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Endocrinology Vol. 148, No. 5 1987-1995
Copyright © 2007 by The Endocrine Society

Follice-Stimulating Hormone Receptor Forms Oligomers and Shows Evidence of Carboxyl-Terminal Proteolytic Processing

Richard M. Thomas, Cheryl A. Nechamen, Joseph E. Mazurkiewicz, Marco Muda, Stephen Palmer and James A. Dias

Wadsworth Center (R.M.T., C.A.N., J.A.D.), David Axelrod Institute for Public Health, New York State Department of Health, and Center for Neuropharmacology and Neuroscience (J.E.M.), Albany Medical College, Albany, New York 12208; Serono Reproductive Biology Institute (M.M., S.P), Serono Inc., Rockland, Massachusetts 02370; and Department of Biomedical Sciences (J.A.D.), State University of New York at Albany, Albany, New York 12201

Address all correspondence and requests for reprints to: James A. Dias, Ph.D., Wadsworth Center, David Axelrod Institute for Public Health, New York State Department of Health, 120 New Scotland Avenue, Albany, New York 12208. E-mail: james.dias{at}wadsworth.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
FSH receptor (FSHR), a member of the G protein-coupled receptor superfamily, is present in the plasma membrane of ovarian granulosa cells and testicular Sertoli cells. FSH regulates normal ovarian follicle development and spermatogenesis through FSHR. The extracellular domain of FSHR is a weakly associated homodimer in the recently solved crystal structure of FSH in complex with the extracellular domain of FSHR. However, there is currently no biochemical data that demonstrate that FSHR exists as a dimer or higher-order oligomer in cell membranes. A fluorescence resonance energy transfer assay was used to determine whether full-length native FSHR is an oligomer. FSHR-specific monoclonal antibody or Fab fragments, labeled with two different fluorophores, allowed the study of nontagged receptor in situ. Unoccupied FSHR exhibited strong fluorescence resonance energy transfer profiles in situ. Complementary coimmunoprecipitation experiments of myc- or FLAG-tagged FSHR indicated that FSHR forms oligomers early in receptor biosynthesis. No effect of FSH treatment was observed. Thus, immature forms of FSHR, not yet fully processed, were observed to coimmunoprecipitate. An unexpected observation was made that the C-terminal epitope tags are removed from FSHR before arrival at the cell surface. These results provide the first evidence for oligomers of full-length FSHR in situ and for C-terminal proteolytic processing of FSHR and that both events take place during biosynthesis. This may explain how heterozygous mutations in the FSHR gene that affect receptor trafficking may be ameliorated by oligomer formation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
FSH IS REQUIRED for follicle development in the ovary and normal spermatogenesis in the testis (for review, see Ref. 1). The glycoprotein hormone receptors, which include FSH receptor (FSHR), LH receptor (LHR), and TSH (TSHR), are composed of a large extracellular domain (ECD) and a heptahelical transmembrane (TM) domain. Structural organization of FSHR at the cell membrane is not fully understood. Recently, a crystal structure of FSH in complex with the ECD of FSHR (FSHRECD) shows that FSHRECD forms weakly associating dimers with each molecule being occupied by one molecule of FSH (2). That observation is of interest because homo- or hetero-oligomerization of receptors has been shown to influence trafficking, ligand binding, and signaling (for review, see Refs. 3 and 4).

In fact, oligomerization has been demonstrated among some of the glycoprotein hormone receptors but not FSHR, the only type 1A G protein-coupled receptor (GPCR) for which there is a crystal structure available. Binding of human chorionic gonadotropin (hCG) enhances the constitutive self association of LHR seen by coimmunoprecipitation (5). Clustering of LHR, which appears to require functional LHR (6), is induced by the binding of LH or hCG (7). In addition, LHR appears to move into lipid rafts (8, 9) and to form large complexes upon desensitization (10).

It should be noted that although the term cluster is often used in fluorescence resonance energy transfer (FRET) studies and the term oligomer is used in biochemical experiments, in this paper, the terms are used interchangeably to designate closely associated FSHR.

TSHR appears to form constitutive self-associated complexes. In contrast to what is observed with LHR, binding of TSH causes a dose-dependent decrease in oligomeric forms, as measured by FRET (11).

In the case of FSHR, the evidence is suggestive of oligomerization. Osuga et al. (12) constructed a chimera of the FSHRECD fused to the TM portion of LHR and an LHR mutant truncated at TM segment V. Neither construct alone induced cAMP in response to hCG, but coexpression restored partial signaling. That study suggested either that the TM I domain is the site of interaction in LHR oligomers or that they associated via the ECD as in the FSHRECD crystal structure. Recent biochemical gain-of-function experiments further substantiate that self-association can occur, and through a process coined transactivation, a binding-defective receptor can be activated by a binding-competent, signaling-defective receptor (13).

Alternate splicing of FSHR, in which six of the seven naturally occurring TM domains are removed, produces an FSHR species that is anchored in the membrane and binds FSH but does not signal through adenylate cyclase (14). Coexpression of the splice variant with wild-type (wt) FSHR prevents a cAMP response to FSH, implying oligomerization between wt and the splice variant (15). A study in which binding- and signaling-defective mutants of FSHR were coexpressed demonstrated the partial restoration of signaling via either adenylate cyclase or phospholipase C, but not both (13).

In this study, using the complementary techniques of imaging FRET of native receptor and coimmunoprecipitation of epitope-tagged receptor, we report the first direct evidence of oligomerization of full-length FSHR at the cell surface. Analysis of intracellular receptors revealed that oligomerization occurs as a normal part of processing and, unlike LHR and TSHR, is not affected by hormone. Another novel finding was that the C terminus of tagged FSHR appears to undergo proteolytic processing before arrival at the cell surface.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Imaging FRET analysis
The efficiency of energy transfer (E) from donor to acceptor, which is highly dependent on the distance between the fluorophores, is often reported as FRET efficiency (E %). The most direct method by which to quantify the absolute efficiency of transfer is to measure donor emission before and after photobleaching of the acceptor. This increase in emission (dequenching) of the donor is a direct measure of the FRET efficiency and is calculated from: E % = [1 – (donor emission before acceptor photobleach)/(donor emission after acceptor photobleach)] x 100. In this study, Alexa 568 was used as the donor fluorophore and Alexa 647 as the acceptor fluorophore. This fluorophore pair meets the requirement for producing FRET in which the emission spectrum of the donor overlaps the absorption spectrum of the acceptor.

