MOLECULAR AND CELLULAR BIOLOGY, Mar. 1994, p. 1956-1963
Copyright ©) 1994, American Society for Microbiology
Characterization of a Novel 23-Kilodalton Protein of Unactive
Progesterone Receptor Complexes
JILL L. JOHNSON,'* THOMAS G. BEITO,2 CHRISTOPHER J. KRCO,2 AND DAVID 0. TOFT'
Department of Biochemistry and Molecular Biology' and Department of Immunology, 2
Mayo Graduate School, Rochester, Minnesota 55905
Received 14 September 1993/Returned for modification 15 October 1993/Accepted 2 December 1993
Immunoprecipitation of unactivated avian progesterone receptor results in the copurification of hsp90,
hsp70, and three additional proteins, p54, p50, and p23. p23 is also present in immunoaffinity-purified hsp90
complexes along with hsp7O and another protein, p60. Antibody and cDNA probes for p23 were prepared in an
effort to elucidate the significance and function of this protein. Antibodies to p23 detect similar levels of p23
in all tissues tested and cross-react with a protein of the same size in mice, rabbits, guinea pigs, humans, and
Saccharomyces cerevisiae, indicating that p23 is a conserved protein of broad tissue distribution. These
antibodies were used to screen a chicken brain cDNA library, resulting in the isolation of a 468-bp partial
cDNA clone encoding a sequence containing four sequences corresponding to peptide fragments isolated from
chicken p23. This partial clone was subsequently used to isolate a full-length human cDNA clone. The human
cDNA encodes a protein of 160 amino acids that does not show homology to previously identified proteins. The
chicken and human cDNAs are 88% identical at the DNA level and 96.3% identical at the protein level. p23 is
a highly acidic phosphoprotein with an aspartic acid-rich carboxy-terminal domain. Bacterially overexpressed
human p23 was used to raise several monoclonal antibodies to p23. These antibodies specifically immunopre-
cipitate p23 in complex with hsp90 in all tissues tested and can be used to immunoaffinity isolate progesterone
receptor complexes from chicken oviduct cytosol.
Unactivated steroid hormone receptors exist in low-salt
tissue extracts as 8 or 9S complexes that are composed of
distinct cellular proteins in addition to the hormone receptor
itself. In the presence of hormone, these complexes dissociate,
leaving the 4S form of the receptor that is capable of dimer-
ization and binding to DNA. In the absence of hormone, the
native avian progesterone receptor (PR) associates with hsp9o,
hsp7o, and three additional proteins, p54, p50, and p23 (30).
The binding of these proteins is salt dependent and stabilized
by molybdate (30).
As the components of the various steroid receptor com-
plexes have been examined (reviewed in references 22 and 34),
hsp90 has been found in association with glucocorticoid,
androgen, estrogen, and mineralocorticoid receptors, as well as
PR. Comparative studies on the other receptor-associated
proteins are less complete. hsp70 has been observed with
chicken and mammalian PR (12, 20) and with some forms of
the glucocorticoid receptor (27). A 59-kDa heat shock protein
(p59, hsp56) (26) associates with mammalian progesterone,
estrogen, androgen, and glucocorticoid receptors (37). p59 has
been shown to bind the immunosuppressant drug FK506 (14,
36, 39). It belongs to the family of FK506-binding proteins
(FKBPs) and has also been called FKBP52 (21). The p54 and
p50 proteins seen in the chick PR complex have recently been
shown to belong to the same family of FKBPs, with p50 more
closely related to p59 (29). Thus, with the exception of p23, the
major receptor-associated proteins have been identified as
either heat shock proteins or immunophilins even though their
significance to receptor structure and function remains un-
In this report we characterize p23, the 23-kDa protein
*Corresponding author. Mailing address: Department of Biochem-
istry and Molecular Biology, Mayo Clinic, Rochester, MN 55905.
Phone: (507) 284-3074. Fax: (507) 284-2053.
originally found in the chicken PR complex. p23 is also present
in some hsp90 complexes. p23 is a highly conserved, novel
protein with no structural homology to known proteins. In
addition, antibodies which specifically immunoprecipitate p23
coprecipitate the chicken PR along with hsp90 and hsp70.
MATERIALS AND METHODS
Preparation of tissue cytosol. Oviduct cytosol was freshly
prepared from estradiol-stimulated chicks (31). Soluble tissue
homogenates from chicken brain, liver, oviduct, and spleen;
mouse liver; and guinea pig liver were prepared in 4 volumes of
50 mM potassium phosphate (pH 7.4)-10 mM thioglycerol
homogenization buffer as previously described (31). Yeast
(Saccharomyces cerevisiae) extract was prepared by vortexing
the yeast pellet in 10 volumes of sodium dodecyl sulfate (SDS)
sample buffer in the presence of glass beads. HeLa cell S100
extract was a gift from Jay Tichelaar. Untreated rabbit reticu-
locyte lysate was purchased from Green Hectares (Oregon,
Antibodies. Mouse monoclonal immunoglobulin G (IgG)
antibody PR22 against the avian PR has been described (35).
