Division of Hormone Research (H.L., B.D., Z.-X.Y., V.P.), Departments of Cell Biology, Pharmacology, and Neuroscience, Georgetown University School of Medicine, Washington, DC 20007; and Institute of Medical Biochemistry (D.T., K.T.), University of Oslo, N-0317 Oslo, Norway
Address all correspondence and requests for reprints to: Dr. Vassilios Papadopoulos, Division of Hormone Research, Department of Cell Biology, Georgetown University School of Medicine, 3900 Reservoir Road, Washington, DC 20007. E-mail: papadopv{at}georgetown.edu
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ABSTRACT |
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INTRODUCTION |
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The primary point of control in the acute stimulation of steroidogenesis by peptide hormones and cAMP involves the first step in this biosynthetic pathway where cholesterol is converted to pregnenolone by the C27 cholesterol side-chain cleavage cytochrome P-450 enzyme (P-450scc) and auxiliary electron transferring proteins, localized on inner mitochondrial membranes (IMM) (2, 3). Detailed studies have shown that the rate-determining step in the hormone-stimulated steroid biosynthesis is the transport of the precursor, cholesterol, from intracellular sources to the IMM (2, 3). In our search for the structural elements participating in the mitochondrial uptake and transfer of cholesterol, we identified the peripheral-type benzodiazepine receptor (PBR) (9).
PBR is an 18-kDa protein, which was originally discovered because it binds the benzodiazepine diazepam with relatively high affinity (9). PBR, although present in all tissues examined, was found to be particularly high in steroid-producing tissues, where it was localized primarily in the outer mitochondrial membrane (OMM) (10). It was then demonstrated that PBR is a functional component of the steroidogenic machinery (11) mediating cholesterol delivery from the outer to the IMM (12). Further studies demonstrated that targeted disruption of the PBR gene in Leydig cells resulted in the arrest of cholesterol transport into mitochondria and steroid formation; transfection of the PBR-disrupted cells with a PBR cDNA rescued steroidogenesis (13). The role of PBR in cholesterol transport was further clarified by studies employing site-directed mutagenesis of PBR and in vitro expression (14). From these studies a region of the cytosolic carboxyl terminus of the receptor was identified as a cholesterol-binding site (14, 15). In vivo studies, in which adrenal and ovarian PBR levels were pharmacologically (16, 17) or developmentally (18) modulated, further demonstrated that the levels of PBR correlated with the ability of the steroidogenic tissues to form steroids.
The functional mitochondrial PBR is a multimeric receptor complex. It is composed of at least the 18-kDa isoquinoline binding protein, the 34-kDa voltage-dependent anion channel, and the adenine nucleotide carrier (19, 20). Further studies on the structure of the receptor indicated that the 18-kDa mitochondrial PBR protein is organized in clusters of four to six molecules. Addition of hCG to Leydig cells induces a rapid increase in PBR ligand binding (21) and morphological changes, namely, redistribution of PBR molecules in large clusters (22). These hCG-induced changes and steroid formation are inhibited by a PKA inhibitor (21, 22), suggesting the presence of a cAMP-inducible element regulating the PBR structure and function. In subsequent studies, using the R2C Leydig cell line, which produces high levels of steroids in a constitutive manner, we also observed that a cytosolic proteinaceous component regulated the ligand binding ability and function of the mitochondrial PBR (23).
Considering these observations, it seemed highly likely that other,
probably cytoplasmic, proteins, may participate in or induce the
formation of the active receptor complex. In this study, we applied the
yeast two-hybrid technique and screened a mouse testis cDNA library
using PBR as bait. We identified several proteins [PBR-associated
proteins (PAPs)] that interacted with PBR. One such clone, named PAP7,
showed high affinity for PBR, no sequence homology to any known entity,
and a tissue distribution close to that of PBR. Interestingly, when
screening a human lymphocyte library with the regulatory subunit RI
of PKA as bait, we also isolated PAP7 as a protein with in
vivo selectivity for PKA-RI
. This finding suggests the presence
of a signal transduction mechanism involving targeting of PKA by PAP7
to mitochondria rich in PBR. Furthermore, our results provide a good
platform from which we can formulate a pathway by which PKA
activity regulates steroidogenic proteins, such as the steroidogenic
acute regulatory protein StAR (24), and reorganization of
PBR topography and function, leading to cholesterol uptake and
transport to the IMM. The cloning, characterization, tissue
distribution, and function of PAP7 in the hormone-stimulated
steroidogenesis are presented herein.
