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Department of Physiology and Biophysics, The University of Iowa Carver College of Medicine, Iowa City, Iowa 52242
Address all correspondence and requests for reprints to: Deborah L. Segaloff, Ph.D., Department of Physiology and Biophysics, Roy J. and Lucille A. Carver College of Medicine, The University of Iowa, Iowa City, Iowa 52242. E-mail: deborah-segaloff{at}uiowa.edu.
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ABSTRACT |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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In recent years both activating and inactivating naturally occurring mutants of each of the glycoprotein hormone receptors have been reported (3, 4, 5, 6, 7). Whereas activating mutants of the receptors result in a gain-of-function of the target cells due to constitutive activity of the receptor in the absence of hormone, inactivating mutants result in a loss of function of the target cells. Although in some cases the decreased hormone responsiveness is due to decreased coupling of the mutant receptor to G protein, in most cases it is due to decreased cell surface expression of the mutant receptor. The impairment in trafficking of the mutant receptor to the cell surface results from the intracellular retention of the mutant receptor in the endoplasmic reticulum (ER). These observations with naturally occurring mutations of the glycoprotein hormone receptors are reminiscent of those made with mutations of the glycoprotein hormone receptors of laboratory design where many were similarly found to result in intracellular retention of the mutant receptor (see Ref. 8 and references therein).
Presumably, the intracellular retention of misfolded mutant glycoprotein hormone receptors occurs as a result of the quality control mechanisms within the ER that detect misfolded proteins and prevent them from exiting the ER. The ER contains a number of folding enzymes and molecular chaperones that both facilitate the folding and export of newly synthesized proteins and recognize misfolded proteins and prevent their transport from the ER to the Golgi (see Refs. 9, 10, 11 for reviews). Key chaperones for glycoproteins are calnexin and calreticulin, which assist in the folding of proteins containing N-linked oligosaccharides and presumably prevent the exit of misfolded glycoproteins from the ER. Another set of chaperones present in the ER, Grp94 and BiP (also referred to in the literature as GRP78) function by preventing aggregation of newly synthesized proteins. Members of the disulfide isomerase family protein disulfide-isomerase (PDI) and Erp57 enzymatically catalyze rate-limiting steps in the folding pathway of polypeptides.
In an effort to understand the processing of the glycoprotein receptors in the ER and what goes awry with misfolded inactivating mutants of the glycoprotein hormone receptors, this study examined the potential association of human (h)LHR, hFSHR, and hTSHR with calnexin, calreticulin, BiP, Grp94, ERp57, and PDI. We also examined chaperone associations with two misfolded loss-of-function mutants of the hLHR that are retained intracellularly. We describe the association of the immature forms of the wild-type (wt) glycoprotein hormone receptors with calnexin, calreticulin, and PDI. Significantly, we also report a different pattern of chaperone protein associations for the intracellularly retained mutants of the hLHR as compared with the wt receptor and also different patterns of chaperone associations between the two mutants.
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RESULTS |
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The results of a Western blot of 293 cells stably transfected with the wt forms of myc-hFSHR, myc-hLHR, or myc-hTSHR are shown in Fig. 1
. To facilitate the identification of mature vs. immature forms of the glycoprotein hormone receptors as shown in Fig. 1, the cell lysates were left untreated or were treated with endoglycosidase H (endoH), which cleaves high mannose-containing N-linked carbohydrates from immature glycoproteins in the ER, or neuraminidase, which cleaves sialic acids from mature glycoproteins. The analysis of the hTSHR on Western blots is more complex than that of the hFSHR and hLHR because some mature hTSHR is cleaved, causing the release of a portion of the N terminus of the receptor, designated fragment A, under reducing conditions (14). Because the myc epitope tag was placed at the N terminus of each receptor, the immunoblot of the hTSHR, therefore, reveals the immature and mature hTSHR as well as the 53-kDa fragment A.