To study native FSHR, FRET was performed using the monoclonal antibody (mAb) 106.105, directed against FSHRECD (16), that had been labeled with fluorophores. The mAb 106.105 or Fab fragments of mAb 106.105 were directly labeled with fluorescent dye Alexa 568 or Alexa 647 (Molecular Probes, Eugene, OR) according to the manufacturer’s instructions. Fab fragments were prepared according to the manufacturer’s instructions (Pierce, Rockford, IL).

Human embryonic kidney (HEK) 293 cells or HEK 293 cells stably transfected with FSHR (HEK 293/FSHR) (17) were plated in 35-mm petri dishes with glass coverslip bottoms (MatTek Corp., Ashland, MA). The cells were labeled with the fluorescently tagged antibodies essentially as described (18). Briefly, a dish was placed on ice and the tissue culture medium was replaced with ice-cold PBS supplemented with 0.9 mM CaCl2, 0.52 mM MgCl2, and 0.16 mM MgSO4 (PBS2+) containing 1% BSA and allowed to equilibrate at 4 C. Individual cell cultures were then incubated with each labeled antibody or Fab fragment diluted in PBS2+ for 30 min at 4 C. The cells were washed with ice-cold PBS (twice for 5 min each) and then fixed with 4% formaldehyde in PBS, freshly prepared from a 16% formaldehyde solution (Electron Microscopy Sciences, Hatfield, PA). After fixation, the cells were washed in PBS at room temperature (twice for 5 min each). As a control for specificity of the primary antibody and Fab fragments, HEK 293 cells were incubated with the fluorescently tagged antibodies and imaged as above.

The cells were then covered with fresh PBS and imaged on a Zeiss LSM 510 META confocal microscope system on an Axiomat 200 M inverted microscope equipped with a x63 1.4 NA, oil-immersion differential interference contrast (DIC) lens. Alexa 568 was excited with the 543-nm line of a HeNe laser, and emission was detected with a 545 dichroic and a 565- to 615-nm band pass filter. Alexa 647 was excited with the 633-nm laser line of a HeNe laser, and emission was detected with a 545 dichroic and a 650- to 710-nm band pass filter. The pinhole was set at 1.32 Airy units and a Z resolution of approximately 2.0 µm. Images were collected at 12-bit intensity resolution over 512 x 512 pixels at a pixel dwell time of 6.4 µsec.

FRET was detected as an increase in donor (mAb 106.105-Alexa 568) intensity after photobleaching of acceptor molecules (mAb 106.105-Alexa 647). A region of interest (ROI) traced on an image of the cells was subjected to photobleach with the 633-nm HeNe laser line for 30–90 sec at maximum power. This irradiation resulted in greater than 95% photodestruction of the acceptor fluor. Four images were collected: 1) an image of the Alexa 568 fluorescence (in the presence of Alexa 647), 2) an image of the Alexa 647 fluorescence, 3) an image of Alexa 568 fluorescence after destruction of acceptor fluorescence by photobleaching, and 4) an image of the Alexa 647 after the Alexa 647 photobleach.

The images were analyzed using the LSM FRET Tool (version 1.5) that is integrated with the LSM 510 collection of software (Zeiss, Inc., Thornwood, NY). FRET analyses were performed on ROI drawn directly on captured confocal images. The analysis uses a cell or its subregion as its own internal standard after photobleaching (19). GraphPad PRISM version 3.0 (GraphPad Software Inc., San Diego, CA) was used for the linear regression analyses of the FRET data.

Plasmid constructions
FSHR was inserted into the mammalian expression vector pRK5 and a FLAG (DYKDDDDK) or c-myc (EQKLISEEDL) epitope tag was inserted C-terminal to the FSHR sequence. The wt FSHR was inserted into the mammalian expression vector pShuttle-CMV (Stratagene, La Jolla, CA).

Cell culture and transfection
HEK 293 cells and HEK 293/FSHR cells were maintained in Eagle’s medium containing 10% fetal bovine serum. Subconfluent 60-mm dishes of HEK 293 cells were transfected (Lipofectamine Plus; Invitrogen, Carlsbad, CA) with 1 µg each of plasmid encoding either FLAG-tagged FSHR or myc-tagged FSHR and incubated for an additional 24–48 h. Cells were treated with 1.2 nM human pituitary FSH in serum-free Eagle’s medium for the indicated times.

Radioreceptor assays (RRAs)
Human pituitary FSH was radiolabeled with I125 (PerkinElmer Life Sciences, Boston, MA) as described (20). Transfected cells were harvested in PBS/EDTA, pelleted, and resuspended in RRA buffer [50 mM Tris (pH 7.5), 25 mM MgCl2, and 0.1% BSA] at a density of 250,000 cells/0.1 ml. [I125]FSH was added at 200,000 cpm/0.1 ml RRA buffer in the presence or absence of 1 µg unlabeled recombinant single-chain FSH essentially as described (20, 21). Bound [I125]FSH was determined using an LKB/Wallac {gamma}-counter (Gaithersburg, MD).

Immunoprecipitations
HEK 293 cells cotransfected with FSHR-FLAG and FSHR-myc were harvested in Igepal-DOC lysis buffer [10 mM Tris (pH 7.5), 1% Igepal, 0.4% deoxycholate, 140 mM NaCl, 5 mM EDTA] supplemented with protease inhibitors (10 µg/ml pepstatin A, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 16 µg/ml benzamide, 10 µg/ml 1,10 phenanthroline, 1 mM phenylmethylsulfonyl fluoride). Protein concentration was determined by BCA assay (Pierce), and equal amounts of cell extract (~0.5 mg) were immunoprecipitated as described in Ref. 17 .

Biotinylation of cell surface proteins
Transfected cells were transferred to ice and washed once with ice-cold PBS. Cell surface proteins were biotinylated using 0.55 mg/ml cell-impermeant, Sulfo-NHS-LC-biotin (Pierce) in PBS for 30 min on ice. Unreacted biotin was quenched and removed with two washes of ice-cold PBS supplemented with 10 mM glycine. The cells were washed once with ice-cold PBS and harvested in 1 ml ice-cold Igepal-DOC lysis buffer.

Cell lysates were immunoprecipitated with mAb 106.105, specific for FSHR, FLAG M2 mAb (Sigma Chemical Co., St. Louis, MO), or myc mAb clone 9E10 (American Type Culture Collection, Manassas, VA).