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RESULTS |
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We then cotransformed Y190 yeast cells with pGAD10-PAP7 together with
the pAS2.1 vector fused with either RI, RIß, RII
, or empty
pAS2.1 Yeast transformed with both plasmids were selected by growth on
Leu-/Trp- plates. Double
positive clones were then transferred by colony lift assay to selective
medium
(His-/Leu-/Trp-,
with 20 mM 3-aminotriazole) to determine positive protein
interaction. Further confirmation of protein interaction was provided
by use of the ß-galactosidase assay. As seen from Fig. 2
, all plasmids were successfully
introduced into Y190 yeast. Growth in His-
plates was seen only in yeast containing PAP7-RI
(not shown).
Positive interaction was also assessed using the ß-galactosidase
assay from a colony lift directly from
Leu-/Trp- plates (Fig. 2
). As seen, only yeast containing the PAP7-RI
showed positive
interaction. Yeast containing RIß or RII
showed growth in
Leu-/Trp- but were not
positive on His- plates, or the
ß-galactosidase assay, indicating that, in yeast, the PAP7 does not
bind to these subunits. Thus, in the two-hybrid assay PAP7 bound
primarily to the RI
subunit of PKA and not to RIß or RII
.
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To determine the in vitro characteristics of PAP7 binding to
the different regulatory subunits of PKA, we extended these initial
GST-precipitation experiments. Recombinant PAP7-GST was purified from
isopropyl-ß-D-thiogalactopyranoside-stimulated
E. coli and incubated with 50 nM of
different recombinant R subunits. Equal loading of PAP7-GST and GST
alone was determined from Coomassie blue-stained gels. As shown in Fig. 4, A and B, GST-PAP7, but not GST,
precipitated RI
and RII
.
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Immunoprecipitation of PAP7 from Human Testis Tissue
To address the in vivo selectivity of R subunit interaction with
PAP7, we performed immunoprecipitation of PAP7 from human testis tissue
followed by immunoblot analysis for specific R subunits. As shown in
Fig. 4, C and D, RI
is coimmunoprecipitated with PAP7 from human
testis tissue. The reason for the different mobility of the recombinant
RI
used as the standard and the immunoprecipitated RI
by the
anti-PAP7 antibody is that the recombinant protein has a 17-amino acid
extension, which changes the apparent SDS-PAGE mobility. In Fig. 4D
the
signal seen in the NRS lane is of lower size than PAP7 and probably
corresponds to the light chain of the antibody used in the experiment.
No coimmunoprecipitation of RII
was seen in agreement with the
observations in the yeast-two hybrid system (not shown).
Tissue and Cell Expression of PAP7 Examined by RNA and Immunoblot
Analyses
By dot blot analysis, PAP7 was present at high levels in brain,
eye, submaxillary gland, testis, and ovary. Interestingly, PAP7 mRNA
was present in embryos and decreased before birth (Fig. 5A). Consistent with these data, PAP7
mRNA was found by Northern blot analysis in adrenal, brain, heart,
liver, testis, and ovarian tissues. PAP7 has a 3-kb major mRNA
transcript in these tissues and an additional 1.7-kb transcript found
only in testis (Fig. 5B
). Preliminary data indicate that the 1.7-kb
transcript is a spliced form present in the germ cells of the testis
(data not shown). PAP7 mRNA was also abundant in three cell lines, C6
glioma, MA-10 Leydig, and Y1 adrenocortical (Fig. 5B
), which have been
widely used for studying the mechanisms regulating steroid
biosynthesis. All three cell lines expressed the 3-kb PAP7 transcript.
The PAP7 expression level in these cell lines was proportionally
correlated with their steroidogenic ability. The PBR mRNA levels were
also examined in these same tissues and cell lines. The levels of the
3-kb PAP7 message in the steroidogenic cell lines, MA-10, Y-1, and C6,
parallel the PBR mRNA expression pattern (Fig. 5C
). However,
considering the cell-specific localization of PBR and PAP7 mRNAs, a
correlation of their tissue expression levels cannot be determined by
RNA (Northern) blot analysis.
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Tissue and Cell Distribution of PAP7 Examined by
Immunohistochemistry and in Situ Hybridization
PAP7 protein expression in different tissues was investigated by
immunohistochemistry (Fig. 6). PAP7 was
present in both Leydig and germ cells of the testis (Fig. 6D
), in
fasciculata reticularis and glomerulosa cells of the adrenal gland
(Fig. 6N
), and theca and granulosa cells of the ovary (Fig. 6L
). In
brain, PAP7 immunoreactivity was very strong in the hippocampus (Fig. 6F
) and specific neuronal and glial cells of the cortex (Fig. 6B
).
Strong immunoreactivity was also found in the paraventricular (Fig. 6H
)
and superoptic nuclei (Fig. 6J
) regions of the hypothalamus. Liver
(Fig. 6V
) and kidney (Fig. 6R
) expressed low levels of PAP7 protein.
