help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rozell, T. G.
Right arrow Articles by Segaloff, D. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rozell, T. G.
Right arrow Articles by Segaloff, D. L.
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.



View larger version (39K):
[in this window]
[in a new window]
  		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.



View larger version (40K):
[in this window]
[in a new window]
  		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.



View larger version (36K):
[in this window]
[in a new window]
  		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.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Schertler GFX, Villa C, Henderson R 1993 Projection structure of rhodopsin. Nature 362:770–772[CrossRef][Medline]
  2. Unger VM, Schertler GFX 1995 Low resolution structure of bovine rhodopsin determined by electron cryo-microscropy. Biophys J 68:1776–1786[Abstract/Free Full Text]
  3. Schertler GFX, Hargrave PA 1995 Projection structure of frog rhodopsin in two crystal forms. Proc Natl Acad Sci USA 92:988–929[Abstract/Free Full Text]
  4. Unger VM, Hargrave PA, Baldwin JM, Schertler GFX 1997 Arrangement of rhodopsin transmembrane {alpha}-helices. Nature 389:203–211[CrossRef][Medline]
  5. Henderson R, Baldwin JM, Ceska TA, Zemlin F, Beckmann E, Downing KH 1990 Model of the structure of bacteriorhodopsin based on high-resolution electron cryo-microscopy. J Mol Biol 213:988–929
  6. Pebay-Peyroula E, Rummel G, Rosenbusch JP, Landau EM 1997 X-ray structure of bacteriorhodopsin at 2.5 Angstroms from microcrystals grown in lipidic cubic phases. Science 277:1676–1681[Abstract/Free Full Text]
  7. Kimura Y, Vassylyev DG, Miyazawa A, Kidera A, Matsushima M, Mitsuoka K, Murata K, Hirai T, Fujiyoshi Y 1997 Surface of bacteriorhodopsin revealed by high-resolution electron crystallography. Nature 389:206–211[CrossRef][Medline]
  8. Baldwin JM 1993 The probable arrangement of the helices in G protein-coupled receptors. EMBO J 12:1693–1703[Medline]
  9. Baldwin JM 1994 Structure and function of receptors coupled to G proteins. Curr Opin Cell Biol 6:180–190[CrossRef][Medline]
  10. Baldwin JM, Schertler GFX, Unger VM 1997 An {alpha}-carbon template for the transmembrane helices in the rhodopsin family of G-protein-coupled receptors. J Mol Biol 272:144–164[CrossRef][Medline]
  11. Sprengel R, Braun T, Nikolics K, Segaloff DL, Seeburg PH 1990 The testicular receptor for follicle stimulating hormone: structure and functional expression of cloned cDNA. Mol Endocrinol 4:525–530[CrossRef][Medline]
  12. McFarland KC, Sprengel R, Phillips HS, Kohler M, Rosemblit N, Nikolics K, Segaloff DL, Seeburg PH 1989 Lutropin-choriogonadotropin receptor: an unusual member of the G protein-coupled receptor family. Science 245:494–499[Abstract/Free Full Text]
  13. Kobe B, Deisenhofer J 1993 Crystal structure of porcine ribonuclease inhibitor, a protein with leucine-rich repeats. Nature 366:751–756[CrossRef][Medline]
  14. Kobe B, Deisenhofer J 1994 The leucine-rich repeat: a versatile binding motif. Trends Biochem Sci 19:415–421[CrossRef][Medline]
  15. Thomas D, Rozell TG, Liu X, Segaloff DL 1996 Mutational analyses of the extracellular domain of the full-length lutropin/choriogonadotropin receptor suggest leucine-rich repaets 1–6 are involved in hormone binding. Mol Endocrinol 10:760–768[Abstract]
  16. Segaloff DL, Ascoli M 1993 The lutropin/choriogonadotropin (LH/CG) receptor-4 years later. Endocr Rev 14:324–347[CrossRef][Medline]
