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INTRODUCTION |
The atrial natriuretic peptide
(ANP)1 receptor designated
GC-A is a single transmembrane protein that functions as a
ligand-activated guanylate cyclase (reviewed in Ref. 1). This receptor
is important in cardiovascular homeostasis; mice lacking the ANP
receptor are hypertensive (1). The binding of ANP to the extracellular
domain of GC-A results in the activation of the intracellular guanylate cyclase catalytic domain and production of the second messenger, cyclic
GMP (1). The intracellular portion of the ANP receptor contains, in
addition to a cyclase domain, a protein kinase-like domain (1). This
domain lacks protein kinase activity, and its function is incompletely
understood. It has an autoinhibitory role, however, repressing cyclase
activity in the absence of bound hormone (2, 3). In the course of our
studies of the ANP receptor, which is regulated by phosphorylation, we
used the yeast two-hybrid system to identify a protein-serine/threonine
phosphatase that binds specifically to the kinase-like domain of the
receptor (4). This phosphatase, designated PP5, may mediate ANP
receptor desensitization.
Heat shock protein 90 (hsp90) and heat shock protein 70 (hsp70) are
molecular chaperones required for the proper folding and trafficking of
many proteins involved in signal transduction (5-7). In general, this
process involves the sequential formation of complexes between newly
synthesized proteins and hsp70 and then with hsp90. A variety of
co-chaperones is also present in the complexes with heat shock
proteins; which co-chaperones are present depends on the protein being
folded. Mature steroid receptor complexes with hsp90, for example,
typically contain a large immunophilin and the small acidic protein p23
as co-chaperones. Protein kinase complexes with hsp90, on the other
hand, typically contain the co-chaperone designated p50cdc37
(5-7). The co-chaperones present in the final hsp90 heterocomplex can
be critical to the function of the hsp90 client protein. For example,
which large immunophilin is present in glucocorticoid receptor·hsp90
complexes determines receptor binding affinity (8, 9), and
p50cdc37 is required for proper folding of some protein kinases
(10). Although the hsp90 folding pathway has been best
characterized in studies of the maturation of newly synthesized steroid
receptors and protein kinases, a number of other signaling proteins
that make use of this pathway have been identified recently (7). Such
discoveries have in large part been made possible by the availability
of the drug geldanamycin, which interferes with hsp90 function by
blocking its binding site for ATP (11).
We have previously reported that PP5 binds to hsp90 and that it is a
major component of glucocorticoid receptor·hsp90 heterocomplexes (12,
13). This raised the question of whether the association of PP5 with
the kinase-like domain of the ANP receptor in the yeast two-hybrid
system was direct or whether it might be mediated by the binding of
hsp90 to both the ANP receptor and PP5. We set out, therefore, to test
whether the ANP receptor was associated with the hsp90 complexes. We
report here that the ANP receptor is associated with hsp90 and the
co-chaperone p50cdc37 via its kinase-like domain and that this
association is important for its function.
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EXPERIMENTAL PROCEDURES |
Materials--
Geldanamycin was obtained from the Drug Synthesis
and Chemistry Branch of the National Cancer Institute. Monoclonal and
polyclonal antibodies to cdc37 (14) and polyclonal antibodies to PP5
(12) have been described previously. Monoclonal antibodies to Hop (F5) and rabbit antisera to FKBP51 and FKBP52 were obtained from Dr. D. Smith (Mayo Clinic, Scottsdale, AZ). The JJ3 monoclonal antibody to p23 was obtained from Dr. D. Toft (Mayo Clinic). A monoclonal antibody to hsc70/hsp70 (SPA-820) was from Stressgen. Antibodies directed against hsp90 (AC-16) and the FLAG epitope (M2) were obtained
from Sigma. 125I-Labeled cyclic GMP was obtained from
Biochemical Technologies.