Gel electrophoresis and Western immunoblotting
Samples (1/4 of immunoprecipitate) were resolved on SDS-PAGE in a discontinuous buffer system (22). After separation, the proteins were transferred to Immobilon-P membranes according to the method of Towbin et al. (23). Membranes were blocked in Tris-buffered saline with Tween 20 (TBST) [10 mM Tris-HCl (pH 7.2), 150 mM NaCl, 0.5% Tween 20] with 5% nonfat dry milk overnight at 4 C. Blots were washed briefly in TBST and probed with mAb 106.105 (5 µg) for 1 h at room temperature. All antibody incubations were done in a volume of 10 ml. Blots were washed and incubated with goat antimouse Ab (1:10,000) conjugated to horseradish peroxidase (HRP) (Biosource, Camarillo, CA) for 1 h at room temperature. In the case of myc mAb, blots were blocked and probed in TBST with 3% BSA. Blots were probed with 10 µg biotinylated myc mAb, prepared according to the manufacturer’s instructions for EZ Link Sulfo-NHS-LC-biotin (Pierce). Blots were then washed and incubated with 10 µg HRP-streptavidin (Pierce). Alternatively, blots were probed with HRP-conjugated FLAG M2 mAb (Sigma) (1:5000) for 1 h at room temperature. Signal was developed using enhanced chemiluminescence Western blot detection reagents (Pierce).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
FSHR is constitutively self-associated on the cell surface
FRET experiments were performed to determine whether FSHR is an oligomer on the surface of cells. FRET occurs over intermolecular distances between 1.0 and 10.0 nm and thus provides a means of assessing protein-protein interactions and of detecting even very small clusters of proteins in the plasma membrane of a cell (19, 18, 24).

The plasma membrane of HEK 293/FSHR cells displayed labeling after incubation with mAb 106.105 conjugated to either Alexa 568 (mAb 106.105-Alexa 568) or to Alexa 647 (mAb 106.105-Alexa 647). HEK 293 cells, which do not express FSHR, showed no specific labeling when incubated with these fluorescently labeled antibodies (data not shown).

In an initial FRET experiment (Fig. 1Go), HEK 293/FSHR cells were incubated with a 1:1 mixture of mAb 106.105-Alexa 568 (donor) and mAb 106.105-Alexa 647 (acceptor). Pre-bleach Alexa 568 and Alexa 647 images were collected (Fig. 1Go, A and C). Selected ROI on the plasma membrane were photobleached, and post-bleach images were collected (Fig. 1Go, B and D). In this experiment, acceptor photobleaching in the ROI resulted in a 23.06% increase in donor fluorescence (Fig. 1BGo) and a 95% decrease in acceptor fluorescence (Fig. 1DGo).


Figure 1
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  		FIG. 1. Oligomerization of FSHR is demonstrated by FRET. Confocal images of a 2-µm optical slice through an HEK 293/FSHR cell stained with mAb 106.105-Alexa 568 (donor), and 106.105-Alexa 647 (acceptor) were collected. A, Donor fluorescence pre-acceptor bleach; B, donor fluorescence post-acceptor bleach; C, acceptor fluorescence pre-bleach; D, acceptor fluorescence post-bleach. Alexa 568 fluorescence is pseudocolored green, and acceptor fluorescence is pseudocolored red. An area of interest was selected (box in D) and pre-bleach images were collected (A and C). Acceptor fluorescence in the area of interest was photobleached with 633-nm laser light, and post-bleach images were collected (B and D).

 
Acceptor photobleaching experiments were performed on selected ROI on the plasma membrane of 30 cells incubated with various donor/acceptor ratios (D:A). In the absence of cellular treatment, a robust average FRET efficiency of 21.89 ± 9.38 was observed for the FSHR, indicating that the receptors are within 10.0 nm of one another in unstimulated cells. These data provide direct evidence that the receptors are oligomerized in the plasma membrane in the absence of FSH.

It should be pointed out, however, that purely random proximity of fluorophores can lead to substantial FRET, especially when the donor and acceptor fluors are confined to the surface and are present in high concentrations. Simple models for analysis of FRET on membranes have been developed and have been used to distinguish between FRET due to a random distribution of high concentrations of donors and acceptors in the membrane and FRET that is due to clustering of donors and acceptors (18, 25, 26, 27). According to these models, if donor and acceptor fluorophores (FSHR) are clustered, then E % is independent of acceptor density, in contrast with a randomly distributed population wherein E % will increase with an increase in acceptor density. Furthermore, for a random distribution, E % will trend to zero at low acceptor density values. A mixed population of random and clustered distributions will exhibit features of both. Figure 2AGo shows the relationship between FRET efficiency and acceptor density expressed as acceptor fluorescence (arbitrary units) at the average D:A of approximately 1.4 (range, 1.18–1.59) and approximately 2.1 (range, 1.60–2.60). At the average D:A, approximately 1.4, E % is dependent upon acceptor density, increasing with increasing acceptor density and trending to zero at low acceptor densities. In contrast, at the average D:A, approximately 2.1, E % is independent of acceptor density. Apparently, a mixed population of random and clustered receptors was uncovered using a bivalent IgG probe.


Figure 2
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  		FIG. 2. FSHR appears to be distributed as a mix of clustered and randomly associated receptors when FRET is performed with intact mAb, whereas only clustered FSHR is detected when FRET is done with labeled Fab fragments. A and B, Cells were labeled with 1:2, 1:1, or 2:1 ratios of intact mAb 106.105-Alexa 568 (donor, D):intact mAb 106.105-Alexa 647 (acceptor, A) (A) or with 1:1 ratio of Fab-mAb 106.105-Alexa 568 and Fab-mAb 106.105-Alexa 647 (B). Experimentally determined D:A ratios ranged from 0.2–1.2 (A) or 1.18–2.6 (B). E% was plotted as a function of acceptor fluorescence intensity (arbitrary units, AU). Curve fitting is by simple linear regression.

 
The mAb used in this study is specific to a discrete linear peptide epitope in the ECD of human FSHR (16). It is possible that the divalent whole antibody molecule could cross-link two FSHR in the plasma membrane, although it is unlikely that such cross-linking would result in the robust FRET signal observed. At most, the results would indicate that FSHR dimers can come close enough for FRET to occur between the fluorescently labeled antibodies. However, to directly test that possibility, monovalent donor and acceptor fluorescently labeled Fab fragments of mAb 106.105 were generated. Each individual Fab probe bound to the surface of HEK 293/FSHR cells and could be blocked by preincubation with intact mAb 106.105 (data not shown).