Heart (Fig. 6P
) and spleen (Fig. 6T
) did not show any immunoreactivity
for PAP7. Each specimen examined was also immunostained with the
antibody preabsorbed with the peptide used to generate and isolate the
antibody, as negative control (Fig. 6
, A, C, E, G, I, K, M, O, Q, S,
and U).
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DISCUSSION |
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The distribution and expression of PAP7 were examined in several mouse tissues such as brain, testis, ovary, adrenal, and kidney, as well as steroidogenic cell lines. The PAP7 expression pattern is similar to the broader expression profile of PBR. In the testis, the Leydig cells are the sites of T biosynthesis. In the ovary, the theca and granulosa cells synthesize progestins and E. Glucocorticoids, androgen, and mineralocorticoids are produced by the zona fasciculata and glomerulosa cells of the adrenal. In the brain, glial cells (29, 30) and some neurons (31, 32) are able to synthesize neurosteroids. Interestingly, these are the sites at which PBR expression is very high in the body (9). In the brain, we found that PAP7 is abundant in the hippocampus and olfactory bulb. We have also found high levels of PBR in these areas (our unpublished data). The presence of steroidogenic enzymes in the olfactory bulb was recently shown (33), although its ability to synthesize steroids is unknown. Moreover, it was recently shown that hippocampal neurons express steroidogenic enzymes and are able to synthesize specific neurosteroids (32). Our observations on the high level of PAP7 expression in these two brain areas complement these recent studies. In addition, high levels of PAP7 mRNA were found in rat C6 glioma cells, mouse MA-10 Leydig cells, and mouse Y1 adrenocortical cells, some of the most widely used cell models with which to study steroid biosynthesis. Because PAP7 is also highly expressed in these steroidogenic tissues, and considering its interaction with PBR, it is highly probable that PAP7 is involved in the regulation of steroid biosynthesis by changing the formation or the conformation of the mitochondrial PBR complex. In the testis, a second smaller PAP7 transcript, possibly due to alternative splicing or alternative use of polyadenylation site signals, was found. The presence of alternatively spliced genes is a common phenomenon in the testis (34, 35). Characterization of this transcript is under investigation in our laboratory.
It was recently reported that a protein named PRAX-1 specifically interacts with the carboxy terminus of PBR (36). PRAX-1 showed localization restricted to brain and thymus. Steroidogenic tissues, rich in PBR, were devoid of PRAX-1. The only similarity between PAP7 and PRAX-1 is that both proteins contain glutamic acid stretches. Whether these stretches are critical for interacting with PBR remains to be determined. A part of PAP7 shares quite high homology with a C. elegans gene that has an unknown function (25). Interestingly, cholesterol is required for C. elegans cell culture (37). Considering that PBR is a cholesterol binding and channel-like protein (14, 15, 38, 39) and the PBR gene is highly conserved in all type of organisms, these data suggest that PAP7 expression may be needed to meet basic requirements for cell survival and growth. PAP7 also shares limited homology with RALBP, a hydrophobic ligand-binding protein that functions in intracellular retinoid transport (26).
PBR is a hydrophobic, integral protein of the OMM. Myristoylation and subsequent OMM translocation is one mechanism that, if present, could enable PAP7 to interact with PBR to transmit the signal generated by cAMP. Interestingly, the only known endogenous PBR ligand, the polypeptide diazepam binding inhibitor (9), is also an acyl-CoA-binding protein (40), suggesting that PAP7 may act as an endogenous PBR protein ligand. However, preliminary studies using MA-10 cells overexpressing PAP7 failed to show differences in PBR ligand binding (data not shown). The observation that PAP7 has potential protein kinase phosphorylation sites raises the possibility that PAP7 phosphorylation could facilitate its interaction with PBR. Whether PAP7 phosphorylation is involved in its interaction with PBR is under investigation.
The finding that the regulatory subunit RI of PKA also interacts
with PAP7 in the yeast two-hybrid system indicated a link between cAMP,
PKA, and PBR. The human PAP7 cDNA isolated in the yeast two-hybrid
system screening using RI
was partial and encompassed amino acids
212369, indicating that the binding domain for PKA resides in this
area. Both RI
and RII
bind PAP7 in in vitro solution
binding and filter overlay assays. However, results obtained from the
GST-PAP7 (216445) pull-down assays with MA-10 Leydig cell cytosolic
fractions, immunoprecipitation of PAP7 from testis, as well as
reconstitution in the two-hybrid system, showed interaction with RI
but not RII
or RIIß. AKAP proteins normally interact with the RII
subunit of PKA via an amphipathic helix region, and several such
regions can be predicted in this area of PAP7. However, a further
mutation/deletion analysis will be required for mapping of the
interaction domain and understanding PKA isoenzyme selectivity,
which may identify some determinants that favor interaction with RI
as recently described for other AKAP binding sites (41, 42).