Antibody D7oc against avian hsp90 was prepared by Brugge
and coworkers (3). Antibodies to p23 were prepared as de-
scribed below. These include an IgM mouse monoclonal
antibody, R3; four mouse monoclonal IgG antibodies, JJ3, JJ5,
JJ6, and JJ1O; and a rabbit antiserum against a p23 peptide,
Immunoprecipitation of PR, hsp9O, and p23 complexes.
PR22 or D7a monoclonal antibody (20
monoclonal antibody ascites was preincubated with 25 ,ul of
protein A-Sepharose CL-4B (Pharmacia, Piscataway, N.J.) in
100 mM Tris-HCl (pH 8.0) for 30 min at room temperature
and then washed with homogenization buffer. One milliliter of
cytosol was added to each 25-pA resin pellet. After 1 h on ice,
with gentle resuspension every 5 min, the pellet was washed
p.g) or 10RIof p23
Vol. 14, No. 3
A 23-kDa PROTEIN OF PR COMPLEXES
four times with 1 ml of homogenization buffer, and extracted
dire tly into SDS sample buffer before analysis by SDS-
polyacrylamide gel electrophoresis (PAGE). For the peptide
competition studies with the JJ3 and JJ5 antibodies, 75
peptide was added to the cytosol during the 1-h immunopre-
Gel electrophoresis and Western blot analysis. Samples
were extracted into SDS sample buffer containing 2% SDS and
3-mercaptoethanol. SDS-polyacrylamide gels were
pared as described previously (13). Gels were stained with
Coomassie brilliant blue R-250. For Western blotting (immu-
noblotting), SDS-polyacrylamide gels were transferred to poly-
Bedford, Mass.) as described elsewhere (12). For blotting p23,
the membranes were incubated with primary antibody at a
1:2,000 dilution overnight at 4°C, washed, incubated with the
appropriate alkaline phosphatase-conjugated goat anti-mouse
second antibody for 30 min, washed, and stained with 5-bromo-
4-chloro-3-indolyl phosphate and nitroblue tetrazolium. The
p23t2 antiserum was used at a 1:1,000 dilution with alkaline
phosphatase-conjugated goat anti-rabbit IgG second antibody.
Obtaining peptide sequence for p23. Immunoprecipitated
PR and hsp9o complexes from chicken oviduct cytosol were
resolved on a 10% gel. After the position of p23 was deter-
mined by staining the edges of the gel with Coomassie blue,
p23 was excised and electroeluted with either a Centricon
Electroeluter (Amicon, Beverly, Mass.) or a Six-Pac Electro-
eluter (Hoefer Scientific Instruments) according to the manu-
facturer's suggestions. The electroeluted protein was dialyzed
into 5 mM NH4HCO3 and precipitated with either ethanol
(40) or chloroform-methanol (38). The pellet was resuspended
and digested with either trypsin or cyanogen bromide (CNBr)
essentially as described previously (33).
Microsequencing of peptide fragments. Protein sequencing
and peptide synthesis were conducted in The Protein Sequenc-
ing/Peptide Synthesis Resource Laboratory, Mayo Clinic, di-
rected by Daniel J. McCormick. After treatment of p23 with
trypsin or CNBr, peptide fragments were resolved by high-
performance liquid chromatography with an ABI Aquapore
OD-300 C18 column. Individual peptides were sequenced
from the amino terminus by gas-phase Edman degradation.
Synthetic peptide synthesis. Peptides were synthesized by
solid-phase methods, using the 9-fluorenylmethyloxycarbonyl
protection scheme. The two internal peptides, p23t2 (residues
49 to 63) and surfI (residues 60 to 75), were synthesized on
Rink resin, and the C-terminal peptide, surf2 (residues 143 to
160), was synthesized on Wang resin. Cysteines were added to
the C terminus of the purified internal peptides and the N
terminus of the C-terminal peptide for conjugation to male-
imide-activated keyhole limpet hemocyanin (Pierce Chemical,
Development of polyclonal antisera to p23. The synthetic
peptide p23t2, LNEIDLFSNIDPNE, was conjugated to key-
hole limpet hemocyanin to raise rabbit polyclonal antiserum.