Preparation of a Stable Cell Line Expressing FLAG-tagged
GC-A--
293 cells were obtained from the American Type Culture
Collection and maintained in Dulbecco's modified Eagle's
medium containing 10% fetal bovine serum and 50 µg/ml
gentamicin. The previously described FLAG-tagged rat GC-A construct in
pSP72 (2) was excised as a SalI/SmaI fragment and
subcloned into the SalI/EcoRV sites of
pcDNA1/Neo (Invitrogen). The resulting construct,
pcDNA1/Neo-FLAG-GC-A, was transfected into the 293 cells using
Lipofectin (Life Technologies, Inc.), and stable transfectants were
selected by growth in the presence of G418 (750 µg/ml). After
isolation with cloning cylinders, individual clones were expanded and
screened by metabolic labeling with
[35S]methionine/cysteine and immunoprecipitation with the
M2 antibody, followed by SDS-PAGE and fluorography as described
previously (15). Several clones were analyzed further by measuring
125I-ANP binding and ANP-stimulated production of cyclic
GMP in intact cells, using previously described methods (2, 15). The
clone having the highest levels of FLAG-tagged GC-A, as determined by all three methods, was used in these studies after subcloning by
limiting dilution. Based on 125I-ANP binding studies, these
cells contain ~50,000 receptors/cell. Cells were maintained as
described above for the control 293 cells except that the medium was
supplemented with 400 µg/ml G418.
Cyclic GMP Production in Intact Cells--
Cells in 24-well
plates were treated in triplicate for ~16 h with the indicated
concentrations of geldanamycin or with a vehicle (Me2SO).
Treatment with 1 µM ANP for 10 min at 27 °C was
performed, and radioimmunoassay of cell extracts was then performed as
described previously (15).
Coimmunoprecipitation Experiments--
Cultures in 10-cm plates
were treated for ~16 h with the indicated concentrations of
geldanamycin or with a vehicle (Me2SO). Cells were then
washed with phosphate-buffered saline and lysed in 50 mM
Tris-HCl, pH 7.4, 150 mM NaCl, 20 mM
Na2MoO4, 0.09% Nonidet P-40, 5 µg/ml
leupeptin, and 5 µg/ml aprotinin. After the clarification of lysates
by centrifugation at 15,000 × g for 20 min at 4 °C, the immunoprecipitation of FLAG-tagged proteins was performed using
either M2 beads (Sigma) or M2 antibody prebound to anti-mouse IgG-agarose beads as described previously (12). After washing five
times with the lysis buffer, the beads were heated in SDS sample
buffer. In one experiment (Fig. 4, lower panel), the
immunoprecipitated proteins were eluted from M2 beads using 200 µg/ml
FLAG peptide (DYKDDDDK) in phosphate-buffered saline to reduce
background staining of the IgG heavy chain. Aliquots of the
immunoprecipitates were then subjected to SDS-PAGE and immunoblotting
with the indicated antibodies as described previously (12).
Expression of GC-A Deletion Mutants--
COS-7 cells in 10-cm
plates were transfected with the indicated plasmids using the Superfect
reagent (Qiagen) according to the manufacturer's instructions. The
GC-A constructs lacking the kinase-like domain (FLAG-
KIN), the
guanylate cyclase domain (FLAG-
CYC), and the intracellular domain
(FLAG-
KC) and the construct consisting of the soluble intracellular
domain (FLAG-IN) have been described previously (15). Two days after
transfection, cells were processed for immunoprecipitation and
immunoblotting as described above.
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RESULTS |
Effect of Geldanamycin on ANP-stimulated Cyclic GMP
Production--
To test whether hsp90 was involved in the processing
of the ANP receptor, GC-A, we treated 293 cells overexpressing
FLAG-tagged GC-A with geldanamycin, an inhibitor of hsp90 function. We
have previously shown that FLAG-tagged GC-A is functionally
indistinguishable from the untagged receptor (15). Cells were treated
overnight with the indicated concentrations of geldanamycin, and GC-A
function was assessed by measuring ANP-stimulated cyclic GMP production in intact cells. As shown in Fig. 1, a
brief treatment with ANP led to a robust cyclic GMP response. Treatment
with geldanamycin led to a concentration-dependent
reduction in ANP-stimulated accumulation of cyclic GMP. Approximately
50% reductions in the ANP response were observed in several
experiments following treatment with 10 µM geldanamycin.
These results suggested that hsp90 is important for GC-A function.

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Fig. 1.