Overexpression of the full-length PAP7 protein increased the
hCG-stimulated steroid synthesis by MA-10 Leydig cells. However,
overexpression of the PAP7 (228445) fragment, which includes the PBR
and PKA-RI binding domains, inhibited hCG-stimulated progesterone
formation in MA-10 Leydig cells. These data suggest that the
overexpressed PAP7 (228445) fragment acts as a competitor of
endogenous PAP7 having a dominant-negative effect. Since this
transfected PAP7 (228445) fragment has a PBR and PKA-RI
binding
domain, it may prevent PBR and/or PKA-RI from interacting with
endogenous PAP7, thus inhibiting cholesterol accumulation into
mitochondria and subsequent steroid formation. This also suggests that
the transfected fragment of PAP7 (228445) lacks, or has an
ineffective, functional domain present in the wild-type protein.
Detailed studies on the effect of PAP7 on the PBR ligand binding
characteristics in response to hCG are in progress. In the same
experiments, overexpression of the PAP7 (228445) fragment did not
alter the hCG-stimulated cAMP accumulation, indicating that the
dominant negative effect seen is downstream of cAMP synthesis. This
effect was subsequently localized at the level of cholesterol transport
to P450scc. Overexpression of the full-length PAP7 increased and
overexpression of the PAP7 (228445) decreased the amount of
cholesterol transported into the IMM, in response to hCG, available to
P450scc for pregnenolone synthesis. The role of PAP7 in the
hormone-induced steroid formation was further demonstrated using
oligonucleotides antisense to PAP7, which specifically inhibited the
hCG-stimulated progesterone formation by MA-10 cells.
These studies demonstrate the presence of a cytosolic protein (PAP7)
involved in the hormonal regulation of steroid formation, which
interacts with both the cytosolic RI subunit of PKA and the
mitochondrial PBR. The association of PKA-RI with steroidogenic
mitochondria has been reported (43). More specifically, it
was shown in porcine ovaries that PKA activity is higher in
mitochondria than cytosol. PKA-RI was then found to be predominant in
the mitochondria whereas PKA-RII was predominant in the cytosol.
Moreover, the mitochondrial PKA-RI to PKA-RII ratio was higher in
corpora lutea, where there is maximal output of progesterone by the
ovary (43). The importance of PKA-RI in Leydig cell
steroidogenesis was also shown by Moger (44), who
suggested that PKA type I is compartmentalized in Leydig cells so that
it has preferential access to endogenously produced cAMP. The
compartmentalization of PKA, mediated through the specific binding of R
subunits to various organelles, has been recently proposed as a
mechanism to target the response to cAMP (45). A dual
RI/RII specificity PKA anchoring protein, which targets PKA to either
mitochondria or endoplasmic reticulum, was recently described
(45). In this article, we present evidence suggesting that
PAP7 is a PKA-RI
anchoring protein targeting the kinase to
mitochondria. Anchoring to the mitochondria could be accomplished via
PAP7 myristoylation. There, PKA could phosphorylate specific
protein substrates, such as StAR (24). StAR is a
cytosolic hormone-induced protein implicated in the hormone-induced
cholesterol transport into mitochondria (24).
Phosphorylation of StAR has been shown to be responsible, in part, for
the regulation of steroidogenesis by hormones (46).
Although initial studies suggested that StAR needs to enter the
mitochondria to exert its activity (47), subsequent
studies demonstrated that its site of action resides outside the
mitochondrion (48). The recent findings that the
mitochondrial PBR is a high-affinity cholesterol-binding protein
(49) and that StAR and PBR are closely associated in the
OMM (50) suggest that a StAR-PBR interaction might be the
key to the initiation of cholesterol transport into mitochondria. At
the same time, PAP7 may serve as an anchoring protein to bring together
many PBR molecules (22), thus allowing the creation of
contact sites of the OMM and IMM and the translocation of cholesterol
from the OMM, where cholesterol may be harbored in the PBR channel
(14, 15, 38, 39), to the IMM where the cytochrome P450scc
is located. Thus, with the identification of PAP7, we may now have an
explanation of how a small, transient increase in cAMP levels may
trigger maximal activation of steroid formation: upon its
targeting/anchoring via PAP7. The activated PKA, targeted by PAP7,
would be able to act at critical subcellular locations, such as
PBR-rich sites of the OMM. The phosphorylation of specific target
proteins will initiate cholesterol transfer into mitochondria.