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Coimmunoprecipitation of hLHR with Chaperone Proteins
We had previously shown that the immature form of the rat LHR is associated with calnexin (18). We initially sought to determine whether similar results would be observed with the hLHR following the same experimental protocol. As shown in Fig. 2, the immature form of the hLHR is indeed coimmunoprecipitated with calnexin, demonstrating their physical association. As would be expected, no association of the mature form of the hLHR was observed with calnexin.
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, lanes 13 and 14). With regard to calreticulin, although nonspecific background was observed in some cases, most notably when apyrase was added to the lysis buffer, the signal from the myc-hLHR cells was far stronger than that of the empty vector cells (cf. Fig. 3, lanes 4 and 11), confirming a specific association of calnexin with the hLHR under those conditions. PDI was found to associate strongly with the immature hLHR when either no additions were made to the lysis buffer or when EDTA/EGTA was present (Fig. 3, lanes 9 and 10). A weak association of PDI with the immature hLHR was seen in the presence of the cross-linking reagent DSP (Fig. 3, lanes 13 and 14). We did not detect associations of Grp94, BiP, or ERp57 with the immature hLHR under any of the conditions tested.
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and 4). In either case, one observes an association of the immature form of the hLHR with calnexin, calreticulin, and PDI, but not with Grp94, BiP, and ERp57. Importantly, the data in Fig. 4 also show that the same pattern of chaperone protein associations is observed with the immature forms of each of the wt glycoprotein hormone receptors.
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, the treatment of cells with castanospermine caused the immature forms of each glycoprotein hormone receptor to migrate with a slightly higher molecular weight than those observed in untreated cells. This would be consistent with the larger oligosaccharides present on them as a result of castanospermine treatment. Castanospermine treatment, however, did not prevent the maturation of the glycoprotein hormone receptors because the mature form of each is still observed. Nor did castanospermine fully inhibit their association with calnexin. Concentrations of castanospermine up to 200 µg/ml were also tested and gave similar results (concentrations above this could not be evaluated due to their toxicity). These data suggest that the nature of the association of calnexin and the glycoprotein hormone receptors is not entirely based on the lectin properties of this chaperone.
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, the predominant form of each mutant on Western blots was an endoH-sensitive immature receptor that comigrated with the immature form of the wt hLHR. The S616 mutant is also visible as a mature protein on the Western blot, but in very low abundance. Confocal microscopy shows the wt hLHR to be expressed predominantly at the cell surface (Fig. 6B). In contrast, little (S616Y) or no (A593P) cell surface expression of either mutant is observed, and there is a large intracellular accumulation of each. The data from Fig. 6, A and B, confirm that the A593P and S616Y hLHR loss-of-function mutants are expressed primarily as immature proteins and are retained intracellularly in the ER, consistent with these mutations causing misfolding of the receptors.
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), we examined the associations of the different chaperone proteins with these two misfolded hLHR mutants. The results, shown in Fig. 7, depict that region of the gel to which each chaperone migrates. Similar to the wt hLHR, calnexin and calreticulin associate with the misfolded hLHR mutants as well. PDI, which associates with hLHR(wt), is also clearly detected as associated with hLHR(S616Y). Little or no PDI, however, appears to be associated with hLHR(A593P). Interestingly, two chaperone proteins that do not associate with the hLHR(wt) associate with one or both misfolded hLHR mutants. Thus, BiP is associated with both A593P and S616Y and Grp94, although not detected as associated with S616Y, is clearly associated with A593P. Altogether, the data in Fig. 7 suggest that not only are the patterns of chaperone proteins with wt vs. misfolded mutants of the glycoprotein hormones different (using the hLHR as an example), but that the chaperone associations between two different mutants of the same receptor are not necessarily the same. For this reason, we did not examine misfolded mutants of the hFSHR and hTSHR because any observed differences in chaperone associations could not necessarily be attributed to differences between the glycoprotein hormone receptors, but could, similarly, be mutation specific.
