Regulation of the Atrial Natriuretic Peptide Receptor by Heat Shock Protein 90 Complexes*

Rajinder KumarDagger , Nicholas Grammatikakis§, and Michael ChinkersDagger

From the Dagger  Department of Pharmacology, University of South Alabama, Mobile, Alabama 36688 and the § Lankenau Institute for Medical Research, Wynnewood, Pennsylvania 19096

Received for publication, November 20, 2000, and in revised form, December 21, 2000



    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Heat shock protein 90 (hsp90) is a chaperone required for the proper folding and trafficking of many proteins involved in signal transduction. We tested whether hsp90 plays a role as a chaperone for GC-A, the membrane guanylate cyclase that acts as a receptor for atrial natriuretic peptide (ANP). When cultured cells expressing recombinant GC-A were treated with geldanamycin, an inhibitor of hsp90 function, the ANP-stimulated production of cyclic GMP was inhibited. This suggested that hsp90 was required for GC-A processing and/or stability. A physical association between hsp90 and GC-A was demonstrated in coimmunoprecipitation experiments. Treatment with geldanamycin disrupted this association and led to the accumulation of complexes containing GC-A and heat shock protein 70 (hsp70). Protein folding pathways involving hsp70 and hsp90 include several pathway-specific co-chaperones. Complexes between GC-A and hsp90 contained the co-chaperone p50cdc37, typically found associated with protein kinase·hsp90 heterocomplexes. GC-A immunoprecipitates did not contain detectable amounts of Hop, FKBP51, FKBP52, PP5, or p23, all co-chaperones found in hsp90 complexes with other signaling proteins. The association of hsp90 and p50cdc37 with GC-A was dependent on the kinase homology domain of this receptor but not on its ANP-binding, transmembrane, or guanylate cyclase domains. The data suggest that GC-A is regulated by hsp90 complexes similar to those involved in the maturation of protein kinases.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


    EXPERIMENTAL PROCEDURES
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INTRODUCTION
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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-Delta KIN), the guanylate cyclase domain (FLAG-Delta CYC), and the intracellular domain (FLAG-Delta 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.


    RESULTS
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INTRODUCTION
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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.

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.

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.

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.

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).



    DISCUSSION
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INTRODUCTION
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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.


    ACKNOWLEDGEMENTS

We thank Drs. David Smith and David Toft for generous gifts of antibodies.


    FOOTNOTES

* This work was supported by National Institutes of Health Grant HL 47063 (to M. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 334-460-6782; Fax: 334-460-6798; E-mail: michaelc@jaguar1.usouthal.edu.

Published, JBC Papers in Press, January 4, 2001, DOI 10.1074/jbc.M010480200


    ABBREVIATIONS

The abbreviations used are: ANP, atrial natriuretic peptide; PAGE, polyacrylamide gel electrophoresis.


    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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