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Endocrinology Vol. 139, No. 4 1588-1593
Copyright © 1998 by The Endocrine Society

ARTICLES

Association of Gonadotropin Receptor Precursors with the Protein Folding Chaperone Calnexin1

Tim G. Rozell2,3, David P. Davis2, Yaohui Chai and Deborah L. Segaloff

Department of Physiology and Biophysics, The University of Iowa College of Medicine, Iowa City, Iowa 52246

Address all correspondence and requests for reprints to: Dr. Deborah L. Segaloff, Department of Physiology, The University of Iowa College of Medicine, Iowa City, Iowa 52242. E-mail: deborah-segaloff{at}uiowa.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The lutropin/choriogonadotropin receptor (LHR) and follitropin receptor (FSHR) are members of the superfamily of G protein-coupled receptors. The carboxyl half of each receptor is composed of the classical seven membrane spanning regions connected by intracellular and extracellular loops. In addition, each receptor contains a large extracellular domain. Despite the complexity of the structure of G protein-coupled receptors, little is known about how these receptors assume their correct conformations during biosynthesis. Although the role of chaperone proteins in the folding of other proteins has been well documented, their role in the folding of G protein-coupled receptors has been an enigma. To better understand the folding of the LH and FSH receptors, we examined their association with the general chaperone proteins calnexin, binding protein (BiP), and the 94-kDa glucose-regulated protein (GRP94). Clonal 293 cell lines expressing comparably high levels of each receptor were solubilized, and the extracts were incubated with the appropriate antibody bound to Protein A-sepharose beads. Experiments were performed using two approaches: 1) coimmunoprecipitation of receptor/chaperone complexes with one of the antireceptor antibodies, then SDS-PAGE and Western blotting using either anticalnexin or anti-KDEL (which recognizes BiP and GRP94) antibodies; or 2) coimmunoprecipitation of receptor/chaperone complexes with anticalnexin or anti-KDEL, then Western blotting with one of the antireceptor antibodies. Using these protocols, we found that the immature forms of both the rLHR and rFSHR are associated with calnexin, but little or no association was observed for either receptor with BiP or GRP94. These experiments show that the precursor forms of the wild-type LHR and FSHR can associate with calnexin, raising the possibility that this chaperone protein may facilitate in the folding of the gonadotropin receptors.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE LUTROPIN/CHORIOGONADOTROPIN receptor (LHR) and follitropin receptor (FSHR) are G protein-coupled receptors composed of two distinct regions of complex structural organization. One is the carboxyl half, which contains the seven transmembrane helices arranged in a bundle-like structure interconnected by three intracellular and three extracellular loops. Unfortunately, a high resolution crystal structure of this domain has not been solved yet for any G protein-coupled receptors. However, low density maps of the photoreceptor rhodopsin (1, 2, 3, 4), which interacts with the G protein transducin, and high density maps of bacteriorhodopsin (5, 6, 7), a bacterial membrane ion pump that does not interact with G proteins but contains seven similarly arranged transmembrane helices, have provided the conceptual basis for the structural models of hormone-regulated G protein-coupled receptors (8, 9, 10). The other structural domain of the gonadotropin receptors is the amino-terminal extracellular domain, which contains several leucine-rich repeat motifs (11, 12). The crystal structure for ribonuclease inhibitor, a protein composed of leucine-rich repeats, suggests that it assumes a horseshoe-like structure in which each leucine-rich repeat consists of a ß-{alpha} conformation that creates a loop extending from the horseshoe-like inner framework (13, 14). The interior of the horseshoe-like structure consists of closely spaced ß-sheets contributed by each leucine-rich repeat, whereas the outer surface of each loop consists of an {alpha}-helix contributed by a leucine-rich repeat. As such, the leucine-rich repeats generate a surface composed of loop-like structures.