  17. Wang H, Jaquette J, Collison K, Segaloff DL 1993 Positive charges in a putative amphiphilic helix in the carboxyl-terminal region of the third intracellular loop of the luteinizing hormone/chorionic gonadotropin receptor are not required for hormone-stimulated cAMP production but are necessary for expression of the receptor at the plasma membrane. Mol Endocrinol 7:1437–1444[Abstract]
  18. Wang Z, Wang H, Ascoli M 1993 Mutation of a highly conserved acidic residue present in the second intracellular loop of G-protein-coupled receptors does not impair hormone binding or signal transduction of the luteinizing hormone/chorionic gonadotropin receptor. Mol Endocrinol 7:85–93[Abstract]
  19. Zhu H, Wang H, Ascoli M 1995 The lutropin/choriogonadotropin receptor is palmitoylated at intracellular cysteine residues. Mol Endocrinol 9:141–150[Abstract]
  20. Abell A, Liu X, Segaloff DL 1996 Deletions of portions of the extracellular loops of the lutropin/choriogonadotropin receptor decrease the binding affinity for oLH, but not hCG, by preventing the formation of mature cell surface receptor. J Biol Chem 271:4518–4527[Abstract/Free Full Text]
  21. Rozell TG, Wang H, Liu X, Segaloff DL 1995 Intracellular retention of mutant gonadotropin receptors results in loss of hormone binding activity of the follitropin receptor but not the lutropin/choriogonadotropin receptor. Mol Endocrinol 9:1727–1736[Abstract]
  22. Jaquette J, Segaloff DL 1997 Temperature sensitivity of some mutants of the lutropin/choriogonadotropin receptor. Endocrinology 138:85–91[Abstract/Free Full Text]
  23. Kremer H, Kraaij R, Toledo SPA, Post M, Fridman JB, Hayashida CY, van Reen M, Milgrom E, Ropers H-H, Mariman E, Themmen APN, Brunner HG 1995 Male pseudohermaphroditism due to a homozygous missense mutation of the luteinizing hormone receptor gene. Nature Gen 9:160–164[CrossRef][Medline]
  24. Laue L, Wu SM, Kudo M, Hsueh AJW, Cutler Jr GB, Griffin JE, Wilson JD, Brain C, Berry AC, Grant DB, Chan WY 1995 A nonsense mutation of the human luteinizing hormone receptor gene in Leydig cell hypoplasia. Hum Mol Gen 4:1429–1433[Abstract/Free Full Text]
  25. Laue LL, Wu SM, Kudo M, Bourony CJ, Cutler GB, Hsueh AJW, Chan WY 1996 Compound heterozygous mutations of the luteinizing hormone receptor gene in Leydig cell hypoplasia. Mol Endocrinol 10:987–997[Abstract]
  26. Aittomaki K, Lucena JLD, Pakarinen P, Sistonen P, Tapanainen J, Gromoll J, Kaskikari R, Sankila E-M, Lehfaslaiho H, Engel AR, Nieshlag E, Huhtaniemi I, de la Chapelle A 1995 Mutation in the follicle-stimulating hormone receptor gene causes hereditary hypergonadotropic ovarian failure. Cell 82:959–968[CrossRef][Medline]
  27. Bergeron JJM, Brenner MB, Thomas DY, Williams DB 1994 Calnexin: a membrane-bound chaperone of the endoplasmic reticulum. Trends Biochem Science 19:124–127[CrossRef][Medline]
  28. Ruddon RW, Bedows E 1997 Assisted protein folding. J Biol Chem 272:3125–3128[Free Full Text]
  29. Hendrick JP, Hartl FU 1993 Molecular chaperone functions of heat-shock proteins. Ann Rev Biochem 62:349–384[CrossRef][Medline]
  30. Hurtley SM, Bole DG, Hoover-Litty H, Helenius A, Copeland CS 1989 Interactions of misfolded influenza virus hemagglutinin with binding protein (BiP). J Cell Biol 108:2117–2126[Abstract/Free Full Text]
  31. Melnick J, Argon Y 1995 Molecular chaperones and the biosynthesis of antigen receptors. Immunol Today 16:243–250[CrossRef][Medline]
  32. Sanchez-Yague J, Rodriguez MC, Segaloff DL, Ascoli M 1992 Truncation of the cytoplasmic tail of the lutropin/choriogonadotropin receptor prevents agonist-induced uncoupling. J Biol Chem 267:7217–7220[Abstract/Free Full Text]