When the labeled Fab fragments were mixed at a molar ratio of 1:1 and incubated with HEK 293/FSHR cells, both probes bound to the cell surface and elicited a robust average FRET efficiency of 11.22 ± 6.05%. Although a only a single 1:1 mixture of Fab-mAb 106.105-Alexa 568 and Fab-mAb 106.105-Alexa 647 was used, the actual D:A that are obtained from the experimental data showed a normal distribution of D:A around a mean of 0.62 ± 0.19 with a range of D:A of 0.2–1.2. When the E % is plotted against acceptor density for this range of actual D:A, E % is independent of acceptor density (Fig. 2BGo). Therefore, when Fab fragments are used as a probe, only clustered FSHR appears to be detected by FRET.

Biogenesis of self-associated FSHR
HEK 293 cells transiently transfected with myc-tagged FSHR or FLAG-tagged FSHR bound comparable levels of [I125]FSH with high specificity in a radioreceptor assay (data not shown), indicating that neither epitope tag interferes with the trafficking of receptor to the plasma membrane or with the ability of FSHR to bind FSH.

The myc- and FLAG-tagged FSHR were coexpressed in HEK 293 cells, and cell extracts were immunoprecipitated with FLAG mAb. These samples were then assessed for the presence of myc-tagged FSHR by probing immunoblots with myc mAb. Both FLAG- and myc-tagged FSHR immunoprecipitate with FLAG mAb (Fig. 3AGo), confirming that FSHR forms oligomers in HEK 293 cells. Three forms of FSHR were visible on the immunoblots: relative molecular mass (Mr) 75,000, Mr 175,000, and a high-molecular-mass form of Mr >175,000 (thick arrowhead). These molecular masses are approximate and vary depending upon the markers used.


Figure 3
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  		FIG. 3. FLAG- and myc-tagged FSHR are constitutively oligomerized. HEK 293 cells were cotransfected with FLAG- and myc-tagged FSHR or mock transfected and were then treated with 1.2 nM FSH for the indicated times. A and C, Cell lysates were immunoprecipitated with FLAG mAb; immunoblots were probed with biotinylated myc mAb and HRP-conjugated streptavidin (A) to detect myc-tagged FSHR in FLAG immunoprecipitates or probed with HRP-conjugated FLAG mAb (C). B and D, Cell lysates were immunoprecipitated with myc mAb; immunoblots were probed with HRP-conjugated FLAG mAb (B) to detect FLAG-tagged FSHR in myc immunoprecipitates or probed with biotinylated myc mAb and HRP-conjugated streptavidin (D).

 
The Mr 75,000 band represents immature FSHR localized to the endoplasmic reticulum (ER) (17). The Mr 175,000 and Mr >175,000 bands may represent myc-tagged FSHR complexed with a chaperone that is not dissociable by SDS. Alternatively, the Mr 175,000 and Mr >175,000 bands may be FSHR targeted for degradation. These complexes are likely to have been oligomerized with FLAG-tagged FSHR, in an SDS-dissociable form, in order for myc-tagged FSHR to be detected in a FLAG immunoprecipitate.

Similar results were observed when immunoprecipitates were solubilized in sample buffer containing dithiothreitol rather than ß-mercaptoethanol or when samples were analyzed on urea gels (data not shown). This suggests that the FSHR is either complexed with another protein or is not completely dissociated by SDS, as suggested for other GPCR, including the receptors for V2 vasopressin (28), dopamine D2 (29), melatonin (30), and TSH-releasing hormone (31).

Oligomers of FLAG- and myc-tagged FSHR are also detectable when cell extracts are immunoprecipitated with myc mAb and then probed with FLAG mAb (Fig. 3BGo). It is apparent that FSHR forms oligomers in the absence of FSH and that treatment with FSH has little effect. The same three forms of FSHR are observed when extracts from cells cotransfected with FLAG- and myc-tagged FSHR are immunoprecipitated with FLAG mAb and then probed with FLAG mAb or immunoprecipitated with myc mAb and then probed with myc mAb (Fig. 3Go, C and D).

To ensure that the oligomeric interactions observed between tagged receptors were specific and not due to aggregation, extracts from cells individually expressing either FLAG- or myc-tagged FSHR were mixed before immunoprecipitation. No FSHR-FLAG was observed in myc immunoprecipitates from mixed lysates (Fig. 4Go) and vice versa (data not shown). Accordingly, the interaction between receptors occurs within the cell rather than during sample preparation.


Figure 4
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  		FIG. 4. High-molecular-weight FSHR-specific bands are not due to aggregation. HEK 293 cells were singly transfected with myc-tagged FSHR (lane 1) or with FLAG-tagged FSHR (lane 2) or cotransfected with FLAG- and myc-tagged FSHR (lane 3). In lane 4, extracts from cells singly transfected with myc- or FLAG-tagged FSHR were mixed together before immunoprecipitation. Cell lysates were immunoprecipitated with myc mAb, immunoprecipitates were analyzed by SDS-PAGE, and the immunoblot was probed with HRP-conjugated FLAG mAb.

 
Epitope-tagged FSHR shows evidence of C-terminal processing
Interestingly, the mature fully glycosylated form of FSHR, Mr 80,000 (17), is not detectable when myc- or FLAG-tagged FSHR is immunoprecipitated with myc or FLAG mAb, respectively, and immunoblots are probed with myc or FLAG mAb (Fig. 3Go), suggesting that the myc and FLAG epitopes are present in immature, but not mature, FSHR. In contrast, mAb 106.105 (specific for the ECD) recognizes both immature and mature FSHR (Fig. 5Go, compare lane 3 vs. lane 4 and lane 5 vs. lane 6).


Figure 5
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  		FIG. 5. Myc epitope is present only in immature FSHR. HEK 293 cells were transfected with wt FSHR or with myc- and FLAG-tagged FSHR, and cell lysates were immunoprecipitated with either mAb 106.105 (105 IP) or with myc mAb. Immunoblots were probed with biotinylated myc mAb and HRP-conjugated streptavidin or with mAb 106.105 and HRP-conjugated goat antimouse Ab as indicated. Lane 1 is a longer exposure of lane 2 to highlight the Mr 175,000 and Mr >175,000 bands.

 
Immunoprecipitation of untagged receptor with mAb 106.105 (Fig. 5Go) shows that untagged FSHR exhibits three forms analogous to that seen with the tagged receptors, although the monomer form of FSHR migrates as a broad band, including the immature Mr 75,000 and the mature fully glycosylated Mr 80,000 species, rather than solely the Mr 75,000 species observed in myc or FLAG immunoprecipitates of tagged receptor. A background band (Mr 60,000), occasionally observed with mAb 106.105, is seen in lanes 1 and 2. A Mr 50,000 band is also seen in lanes 1 and 2 and represents the heavy chain of mAb 106.105. This band is not visualized when HRP-106.105 is used (Fig. 5Go).