Effect of geldanamycin on ANP-stimulated
cyclic GMP production. Cells stably expressing GC-A were incubated
overnight with the indicated concentrations of geldanamycin, then
treated for 10 min with ANP as described under "Experimental
Procedures." Cell-associated cyclic GMP was then measured by
radioimmunoassay. Results are presented as mean ± S.D. for a
representative experiment. GA,
geldanamycin.
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Coimmunoprecipitation of hsp90 with GC-A--
Although the
inhibition of ANP-stimulated cyclic GMP production by geldanamycin
indicated a role for hsp90 in GC-A function, it did not
demonstrate a direct role. It would be possible for geldanamycin
to inhibit GC-A function indirectly. For example, hsp90 could be
required for the processing of another protein that in turn regulated
GC-A activity. To test whether GC-A was physically associated with
hsp90, we performed coimmunoprecipitation experiments. Detergent
lysates of the control 293 cells or of 293 cells expressing FLAG-tagged
GC-A were subjected to immunoprecipitation with the M2 antibody to the
FLAG epitope. Immunoprecipitated proteins were analyzed by Western
blotting with different antibodies. In control blots with M2 antibody,
the expected GC-A doublet of 130 and 110 kDa was seen in
immunoprecipitates from the FLAG-GC-A cell line but not in
immunoprecipitates from the control 293 cells (Fig.
2). hsp90 was coimmunoprecipitated with
the FLAG-tagged GC-A in the absence of geldanamycin, but the complex
between GC-A and hsp90 was disrupted following treatment with
geldanamycin (Fig. 2). These data indicated that GC-A was present in a
geldanamycin-sensitive complex with hsp90.

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Fig. 2.
Coimmunoprecipitation of hsp90 with
GC-A. Lysates from the indicated cultures were subjected to
immunoprecipitation with the M2 antibody prebound to goat anti-mouse
IgG beads. Immunoprecipitates were then analyzed by SDS-PAGE and
immunoblotting with the indicated antibodies. Cells were treated
overnight with 5 µM geldanamycin or with
Me2SO vehicle. C, control.
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Association of GC-A with hsp70--
The folding pathway for
proteins chaperoned by hsp90 normally begins with an association
between newly synthesized proteins and hsp70. When the transfer of
these proteins to an hsp90 complex is blocked by geldanamycin,
complexes with hsp70 accumulate (6). We tested whether this pathway was
involved in the folding of GC-A by using the coimmunoprecipitation
approach described above. FLAG-tagged GC-A was immunoprecipitated from
cell lysates with the M2 antibody, and immunoprecipitates were analyzed
by Western blotting with an antibody to hsp70. As shown in Fig.
3, hsp70 was not immunoprecipitated with
the M2 antibody from untreated cells or from control cells lacking
FLAG-tagged GC-A. After treatment with 10 µM
geldanamycin, however, we observed coimmunoprecipitation of hsp70 with
GC-A. The accumulation of hsp70·GC-A complexes in the presence of
geldanamycin suggested a folding pathway much like that observed for
other proteins in which first hsp70 and then hsp90 act as
chaperones.

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Fig. 3.
Coimmunoprecipitation of p50cdc37
and hsp70 with GC-A. Lysates from the indicated cultures were
subjected to immunoprecipitation with the M2 antibody prebound to goat
anti-mouse IgG beads. Immunoprecipitates were then analyzed by SDS-PAGE
and immunoblotting with monoclonal antibodies directed against
p50cdc37 or hsp70. Cells were treated overnight with 10 µM geldanamycin or with Me2SO vehicle.
C, control.
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Presence of cdc37 in hsp90·GC-A Complexes--
At least 10 different co-chaperones associated with hsp90 complexes have been
identified (7). Which co-chaperones are associated with hsp90 is
dependent upon which hsp90 client protein is present in the
heterocomplex. In the steroid receptor pathway, Hop is thought to
recruit hsp90 to complexes between newly synthesized receptors and
hsp70. Mature receptors are found in complexes with hsp90 containing
one of the large immunophilins (CyP-40, FKBP51, or FKBP52) or PP5 and
the acidic protein p23 (5). Mature hsp90 complexes with protein
kinases, on the other hand, contain p50cdc37 rather than a
large immunophilin or PP5 (6, 7). We tested which of these
co-chaperones was associated with GC-A by using coimmunoprecipitation
and Western blotting (Figs. 3 and 4). As shown in Fig. 4, FKBP51, FKBP52, Hop, PP5, and p23 were undetectable in
FLAG-GC-A immunoprecipitates in the absence or presence of geldanamycin, although the proteins were readily detected in cell lysates. p50cdc37, however, specifically coimmunoprecipitated
with GC-A (Fig. 3). The association between p50cdc37 and GC-A
was disrupted following geldanamycin treatment, much like that between
hsp90 and GC-A (Fig. 3). In that they contained p50cdc37 and
none of the other co-chaperones tested, the hsp90 complexes with GC-A
were similar to those associated with protein kinases.