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DISCUSSION |
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There is very little known about the role of chaperone proteins in the folding of GPCRs (41), much less the glycoprotein hormone receptors (18, 42). An earlier study from our laboratory showed that the immature forms of the rat LHR and FSHR could be detected as associated with calnexin (18). Other chaperone proteins were not examined, and it was not known whether these results would be applicable to the human forms of the receptors. A recent study by Siffroi-Fernandez et al. (42) reported associations of calnexin and calreticulin with the immature form of the hTSHR. The present study was undertaken to examine a number of different chaperone proteins (calnexin, calreticulin, Grp94, BiP, Erp57, and PDI) to determine which ones associate with the nascent forms of each of the human glycoprotein hormone receptors. In addition to determining whether the repertoire of chaperone proteins associating with each of the human glycoprotein hormone receptors was the same or not, we also wished to examine whether the repertoire was altered between a wt glycoprotein receptor and misfolded mutants thereof. The data presented indicate a similar pattern of chaperone protein associations between the wt hLHR, hFSR, and hTSHR. Thus, the immature forms of each of the wt glycoprotein hormone receptors can be coimmunoprecipitated with calnexin, calreticulin, and PDI, but not with Grp94, BiP, and ERp57. Although we cannot rule out the possibility that the newly synthesized wt forms of the glycoprotein hormone receptors also interact with the ER chaperones GRP94, BiP, and ERp57, our studies failed to detect associations with these proteins using a variety of experimental approaches. In this respect, our results differ from those of Siffroi-Fernandez et al. (42), who observed an association of BiP with the immature hTSHR when expressed in K652 cells. However, there are a number of technical differences between the two studies that could easily account for the apparently discrepant results regarding BiP and the wt hTSHR. One notable difference is that different cell lines were used in the two studies. This may be relevant given that redox potentials vary between cells, and chaperone protein associations are dependent upon the redox potential (43). Also, Siffroi-Fernandez overexpressed BiP to better enable the detection of its association with the hTSHR. Even with the same cells, we have shown that different lysis conditions can have marked effects on the ability to detect a given chaperone association (Fig. 3). Therefore, different lysis conditions may also be a factor to consider. For these reasons, we would argue that the direct comparison between the wt glycoprotein hormone receptors and mutants thereof, using the same cell line and the same lysis conditions for a given chaperone association, better enable one to compare the chaperone associations between the glycoprotein hormone receptors. Our data show that in HEK293 cells, the chaperone proteins calnexin, calreticulin, and PDI physically associate with newly synthesized glycoprotein hormone receptors, suggesting a role for these chaperones in the folding of these receptors.
In spite of the wealth of information regarding the role of N-linked oligosaccharides in the binding of calnexin and calreticulin to newly synthesized glycoproteins, there is increasing data to suggest that these chaperone proteins can also interact with proteins independent of their N-linked carbohydrates (44, 45). Our study examined this issue by determining whether calnexin associated with the immature forms of the glycoprotein hormone receptors when cells were treated with castanospermine. Under these conditions, the monoglucosylated form of the glycoprotein is not generated and, therefore, binding of calnexin to the glycoprotein via interactions with the carbohydrate portion of the molecule is inhibited. Importantly, our data show that interactions of the immature forms of the hFSHR, hLHR, and hTSHR with calnexin are still observed under these conditions, suggesting that calnexin can interact with the glycoprotein hormone receptors independent of N-linked carbohydrates (cf. Fig. 5). Although we used concentrations of castanospermine comparable to those reported by others for similar studies and higher concentrations were toxic to the cells, we cannot formally rule out the possibility that monoglucosylation was not entirely prevented under the conditions used. Assuming that monoglucosylation was completely blocked, our data would support the model of a dual-binding mode for calnexin and calreticulin in which unfolded glycoproteins interact with these chaperones through both a lectin site and a polypeptide site (44, 46, 47).
Although calnexin and calreticulin were found to physically associate with the immature forms of the wt glycoprotein hormone receptors, we did not observe an association with ERp57. This was somewhat surprising given that ERp57, a thiol-disulfide oxidoreductase that catalyzes disulfide bond formation and isomerization, is thought to be a cochaperone that physically associates with calnexin and calreticulin when they are bound to the nascent polypeptide (44, 48, 49). Instead, we observed the association of PDI, another member of the thiol oxidoreductase family, with the immature wt glycoprotein hormone receptors. However, previous studies have shown that PDI and ERp57 bind to newly synthesized proteins with different specificities (50). These considerations, taken together with the observation that PDI could be coimmunoprecipitated with each of the glycoprotein hormone receptors, but not cross-linked to the receptor itself, suggest that, for the glycoprotein hormone receptors, PDI may be acting as a cochaperone with calnexin and calreticulin.