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MATERIALS AND METHODS |
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Strains and Media
The genotype of the Saccharomyces cerevisiae reporter
strain HF7c is MATa, ura352, his3200, lys2801, ade2101,
trp1901, leu23, 112, gal4542, gal80538, LYS2::GAL-
HIS3, URA3::(GAL4 17-mers)3-CYC1-lacZ
(CLONTECH Laboratories, Inc.). Yeast strains were grown at
30 C in standard liquid YPD medium or minimal SD synthetic medium with
appropriate supplement amino acids (CLONTECH Laboratories, Inc.).
Plasmids and Construction
The mouse PBR cDNA coding sequence was subcloned into pGBT9
(CLONTECH Laboratories, Inc.) at EcoRI and
BamHI sites (pGBT-PBR). The fusion site was verified by
sequencing. Functional fusion PBR protein, expressed in yeast cells,
was verified by PBR ligand binding assay. Mouse testis cDNA library was
constructed in pGAD10 [LEU2, GAL4 (768881)] (CLONTECH Laboratories, Inc.). Amplification of premade libraries was
performed by growing the transformants on LB-agar-ampicillin and
purifying the plasmids DNA with the Plasmid Giga kit
(QIAGEN, Valencia, CA). In the transfection experiments,
PAP7 partial sequence (including 217-amino acid C-terminal sequence,
from 228445) and full-length PAP7 were inserted into pSVzeo vector
(Invitrogen, Carlsbad, CA) at EcoRI and
BamHI sites and named as pSVPAP7p and pSVPAP7f
accordingly.
Yeast Two-Hybrid Screening
The MATCHMAKER two-hybrid system (CLONTECH Laboratories, Inc.) was applied in this study (detailed in manufacturers
instruction book). Briefly, the yeast reporter host strain HF7c was
simultaneously cotransformed with both pGBT-PBR and the mouse testis
cDNA library in pGAD10 plasmid by using the lithium acetate
high-efficiency method (51). Screening of a normal human
lymphocyte library was also performed using the CLONTECH Laboratories, Inc. MATCHMAKER two-hybrid system. The
full-length RI subunit of PKA was subcloned into pAS2.1 and
cotransformed together with the cDNA library (CLONTECH Laboratories, Inc., catalog no. HL4014AB) into Y190 yeast cells.
HIS positive clones were further selected by colony lift filter assay
for ß-galactosidase activity. Plasmid DNA was rescued in E.
coli DH5
from yeast cells. Plasmids were retransformed into
yeast HF7c cells with plasmid pGBT-PBR to test for histidine
prototrophy and ß-galactosidase activity (CLONTECH Laboratories, Inc. manual). The cDNA inserts from the positive
clones were sequenced. The full-length PAP7 cDNA was obtained by using
the 5'- and 3'-RACE kit from CLONTECH Laboratories, Inc.
Sequence Analysis
The ABI PRISM dyes terminator cycle sequencing ready reaction
kit and sequencer (both from PE Applied Biosystems, Foster
City, CA) were used for sequencing at the Lombardi Cancer Center
Sequencing Core Facility (Georgetown University). DNA sequences were
analyzed by using Entrez and BLAST program against GenBank Database
(National Center for Biotechnology, National Library of Medicine, NIH,
Bethesda, MD).
Cell Culture and Transient Transfection
Mouse MA-10 cells were grown in modified Waymouths MB752/1
medium containing 15% horse serum, as described previously
(11). Rat C6 glioma and mouse Y1 adrenal cortical cells
were cultured in DMEM and DMEM F12, respectively, with 10% FBS
(12, 29). MA-10 cells were transiently transfected by
electroporation (52). Each Genepulser cuvette (0.4-cm gap,
Bio-Rad Laboratories, Inc.) contained 8 x
106 cells in 350 µl antibiotic-free complete
Waymouths growth medium (see above), plus 30 µg plasmid DNA in 50
µl of 0.1x Tris-EDTA. Cells in electroporation cuvettes were
electroshocked at 330 V and at a capacitance of 950 µFarads generated
from Genepulser (Bio-Rad Laboratories, Inc.). The cells
were placed immediately on ice for 10 min before plating into 96-well
plates. Transfection efficiency was monitored by ß-galactosidase
staining and varied from 50 to 90% of the cells attached to the
dishes.
PC12 rat pheochromocytoma cells were grown and maintained as we previously described (53). PC12 cells were transfected with either pSVzeoPAP7 full-length cDNA or empty vector using the lipofectAMINE 2000 reagent following the instructions suggested by the manufacturer (Life Technologies, Inc.).
Radioligand Binding Assays
3H-PK11195 and
3H-Ro54864 binding studies were performed as we
described previously (11). The dissociation constant
(Kd) and the number of binding sites
(Bmax) were determined by Scatchard plot analysis
of the data using the LIGAND program (54).
