tissue source for the receptor. An identical
time course of accumulation was found by using immunoblotting to estimate receptor protein content in both
soluble and aggregated forms. The steroid-binding
specificity of the soluble or reconstituted androgen receptor
expressed in insect cells was very similar to that of the
native receptor in its physiological
target tissues (33), in
that the rank order of competition was androgen
>
estradiol > progesterone
> glucocorticoid. The equilibrium dissociation constant for [3H]mibolerone binding
(5 nmol/L) was somewhat higher for the recombinant
protein purified to homogeneity than that reported for
the physiologically
occurring
receptor form-which
may
indicate that some auxiliary
proteins are required for
this event. However, in another study, no such protein
was needed for a truncated androgen receptor expressed
in prokaryotic cells to bind its cognate ligand with high
affinity
(10).
An additional criterion to evaluate functional properties of the recombinant receptor is its ability to interact
with specific DNA motifs. We have used for this purpose
a glucocorticoid-responsive
element (GRE) of the tyrosine aminotransferase
gene, which has been previously
shown to confer androgen responsiveness
upon a reporter gene (34), and an ARE present in the first intron
of the rat C3(1) gene (35). Soluble extracts from’ AcrARinfected cells, or samples reconstituted from 6 mol/L
GdnHC1 in the presence of ZnCl2, interacted in a nudeotide sequence-specific
fashion with the two DNA elements, as revealed by band-shift experiments
(15). Samples that were prepared and incubated
in the presence of
50 nmolJL 5a-dihydrotestosterone
yielded results idenwith those studied
in the absence
of the steroid.
The mutant receptor with a C-terminal deletion (787902, Figure 2) bound to ARE sequences as well as the
full-length receptor. DNA-binding
experiments
conducted with the deletion mutants M6-408
and 38-296
revealed that a partial removal of the A/B domain did
not abolish the ability of these variant receptors to
interact with specific DNA sequences; however, the
apparent affinity of the two mutants for ARE sequences
was not as high as that of the full-length receptor
(Palvimo et al., unpublished observations). Properties of
the other variant receptors depicted in Figure 2 have
not yet been studied in this respect.
Several
antisera
that we have raised (15, 36) in
rabbits against defined sequences corresponding to various regions of the androgen receptor have proven very
useful reagents.
The locations of these sequences in the
human receptor are illustrated in Figure 3. Three of the
antisera were particularly valuable: antibodies against
androgen receptor peptide 3 (N-terminal),
peptide 4
(mid-region of the A/B domain), and peptide 5 (hinge
region). The first antiserum,
corresponding
to residues
14-32 of the human receptor,
reacted
well with all
recombinant
receptors
(Figure
2) so far produced
in
tical
CLINICAL CHEMISTRY, Vol. 39, No. 2, 1993
349
A/B
C
559
D
E
624 676
919
MODULATOR
f
IMMUNOBLOUiNG:
ARP3
ARP4
[1 4-321
1250-2681
.++
++
++
+++
DNABINDING:
t
..
ARPI
1479-4951
+
t
ARP5
[626-644J
At
ARP2
[770.786J
++
‘P
Fig. 3. Locationof the peptidesequencesusedto raiseandrogenreceptorantibodies
Schematicrepresentation of the human receptorstructure is based on the sequence published by Lubahnet aI. (17).
Properties
of these antiserahave been
describedin detail in previous publications from this laboratory (15, 36). The number of + signsin immunoblottingindicates how wellan antiserum performed in
Western blotting
and (or) immunohistochemistry. In the DNA-bindingexperiments, + refers to stabilizationand up-shifting of ARE-receptor compiexes, - refers
to inhibitionof ARE-receptor interaction, and ? indicatesthatthis function
has notyetbeen testedby gel-retardation
experiments
insect cells, indicating that the synthesis of these proteins indeed commences at the authentic AUG codon of
the receptor mRNA. This antibody
along with that
raised against androgen receptor peptide 4 served as
excellent specificity controls in band-shift experiments,
because
they both supershifted
and stabilized AREreceptor complexes (Figure 4). The fact that responsive
element-receptor
complexes
were significantly
stabilized by these antibodies
may be interpreted
to indicate
that accessory proteins are involved in the formation of
this complex under in vivo conditions. The stabilization
effect appeared to be more dramatic
with the deletion
Ab:+-
Ab-AR
+
+
+
.
mutants than with the full-length recombinant receptor. By contrast, the hinge-region antiserum (against
androgen receptor peptide 5) inhibited the interaction of
both soluble and renatured receptor preparations with
specific DNA elements (Figure 4). Two possible mechanisms may account for this effect: (a) direct masking by
the antibody of the zinc finger region in the receptor,
and (b) inhibition of receptor dimerization,
which may
be important
for the formation of specific DNA-receptor
complexes (2-6). It remains to be elucidated whether
this particular
antiserum
also blunts androgen
receptor-dependent transcription
in vitro, in a fashion similar
to an antiserum
directed against the DNA-binding domain of the estrogen
receptor,
which prevented
both
estrogen response element-receptor
interaction and estrogen receptor-mediated
transcription in a cell-free
system (37).