Due to the complex structure of the gonadotropin receptors, it is not altogether surprising that strict cellular mechanisms exist to ensure that only properly folded receptors are inserted into the plasma membrane. There have been numerous reports demonstrating that mutations introduced into the rLHR or rFSHR by site-directed mutagenesis result in the intracellular retention of the mutant receptors (15, 16, 17, 18, 19, 20, 21, 22). For the mutants that have been analyzed thus far, it appears that they are trapped in the endoplasmic reticulum because they remain in an endoglycosidase H-sensitive form (21). In addition, the naturally occurring inactivating mutations of the hLHR and hFSHR that have been described result in extremely reduced levels of cell surface receptors (23, 24, 25, 26). Although it has not been determined in most cases whether the decreased cell surface expression is due to increased degradation of the mutant receptor and/or increased intracellular retention, both processes would necessitate intracellular quality control mechanisms that enable the cell to recognize the mutant receptors as being misfolded and prevent their transport to the plasma membrane.

Chaperone proteins are known to interact with a wide variety of glycosylated proteins and are thought to serve as a mechanism of quality control by binding to misfolded proteins and causing them to be retained in the endoplasmic reticulum (27, 28). A proposed mechanism for this quality control is that chaperones bind proteins that are in an intermediate folding conformation, thus preventing misfolding and aggregation of these intermediates and making the folding process much more efficient (29). Thus, within the biosynthesis of normal proteins, chaperone proteins are involved in ensuring that only the properly folded intermediates proceed further in the biosynthetic and processing pathway. In addition, chaperones have been found to interact longer with mutated proteins that cannot attain a normal conformation, leading to retention of these misfolded proteins in the ER (30).

Ultimately, it is our goal to understand the process whereby the gonadotropin receptors attain their proper mature conformation. Toward this goal, we undertook the present study to determine whether any of the commonly used protein-folding chaperones were physically associated with the rLHR or rFSHR precursor proteins. Because there are no reports yet of chaperone proteins associated with any other G protein-coupled receptors, we chose to examine three ubiquitous chaperone proteins commonly associated with other proteins: calnexin, BiP, and GRP94 (31). In this report, we describe the association of the immature forms of both the rLHR and rFSHR with the protein-folding chaperone calnexin, but not with BiP or GRP94.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and antibodies
Clonal cell lines of human embryonic kidney 293 cells (293 cells; ATCC CRL 1573) expressing either the wild-type rLHR (rLHR-wt1; approximately 500,000 receptors per cell) (32) or wild-type rFSHR (rFSHR-wt1; approximately 100,000 receptors per cell) (33) were used for all coimmunopreciptation protocols. 293 cells stably transfected with empty pcDNA I/neo vector (34) (empty vector) were used as controls.

Monoclonal anticalnexin (AF8) (35) was generously provided by Dr. Michael Brenner (Harvard University, Cambridge, MA), and monoclonal anti-KDEL (which recognizes BiP and GRP94) was obtained from StressGen (Victoria, British Columbia, Canada). Anti-rLHR02 and anti-rFSHR (anti-F) have previously been described (36, 37). All reagents, unless otherwise indicated, were obtained from Sigma Chemical Co. (St. Louis, MO).

Protein extractions
Human embryonic 293 cells expressing rLHR-wt1, rFSHR-wt1, or empty vector were solubilized by incubating in lysis buffer (0.5% NP-40, 0.9% NaCl, 0.02 M HEPES, 1 mM EDTA, 200 mM phenylmethylsulfonyl fluoride, 230 µM leupeptin, 5 µM pepstatin A) for 15 min on ice. Extracts were assayed for total protein by the Bradford assay (38).

Coimmunoprecipitations
All coimmunoprecipitations were performed by first binding antichaperone or antireceptor antibodies to protein A-sepharose beads (39). For each coimmunoprecipitation, 100 µl of a 50% suspension of protein A-sepharose in lysis buffer containing 1% BSA and 1 mM NaN3 were added per tube; tubes were centrifuged briefly, and beads were washed with 500 µl lysis buffer. Antichaperone antibodies were then added to give a final dilution of 1:50 or 1:100 in 100 µl lysis buffer, and antireceptor antibodies were added to give a final concentration of 1.5 µg IgG/µl in 100 µl lysis buffer. The antibodies were allowed to bind to the protein A-sepharose beads overnight at 4 C while rotating, after which they were centrifuged briefly and washed with 750 µl lysis buffer. For each immunoprecipitation, 2 mg of total protein from each extract was added to the protein A-antibody-linked beads in a final volume of 750 µl lysis buffer, and tubes were rotated at 4 C for 90 min. Each sample was washed 4 times with 1.5 ml of lysis buffer and prepared for Western blotting as described below. Identities of each of the chaperone proteins were verified by comigration with purified chaperones (data not shown).