The second test bleed was reactive with p23 on a Western blot
of chick oviduct cytosol. The polyclonal antiserum was affinity
purified on an antigen column. Briefly, bovine serum albumin-
conjugated peptide was covalently cross-linked to ACTIGEL-
ALD according to the manufacturer's instructions (Sterogene
Bioseparations, Arcadia, Calif.). Polyclonal antiserum diluted
1:10 in 10 mM Tris-Cl (pH 7.5) was bound to the column and
washed extensively with 10 mM Tris-Cl (pH 7.5) and then with
500 mM NaCl-10 mM Tris-Cl (pH 7.5). The p23-specific
antibodies were eluted into
glycine-Cl (pH 2.5) and then with 100 mM triethylamine-Cl
I M Tris-Cl (pH 8.0) with 100 mM
(pH 11.5), pooled, and dialyzed into phosphate-buffered saline
by the method of Harlow and Lane (6).
Immunoscreening of cDNA expression libraries. A Clontech
CL1I016b) was screened with R3 antibody and purified poly-
clonal antiserum by conventional methods. Plaques positive on
tertiary screens were used for large-scale phage preparations
performed by the glycerol gradient step method (25). The
phage DNA was digested with EcoRI, and the insert was
subcloned into pGEM 7zf(-) (Promega, Madison, Wis.) by
conventional methods (25). Double-stranded sequencing was
performed on two independent subclones by using [35S]dATP
and the Sequenase 2.0 kit (U.S. Biochemicals).
Library screening with labeled RNA probe from a partial
clone. A Clontech human testis cDNA library (catalog number
HL101Ob) was screened with an [ot_-32P]GTP-labeled RNA
probe synthesized from the partial chicken clone. The prehy-
bridization and hybridization of the labeled RNA probe to
nylon filters (Amersham, Inc.) were carried out in 5 x SSPE
(1 x SSPE is 0.18 M NaCl, 0.01 M NaH2PO4, and 0.001 M
EDTA), 50% formamide, 5 x Denhardt's solution (1 x Den-
hardt's solution is 0.02% [wt/vol] bovine serum albumin, 0.02%
[wt/vol] Ficoll [Pharmacia], and 0.02% [wt/vol] polyvinylpyrro-
lidone), 0.5% (wt/vol) SDS, and 0.25 mg of denatured soni-
cated salmon sperm DNA at 40°C according to the manufac-
Bacterial expression of human p23. A full-length human p23
PCR product was generated by using the forward p23-specific
a reverse vector-specific SP6 primer. The 650-bp PCR product
was digested with BamHI and subcloned into the Invitrogen
(San Diego, Calif.) pTrcHis expression vector. The resulting
26-kDa fusion protein replaces the original two amino acids
of the native sequence with the additional amino-terminal
DDKD. Escherichia coli TOPIO (Invitrogen) containing the
expression vector was grown at 37°C to an optical density at
600 nm of 0.3, induced with 1 mMisopropylthio-p-D-galacto-
side and harvested 4 h later. Soluble cell extract was prepared
by resuspending and sonicating the pellet in 20 mM sodium
phosphate-500 mM sodium chloride (pH 7.8). Recombinant
p23 elutes from a DEAE-cellulose column during a 0.4 M KCI
step elution as a highly purified protein. Collection and dialysis
of peak fractions yield approximately 10 mg of 50 to 70% pure
p23 per liter of culture.
Monoclonal antibody production. Mouse monoclonal anti-
body R3 was prepared by the intrasplenic immunization
method of Nilsson and Larsson (19). PR complexes immuno-
precipitated from chick oviduct cytosol were resolved by
SDS-PAGE, transferred to an Immobilon membrane (Milli-
pore), and stained with Coomassie blue. The p23 band was
excised and inserted directly into the spleens of three mice.
This was repeated after
formed 5 days later. Hybridoma culture supernatants were
tested for p23 antibodies by Western blotting against prepara-
tions of PR complex. Additional antibodies were prepared by
conventional subcutaneous injection with p23 expressed in E.
coli and purified on DEAE-cellulose. Hybridoma supernatants
were tested on enzyme-linked immunosorbent assay plates
coated with recombinant p23, yielding nine positive hybrido-
Reconstitution ofPR complex with 35S-labeled p23. [35S]me-
thionine-labeled p23 was produced by using the TNT T7-
coupled reticulocyte lysate system (Promega, Madison, Wis.)
according to the manufacturer's instructions. Briefly, 0.5 p.g of
the Hp23C clone was transcribed and translated in Promega
1 month, and cell fusion was per-
VOL. 14, 1994
1958JOHNSON ET AL.