The detection of only immature FSHR by the myc and FLAG mAbs is suggestive of proteolytic clipping of the C terminus of tagged FSHR because the myc and FLAG mAbs recognize a C-terminal epitope tag. Biotinylation experiments were carried out to solidify this result.

Biotinylation of FSHR oligomers
The evidence presented up to this point indicates that immature FSHR oligomerizes in the ER before final glycosylation processing. To distinguish between intracellular receptor and cell surface receptor, cell surface proteins were labeled with cell-impermeant biotin in HEK 293 cells transfected with myc- or FLAG-tagged FSHR. Cell extracts were immunoprecipitated with myc, FLAG, or 106.105 mAb, and biotin-labeled proteins were detected on immunoblots with HRP-streptavidin (Fig. 6Go).


Figure 6
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  		FIG. 6. Mature Mr 80,000 FSHR is the only form of FSHR biotinylated by cell-impermeant biotin. HEK 293 cells were transfected with myc- or FLAG-tagged FSHR or mock transfected. Surface proteins were biotinylated (A and C) or treated with vehicle (B and D). Cell lysates were immunoprecipitated with 106.105, myc, or FLAG mAb, immunoprecipitates were analyzed by SDS-PAGE, and immunoblots were probed with HRP-streptavidin (A and B). Immunoblots were reprobed with mAb 106.105 (C and D).

 
The mature Mr 80,000 band (compare Fig. 6Go, A and B) is labeled by biotin, indicating that this is the only species of FSHR on the cell surface. The Mr 75,000, Mr 175,000 and Mr >175,000 proteins, detectable when the blot is reprobed with mAb 106.105 (compare Fig. 6Go, A and C), appear to be intracellular forms of FSHR because these forms are not labeled by biotin.

The immunoprecipitation of low levels of myc- and FLAG-tagged mature FSHR at the cell surface (Fig. 6AGo) indicates that a fraction of mature FSHR on the cell surface retains the C-terminal epitope tag, either because the clipping occurs at the cell surface so that tagged and untagged FSHR are detectable or because overexpression of the tagged receptor has overwhelmed the quality control mechanism of the cell allowing some FSHR to evade proteolytic processing.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The evidence that constitutive oligomerization of FSHR occurs in the ER is strong. Myc mAb immunoprecipitates three forms of FLAG-tagged FSHR (Mr 75,000, Mr 175,000, and Mr >175,000) in cells cotransfected with myc- and FLAG-tagged FSHR and vice versa. The detection of ER-localized immature FSHR (Mr 75,000) in oligomers suggests that FSHR forms oligomers at an early stage of biosynthesis. In addition, the three forms of FSHR involved in oligomerization are found intracellularly, as shown by the observation that only the mature Mr 80,000 form of FSHR is labeled with cell-impermeant biotin.

Constitutive oligomerization has been demonstrated in a number of other GPCR, including the receptors for TSH-releasing hormone (31, 32), GABAB (33), melatonin (30), dopamine D2 (34, 35), {alpha}-factor (36, 37), vasopressin (38), and serotonin 5-HT2C (39). Constitutive oligomers have also been observed for the {delta}-opioid receptor (40) and ß2-adrenergic receptor (41, 42).

The results reported in this study differ from that seen with LHR in that LHR oligomers appear to exist on the cell surface in stable cell lines but were not demonstrable in transiently transfected cells (5). Furthermore, LHR oligomer formation increases with high concentrations of hCG. Interestingly, both LHR (5) and FSHR occur in high-molecular-mass SDS-resistant forms, Mr 166,000 and 240,000 in the case of LHR and Mr 175,000 and >175,000 in the case of FSHR. These high-molecular-mass forms may represent gonadotropin receptor covalently cross-linked or otherwise irreversibly associated with a protein, either a chaperone or a cargo protein. The observation of these forms in two different receptors suggests a general mechanism may be revealed by determination of the nature of these forms, a current goal of the lab. Another difference worth noting is that Tao et al. (5) did not observe monomers of LHR in coimmunoprecipitation experiments in contrast to the monomers of FSHR reported here. This is likely due to differences in transfection efficiencies and/or sample processing.

In contrast to the abundant evidence that LHR self-associates in response to agonist binding (6, 7, 8, 10), the results reported here indicate that FSH has little impact on FSHR oligomerization. This is not surprising in light of the observation that FSHR forms oligomers in the ER and remains in a complex on the cell membrane. However, it points to a significant difference between these two otherwise similar receptors. Oligomerization in the ER could assist in trafficking of receptor to the cell membrane, as is seen for ß2-adrenergic receptor (43), by shielding ER retention signals or by ensuring correct folding. On the other hand, formation of oligomers in the ER does not preclude additional oligomerization occurring in response to hormone that cannot be detected by methods reported herein.

Roess and Smith (6) found that wt LHR has a lower lateral mobility and higher self-association than does signaling-defective LHR, suggesting that self-association depends on functional receptor. In addition, desensitized LHR is organized into bigger complexes than is actively signaling LHR (10), suggesting a progression from small clusters to more complex arrays as unoccupied LHR binds hormone and becomes desensitized. These studies suggest that LHR has a greater propensity to form higher-order clusters than does FSHR.

In contrast to what is observed with LHR and FSHR, oligomerization of TSHR is significantly reduced by TSH treatment (11). Dissociation of constitutive TSHR oligomers induced by TSH may serve to release TSHR from an inactive conformation. Alternatively, post-binding proteolytic clipping of the TSHR ectodomain may result in destabilization of higher-order oligomers.

Because imaging FRET was conducted with wt FSHR, the possibility of variable expression levels of tagged FSHR was not an issue. In addition, the novel use of differentially labeled mAb or Fab fragments of the mAb specific for FSHR in FRET obviated the need for C-terminal fusion proteins.

A note of caution should be interjected here. These experiments demonstrate that the valency of the FRET probes is an important consideration. Use of the bivalent mAb probes or the monovalent Fab probes produced a robust FRET signal, but upon quantitative FRET analysis, the distributions of FSHR were different with the two different probes.