SDS-PAGE and Western blotting
All Western blotting reagents were obtained from Bio-Rad (Richmond, CA), unless otherwise indicated. Following final washes for each of the immunoprecipitations, 150 µl Laemmli sample buffer (40) containing reducing agents was added to each pellet. Samples were vortexed 15 min, centrifuged at 12,000 x g for 2 min, and electrophoresed on a 7.5% SDS-polyacrylamide gel. Proteins were then transferred to PVDF membranes, blocked for 2 h in blocking solution (10% nonfat powdered milk, 10% glycerol, 0.2% Tween-20 in PBS), and incubated overnight at 22 C with the appropriate antibody. Anticalnexin AF8 was used at a final dilution of 1:750,000, anti-KDEL at a final dilution of 1:500, anti-rLHR02 IgG at a final concentration of 2 µg/ml, and anti-F IgG at a final concentration of 1 µg/ml. After washing 5 times in blocking solution, membranes were incubated with goat antimouse IgG conjugated to horseradish peroxidase (1:40,000) for detection of monoclonal antibodies against calnexin or KDEL, or goat antirabbit HRP (1:30,000) for detection of anti-rLHR02 or anti-F. Specific protein bands were visualized using enhanced chemiluminescence (Amersham, Arlington Heights, IL).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
To examine the association of the gonadotropin receptors with chaperone proteins, we asked whether a given chaperone protein could be coimmunoprecipitated with the rLHR or rFSHR. This experimental approach has been widely used in a number of other systems (see Refs. 41–46 for examples). Because gonadal cells express relatively low levels of LH and FSH receptors, they could not be used for these experiments. Instead, human embryonic 293 cells stably expressing higher levels of rLHR and rFSHR (500,000 and 100,000 receptors per cell, respectively) were used. Two distinct protocols of coimmunoprecipitation were employed. In the first protocol, potential receptor-chaperone complexes were first coimmunoprecipitated with antireceptor antibodies, then the complexes were dissociated and the chaperone proteins were visualized on Western blots probed with either anticalnexin antibodies or anti-KDEL antibodies (which recognize both BiP and GRP94). As shown in Fig. 1AGo, calnexin expression was approximately equal in nonimmunoprecipitated extracts from 293 cells expressing either the rFSHR, rLHR or empty vector. In addition, immunoprecipitation with anti-rLHR or anti-rFSHR resulted in a visible protein band at the same molecular weight as calnexin, and only in extracts from cells expressing the rLHR or rFSHR, respectively. Although it appears that a greater amount of calnexin is associated with the rFSHR than the rLHR in Fig. 1AGo, it is difficult to make this conclusion because the anti-rFSHR and anti-rLHR antibodies used for the immunoprecipitations likely have different efficiencies.



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  		Figure 1. Detection of calnexin, BiP, or GRP94 after coimmunoprecipitation with anti-rLHR or anti-rFSHR. A, Extracts of 293 cells stably transfected with either the rLHR, rFSHR, or empty pcDNA I/neo vector were subjected to immunoprecipitation with anti-rLHR or anti-rFSHR, then probed on a Western blot with anticalnexin as described in Materials and Methods. Lanes 1 through 3 are nonimmunoprecipitated extracts; lanes 4 and 5 are extracts from cells expressing either the rLHR or empty pcDNA I/neo vector and subjected to immunoprecipitation with anti-rLHR; lanes 6 and 7 are extracts from cells expressing either the rFSHR or pcDNA I/neo (empty vector) subjected to immunoprecipitation with anti-rFSHR. B, As described for as panel A, except that the Western blot was probed with anti-KDEL (which recognizes BiP and GRP94).