12 3 4 5
p23 Peptide Sequence
Trypsin Fragmen t #1
F F DR F S (i NI
KG E SGQ A WP R
L N E I D) L FS N IDPN E
LT l IS X 1.i
FIG. 1. p23 is part of the unactivated chicken PR complex. Immu-
nopurified complexes were isolated from chicken oviduct cytosol as
described. (Left panel) Immune isolation of chicken PR complex with
antibody PR22; (right panel) immune isolation of hsp90 complex with
antibody D7c. The protein A pellets were washed with homogeniza-
tion buffer, boiled in sample buffer, run on an SDS-10% polyacryl-
amide gel, and stained with Coomassie blue. The positions of molec-
ular mass standards (Diversified Biotech) are shown on the right. K,
lysate with T7 polymerase in a 50-,u volume. After 2 h the
lysate was divided into three reaction mixtures, and Promega
untreated lysate (catalog number L4150) was added to a
volume of 200RI.An ATP regenerating system (32) was also
added at this time.
Chick oviduct cytosol was prepared as described above,
adjusted to 0.5 M KCl, and incubated on ice for 30 min. One
and a half milliliters of this salt-stripped cytosol was added to
each 25-plA protein A-PR22 antibody resin mixture. After a 1-h
incubation on ice, the pellets were washed four times (1 ml
each wash) in 10 mM Tris-Cl (pH 7.5). The reticulocyte lysate
mixture was added to the PR resin pellet and incubated at 30°C
for 30 min with mixing every 5 min. To activate the receptor, 2
X 10-7 M (final concentration) progesterone was added as
indicated and the 30°C incubation was continued for an
additional 30 min. The samples were washed five times (1 ml
each wash) with 10 mM Tris-Cl (pH 7.5) and analyzed by
Computer analysis of sequence. All computer analyses of
DNA and protein sequences were done with the Genetics
Computer Group (GCG; Madison, Wis.) sequence analysis
Nucleotide sequence accession number. The human p23
cDNA and chicken p23 cDNA sequences have been submitted
to GenBank with the accession numbers L24804 and L24898,
p23 was first observed as a component of the unactivated
complex of the avian PR (30). However, its existence is not
confined to receptor complexes, since it has also been isolated
in complexes with the 90-kDa heat shock protein, hsp90 (33).
A typical immunoprecipitation of chick oviduct PR with the
monoclonal antibody PR22 is shown in Fig. 1 (left panel). The
PR22 antibody recognizes the B and A forms of the receptor
(35). Five additional proteins, hsp9O, hsp7O, p54, p50, and p23,
FIG. 2. Peptide fragments of p23 and development of polyclonal
antiserum to p23. One CNBr fragment and three trypsin fragments
obtained from gel-purified p23 were sequenced. Polyclonal antiserum
p23t2 was raised against the second trypsin fragment. Chick oviduct
cytosol was run on a 10% gel, transferred to Immobilon, and Western
blotted with antibodies against p23. Lane 1, anti-mouse IgM second
antibody alone; lane 2, IgM monoclonal antibody R3; lanes 3 to 5,
1:1,000 dilution of rabbit serum; lane 3, rabbit preimmune serum; lane
4, reactive p23t2 polyclonal antiserum; lane 5, 23t2 polyclonal anti-
serum inhibited with 100 pLg of peptide.
have been shown to specifically copurify with the PR (30). The
positions ofPR B and A, hsp90, hsp70, and p23 are shown. The
identities of these proteins were confirmed by Western blotting
(data not shown). The heavy chain of the antibody obscures the
p54 and p50 proteins. From densitometric analysis of Coo-
massie blue-stained gels, the molar ratio of p23 to PR appears
to be approximately 1:1 (30). The proteins copurifying with the
chick oviduct hsp90 immunoprecipitated with D7a
shown in Fig.
identified on Western blots as hsp7o, p60, and p23 (33; data
not shown). The major proteins that specifically copurify with
hsp90 are hsp7o and a stress-related protein termed p60 (33).
p60 is a homolog of the yeast stress protein, STI1 (18), and this
protein has also been shown to be up-regulated by viral
transformation of human cells (9). p23 is less abundant than
the above proteins but is still very evident.
Immunoscreening of chicken cDNA expression libraries.
chicken p23 digested with trypsin or CNBr are shown in Fig. 2
(left panel). These peptide fragments indicated that p23 was a
novel protein. p23 in chick oviduct cytosol can be detected on
Western blots with the mouse IgM monoclonal antibody, R3,
which was raised against gel-purified p23 (Fig. 2, lane 2). Initial
screening of Clontech 5'-stretch liver and brain libraries with
antibody R3 resulted in the isolation of numerous false-
positive clones, prompting us to generate polyclonal antiserum
against the longest trypsin fragment, LNEIDLFSNIDPNE.
This antiserum, p23t2, specifically recognizes p23 on a Western
blot with chick oviduct cytosol, and this interaction can be
inhibited by free peptide (Fig. 2, lanes 3 to 5). Western blotting
of a two-dimensional gel confirmed that the peptide antiserum
was specific for p23 (data not shown). After peptide affinity
column purification to remove unrelated antibodies, this anti-
serum was used to screen a Clontech 5'-stretch brain library.