To examine the ramifications of misfolding on the associations of chaperone proteins with the glycoprotein hormone receptors, we focused on two hLHR mutants, A593P and S616Y, that are retained intracellularly (cf. Fig. 6). We had expected to observe a change in the repertoire of chaperone proteins associating with the misfolded hLHR mutants as compared with the immature wt hLHR. This was indeed observed. Therefore, although the mutants still associated with calnexin and calreticulin [although the association of calnexin with hLHR(A593P) appeared to be reduced as compared with hLHR(S616Y) or the wt receptor], both mutants associated with BiP, a chaperone protein that was not observed associated with any of the wt glycoprotein hormone receptors. Our data showing an association of BiP with the misfolded hLHR mutants, but not the wt hLHR precursor, are consistent with the presumed actions of BiP (as reviewed in Ref. 11). Thus, BiP promotes the proper folding of newly synthesized polypeptides by inhibiting their aggregation as they undergo different folding conformations. Whereas it interacts transiently with properly folded proteins, it remains stably associated with proteins that are misfolded. In the latter case, it may mediate their retrograde translocation for proteosomal degradation. It is likely, therefore, that if BiP were interacting with the newly synthesized wt hLHR, the interactions are too transient to be detected. In contrast, our detection of interactions of BiP with the hLHR mutants suggests more stable associations between these proteins.
Although both hLHR mutants were found to associate with BiP, differences between the mutants were observed with regard to PDI and Grp94 interactions. Whereas PDI was observed to be associated with hLHR(S616Y), similar to the wt hLHR receptor precursor, it was not detected in association with hLHR(A593P). Furthermore, Grp94 was observed to associate with hLHR(A593P), but not with hLHR(S616Y). Grp94, a member of the heat shock protein 90 class of proteins, is found in some cases in ternary complexes with BiP and nascent polypeptides (as reviewed in Ref. 11). Similar to BiP, its interactions with misfolded proteins are more prolonged. It has been suggested that Grp94 interacts with more advanced folding intermediates than BiP (25). Our observations are not in agreement because we observe a greater association of Grp94 with hLHR(A593P), where no mature mutant receptor is detected (Fig. 6), than with hLHR(S616Y), where a small amount of mature mutant receptor is detected. Regardless, though, the differences in the patterns of chaperone protein associations with two different misfolded mutants of the hLHR argue that despite the net effect of these mutations being to cause their intracellular retention in the ER (and presumably ultimately their degradation), their conformations are most likely distinct and they probably progress to different stages of folding maturation (and hence different chaperone proteins) before being targeted for degradation. The degree to which a given misfolded mutant advances in the folding process may be relevant in terms of differential abilities to rescue misfolded mutants of GPCRs by decreased temperature (8) or pharmacological chaperones (51, 52, 53).
In summary, we have identified key ER chaperone proteins that associate with the immature forms of the glycoprotein hormone receptors and with misfolded mutants thereof. Given that most loss-of-function mutations of the glycoprotein hormone receptors are a result of misfolding of the receptors that lead to intracellular retention of the receptors and, therefore, decreased cell surface expression, future studies on how the folding of these proteins is regulated and the fate of misfolded glycoprotein hormone receptors are clearly important.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Epitope-Tagged Receptor Constructs
The wt hLHR and hFSHR cDNA were kindly provided by Ares Advanced Technology (Ares-Serono Group, Randolph, MA). The wt hTSHR was generously given to us by Dr. Gilbert Vassart (Institut de Recherche Interdisciplinaire en Biologie Humaine et Nuléaire and Service de Genetique Medicale, Université Libre de Bruxelles, Campus Erasme, Brussels, Belgium). Each receptor cDNA was subcloned into pcDNA3.1(neo) (Invitrogen, Carlsbad, CA). A c-myc epitope tag was inserted after the signal peptide of each receptor such that the tag would be on the N terminus of the mature protein by using the PCR overlap extension method (54, 55). The epitope-tagged receptors are referred to herein as myc-hLHR, myc-hFSHR, and myc-hTSHR. Automated DNA sequencing, performed by the DNA core of the Diabetes and Endocrinology Research Center of the University of Iowa, was performed to ensure the fidelity of each modified receptor construct. Preliminary experiments verified that the placement of a myc epitope tag at the N terminus of the wt receptors did not adversely affect protein function in terms of cell surface expression, hormone binding activity, or hormone-stimulated cAMP production.