 
In contrast to the results with anticalnexin, there appeared to be very little coimmunoprecipition of either BiP or GRP94 with either the rLHR or rFSHR (Fig. 1BGo). The amount of BiP and GRP94 in nonimmunoprecipitated extracts from cells expressing the rLHR, rFSHR or empty pcDNA I/neo vector was approximately equal (lanes 1, 2 and 3 of Fig. 1BGo), indicating that expression of these chaperones was not cell-line dependent.

A second protocol for immunoprecipitation was used to preclude the possibility that immunoprecipitation with the antireceptor antibodies somehow prevented or disrupted the association of the receptors with any of the chaperone proteins. In this case, the chaperone proteins were first coimmunoprecipitated from stably transfected 293 cells using either anticalnexin or anti-KDEL, then probed on a Western blot using antireceptor antibodies. As shown in Fig. 2AGo, a specific band was visible at 68 kDa from extracts of cells expressing the rLHR that were subjected to immunoprecipitation with anticalnexin. This band was not visible in immunoprecipitates of extracts from cells transfected with empty vector, or when extracts were immunoprecipitated with normal mouse serum. Likewise, a band was visible at 82 kDa when extracts from cells expressing the rFSHR were subjected to immunoprecipitation with anticalnexin and probed with anti-rFSHR on a Western (Fig. 2BGo). As with the rLHR shown in Fig. 2AGo, this specific band was not present in extracts from cells transfected with empty vector or when extracts were immunoprecipitated with normal mouse serum.



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  		Figure 2. Detection of immature forms of the rLHR and rFSHR after coimmunoprecipitation with calnexin. A, Extracts of 293 cells stably transfected with either the rLHR or empty vector were subjected to immunoprecipitation with anticalnexin or normal mouse serum (NMS), then probed on a Western blot with anti-rLHR as described in Materials and Methods. The first lane for either rLHR or empty vector represents nonimmunoprecipitated extracts. Anticalnexin was used at a final dilution for immunoprecipitation of either 1:100 or 1:50 and is represented in lanes 2 and 3, respectively, for each extract. B, As described for panel A, except 293 cells stably expressing the rFSHR or empty vector were subjected to immunoprecipitation with anticalnexin, then probed on a Western blot using anti-rFSHR.

 
Both the rLHR and rFSHR run on gels as two distinct molecular mass species, with the mature form of the receptors running at approximately 89 kDa and the immature forms at approximately 68 or 82 kDa, respectively (34). For both receptors, the only visible bands after coimmunoprecipitation with anticalnexin migrated at the approximate molecular mass of the immature forms of each receptor, as compared with nonimmunoprecipitated extracts run on the same gel (Fig. 2Go).

Extracts from cells expressing either the rLHR or rFSHR were also subjected to immunoprecipitation with anti-KDEL, then probed on a Western blot with antireceptor antibodies (Fig. 3Go). Neither the rLHR (Fig. 3AGo) or rFSHR (Fig. 3BGo) appeared to associate with BiP or GRP94, as little or no receptor bands were visible following this coimmunoprecipitation protocol. Although it is possible that the amount of anti-KDEL used for the immunoprecipitation was inadequate for visualization of BiP or GRP94 on a Western blot, the concentration of antibody used was at least 10-fold higher than that required for visualization of these chaperones when probing a Western blot. In addition, BiP and GRP94 were clearly visible from nonimmunoprecipitated extracts on a Western blot probed with anti-KDEL, whereas no bands were visible after immunoprecipitating the same extracts with antireceptor antibodies. These immunoprecipitations and Western blotting conditions were identical for anti-KDEL, anticalnexin, anti-rLHR and anti-FSFH antibodies. Thus, we can conclude that, under the same experimental conditions, the immature forms of both the rLHR and rFSHR associate with calnexin, but not with BiP or GRP94.