One clone, c23e, that was positive and competed with free
peptide was isolated. This clone was also detected with anti-
1 (right panel). These proteins have been
MOL. CELL. BIOL.
A 23-kDa PROTEIN OF PR COMPLEXES
CGCCGCCAGAGAGCCCGCCCACCAGTT CGC CCGTCCCCTGCAGCAGCC
ASA K W
36 AGGAATTFAGCT¶A&G TGAT
D W E
FIG. 3. Human p23 cDNA sequence. The 782-bp full-length p23
cDNA clone encodes the complete 160-amino-acid p23 open reading
frame. The human sequences corresponding to the peptide fragments
obtained from gel-purified chicken p23 are underlined. The DNA
homology between chicken and human p23 DNA is 88%.
p23 cDNA and protein sequence. The 468-bp partial clone,
c23e, encodes an open reading frame of 145 amino acids,
including the four peptides obtained from sequenced frag-
ments of chicken p23 (data not shown). An [a-32P]GTP-
labeled c23e RNA probe was used to screen a human testis
cDNA library. Three clones, all with inserts of approximately
the same size, were isolated. One of these clones was selected
for further characterization and found to contain an insert 782
nucleotides in length. This clone, Hp23C, represents the
complete coding sequence of p23 and also contains 232 bp of
5' untranslated sequence and 70 bp of 3' untranslated se-
quence (Fig. 3).
The human cDNA encodes an open reading frame of 160
amino acids (Fig. 3). The four peptide sequences obtained
from chicken p23 (Fig. 2) correspond closely to the human
sequence and are underlined in Fig. 3. p23 is highly conserved,
with the chicken and human sequences being 96.3% identical
at the amino acid level (Fig. 4). p23 is a very hydrophilic
protein with an aspartic acid-rich carboxy terminus. The pre-
dicted pl of 4.2 is in good agreement with the observed pl of
4.5 to 5.2 from SDS-polyacrylamide and isoelectrofocusing gels
(30). The predicted molecular mass of p23 is only 18.7 kDa,
whereas p23 migrates at approximately 23 kDa on SDS-
polyacrylamide gels. To determine whether we had correctly
identified the amino terminus of p23, we obtained amino-
terminal peptide sequence from SDS-PAGE-purified chicken
and rabbit p23. The chicken and rabbit amino-terminal p23
sequences are identical to the derived amino terminus of the
human p23 clone (Fig. 4). Thus, the size discrepancy is due to
either decreased mobility of p23 on SDS-polyacrylamide gels
or posttranslational modification.
A search against GenBank (1) nucleic acid sequences re-
NOPASAKWYD RRDYVFIEFC VEDSKDVNVN
MIPASAKWYD RRDYVFIEFC VZSKDVNVN
FNKSKLTFSC LGGSDNFKHL NEIDLFHCID
FEKSKLTFSC LGGSDNFRHL NEIDLFNNID
KEEAKLNWLS VDFNNWKDWE DDSDELMSNF
KERAKLNWLS VDFNNWBIDWE DDSDEDKSNF
DRFSEOI GGD13DVDLPB VDGADDDSQD
DRFSEANNK GGDDDVDLPE VDGADDDSPD
FIG. 4. p23 protein sequence
quences for rabbit, human, and chicken p23 are shown. The full-length
human coding sequence is deduced from the cDNA sequence. The
chicken sequence is a composite of amino-terminal sequence and
partial cDNA sequence (residues 16 to 160). The rabbit sequence was
obtained by amino-terminal sequencing of gel-purified rabbit p23
transferred to Problott. The chicken and human sequences are 96.3%
identical, with the differences marked with asterisks. Searches revealed
no homologies with known proteins in either the Swiss-Prot or
NBRF-PIR data bases.
is highly conserved. Protein se-
vealed extensive homology between the Hp23C clone and the
opposite strand of the 5' untranslated region ofthe human zinc
finger 6 (ZNF6) locus (16) (GenBank accession number
X56465). As published, this sequence contains multiple frame-
shifts in the p23 coding sequence. Relative to the 3' end of the
p23 coding sequence, the ZNF6 sequence and the Hp23C
clone are >98% identical, with only 7 nucleotide differences
over 565 nucleotides, at which point the sequences diverge.
The location of the p23 sequence in the untranslated region of
the ZNF6 locus raises the possibility that the sequence repre-
sents a p23 pseudogene. Because the homology is over 98%
identical throughout the p23 coding region and the sequences
have a distinct divergence point, the ZNF6 locus most likely
represents a cloning artifact in which the p23 cDNA and the
ZNF6 cDNA became ligated in a head-to-head manner,
although the possibility of the ZNF6 representing a pseudo-
gene has not been eliminated. Portions of the p23 coding
sequence were recently submitted to GenBank (accession
number T11431) as an expressed sequence tag isolated from
human pancreatic islet.