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Fig. 4.
Immunoblotting of GC-A immunoprecipitates
with antibodies to other hsp90 co-chaperones. A,
lysates from the indicated cultures were subjected to
immunoprecipitation with M2 beads diluted 10-fold with Sepharose 4B.
After washes, samples were eluted by heating in SDS sample buffer.
Immunoblotting was then performed using monoclonal antibodies to the
FLAG epitope (M2), Hop, or p23 and rabbit antisera to FKBP51 or FKBP52.
B, lysates from the indicated cultures were subjected to
immunoprecipitation with undiluted M2 beads. After washes, samples were
eluted at 4 °C using the FLAG peptide to minimize the background
staining of the IgG heavy chain by the anti-PP5 serum. Immunoblotting
was then performed using monoclonal antibodies to the FLAG epitope (M2)
or a rabbit antiserum to PP5. For the lysate controls in both panels,
an aliquot of the untreated FLAG-GC-A 293 cell lysate was used.
C, control.
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Role of the Protein Kinase-like Domain of GC-A in Binding cdc37 and
hsp90--
GC-A and other membrane guanylate cyclases contain an
intracellular protein kinase-like domain that, although it lacks
protein kinase activity, contains most of the conserved residues found in authentic protein kinases (1). Because p50cdc37 generally
functions as a co-chaperone for protein kinases, we hypothesized that
the interaction of p50cdc37 with hsp90 complexes containing
GC-A might be mediated by the kinase-like domain. We tested this
hypothesis by examining the coimmunoprecipitation of p50cdc37
with full-length GC-A and with several GC-A deletion mutants. The
FLAG-tagged proteins were expressed in COS-7 cells and
immunoprecipitated from detergent lysates with the M2 antibody to the
FLAG epitope. Immunoprecipitates were then subjected to SDS-PAGE and
immunoblotting with the indicated antibodies. As shown in Fig.
5A, p50cdc37
coimmunoprecipitated with full-length GC-A, with GC-A lacking its
guanylate cyclase catalytic domain, or with the soluble intracellular domain of GC-A. Immunoprecipitates of deletion mutants lacking the
kinase-like domain or lacking the entire intracellular domain did not
contain p50cdc37 (Fig. 5A). Thus, the kinase-like
domain appears to be required for the binding of GC-A to
p50cdc37. The extracellular, transmembrane, and guanylate
cyclase domains of GC-A were not required for p50cdc37 binding.
The expression of the wild-type GC-A and each of the deletion mutants
was confirmed by immunoblotting with M2 (Fig. 5A). Because
of a background band of ~90 kDa in Western blots in COS cells, we
examined the association of hsp90 with the various GC-A mutants by
metabolic labeling with [35S]methionine and
immunoprecipitation. As for p50cdc37, coimmunoprecipitation of
hsp90 with GC-A was dependent on the presence of the protein
kinase-like domain (Fig. 5B). Based on these results, we
conclude that the binding of GC-A to both hsp90 and p50cdc37 is
mediated by the protein kinase-like domain of GC-A.

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Fig. 5.
Mapping of the p50cdc37 and hsp90
binding sites on GC-A. M2 immunoprecipitates of detergent lysates
prepared from untransfected COS cells or COS cells transfected with the
indicated GC-A deletion mutants were analyzed by SDS-PAGE and
immunoblotting with the indicated antibodies (A) or
autoradiography of immunoprecipitates from cells metabolically labeled
with [35S]methionine
(B).