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  		Figure 3. Lack of detection of the rLHR and rFSHR after coimmunoprecipitation with BiP or GRP94. A, Extracts of 293 cells stably transfected with either the rLHR or empty vector were subjected to immunoprecipitation with anti-KDEL or normal mouse serum (NMS), then probed on a Western blot with anti-rLHR as described in Materials and Methods. The first lane for either rLHR or empty vector represents nonimmunoprecipitated extracts. Anti-KDEL was used at a final dilution for immunoprecipitation of either 1:50 or 1:100 and is represented in lanes 2 and 3, respectively, for each extract. B, As described for panel A, except the extracts were obtained from 293 cells stably expressing the rFSHR or empty vector and subjected to immunoprecipitation with anti-KDEL, then probed on a Western blot using anti-rFSHR.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The LHR and FSHR are members of the superfamily of G protein-coupled receptors and thus contain seven transmembrane helices connected by extra- and intracellular loops. In addition, the gonadotropin receptors also possess large amino-terminal extracellular domains that are glycosylated. Little is known, however, about how the gonadotropin receptors, or other G protein-coupled receptors, fold into their proper conformation during and after biosynthesis. In recent years, it has become increasingly clear that chaperone proteins play important roles in the folding of newly synthesized proteins. Chaperone proteins are thought to assist in the folding of newly synthesized proteins primarily by preventing the the aggregation of misfolded intermediates. As such, through the actions of chaperone proteins, the yield of correctly folded protein is increased (47, 48). Three of the most widely studied chaperone proteins are calnexin, BiP, and GRP94 (31). The present studies were undertaken to determine if any of these ubiquitous chaperone proteins were involved in the biosynthesis of the gonadotropin receptors. As has been done with other protein systems, we addressed this question by examining whether the gonadotropin receptors could be coimmunoprecipitated with a given chaperone protein (41, 42, 43, 44, 45, 46). A positive result of this nature would suggest that in intact cells, the receptor at some point in its biosynthesis physically associates with the chaperone protein. Unfortunately, the low levels of LH and FSH receptors present in gonadal cells precluded us from using these cells for the coimmunoprecipitation studies. Instead, 293 cells stably expressing relatively high levels of LH or FSH receptors were used. In this system we show that calnexin, but not BiP or GRP94, is associated with the precursor forms of the LHR and FSHR. Of course, there is the potential that the association of the LHR and FSH precursors with calnexin may have been due to the high expression of the receptors in these cells. At the moment we cannot readily sort out this issue because it is not technically feasible to perform receptor immunoprecipitations with cells expressing lower levels of receptor. Nonetheless, we can conclude that, under the conditions used, there is a physical association of the LHR and FSHR precursors with calnexin, but not with BiP or GRP94. Thus, the LHR and FSHR precursors are capable of associating with calnexin, suggesting that calnexin may play a role in the folding of these receptors.

Calnexin is a transmembrane protein in which the C-terminal portion contains an ER retention signal, and the N-terminal portion extends into the ER lumen (27), where it presumably interacts with a number of different proteins. Through its quality control function, calnexin has been shown to play a major role in a number of human diseases, including cystic fibrosis. Both the wild type and mutant {Delta}F508 form of the cystic fibrosis transmembrane conductance regulator (CFTR) have been shown to interact with calnexin; however, only the wild-type form of the CFTR can dissociate from calnexin and be transported to the cell membrane (27). Calnexin has been found to associate with a wide variety of glycoproteins, including the {alpha}, ß, CD3{delta}, and {epsilon} subunits of the T cell receptor, and the major histocompatibility (MHC) antigen class I {alpha}, and class II {alpha}, ß, and Ii molecules (31). This is the first report to show members of the G protein-associated receptor family associating with calnexin during their biosynthesis.

GRP94 is a less well characterized member of the chaperone family but has been shown to associate with unassembled immunoglobulin chains, MHC class II, and a mutant viral protein (31, 49). GRP94 contains the ER retention signal, KDEL, on the C-terminus, but it is possible that both transmembrane and soluble forms exist (50).