Comparisons of p23 sequence to protein sequences listed in
the Swiss-Prot and NBRF-PIR data bases do not reveal any
significant homologies to known proteins, indicating thatp23is
a novel protein. p23 has been observed to be aphosphoprotein
(4), and multiple conserved candidate phosphorylation sites
are present, as detected by the GenBank Motifs (1) program.
No other common structural motifs were detected.
Tissue and species distribution of p23. Recombinant p23
was expressed in E. coli and used to prepare a series of
monoclonal antibodies (see Materials and Methods). Antibody
VOL. 14, 1994
1960JOHNSON ET AL.
FIG. 5. Tissue and species cross-reactivities of the monoclonal
antibody JJ5. (A) Ten microliters of various tissue and cell cytosols was
run on a 12% gel, transferred to Immobilon, and Western blotted with
the antibody JJ5 (1:5,000 dilution). Lane 1, chicken oviduct cytosol;
lane 2, chicken brain cytosol; lane 3, chicken spleen cytosol; lane 4,
chicken liver cytosol; lane 5, mouse liver cytosol; lane 6, guinea pig
liver cytosol; lane 7, HeLa cell extract; lane 8, rabbit reticulocyte lysate.
(B) Western blot of S. cerevisiae whole-cell extract with monoclonal
antibodies JJ6 (lane 1) and JJ10 (lane 2) at a 1:500 dilution. Positions
of molecular mass markers are shown on the right. K, kilodalton.
JJ5 was used to screen various chicken and mammalian tissues
to identify the tissue and species distribution of p23. Per gram
of tissue, similar levels of p23 are seen in chicken oviduct,
brain, spleen, and liver cytosols (Fig. SA). In a comparison of
chicken oviduct cytosol versus crude chicken oviduct nuclear
extract, the majority of p23 appears to be present in the
cytosol, although there are detectable levels in the nuclear
extract (data not shown). The JJ5 monoclonal antibody also
cross-reacts with 23-kDa antigens in mouse liver, guinea pig
liver, rabbit reticulocyte lysate, and human HeLa cell extract
(Fig. SA). Although JJ5 does not recognize a 23-kDa protein in
S. cerevisiae (data not shown), two additional monoclonal
antibodies, JJ6 and JJ1O, do recognize a 21 to 22-kDa protein
in S. cerevisiae (Fig. SB). These studies clearly show that p23 is
a protein with a broad tissue and species distribution. In
addition, a panel of antibodies against p23, including the
monoclonal antibodies JJ3, JJ5, and R3, were found not to
share cross-reactivity with any proteins other than p23, sug-
gesting that p23 is a unique protein.
Immunoprecipitation of p23. Immunoprecipitations from
oviduct cytosol with two monoclonal
against p23 are shown in Fig. 6. Upon immunoprecipitation
respectively), the pattern of bands is quite similar to that with
PR22 immunoprecipitation (lane 2). The bands corresponding
to the PR B and A forms, hsp90, and hsp70 have been
confirmed by Western blot analysis (data not shown). Two
peptides, surfl (DPNESKHKRTDRSILC) and surf2 (GADD
were prepared because their
quences were predicted to be highly antigenic. These peptides
were tested for their ability to block the interaction of p23 with
antibodies JJ3 and JJ5. The binding of antibody JJ5 but not
antibody JJ3 to p23 complexes is inhibited by the peptide surf2,
which corresponds to the carboxy-terminal 18 amino acids of
p23 (Fig. 6, lane 8). In addition to inhibiting binding of p23 to
the antibody, the surf2 peptide also specifically eliminates the
copurification of hsp90, PR B and A, hsp70, and additional
JJ3 and JJ5 against p23 (lanes 3 and 6,
FIG. 6. Monoclonal antibodies to p23 immunoprecipitate PR. p23
complexes were immunoaffinity isolated, run on an 11% gel, and
stained with Coomassie blue. Lane 1, mock immune precipitation with
protein A-Sepharose; lane 2, immune isolation with monoclonal
antibody PR22 against the PR; lane 3, immune isolation with the
monoclonal antibody JJ3; lane 6, immune isolation with the monoclo-
nal antibody JJ5. The surf2 peptide corresponding to the 18 carboxy-
terminal amino acids effectively blocked the immune precipitation of
p23 complexes by the JJ5 antibody (lane 8) but had no effect on the JJ3
antibody (lane 5). The surfl peptide (amino acids 60 to 75) had no
effect on either JJ3 (lane 4) or JJ5 (lane 7). Positions of molecular mass
markers are shown on the right. K, kilodalton.