What might account for these observations? FRET occurs when acceptor and donor fluors are in close proximity, typically 1.0–10.0 nm. An intact IgG molecule is reported to be approximately 12 nm in length, and the hinge region connecting the Fab and Fc regions is flexible such that the maximum distance between the ends of the Fab regions in a fully open configuration is approximately 14–15 nm (44). Thus, if an IgG donor and IgG acceptor were each bound to FSHR molecules and they were stretched out in a fully open conformation on the cell surface, these probes could be long enough to give a FRET signal even though the distance between the FSHR might be more than 10 nm. These divalent probes would also show FRET with FSHR that was in fact oligomerized. In this case, a mixed population of randomly associated and clustered FSHR would be observed. In fact, this is what was observed in this study.

In contrast, the Fab fragments are approximately 6 nm in length and bind a single FSHR. Thus, if the FSHR are distributed as oligomers, FRET will occur and it will be independent of increasing acceptor concentration because, in this clustered situation, a donor molecule is already associated with an acceptor molecule. Again, this was found to be the case in this study.

Receptor self association may also assist in signaling as a way of regulating multiple signaling pathways. For instance, angiotensin II type I receptor mutants, defective in ligand binding or signaling, exert a dominant-negative effect on wt angiotensin II type I receptor with regard to G{alpha}q activation (45). ERK activation, however, is not inhibited, suggesting that G{alpha}q signaling, but not ERK activation, is dependent on oligomerization. Receptor dimerization provides an attractive explanation for the transactivation effects observed when binding-defective, signaling-competent receptors are coexpressed with binding-competent, signaling-defective mutants of FSHR (14).

More recently, it has been proposed that receptor dimerization enables negative cooperativity (46). Negative cooperativity in a system ensures that physiological responses occur at subsaturating levels of agonist with accommodation at higher levels of agonist as well. The evidence for negative cooperativity in FSHR suggests that a conformational change of FSH and/or FSHR occurs upon binding (46). In contrast, one would predict, based on the crystal structure of FSH in complex with FSHRECD, that the FSHR hormone-binding domain would not be conformationally flexible due to hydrogen bonding of ß-sheets (2). These two pieces of data are not necessarily in contradiction. Rather, they speak to the need to crystallize the entire FSHRECD, including the last 100 amino acids, which likely play a role in regulating binding in situ (47, 48). Indeed, circular dichroism spectroscopy of FSH in complex with the full-length FSHRECD had demonstrated a conformational change upon hormone binding (21).

The mechanism and extent (dimer, tetramer, etc.) of FSHR oligomerization is not known. The minimal complex is likely to be a dimer, which would be consistent with the observations described in this report. Moreover, the crystal structure of FSHR hormone-binding domain, in complex with FSH, revealed a dimer with a weakly interacting interface (2). This interface is not likely to be the major stabilizing force for dimerization. The area of contact is small and could easily be due to crystal contacts, thus not representing a physiologically relevant protein interaction site for dimerization.

It is likely that other forces are at play, and chimeric receptor experiments suggest association through the TM domains (12). Both mechanisms may be operative here and may affect the quality of the signal generated by offering temporal and spatial constraints upon receptor recycling and internalization (49).

The discovery of C-terminal proteolytic processing of tagged FSHR, as evidenced by the relative lack of C-terminal epitope tags in mature FSHR and in FSHR at the cell surface, was serendipitous. Tools for analyzing the C terminus of FSHR have not previously been available. Although a number of studies have been published in which an epitope tag has been inserted at the N terminus of FSHR, this is the first report in which FSHR was tagged at the C terminus. In contrast, LHR has been tagged at both the N and C termini with myc, FLAG, and green fluorescent protein epitopes with no report of proteolytic processing (5, 50, 51, 52).

Alternatively, the myc and FLAG epitopes may be inaccessible to antibody. However, for that to be the case, the C-terminal epitope must be inaccessible when the protein is in its native state (immunoprecipitations) or denatured (immunoblots) because FSHR that appeared to be lacking the C-terminal epitope tag was detected under both conditions. C-terminal processing seems to be the best explanation for these data at the present time. Protein sequencing of wt FSHR would establish whether this phenomenon is restricted to epitope-tagged FSHR.

Among candidate endoproteases for the C-terminal processing of FSHR are the proprotein convertases (for review, see Refs. 53 and 54). The proprotein convertases cleave proproteins to induce activation. Hormones, growth factors, and membrane receptors are among the substrates of these proteases.

Finally, it is important to consider the physiological implications of receptor oligomerization in the ER in genetic disorders. In the case of heterozygous mutations of the glycoprotein hormone receptor genes that lead to trafficking defects, oligomerization with viable receptor could lead to functional receptor present on the cell surface, as has been so elegantly shown in vitro (12). In addition, the proximity of the two ECD domains offers another level of potential control of hormone binding.


   Acknowledgments
 
George Bousfield (Wichita State University, Wichita, KS) provided a human pituitary crude extract (GTN fraction) from which FSH was purified in our laboratory. Linda O’Keefe, Hiroko Yoshinari, and Gerald Kornatowski in the Tissue Culture Facility, Wadsworth Center, supplied the HEK 293 and HEK 293/FSHR cells. A special thanks is extended to Margarida Barroso (Albany Medical College, Albany, NY) for her insightful discussions and help with the quantitative FRET analyses.


   Footnotes
 
This work was supported by National Institutes of Health Grants HD18407 (to J.A.D.) and RR01976 (to J.E.M.).

First Published Online February 1, 2007

Abbreviations: D:A, Donor/acceptor ratio; E, efficiency of energy transfer; ECD, extracellular domain; ER, endoplasmic reticulum; FRET, fluorescence resonance energy transfer; FSHR, FSH receptor; FSHRECD, ECD of FSHR; GPCR, G protein-coupled receptor; hCG, human chorionic gonadotropin; HEK, human embryonic kidney; HRP, horseradish peroxidase; LHR, LH receptor; mAb, monoclonal antibody; Mr, relative molecular mass; ROI, region(s) of interest; RRAs, radioreceptor assays; TBST, Tris-buffered saline with Tween 20; TM, transmembrane; TSHR, TSH receptor; wt, wild type.

Received December 13, 2006.