  33. Shibata EF, Matsuda JJ, Volk KA, Collison KA, Segaloff DL 1992 Evidence that the FSH receptor itself is not a calcium channel. Endocrinology 131:979–981[Abstract]
  34. Davis D, Liu X, Segaloff DL 1995 Identification of the sites of N-linked glycosylation on the follicle-stimulating hormone (FSH) receptor and assessment of their role in FSH receptor function. Mol Endocrinol 9:159–170[Abstract]
  35. Hochstenbach F, David V, Watkins S, Brenner MB 1992 Endoplasmic reticulum resident protein of 90 kilodaltons associates with the T- and B-cell antigen receptors and major histocampatibility complex antigens during their assembly. Proc Natl Acad Sci USA 89:4734–4738[Abstract/Free Full Text]
  36. Rodriguez MC, Segaloff DL 1990 The orientation of the LH/CG receptor in rat luteal cells as revealed by site-specific antibodies. Endocrinology 127:674–681[Abstract]
  37. Quintana J, Hipkin RW, Ascoli M 1993 A polyclonal antibody to a synthetic peptide derived from the rat follicle-stimulating hormone receptor reveals the recombinant receptor as a 74-kilodalton protein. Endocrinology 133:2098–2104[Abstract]
  38. Bradford MM 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 53:304–308
  39. Hipkin RW, Sanchez-Yague J, Ascoli M 1993 Identification and characterization of an LH/CG receptor precursor in a human kidney cell line stably transfected with the rat luteal LH/CG receptor cDNA. Mol Endocrinol 6:2210–2218[Abstract]
  40. Laemmli U 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680[CrossRef][Medline]
  41. Kuznetsov G, Chen LB, Nigam SK 1997 Multiple molecular chaperones complex with misfolded large oligomeric glycoproteins in the endoplasmic reticulum. J Biol Chem 272:3067–3063
  42. Ou W-J, Cameron PH, Thomas DY, Bergeron JM 1994 Association of folding intermediates of glycoproteins with calnexin during protein maturation. Nature 364:771–776
  43. Hammond C, Helenius A 1994 Folding of VSV G protein: sequential interaction with BiP and Calnexin. Science 266:456–458[Abstract/Free Full Text]
  44. Snijders AJ, Ho SC, Haase VH, Pillai S, Bernards A 1997 A lymphocyte-specific Ltk tyrosine kinase isoform is retained in the endoplasmic reticulum in association with calnexin. J Biol Chem 272:1297–1301[Abstract/Free Full Text]
  45. Balow JP, Weissman JD, Kearse KP 1995 Unique expression of major histocompatibility complex class I proteins in the absence of glucose trimming and calnexin association. J Biol Chem 270:29025–29029[Abstract/Free Full Text]
  46. Gelman MS, Chang W, Thomas DY, Bergeron JJM, Prives JM 1995 Role of the endoplasmic reticulum chaperone calnexin in subunit folding and assembly of nicotinic acetylcholine receptors. J Biol Chem 270:15085–15092[Abstract/Free Full Text]
  47. Xu Z, Horwitz AL, Sigler PB 1997 The crystal structure of the asymmetric GroEL-GroES-(ADP)7 chaperonin complex. Nature 388:741–750[CrossRef][Medline]
  48. Chang W, Gelman MS, Prives JM 1997 Calnexin-dependent enhancement of nicotinic acetylcholine receptor assembly and surface expression. J Biol Chem 272:28925–28932[Abstract/Free Full Text]
  49. Melnick J, Dul J, Argon Y 1994 Sequential interaction of the chaperones BiP and GRP94 with immunoglobulin chains in the endoplasmic reticulum. Nature 370:373–375[CrossRef][Medline]
  50. Kang HS, Welch WJ 1991 Characterization and purification of the 94-kDa glucose-regulated protein. J Biol Chem 266:5643–5649[Abstract/Free Full Text]