Transiently and Stably Transfected Cells
HEK293 cells were maintained at 5% CO2 in a culture medium consisting of DMEM containing 50 µg/ml gentamicin, 10 mM HEPES, and 10% newborn calf serum. Using the calcium precipitation method (56), HEK293 cells were transfected using 20 µg plasmid in 10 ml/100-mm dish. Transiently transfected cells were used for experiments 48 h after transfection. Stably transfected cells were obtained by selecting transfected cells in growth media containing 700 µg/ml G418 (Invitrogen, Carlsbad, CA) for 2 wk. The cell lines were then maintained in the selective media.
Western Blotting of Detergent-Solubilized Cell Extracts
HEK293 cells stably expressing myc-tagged glycoprotein hormone receptors were analyzed by Western blotting as follows. After cells were washed they were detergent solubilized using Lysis Buffer [0.5% Nonidet P-40 in 150 mM NaCl, 20 mM HEPES (pH 7.4), containing 1 mM phenylmethylsulfonylfluoride, 5 µg/ml leupeptin, and 5 nM pepstatin A as protease inhibitors). The extracts were rotated 30 min at 4 C in a 1.5-ml tube rotator. The lysate was then cleared by centrifugation for 15 min at 4 C in a microcentrifuge at 13,000 rpm. The Bradford assay (57) was used to measure protein concentration in the supernatant. A 6-fold concentrate of Laemmli sample buffer containing reducing agents [12% (wt/vol) sodium dodecyl sulfate (SDS), 40% glycerol, 109 mM EDTA, 1.5 M Tris/HCl, 98 mg/ml dithiothreitol, and 6% vol/vol ß-mercaptoethanol) was used to dilute the samples 1:6. Samples were incubated in Laemmli buffer 1 h at room temperature and then used immediately or stored at –20 C. The samples were run on a 7.5% SDS-PAGE gel and transferred to a PVDF membrane. The membrane was probed with anti-myc monoclonal antibody (9E10, 1:500 dilution) (Santa Cruz Biotechnology). Goat anti-mouse antibody conjugated to horseradish peroxidase (Bio-Rad, 1:1000 dilution) was used as the secondary antibody. Bands were detected using an ECL detection system (Amersham Pharmacia Biotech).
Treatment of Solubilized Extracts with Glycosidases
Protein detergent-solubilized extract (100 µg), obtained from stably transfected cells, was incubated 24 h at 37 C with either no additions or with endoglycosidase H (300 mU/ml) or neuraminidase (300 mU/ml).
Various Lysis Conditions and Cross-Linking of Proteins
Stably transfected cell lines were harvested by scraping, and lysed on ice by addition of Lysis Buffer NaCl, 20 mM HEPES, which in some cases was supplemented with either 5 mM EDTA plus 5 mM EGTA, or with 50 U/ml apyrase, 2.5 mM ATP or 2.5 mM MgCl2. When cross-linking was performed, two different protocols were used. For cross-linking during lysis, cells were incubated for 30 min on ice in Lysis Buffer containing 0.2 mM DSP freshly prepared from a 2000-fold frozen stock in dimethyl sulfoxide (23). Unreacted cross-linking reagent was blocked for 10 min by adjusting the lysates to 50 mM Tris/HCl, pH 7.4. For in situ cross-linking of proteins in living cells, cells were incubated for 30 min on ice with 0.2 mM DSP in PBS, pH 7.4 (PBS), adjusted to 50 mM Tris/HCl, and incubated for 10 min. Cells were then scraped, washed, and solubilized in Lysis Buffer supplemented with protease inhibitors as described above.