RNA (Northern) Blot Analysis
Total tissue and cellular RNA was isolated by the acid
guanidinium thiocyanate-phenol-chloroform extraction method using RNA
STAT60 reagent (Tel-Test Inc., Friendswood, TX). RNA was
separated by denaturing electrophoresis and transferred to a Nytran
membrane (Schleicher & Schuell, Inc., Keene, NH). The RNA
blots were hybridized with 32P-labeled PAP7 cDNA
probe generated from random priming (Roche Molecular Biochemicals, Indianapolis, IN) (16, 21).
Autoradiography was performed by exposing Kodak X-Omat AR
films (Eastman Kodak Co., Rochester, NY) to the blots at
-80 C overnight.
Relative Expression Level Analysis
Mouse master blot from CLONTECH Laboratories, Inc.
was used to analyze relative expression levels of PAP7 among 50 tissues
and during development. Hybridization and probe construction were
performed as described above. The density of autoradiographs was
analyzed and quantified using SigmalGel Software (SPSS, Inc., Chicago, IL).
Antibody Generation And Immuno (Western) Blot Analysis
Rabbit anti-PAP7 antibody was prepared by sequential
immunization with a peptide (367391) SSDEEEEEEENVTCEEKAKKNANKP of
PAP7 protein, which was coupled to keyhole limpet hemocyanin. PAP7
antibodies were purified by an affinity resin containing the same
peptide immobilized onto agarose (Bethyl Laboratories, Montgomery, TX).
MA-10 cells were solubilized in sample buffer (25 mM
Tris-HCl (pH 6.8), 1% SDS, 5% ß-mercaptoethanol, 1 mM
EDTA, 4% glycerol, and 0.01% bromophenol blue), boiled for 5 min, and
loaded onto a 15% SDS-PAGE minigel (MiniProtein II System,
Bio-Rad Laboratories, Inc.). Electrophoresis was performed
at 25 mA/gel using a standard SDS-PAGE running buffer (25
mM Tris, 192 mM glycine, and 0.1% SDS). The
proteins were electrophoretically transferred to a nitrocellulose
membrane (Schleicher & Schuell, Inc.). The membrane was
incubated in blocking TTBS (20 mM Tris/HCl, pH 7.5, 0.5
M NaCl, and 0.05% Tween-20) buffer containing 10% nonfat
milk) at room temperature for 1 h, followed by incubation with a
primary antibody against PAP7 (1:2,000) for 2 h. The membrane was
washed with TTBS three times for 10 min each time. After 1 h
incubation with the secondary antibody, goat antirabbit IgG conjugated
with horseradish peroxidase (HRP) (Transduction Laboratories, Inc., Lexington, KY), the membrane was washed with
TTBS three times for 10 min each time. Specific protein bands were
detected by chemiluminescence using the Renaissance Kit
(DuPont-NEN Life Science Products, Wilmington, DE).
Immunocytochemistry
MA-10 cells were cultured on four-chambered SuperCell Culture
Slides (Fisher Scientific, Pittsburgh, PA) and fixed with
methanol at 4 C for 15 min. The fixed cells were incubated with PAP7
antibody (1:250 dilution) with or without PAP7 peptide for 1 h.
After washing, the cells were incubated with HRP-conjugated goat
antirabbit secondary antibody (Transduction Laboratories, Inc.) for 1 h. PAP7 staining was visualized with peroxidase
using 3-amino-9-ethyl carbazole as a chromogen to yield a red reaction
product. After counterstaining with hematoxylin, slides were dehydrated
and permanently mounted.
Immunohistochemistry
Mouse tissues were freshly snap frozen in TISSUE TEK
(Fisher Scientific, Wood Dale, IL) on dry ice.
Specimens were fixed in cold methanol immediately after sectioning. The
slides were then placed in a chamber containing acetone for 1 min at
room temperature to remove the lipid droplets and then incubated in
blocking solution (10% goat serum) (Zymed Laboratories, Inc., South San Francisco, CA) for 10 min. Subsequently, the
slides were incubated with anti-PAP7 antibody (1:100) for 3 h at
37 C in a humid chamber, washed with PBS three times for 5 min each,
incubated with HRP-conjugated goat antirabbit secondary antibody for
1 h at 37 C, and then washed with PBS as before. To amplify the
signal the slides were treated with biotinyl-tyramide working solution
(TSA-Indirect Tyramide Signal Amplification Kit) (NEN Life Science Products) for 10 min at room temperature, and then
incubated with diluted streptavidin-HRP for 30 min at room temperature.
After treatment with 3-amino-9-ethyl carbazole reagent for 1 h at
37 C for color staining, the sections were counterstained with
hematoxylin, dehydrated, and permanently mounted. Slides were viewed
and pictures taken using an BX-40 microscope equipped with a PM20
camera system (Olympus Corp., Melville, NY).