Accepted for publication January 19, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Dias JA, Cohen BD, Lindau-Shepard B, Nechamen CA, Peterson AJ, Schmidt A 2002 Molecular, structural, and cellular biology of follitropin and follitropin receptor. Vitam Horm 64:249–322[Medline]
  2. Fan QR, Hendrickson WA 2005 Structure of human follicle-stimulating hormone in complex with its receptor. Nature 433:269–277[CrossRef][Medline]
  3. Terrillon S, Bouvier M 2004 Roles of G-protein-coupled receptor dimerization. EMBO Rep 5:30–34[CrossRef][Medline]
  4. Kroeger KM, Pfleger KD, Eidne KA 2003 G-protein coupled receptor oligomerization in neuroendocrine pathways. Front Neuroendocrinol 24:254–278[CrossRef][Medline]
  5. Tao YX, Johnson NB, Segaloff DL 2004 Constitutive and agonist-dependent self-association of the cell surface human lutropin receptor. J Biol Chem 279:5904–5914[Abstract/Free Full Text]
  6. Roess DA, Horvat RD, Munnelly H, Barisas BG 2000 Luteinizing hormone receptors are self-associated in the plasma membrane. Endocrinology 141:4518–4523[Abstract/Free Full Text]
  7. Horvat RD, Nelson S, Clay CM, Barisas BG, Roess DA 1999 Intrinsically fluorescent luteinizing hormone receptor demonstrates hormone-driven aggregation. Biochem Biophys Res Commun 255:382–385[CrossRef][Medline]
  8. Roess DA, Smith SM 2003 Self-association and raft localization of functional luteinizing hormone receptors. Biol Reprod 69:1765–1770[Abstract/Free Full Text]
  9. Smith SML, Lei Y, Liu JJ, Cahill ME, Hagen GM, Barisas BG, Roess DA 2006 Luteinizing hormone receptors translocate to plasma membrane microdomains after binding of human chorionic gonadotropin. Endocrinology 147:1789–1795[Abstract/Free Full Text]
  10. Hunzicker-Dunn M, Barisas G, Song JM, Roess DA 2003 Membrane organization of luteinizing hormone receptors differs between actively signaling and desensitized receptors. J Biol Chem 278:42744–42749[Abstract/Free Full Text]
  11. Latif R, Graves P, Davies TF 2002 Ligand-dependent inhibition of oligomerization at the human thyrotropin receptor. J Biol Chem 277:45059–45067[Abstract/Free Full Text]
  12. Osuga Y, Hayashi M, Kudo M, Conti M, Kobilka B, Hsueh AJ 1997 Co-expression of defective luteinizing hormone receptor fragments partially reconstitutes ligand-induced signal generation. J Biol Chem 272:25006–25012[Abstract/Free Full Text]
  13. Ji I, Lee C, Jeoung M, Koo Y, Sievert GA, Ji TH 2004 Trans-activation of mutant follicle-stimulating hormone receptors selectively generates only one of two hormone signals. Mol Endocrinol 18:968–978[Abstract/Free Full Text]
  14. Sairam MR, Jiang LG, Yarney TA, Khan H 1997 Alternative splicing converts the G-protein coupled follitropin receptor gene into a growth factor type I receptor: implications for pleiotropic actions of the hormone. Mol Reprod Dev 48:471–479[CrossRef][Medline]
  15. Sairam MR, Jiang LG, Yarney TA, Khan H 1996 Follitropin signal transduction: alternative splicing of the FSH receptor gene produces a dominant negative form of receptor which inhibits hormone action. Biochem Biophys Res Commun 226:717–722[CrossRef][Medline]
  16. Lindau-Shepard BA, Brumberg HA, Peterson AJ, Dias JA 2001 Reversible immunoneutralization of human follitropin receptor. J Reprod Immunol 49:1–19[CrossRef][Medline]
  17. Nechamen CA, Dias JA 2003 Point mutations in follitropin receptor result in ER retention. Mol Cell Endocrinol 201:123–131[CrossRef][Medline]
  18. Kenworthy AK, Edidin M 1998 Distribution of a glycosylphosphatidylinositol-anchored protein at the apical surface of MDCK cells examined at a resolution of <100 Å using imaging fluorescence resonance energy transfer. J Cell Biol 142:69–84[Abstract/Free Full Text]
  19. Siegel RM, Chan FK, Zacharias DA, Swofford R, Holmes KL, Tsien RY, Lenardo MJ 2000 Measurement of molecular interactions in living cells by fluorescence resonance energy transfer between variants of the green fluorescent protein. Sci STKE 2000:L1
  20. Weiner RS, Dias JA 1992 Biochemical analyses of proteolytic nicking of the human glycoprotein hormone {alpha}-subunit and its effect on conformational epitopes. Endocrinology 131:1026–1036[Abstract]
  21. Schmidt A, MacColl R, Lindau-Shepard B, Buckler DR, Dias JA 2001 Hormone-induced conformational change of the purified soluble hormone binding domain of follitropin receptor complexed with single chain follitropin. J Biol Chem 276:23373–23381[Abstract/Free Full Text]
  22. Laemmli UK 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685[CrossRef][Medline]
  23. Towbin H, Staehelin T, Gordon J 1979 Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76:4350–4354[Abstract/Free Full Text]
  24. Kenworthy AK 2001 Imaging protein-protein interactions using fluorescence resonance energy transfer microscopy. Methods 24:289–296[CrossRef][Medline]
  25. Pentcheva T, Edidin M 2001 Clustering of peptide-loaded MHC class I molecules for endoplasmic reticulum export imaged by fluorescence resonance energy transfer. J Immunol 166:6625–6632[Abstract/Free Full Text]
  26. Wallrabe H, Chen Y, Periasamy A, Barroso M 2006 Issues in confocal microscopy for quantitative FRET analysis. Microsc Res Tech 69:196–206[CrossRef][Medline]
  27. Bhatia S, Edidin M, Almo SC, Nathenson SG 2005 Different cell surface oligomeric states of B7-1 and B7-2: implications for signaling. Proc Natl Acad Sci USA 102:15569–15574[Abstract/Free Full Text]
  28. Zhu XY, Wess J 1998 Truncated V2 vasopressin receptors as negative regulators of wild-type V2 receptor function. Biochemistry 37:15773–15784[CrossRef][Medline]
  29. Ng GY, O’Dowd BF, Lee SP, Chung HT, Brann MR, Seeman P, George SR 1996 Dopamine D2 receptor dimers and receptor-blocking peptides. Biochem Biophys Res Commun 227:200–204[CrossRef][Medline]