Immunoprecipitations
HEK293 cells were stably transfected with myc-tagged receptors, and detergent-solubilized lysates were prepared as described above, using various lysis conditions and cross-linking protocols. The day before the experiment, each given antichaperone antibody (ERp57, 1:10; Grp94, 1:10; BiP, 1:10; PDI, 1:10; calreticulin, 1:10; calnexin, 1:50) or anti-myc antibody (9E10, 1:10) was conjugated to 20 µl protein A-Sepharose by rotating overnight at 4 C. On the day of the experiment, the conjugated antibody resin was washed twice with 1 ml Lysis Buffer, and 500 mg cell extract were added. After rotating 90 min at 4 C, the beads were washed three times with 1 ml Lysis Buffer, and the immunoprecipitate was eluted with 100 µl Laemmli sample buffer containing reducing agents. Each immunoprecipitate (50 µl) was resolved by SDS-PAGE and transferred to a polyvinylidenedifluoride membrane as described above. The membrane was probed with antichaperone antibody (antibody dilutions: ERp57, 1:3,000; Grp94, 1:3,000; BiP, 1:3,000; PDI, 1:3,000; calreticulin, 1:20,000; calnexin, 1:10,000) or anti-myc antibody (9E10, 1:500 or polyclonal anti-myc, 1:500). Secondary antibody (1:1000) against monoclonal or polyclonal antibodies was used to generate immunoreactive bands, which were visualized using an ECL detection system.
Castanospermine Treatment of Transiently Transfected Cells
Castanospermine was solubilized in growth media at a concentration of 100 µg/ml and immediately added to 100-mm plates of cells. Cells were treated with castanospermine 2 h before transfection, and then transfected with receptor constructs using growth media containing castanospermine. After the overnight transfection, the media were then replaced with fresh growth media containing castanospermine, and the cells were allowed to grow an additional 24 h.
Confocal Imaging of the myc-hLHR and Mutants Thereof
Two days before the experiment, cells stably transfected with myc-hLHR(wt), myc-hLHR(A593P), or myc-hLHR(S616Y) were plated onto lysine-coated slides (Biocoat cellware from Falcon). All reagents and incubations for the immunohistochemistry experiment were at room temperature. Cells were washed three times with filtered PBS for immunohistochemistry (PBS-IH), 137 mM NaCl, 2.7 mM KCl, 1.4 mM KH2PO4, 4.3 mM Na2HPO4 (pH 7.4), and then fixed with 4% paraformaldehyde in PBS-IH for 30 min. Cells were permeabilized with 1% Triton X-100 in PBS-IH for 4 min. After incubating with blocking solution (5% BSA in PBS-IH) for 1 h with polyclonal anti-myc antibody (Santa Cruz Biotechnology) diluted 1:100. After washing they were incubated 1 h with fluorescein isothiocyanate-conjugated goat antirabbit IgG diluted 1:1000 in PBS-IH/BSA. Cells were washed with PBS-IH and allowed to dry, after which Vectashield Mounting Media (Vector Laboratories, Inc., Burlingame, CA) and a coverslip were placed on top. Images were collected with a Bio-Rad 1024 laser confocal microscope.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: DSP, Dithiobis(succynimidyl propionate); ECL, enhanced chemiluminescence; endoH, endoglycosidase H; ER, endoplasmic reticulum; FSHR, FSH receptor; GPCR, G protein-coupled receptor; HEK, human embryonic kidney; LHR, LH receptor; PBS-IH, PBS for immunohistochemistry; PDI, protein disulfide-isomerase; SDS, sodium dodecyl sulfate; TSHR, TSH receptor; wt, wild-type.
Received for publication October 20, 2003. Accepted for publication April 12, 2004.
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