GST Precipitation Assay
The PAP7 (216445) partial cDNA encoding interaction domain was
inserted into pGEX-6P-1 bacterial expression vector (Amersham Pharmacia Biotech, Arlington Heights, IL). The GST-fused PAP7
(216445) proteins were expressed in E. coli induced by
isopropyl-ß-D-thiogalactopyranoside and
purified with glutathione-Sepharose (Amersham Pharmacia Biotech). MA-10 mitochondria were prepared as previously
described (12). The cytosol was collected and the
mitochondria were further treated with HEPES/sucrose buffer (10
mM HEPES, pH 7.5, 320 mM
sucrose) containing 0.5% digitonin to partially solubilize PBR.
Cytosol and mitochondria extracts were incubated with purified GST-PAP7
(216445) in 1 ml of PBS (137 mM NaCl, 2.7 mM
KCl, 4.3 mM
Na2HPO4·7
H2O, 1.4 mM
KH2HPO4) overnight at 4 C.
Glutathione-Sepharose 4B was added and rotated for 30 min at 4 C. The
beads were washed with cold PBS five times for 5 min each. The beads
were then boiled in SDS-PAGE buffer (1% SDS, 1% mercaptoethanol, 10
mM Tris-HCl (pH 8.0), 20% glycerol, 0.05% bromophenol
blue), and the proteins were separated on a 816% SDS-polyacrylamide
gel and analyzed by Western blot using anti-PBR (see above) and
anti-PKA-RI antisera (Santa Cruz, CA). Purified PAP7 protein was also
incubated with 2.5 µg of purified recombinant R subunits [RI,
RIß, RII
, RIIß prepared as previously described
(55) in 100 µl of pull-down buffer (300 mM
NaCl, 0.1% Triton X-100, 1 mM phenylmethylsulfonyl
fluoride, 1 mM EDTA, 5 mM benzamidine, 5
mM dithiothreitol (DTT), 10 µg/ml of antipain,
chymostatin, leupeptin, and pepstatin A). Extracts were then incubated
with 50 µl of glutathione beads (1:1 slush in pull-down buffer) for
30 min. Beads were subsequently washed, then boiled in SDS-PAGE buffer,
and Western blot analysis was performed for the presence of R subunits.
Monoclonal antibodies directed against human RI
and human RII
(catalog nos. P53620 and P55120, respectively; K. Tasken in
collaboration with Transduction Laboratories, Inc.,
Lexington, KY) were used at a concentration of 1.0 µg/ml for
immunoblotting.
Immunoprecipitations
Human testis tissue was obtained from patients 65 to 75 yr of
age in whom testes were removed as treatment for prostate cancer at the
Department of Surgery, Ullevaal University Hospital, Oslo, Norway. Less
than 5 min after removal, epididymis and rete testis were removed,
testes were decapsulated, and pieces of testicular tissue were
immediately frozen in liquid nitrogen. Testis tissue was crushed under
liquid nitrogen using a pestle and mortar and suspended in sucrose
buffer (250 mM sucrose, 10 mM
KHPO4, 2 mM EDTA, 0.5% Triton
X-100). Tissue was then homogenized three times for 20 sec and
centrifuged in an Eppendorf centrifuge to remove the
insoluble material. Immunoprecipitation was performed using 500 µg
protein with anti-PAP7 antibody (1:100 dilution) overnight at 4 C.
Twenty-five microliters of protein A/G plus agarose were then added for
1 h, after which the beads were washed, then boiled in SDS-loading
buffer, and precipitates were submitted to Western blot analysis (as
above).
In Situ Hybridization
In situ hybridization was performed according to
Foxs protocol (56) by us and Molecular Histology Labs,
Inc (Gaithersburg, MD). Briefly, slides were rehydrated in 10
mM DTT in PBS (30 min at 45 C), followed by 10
mM DTT-10 mM
iodoacetamide-10 mM N-methyl maleimide
in PBS (10 min at room temperature). Slides were rinsed in PBS (two
times for 3 min), acetylated (0.5% acetic anhydride in 0.1
M triethanolamine, pH 8.0, for 10 min), and
rinsed in 2x sodium citrate/chloride buffer (SSC) for 10 min.