AR
Expression of Other Members of the Steroid Receptor
Superfamily In the Baculovlrus System
During the last few years, several members of the
steroid/thyroid
hormone receptor superfamily
have been
overexpressed
by use of the baculovirus
system. These
include at least the following proteins: mouse and hu-
F14
Fig. 4. Influence of domain-specific androgen receptorantibodieson
the interactionbetweena full-lengthrecombinant receptorprotein
and an androgen-responsiveelement (ARE) of the rat tyrosine
aminotransferasegene, as demonstratedby an electrophoretic
mobilityshift assay
Receptor preparations originated from solubleinsectcell extracts
and were
prepared as describedby Xie et al. (15). F, free [P1ARE; AR, androgen
receptor-[P)ARE complex; Ab-AR, antibody-androgen receptor-(’P1ARE
complex; NAS. normal rabbit serum;
Ab, antiserum; ARP3, ARP4, and ARP5,
antiseraraisedagainstAR peptides3,4,and 5,respectively
(seeFig.3). The
two bendsbetweenfree 1P1ARE and AR-ARE complexes representnonspecific
DNA-proteininteractionof unknownnature
350 CLINICAL CHEMISTRY, Vol. 39, No. 2,
1993
man estrogen receptor (37, 38), human and chicken
progesterone receptors (39, 40), human and rat glucocorticoid receptor
(41, 42), human
mineralocorticoid
receptor (43), rat and human vitamin D receptor (31,
44), and human thyroid hormone receptor 131 (45, 46).
The results from these studies indicate very clearly that
the baculovirus expression system is well suited for a
large-scale production of the ligand-induced
transcription factors, such as those belonging to the steroidl
thyroid
hormone receptor superfamily.
A relatively
high level of expression was achieved in all cases and,
even more importantly,
biologically active full-length
receptors were produced in every instance.
The level of
expression in our studies with the androgen
appeared
to be higher than the expression
receptor
of other
receptors cited above, although accurate comparisons
cannot be made because different methods were used to
extract receptors from infected cells. Furthermore,
we
are not aware of other reports on steroid/thyroid hormone receptor overproduction
with the baculovirus sys-
tern, in which much aggregated
expressed.
of the expressed
proto that in our work has been reported for
tein similar
other
form of the receptor was
However, aggregation
proteins
produced
in insect
cells,
such
as protein
phosphatase 1 (47) and urate oxidase (48). Aggregation
did not necessarily
present a problem in any of these
cases; for instance,
the initial insolubility
permitted
purification of the androgen receptor to virtual homogeneity in a single
gel-filtration
step (15).
Except for the human and chicken progesterone receptors (39, 40), all other intracellular receptors expressed in insect cells were able to interact with specific
DNA elements in a hormone-independent fashion. The
reason for this dissimilarity remains to be elucidated.
Possibly there are inherent differences among the members of this superfamily in their activation process, i.e.,
the mechanisms
by which their DNA-binding
activity is
generated. However, it is equally likely that this disparity relates to the different experimental
conditions used
in different studies.
steroid binding, DNA binding, and immunological
Endocrinol
10. Ohara-Nemoto Y, Nemoto T, Ota M. The Mr 90,000 heat shock
protein-free androgen receptor has a high affinity for steroid, in
contrast to the glucocorticoid receptor. J Biochem 1991;109:113-9.
11. Quarmby VE, Kemppainen JA, Sar M, Lubahn DB, French
FS, Wilson EM. Expression of recombinant androgen receptor in
cultured mammalian cells. Mol Endocrinol 1990;4:1399-407.
12. Tan J-A, Marschke KB, Ho K-C, Perry ST, Wilson EM, French
FS. Response elements of the androgen-regulated
C3 gene. J Biol
Chem 1992;267:4456-86.
13. Smith GE, Fraser MJ, Summers MD. Molecular engineering
of the Autographa californica nuclear polyhedrosis virus genome:
deletion mutations within the polyhedrin gene. J Virol 1983;46:
584-93.
14. Luckow VA, Summers MD. Trends in the development of
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