  51. Kohno K, Normington K, Sambrook J, Gething MJ, Mori K 1993 The promoter region of the yeast KAR2 (BiP) gene contains a regulatory domain that responds to the presence of unfolded proteins in the endoplasmic reticulum. Mol Cell Biol 13:877–890[Abstract/Free Full Text]
  52. Lee AS 1987 Coordinated regulation of a set of genes by glucose and calcium ionophores in mammalian cells. Trends Biochem Sci 12:20–23[CrossRef]
  53. Partaledis JA, Berlin V 1993 The FKB2 gene of Saccharomyces cerevisiae, encoding the immunosuppressant-binding protein FKBP-13, is regulated in response to accumulation of unfolded proteins in the endoplasmic reticulum. Proc Natl Acad Sci USA 90:5450–5454[Abstract/Free Full Text]
  54. Rose MD, Misra LM, Vogel JP 1989 Kar2, a karyogamy gene, is the yeast homologue of mammalian BiP/GRP78. Cell 57:1211–1221[CrossRef][Medline]
  55. Herbert DN, Foellmer B, Helenius A 1995 Glucose trimming and reglucosylation determine glycoprotein association with calnexin in the endoplasmic reticulum. Cell 81:425–433[CrossRef][Medline]
  56. Davis DP, Rozell TG, Liu X, Segaloff DL 1997 The six N-linked carbohydrates of the lutropin/choriogonadotropin receptor are not essential for correct folding, cell surface expression, hormone binding, or signal transduction. Mol Endocrinol 11:550–562[Abstract/Free Full Text]
  57. van Leeuwen JEM, Kearse KP 1996 Calnexin associates exclusively with individual CD3{delta} and T cell antigen receptor (TCR) {alpha} proteins containing incompletely trimmed glycans that are not assembled into multisubunit TCR complexes. J Biol Chem 271:9660–9665[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Pharmacol. Rev.Home page
P. M. Conn, A. Ulloa-Aguirre, J. Ito, and J. A. Janovick
G Protein-Coupled Receptor Trafficking in Health and Disease: Lessons Learned to Prepare for Therapeutic Mutant Rescue in Vivo
Pharmacol. Rev., September 1, 2007; 59(3): 225 - 250.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
R. B. Free, L. A. Hazelwood, D. M. Cabrera, H. N. Spalding, Y. Namkung, M. L. Rankin, and D. R. Sibley
D1 and D2 Dopamine Receptor Expression Is Regulated by Direct Interaction with the Chaperone Protein Calnexin
J. Biol. Chem., July 20, 2007; 282(29): 21285 - 21300.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
L. Zhu, Y. Imanishi, S. Filipek, A. Alekseev, B. Jastrzebska, W. Sun, D. A. Saperstein, and K. Palczewski
Autosomal Recessive Retinitis Pigmentosa and E150K Mutation in the Opsin Gene
J. Biol. Chem., August 4, 2006; 281(31): 22289 - 22298.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S. R. Hawtin
Pharmacological Chaperone Activity of SR49059 to Functionally Recover Misfolded Mutations of the Vasopressin V1a Receptor
J. Biol. Chem., May 26, 2006; 281(21): 14604 - 14614.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
J. Robert, C. Auzan, M. A. Ventura, and E. Clauser
Mechanisms of Cell-surface Rerouting of an Endoplasmic Reticulum-retained Mutant of the Vasopressin V1b/V3 Receptor by a Pharmacological Chaperone
J. Biol. Chem., December 23, 2005; 280(51): 42198 - 42206.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pharmacol.Home page
R. Frenzel, K. Krohn, M. Eszlinger, A. Tonjes, and R. Paschke
Sialylation of Human Thyrotropin Receptor Improves and Prolongs Its Cell-Surface Expression
Mol. Pharmacol., October 1, 2005; 68(4): 1106 - 1113.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
M. Ascoli
Learning New Tricks from an Old Dog: The Processing of the Intracellular Precursor of the Luteinizing Hormone Receptor (LHR) into the Mature Cell-Surface LHR Is a Regulated Process
Endocrinology, August 1, 2005; 146(8): 3221 - 3223.
[Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
S. Yamashita, K. Nakamura, Y. Omori, K. Tsunekawa, M. Murakami, and T. Minegishi
Association of Human Follitropin (FSH) Receptor with Splicing Variant of Human Lutropin/Choriogonadotropin Receptor Negatively Controls the Expression of Human FSH Receptor
Mol. Endocrinol., August 1, 2005; 19(8): 2099 - 2111.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
D. Mizrachi and D. L. Segaloff
Intracellularly Located Misfolded Glycoprotein Hormone Receptors Associate with Different Chaperone Proteins than Their Cognate Wild-Type Receptors
Mol. Endocrinol., July 1, 2004; 18(7): 1768 - 1777.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
K. Nakamura, S. Yamashita, Y. Omori, and T. Minegishi
A Splice Variant of the Human Luteinizing Hormone (LH) Receptor Modulates the Expression of Wild-Type Human LH Receptor
Mol. Endocrinol., June 1, 2004; 18(6): 1461 - 1470.
[Abstract] [Full Text] [PDF]

Home page
 Hum Reprod UpdateHome page
A. Ulloa-Aguirre, J. A. Janovick, A. Leanos-Miranda, and P. M. Conn
Misrouted cell surface GnRH receptors as a disease aetiology for congenital isolated hypogonadotrophic hypogonadism
Hum. Reprod. Update, March 1, 2004; 10(2): 177 - 192.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pharmacol.Home page
V. Chaipatikul, L. J. Erickson-Herbrandson, H. H. Loh, and P.-Y. Law
Rescuing the Traffic-Deficient Mutants of Rat {micro}-Opioid Receptors with Hydrophobic Ligands
Mol. Pharmacol., July 1, 2003; 64(1): 32 - 41.
[Abstract] [Full Text] [PDF]

Home page
 Endocr. Rev.Home page
M. Ascoli, F. Fanelli, and D. L. Segaloff
The Lutropin/Choriogonadotropin Receptor, A 2002 Perspective
Endocr. Rev., April 1, 2002; 23(2): 141 - 174.
[Abstract] [Full Text] [PDF]

Home page
 J. Pharmacol. Exp. Ther.Home page
Y. Nagayama, E. Nishihara, H. Namba, S. Yamashita, and M. Niwa
Identification of the Sites of Asparagine-Linked Glycosylation on the Human Thyrotropin Receptor and Studies on Their Role in Receptor Function and Expression
J. Pharmacol. Exp. Ther., October 1, 2000; 295(1): 404 - 409.
[Abstract] [Full Text]

Home page
 EndocrinologyHome page
L. Fayadat, S. Siffroi-Fernandez, J. Lanet, and J.-L. Franc
Calnexin and Calreticulin Binding to Human Thyroperoxidase Is Required for Its First Folding Step(s) But Is Not Sufficient to Promote Efficient Cell Surface Expression
Endocrinology, March 1, 2000; 141(3): 959 - 966.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
P. Touraine, I. Beau, A. Gougeon, G. Meduri, A. Desroches, C. Pichard, M. Detoeuf, B. Paniel, M. Prieur, J-R Zorn, et al.
New Natural Inactivating Mutations of the Follicle-Stimulating Hormone Receptor: Correlations between Receptor Function and Phenotype
Mol. Endocrinol., November 1, 1999; 13(11): 1844 - 1854.
[Abstract] [Full Text]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rozell, T. G.
Right arrow Articles by Segaloff, D. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rozell, T. G.
Right arrow Articles by Segaloff, D. L.

HOME HELP FEEDBACK</