proteins of lesser abundance: one migrating at approximately
200 kDa, a few proteins in the 35- to 40-kDa range, and two
lower-molecular-mass proteins of about 15 kDa. The identities
of the latter proteins are unknown. The surfl peptide does not
have an effect on immunoprecipitation of p23 with either
In Fig. 7, antibody JJ3 was used to immunoprecipitate p23
from mouse liver, rabbit reticulocyte lysate, chicken brain,
spleen, liver, and oviduct. hsp90 and hsp70 specifically copre-
cipitate with p23 in each tissue tested, although the ratios of
p23 to hsp90 and hsp70 vary depending on the tissue. A
number of other proteins coprecipitate with p23 antibodies in
various tissues, the most prominent being a 42-kDa protein
seen precipitating from chick liver (lane 6). Only the bands
corresponding to hsp90 and hsp70 have been identified by
Western blot (data not shown). A similar pattern of immuno-
precipitation was seen with the JJ5 antibody (data not shown).
Reconstitution of p23 into PR complexes. Immunoaffinity-
isolated chicken PR that has been stripped of associated
proteins in high salt will reassociate with rabbit hsp90, hsp70,
and p23 when incubated in rabbit reticulocyte lysate (31, 32).
This is not a simple binding event but occurs through a
temperature- and ATP-dependent process that is still unclear.
Once reassembled, the addition of progesterone leads to
activation of the PR, evident in the dissociation of hsp90 and
p23 (31, 32). To demonstrate that the full-length cDNA clone
Hp23C is sufficient to produce functional p23, we transcribed
and translated the clone in vitro, producing [35S]methionine-
labeled human p23. The translation mixture was then added to
a standard chicken PR reconstitution (32), shown in Fig. 8. On
the Coomassie blue-stained gel (lanes 1 to 5), lane 1 shows the
proteins in the intact receptor complex isolated from chick
oviduct cytosol, and lane 2 shows the isolated PR after
treatment with 0.5 M KCl to remove hsp9o and p23. When
samples identical to that of lane 2 (salt stripped) were incu-
MOL. CELL. BIOL.
A 23-kDa PROTEIN OF PR COMPLEXES
FIG. 7. Tissue and species specificities of immunoisolated p23
complexes. The antibody JJ3 was used to immunoprecipitate p23 from
different tissues and species. One milliliter of tissue cytosol was used
per 25-,ul protein A-Sepharose pellet. Lane 2, mouse liver cytosol; lane
3, 0.5 ml of rabbit reticulocyte lysate; lane 4, chick brain cytosol; lane
5, chick spleen cytosol; lane 6, chick liver cytosol; lane 7, chick oviduct
cytosol; lane 1, control immunoprecipitation from mouse liver, using
PR22-bound protein A-Sepharose control. The samples were run on
an 11% gel and stained with Coomassie blue. Positions of molecular
mass markers are shown on the right. K, kilodalton.
bated in rabbit reticulocyte lysate at 30°C for 30 min, this
results in the binding of rabbit hsp90 and p23 to the receptor,
as evident in lane 4. The subsequent addition of 2 x 10-7 M
progesterone promotes a partial dissociation of hsp90 and p23
(lane 5). The extent of [s5S]methionine-labeled p23 in these
reconstituted samples was shown by autoradiography (lanes 6
to 8). The [35S]methionine-labeled p23 comigrates with rabbit
p23 and specifically associates with PR in the presence of
reticulocyte lysate (lane 7). Further treatment with progester-
one to activate the PR leads to decreased recovery of both
rabbit p23 in the stained gel and human p23 in the autoradio-
graph (lane 8).
In this report we have identified p23 as a novel, ubiquitous,
and conserved protein that associates with unactivated PR
complexes. Although p23 has only been studied in detail in
relation to PR complexes, it may be a component of other
steroid receptors complexes, as well as other complexes con-
taining hsp9o. A 23-kDa protein associated with mouse L-cell
glucocorticoid receptor (2) cross-reacts with the monoclonal
antibody R3 (10), indicating that it is a related protein. The
22-kDa protein associated with bovine estrogen receptor (24)
is a likely candidate for a p23 homolog, although this has not
yet been tested. Recent studies with the unactivated human PR
from T47D cells also show p23 copurifying with human PR
(17). Despite these reports, p23 has not been recognized as a
major component common to steroid receptor complexes, in
part because of the lack of alternative methods for showing a
relationship between p23 and steroid receptors. The demon-
stration that antibodies to p23 specifically coprecipitate the PR
complements studies showing p23 copurifying with immuno-
precipitated PR, providing further evidence that p23 is indeed
a structural component of the PR complex.
p23 is a highly conserved protein that has been found in
every tissue tested to date. The high amino acid homology
(96.3%) between chicken and human p23 equals that of hsp90,
which shows 96% identity at the amino acid level between
chicken and human forms (8). p23 is not one of the major heat
shock proteins, and its sequence shows no relatedness to those
of the family of 22- to 28-kDa heat shock proteins (15).