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DISCUSSION |
The function of the protein kinase-like domain found in membrane
guanylate cyclases has been elusive since its initial description more
than a decade ago (16). The activation of GC-A by the deletion of this
domain suggests that it served an autoinhibitory function, and
it has been widely assumed, based on its homology to protein kinases,
that this domain contains the allosteric ATP binding site required for
the activation of the wild-type GC-A (1). It is still unclear, however,
exactly how the protein kinase-like domain regulates receptor activity.
More recent evidence has suggested that the protein kinase-like domains
of membrane guanylate cyclases may function as anchoring sites for
regulatory proteins. The protein kinase-like domain of GC-A interacts
with the TPR domain of PP5 in the yeast two-hybrid system (4). Although
we did not detect the coimmunoprecipitation of PP5 and GC-A from
detergent lysates, this does not preclude their association in
vivo; interactions between the TPR proteins and their targets can
be detergent-sensitive. The protein kinase-like domains of the retinal
membrane guanylate cyclases have been suggested to be important for the
binding of GCAPs, which regulate their guanylate cyclase
activity in a Ca2+-dependent manner (17). We
now report that the protein kinase-like domain of GC-A contains a
binding site for p50cdc37·hsp90 complexes. It seems likely
that the association between GC-A and hsp90 is mediated by
p50cdc37 because this protein has been shown to bind directly
to protein kinases (14). We propose that p50cdc37 binds to GC-A
because the structure of the kinase-like domain of GC-A is similar to
that of authentic protein kinases.
The coimmunoprecipitation data presented here are consistent with most
models for the folding of client proteins by hsp70, hsp90, and their
co-chaperones. Similar to other proteins, GC-A appears to form
complexes with hsp70 and then, in a geldanamycin-sensitive manner, to
form a complex with hsp90. As has been shown for other hsp90 client
proteins, the disruption of this complex by geldanamycin treatment
leads to a disruption of GC-A function. We were initially surprised
that we did not detect p23, Hop, or large immunophilins in the
GC-A·hsp90 complexes and that the only hsp90 co-chaperone we detected
was p50cdc37. Because p50cdc37 is thought to be a
kinase-specific hsp90 co-chaperone, its association with a membrane
guanylate cyclase receptor was unexpected. This association, however,
was explained by mapping experiments localizing the site of
p50cdc37 and hsp90 binding to the kinase-like domain of GC-A.
Although the literature tends to draw a clear line between protein
kinases forming complexes with hsp90 and p50cdc37 and other
proteins forming complexes with hsp90 and other co-chaperones, the
reality is probably more complex. A recent report describes the
association of two different protein kinases with hsp90 complexes containing not only p50cdc37 but also the large immunophilin
FKBP52 (18). The precise pathway leading to the final
kinase·p50cdc37·hsp90 complex is not fully understood. Our
data do not establish that these other co-chaperones are not involved
in the processing of GC-A; we can only conclude that under the
conditions described here, we do not detect these proteins in GC-A immunoprecipitates.
Although the processing of proteins via the hsp90 pathway has been best
studied using steroid receptors and protein kinases, in recent years it
has become clear that a variety of other hsp90 client proteins exists.
These include telomerase (19), nitric-oxide synthase (20), G protein
subunits (21), the cystic fibrosis transmembrane regulator (22), and
now GC-A. To our knowledge, GC-A is the first protein that is not a
protein kinase that is associated with p50cdc37 in the hsp90
folding pathway. The role of hsp90 and p50cdc37 in GC-A
processing and the biochemical mechanisms by which these proteins
affect GC-A activity remain to be determined. It is not clear whether
they are involved in the subcellular trafficking of GC-A, in
maintaining GC-A in a hormone-activable conformation, as previously
shown for steroid receptors, or in some other function. In the case of
one Src family protein kinase, it has been shown that hsp90 is
required for the folding of the protein kinase domain but not for the
folding of the SH2 domain (23). It is possible that the same is true of
GC-A, i.e. that hsp90 may be required only for the folding
of the kinase-like domain. We believe that hsp90 and p50cdc37
will be found to play a general role in processing membrane guanylate cyclases. A genetic connection between hsp90 and an olfactory guanylate
cyclase in Caenorhabditis elegans was described recently (24). It will be interesting to determine whether hsp90 and p50cdc37 are associated with other mammalian membrane guanylate
cyclases and to see whether mutations in p50cdc37 in other
model organisms (25) affect guanylate cyclase function.