BiP (also known as GRP78) is a member of the heat shock 70 (hsp70) family of proteins and is found as a soluble protein in the lumen of the ER (31). BiP and its yeast analog KAR2 are inducible by certain stress conditions, one of which is thought to be the accumulation of misfolded and aggregated proteins within the lumen of the ER (51, 52, 53, 54). Like GRP94, BiP also contains the KDEL endoplasmic reticulum retention signal. In the present experiment, the cell lines used produce large quantities of properly folded receptors (100,000 to 500,000 receptors per cell) and, although it appears that BiP and GRP94 are not directly involved in the process of folding of the wild-type rLHR and rFSHR, it nonetheless is possible that BiP and GRP94 might associate with misfolded, intracellularly retained mutant receptors.

The binding of calnexin to glycoproteins has been shown to occur in most cases via interactions of this chaperone protein with N-linked oligosaccharides (55). Previous studies have shown that the rFSHR is glycosylated on two of its three potential N-linked glycosylation sites (34). Furthermore, the rFSHR was found to require the presence of at least one mature carbohydrate before attaining a conformation capable of hormone binding. Thus, tunicamycin-derived, nonglycosylated FSHRs were trapped intracellularly and could not bind FSH (34). In contrast, the rLHR has six consensus sequences for N-linked glycosylation, all of which appear to be glycosylated (56). However, the hLHR does not appear to absolutely require N-linked carbohydrates to fold properly. Tunicaymcycin-treated cells expressing the rLHR were found to bind hCG to cell surface receptors with high affinity and to respond to hCG with increased cAMP production (56). Thus, it appears that the peptide backbone of the rLHR is capable of folding more readily into a mature conformation than the rFSHR, and that N-linked carbohydrates are absolutely necessary for this process only in the rFSHR.

It may at first appear contradictory that calnexin, which requires N-linked carbohydrates to bind to proteins, associates with the rLHR during its biosynthesis, yet a nonglycosylated form of the rLHR can fold properly. These two observations are not, however, necessarily mutually exclusive. First, although calnexin generally associates with proteins in a glycan-dependent manner, there have been reports of proteins which associate with calnexin in a glycan-independent manner (57). Secondly, even if the association of calnexin with the rLHR required N-linked carbohydrates, the folding of a deglycosylated function rLHR may be accomplished in the absence of calnexin binding if the role of calnexin in rLHR folding is facilitative rather than essential. Chaperone proteins are thought to facilitate the folding of proteins and, thus, will generally increase the steady state levels of properly folded protein (29, 47, 48). In fact, the steady-state levels of nonglycosylated rLHR detected in tunicamycin-treated cells is extremely low. Although at least some of this reduction in rLHR expression may be due to the inhibitory properties of tunicamycin on protein synthesis, a reduction in the levels of nonglycosylated rLHR may also be due to the inability of calnexin to associate with the rLHR in the absence of its N-linked carbohydrates.

In conclusion, calnexin associates with both the rLHR and rFSHR precursors in cell lines that produce large numbers of receptors. Because of its well characterized role as a chaperone protein, it is likely that calnexin facilitates the proper folding of these receptors, perhaps by stabilizing the folding intermediates and preventing the formation of misfolded aggregates. These studies provide the basis for future work and the differences, if any, in the role of chaperone proteins with the LHR vs. the FSHR and the role of chaperone proteins in the retention of mutant forms of the gonadotropin receptors.


   Acknowledgments
 
We gratefully acknowledge the gift of anticalnexin from Dr. Michael Brenner, the helpful suggestions regarding immunoprecipitations from Drs. Mario Ascoli and Zheng Wang, and the critical reading of the manuscript by Drs. Mario Ascoli and Mark Stamnes.


   Footnotes
 
1 These studies were supported by NIH Grant HD-22196 (to D.L.S.). The services and facilities provided by the Diabetes and Endocrinology Research Center of the University of Iowa (supported by Grant DK-25295) are gratefully acknowledged. While this study was in progress, D.L.S. was a recipient of a NIH Research Career Award (HD-00968) and T.G.R. was a recipient of a NIH National Research Service Award. Current address of D.P.D. is O-305, Department of Pathology, Committee on Immunology, University of Chicago, Chicago, Illinois 60637. Back

2 Denotes equal contribution by these authors. Back

3 Current address: Department of Animal Sciences, Kansas State University, Manhatten, Kansas 66506. Back

Received September 16, 1997.


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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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