Incubation of minced chick oviduct at 43°C for 1 h produces a
substantial increase in the synthesis ([35S]methionine labeling)
FIG. 8. Reassociation of p23 with PR complexes. [35S]methionine-labeled p23 was transcribed and translated from theHp23Cclone in vitro
and assayed for its ability to associate with PR. Samples were run on an 11% gel and stained with Coomassie blue. Lane 1, native PRcomplex
immunoisolated from chicken oviduct cytosol; lane 2, PR stripped of associated proteins by high salt; lanes 3 to 5, rabbitreticulocyte lysate
containing [35S]methionine-labeled p23 was added to salt-stripped PR; lane 4, PR reconstitution in rabbit reticulocyte lysate; lane 5, PR
reconstitution followed by treatment with progesterone; lane 3, mock reconstitution (resin withoutPR);lanes 6 to 8,autoradiographsof lanes 3
to 5, respectively; lane 9, autoradiograph of the total translation mixture. Positions of molecular mass markers are shown on theright. K,kilodalton.
VOL. 14, 1994
JOHNSON ET AL.
of hsp9o, hsp70, and hsp24, with no increase in the synthesis of
p23 (data not shown).
The common identity of p23 in receptor complexes and in
hsp90 complexes and the protein encoded by the cloned p23
cDNA was demonstrated by uniform recognition by monoclo-
nal antibodies prepared against p23 from chick oviduct or from
bacterially expressed protein. Also, p23 synthesized in vitro
was able to bind to PR and to partially dissociate from the
receptor complex upon progesterone treatment. hsp70 and
hsp9o appear to bind directly to the PR (12, 28), but it is not
known whether p54, p50, and p23 bind to the PR through
hsp90 or hsp70 or whether they bind to the receptor itself.
From the present work, it appears that most p23 in oviduct
cytosol is bound to hsp90, suggesting that p23 is binding to the
receptor in conjunction with hsp90. This does not rule out
direct binding of p23 to the PR.
While it seems likely that p23 is a common component of
function of this protein is completely unknown. No clues are
provided from its sequence, which lacks any identity to known
proteins. The only common functional motifs present in p23
are multiple potential phosphorylation sites. Although p23 is
readily labeled with 32p; in rabbit reticulocyte lysate (4), it has
not yet been determined whether the phosphorylation state of
p23 is important for binding to hsp90 or the PR. We have
found that the simple treatment of extracts with phosphatase
or phosphatase inhibitors does not affect the appearance of
p23 in PR complexes (11).
p23 appears to be structurally unrelated to the two major
classes of proteins previously shown to bind steroid receptors,
those being heat shock proteins (22, 34) and immunophilins
(14, 23, 36, 39). However, the interaction of p23 with hsp90 and
hsp70 observed in a variety of tissues suggests that its function
relates to these two proteins that
chaperones in the folding and processing of proteins (5, 7). In
many tissues, p23 appears to be almost totally bound to hsp90.
On the basis of immunodepletion studies in chick oviduct
cytosol (data not shown), we estimate the concentration of p23
in the cytosol to be 5 to 7 ,ug/ml. By using estimated levels of
receptor (1 ,ug/ml) and hsp90 (150 ,ug/ml), this allows the
rough estimate for the molar ratio of PR/p23/hsp90 at 1:30:
175. The abundance of hsp90 relative to PR and p23 in the
cytosol suggests that p23-associated hsp90 may define a sub-
population of hsp90 that may be directed toward a specialized
function in which the proteins cooperate in the binding,
folding, or processing of a protein substrate, such as steroid
Clues as to the function of p23 might be gained by studies on
the assembly of steroid receptor complexes. Complexes of the
avian PR do not form simply by mixing the individual protein
components. This appears to be an energy-driven process that
requires ATP hydrolysis, hsp70, and additional factors that
have not been identified (32). In a similar vein, we have
recently found that the binding of p23 to hsp90 and hsp70 does
not occur upon mixing these proteins together. However, these
complexes can be disrupted and reformed in rabbit reticulocyte
lysate through a process that is also dependent on ATP (11).
Thus, parallels suggesting that the assembly of the p23 complex
is a key step in the formation of PR complexes exist.
this has not yet been established. The
We thank Jay Tichelaar for providing cultured cells, Joan Brugge for
providing antibody D7a-, and Jeff Salisbury for providing the human
cDNA library. We thank Benjamin Madden and Jane Liebenow for
their participation in peptide sequencing and synthesis. Technical
assistance by Nancy McMahon and Bridget Stensgard
These studies were supported by NIH grant HD 09140.
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