  30. Ayoub MA, Couturier C, Lucas-Meunier E, Angers S, Fossier P, Bouvier M, Jockers R 2002 Monitoring of ligand-independent dimerization and ligand-induced conformational changes of melatonin receptors in living cells by bioluminescence resonance energy transfer. J Biol Chem 277:21522–21528[Abstract/Free Full Text]
  31. Zhu CC, Cook LB, Hinkle PM 2002 Dimerization and phosphorylation of thyrotropin-releasing hormone receptors are modulated by agonist stimulation. J Biol Chem 277:28228–28237[Abstract/Free Full Text]
  32. Kroeger KM, Hanyaloglu AC, Seeber RM, Miles LE, Eidne KA 2001 Constitutive and agonist-dependent homo-oligomerization of the thyrotropin-releasing hormone receptor. Detection in living cells using bioluminescence resonance energy transfer. J Biol Chem 276:12736–12743[Abstract/Free Full Text]
  33. White JH, Wise A, Main MJ, Green A, Fraser NJ, Disney GH, Barnes AA, Emson P, Foord SM, Marshall FH 1998 Heterodimerization is required for the formation of a functional GABAB receptor. Nature 396:679–682[CrossRef][Medline]
  34. Lee SP, O’Dowd BF, Ng GYK, Varghese G, Akil H, Mansour A, Nguyen T, George SR 2000 Inhibition of cell surface expression by mutant receptors demonstrates that D2 dopamine receptors exist as oligomers in the cell. Mol Pharmacol 58:120–128[Abstract/Free Full Text]
  35. Guo W, Shi L, Javitch JA 2003 The fourth transmembrane segment forms the interface of the dopamine D2 receptor homodimer. J Biol Chem 278:4385–4388[Abstract/Free Full Text]
  36. Overton MC, Blumer KJ 2000 G-protein-coupled receptors function as oligomers in vivo. Curr Biol 10:341–344[CrossRef][Medline]
  37. Overton MC, Chinault SL, Blumer KJ 2003 Oligomerization, biogenesis, and signaling is promoted by a glycophorin A-like dimerization motif in transmembrane domain 1 of a yeast G protein-coupled receptor. J Biol Chem 278:49369–49377[Abstract/Free Full Text]
  38. Terrillon S, Durroux T, Mouillac B, Breit A, Ayoub MA, Taulan M, Jockers R, Barberis C, Bouvier M 2003 Oxytocin and vasopressin V1a and V2 receptors form constitutive homo- and heterodimers during biosynthesis. Mol Endocrinol 17:677–691[Abstract/Free Full Text]
  39. Herrick-Davis K, Grinde E, Mazurkiewicz JE 2004 Biochemical and biophysical characterization of serotonin 5-HT2C receptor homodimers on the plasma membrane of living cells. Biochemistry 43:13963–13971[CrossRef][Medline]
  40. Mcvey M, Ramsay D, Kellett E, Rees S, Wilson S, Pope AJ, Milligan G 2001 Monitoring receptor oligomerization using time-resolved fluorescence resonance energy transfer and bioluminescence resonance energy transfer - The human {delta}-opioid receptor displays constitutive oligomerization at the cell surface, which is not regulated by receptor occupancy. J Biol Chem 276:14092–14099[Abstract/Free Full Text]
  41. Mercier JF, Salahpour A, Angers S, Breit A, Bouvier M 2002 Quantitative assessment of ß1- and ß2-adrenergic receptor homo- and heterodimerization by bioluminescence resonance energy transfer. J Biol Chem 277:44925–44931[Abstract/Free Full Text]
  42. Angers S, Salahpour A, Joly E, Hilairet S, Chelsky D, Dennis M, Bouvier M 2000 Detection of ß2-adrenergic receptor dimerization in living cells using bioluminescence resonance energy transfer (BRET). Proc Natl Acad Sci USA 97:3684–3689[Abstract/Free Full Text]
  43. Salahpour A, Angers S, Mercier JF, Lagace M, Marullo S, Bouvier M 2004 Homodimerization of the ß2-adrenergic receptor as a prerequisite for cell surface targeting. J Biol Chem 279:33390–33397[Abstract/Free Full Text]
  44. dePetris S 1978 Immunoelectron microscopy and immunofluorescence in membrane biology. Methods Membr Biol 9:1–201
  45. Hansen JL, Theilade J, Haunso S, Sheikh SP 2004 Oligomerization of wild type and nonfunctional mutant angiotensin II type I receptors inhibits g{alpha}q protein signaling but not ERK activation. J Biol Chem 279:24108–24115[Abstract/Free Full Text]
  46. Urizar E, Montanelli L, Loy T, Bonomi M, Swillens S, Gales C, Bouvier M, Smits G, Vassart G, Costagliola S 2005 Glycoprotein hormone receptors: link between receptor homodimerization and negative cooperativity. EMBO J 24:1954–1964[CrossRef][Medline]
  47. Costagliola S, Panneels V, Bonomi M, Koch J, Many MC, Smits G, Vassart G 2002 Tyrosine sulfation is required for agonist recognition by glycoprotein hormone receptors. EMBO J 21:504–513[CrossRef][Medline]
  48. Nakabayashi K, Kudo M, Kobilka B, Hsueh AJ 2000 Activation of the luteinizing hormone receptor following substitution of Ser-277 with selective hydrophobic residues in the ectodomain hinge region. J Biol Chem 275:30264–30271[Abstract/Free Full Text]
  49. Krishnamurthy H, Kishi H, Shi M, Galet C, Bhaskaran RS, Hirakawa T, Ascoli M 2003 Postendocytotic trafficking of the follicle-stimulating hormone (FSH)-FSH receptor complex. Mol Endocrinol 17:2162–2176[Abstract/Free Full Text]
  50. Hong S, Ji I, Ji TH 1999 The {alpha}-subunit of human choriogonadotropin interacts with the exodomain of the luteinizing hormone/choriogonadotropin receptor. Endocrinology 140:2486–2493[Abstract/Free Full Text]
  51. Richter-Unruh A, Verhoef-Post M, Malak S, Homoki J, Hauffa BP, Themmen AP 2004 Leydig cell hypoplasia: absent luteinizing hormone receptor cell surface expression caused by a novel homozygous mutation in the extracellular domain. J Clin Endocrinol Metab 89:5161–5167[Abstract/Free Full Text]
  52. Horvat RD, Barisas BG, Roess DA 2001 Luteinizing hormone receptors are self-associated in slowly diffusing complexes during receptor desensitization. Mol Endocrinol 15:534–542[Abstract/Free Full Text]
  53. Taylor NA, Van de Ven WJ, Creemers JW 2003 Curbing activation: proprotein convertases in homeostasis and pathology. FASEB J 17:1215–1227[Abstract/Free Full Text]
  54. Molloy SS, Anderson ED, Jean F, Thomas G 1999 Bi-cycling the furin pathway: from TGN localization to pathogen activation and embryogenesis. Trends Cell Biol 9:28–35[CrossRef][Medline]



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