Twenty-five microliters of prehybridization cocktail (2x SSC, 1x
Denhardts, 50 mM phosphate buffer, 50
mM DTT, 500 µg/µl of salmon sperm DNA, 250
µg/µl of tRNA, 5 µg/ml poly(dA), 100 µg/ml poly(A), 0.05
pmol/ml of randomer, 57% dextran/formamide) were applied to individual
slides, which were coverslipped, incubated at room temperature for
1 h, heat denatured at 80 C for 2 min, and submersed in ice-cold
2x SSC. Slides were removed from the ice bath;
35S-labeled cRNA probe was applied, covered,
placed in a sealed humidified chamber, and incubated at 45 C overnight
(hybridization cocktail contained 0.08 µl of probe per µl of
prehybridization cocktail). Coverslips were removed, and slides were
rinsed three times in 2x SCC (5 min each at room temperature). Slides
were then incubated in 76% formamide-0.25x SSC-1.2
mM DTT-0.5 mM EDTA (two
times for 30 min at 37 C), 0.25x SSC (10 min at 37 C), and
ribonuclease (RNase) (RNase A, 25 µg/ml; RNase T1, 5 U/ml) in 0.5
M NaCl-1.2 mM DTT-0.01
M Tris-HCl (pH 7.4) for 40 min at 37 C. Slides
were rinsed in 2x SSC (two times for 5 min), dehydrated through graded
0.3 M ammonium acetate-ethanol, and air dried.
Slides were then dipped in Kodak NTB-3 photographic
emulsion (Eastman Kodak Co., Rochester, NY) at 45 C,
sealed in a light-tight box, and incubated at 4 C for 5 d. Slides
were developed at 15 C in D-19 developer (Kodak) for 4
min, followed by fixation (Kodak) for 6 min. Slides were
counterstained with hematoxylin and eosin, coverslipped with permount,
and evaluated under bright- and dark-field microscopy.
Steroid Biosynthesis
Control or transfected MA-10 cells were plated into 96-well
plates at the density of 2.5 x 104 cells
per well for overnight. The cells were stimulated with 50 ng/ml hCG in
0.2 ml/well serum-free medium for 2 h. The culture medium was
collected and tested for progesterone production by RIA using
antiprogesterone antisera (ICN Biochemicals, Inc., Costa
Mesa, CA), following the conditions recommended by the manufacturer.
Progesterone production was normalized by the amount of protein in each
well. RIA data were analyzed using the MultiCalc software (EG&G
Wallac, Inc., Gaithersburg, MD).
cAMP Assay
MA-10 cells, either empty, transfected with pSVzeo vector alone,
or transfected with PAP7, were treated and incubated as described
above. At the end of the incubation, ethanol (65% final concentration)
was added to the samples. cAMP was measured using the
cAMP[125I] RIA system from Amersham Pharmacia Biotech.
Evaluation of Cholesterol Accumulation in Mitochondria Using
R(+)-p-Aminoglutethimide (AMG)
Cells were incubated in culture medium containing 500
µM AMG and medium with and without hCG (50 ng/ml) for
2 h. Cells were harvested and mitochondria were purified in the
presence of AMG as we previously described (12). Purified
mitochondria were washed in an AMG-free buffer so that P450scc could
recover its function and metabolize the accumulated cholesterol.
Mitochondria, at a concentration of 1 mg/ml, were then incubated in the
presence of 15 mM isocitrate and 5 mM NADP for
15 min at 37 C. The reaction was stopped with ice-cold ethanol, and
pregnenolone was extracted and measured by RIA (12).
Oligonucleotide Treatment
Antisense oligonucleotides and controls (missense and
fluorescein isothiocyante-labeled antisense) directed to PAP7 were
designed and manufactured by Biognostik, Göttingen,
Germany. The sequences were: antisense, TGGCCTGTCCATTAACTG;
randomized mismatch control with same AT/GC ratio (missense),
GTCCCTATACGAAC. Cells were treated for various periods of time with
increasing concentrations of oligonucleotides. At the end of the
incubation cells were washed and treated for 2 h with hCG (50
ng/ml).
Protein Quantification and Statistical Analysis
Proteins were quantified by the dye-binding assay of Bradford
(57) with BSA as the standard. Statistical analysis was
performed by unpaired t test or ANOVA followed by the
Student-Newman-Keuls test or the Dunnett multiple comparisons test
using the Instat (v.3.0) package from GraphPad Software, Inc. (San Diego, CA).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 On leave from the Institute of Pharmaceutical Biology,
Martin-Luther-University Halle-Wittenberg, Halle/Saale,
Germany.
Abbreviations: AKAP, A kinase anchoring protein; AMG,
aminoglutethimide; C subunit, catalytic subunit of PKA; DTT,
dithiothreitol; GST, glutathione-S-transferase; HRP,
horseradish peroxidase; IMM, inner mitochondrial membrane; OMM, outer
mitochondrial membrane; PAP, PBR-associated protein; PBR,
peripheral-type benzodiazepine receptor; RACE, rapid amplification of
cDNA ends; RI, RIß, RII
, RIIß, regulatory subunits of
PKA; SSC, sodium citrate/chloride buffer; StAR, steroidogenic acute
regulatory protein.
Received for publication July 6, 2000. Accepted for publication August 14, 2001.
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REFERENCES |
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