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Biology of Reproduction 62, 1240-1247 (2000)
© 2000 Society for the Study of Reproduction, Inc.

Articles

Deletion of Follicle-Stimulating Hormone (FSH) Receptor Residues Encoded by Exon One Decreases FSH Binding and Signaling in the Rat1

Kathrin I. Manna, Xunxian Liub, and James A. Dias,c

a Department of Biological Sciences, and b Department of Biomedical Sciences, State University of New York at Albany, Albany, New York 12208 c Wadsworth Center, David Axelrod Institute for Public Health, New York State Department of Health, Albany, New York 12208


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The rat FSH receptor (rFSHR) shares considerable homology with the rat LH receptor (rLHR), yet binds human FSH (hFSH) with high fidelity, suggesting that the binding determinant encoded by the rFSHR gene shares no homology with the analogous rLHR primary sequence, thereby affording specificity of ligand binding. Two such regions of primary sequence have been previously identified and studied by peptide challenge tests and immunoneutralization studies. We therefore implemented site-directed mutagenesis to delete the regions S9-N30 and D300-F315 of the mature rFSHR sequence. The mutant receptor ({Delta}rFSHR) cDNAs were expressed in insect cells. The large deletion {Delta}rFSHRS9-N30 and a smaller deletion, {Delta}rFSHRS9-S18, did not bind 125I-hFSH. However, {Delta}rFSHRK19-R29 and {Delta}rFSHRD300-F315 bound 125I-hFSH with an affinity indistinguishable from wild-type rFSHR. The deletion mutants {Delta}rFSHR S9-N30 or {Delta}rFSHRS9-S18 were not detectable on the cell surface by flow cytometry unless cells were sheared. Although 125I-hFSH binding to {Delta}rFSHRK19-R29 was normal, this form of the receptor was defective for signal transduction whereas {Delta}rFSHRD300-F315 was not. Furthermore, neither region seems to be a specificity determinant, since their removal did not result in high-affinity binding of hCG to {Delta}rFSHR.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The pituitary glycoprotein hormone (GPH) FSH is a heterodimer composed of an {alpha} subunit and a ß subunit. FSH action, as with the other GPH, is mediated by cell membrane-bound GPH receptors (GPHR) [1]. Early, slow stages of granulosa cell proliferation and preantral follicle growth occur in the absence of gonadotropins, but FSH is required for normal growth, especially the final rapid stages of development from preantral to preovulatory follicles [2, 3]. Infertility affected one in twelve married women in 1988 [4]. To better understand and treat infertility, which is a considerable public health problem, the interaction of human (h) FSH with its receptor must be characterized.

Elucidation of amino acids in hFSH that are critical for binding to the hFSH receptor (hFSHR) was greatly aided by the determination of the three-dimensional structure of hCG [5, 6], and suggested that the high-affinity interaction is the result of a discrete yet localized subset of residues on the hormone [7]. According to the deduced primary sequence [8], the rat (r) FSHR has a large (> 300-residue) extracellular N-terminal domain in addition to a seven-pass transmembrane domain, and a short (63-residue) intracellular C-terminal tail. The rFSHR gene is divided into 10 exons, with exons 1–9 encoding most of the extracellular domain (ECD) [9]. Importantly, the ECD of rFSHR and hFSHR is necessary and sufficient to bind hFSH [10–13]. Exons 2–8 are a repeating leucine-rich repeat (LRR) motif perhaps arising through exon duplication and shuffling [14]. A question, then, is whether a similar discrete subset of receptor residues enables the high-affinity interaction. In addition to describing the molecular underpinnings of this high-affinity interaction, a better understanding is needed of how, after binding to the receptor, hFSHR transduces the hFSH signal to exert its major effect by activation of adenylyl cyclase [15]. Guided by previously published peptide challenge [16, 17] and immunoneutralization [18, 19] experiments, we tested the hypothesis that the unique rFSHR regions of residues 9–30 and 300–315 are essential hormone binding domains and specificity determinants.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Deletion Mutagenesis

pALTER (formerly pSELECT; Promega Biotech, Madison, WI) containing the wild-type rFSHR sequence was prepared as described previously [20]. Deletion mutagenesis was performed with the Altered Sites in vitro mutagenesis system (Promega Biotech) in the following way: single-strand template generated from the phagemid was annealed to 70-mer oligonucleotides complementary to sequences 35 base pairs upstream and downstream of the deletion site. The sequences of the templates were as follows: S9-N30, 5'-CGG-GAT-CTG-GAT-GTC-ATC-ACT-GGC-TGT-GTC-ATT-GCG-CCA-TTG-AAC-TGA-GGT-TTG-TGC-TCA-CCA-AGC-TTC-3'; K19-R29, 5'-ATT-GCT-CTA-ATA-GGG-TCT-TTC-TCT-GCC-AAG-ACA-GCA-ACG-CCA-TTG-AAC-TGA-GGT-TTG-TGC-TCA-CCA-AGC-3'; and S9-S18, 5'-CGG-GAT-CTG-GAT-GTC-ATC-ACT-GGC-TGT-GTC-ATT-GCA-AGG-TGA-CAG-AGA-TTC-CGA-CCG-ACC-TCC-CCC-GGA-A-3'. Annealing of the oligomer caused the noncomplementary part of the phagemid to loop out and resulted in subsequent deletion of that fragment from the coding strand during plasmid synthesis. Recombinant plasmids were identified by size of polymerase chain reaction (PCR) products and subsequently manually sequenced to confirm that the correct open reading frame was maintained and that no spontaneous mutations had taken place.

Each of the recombinant plasmids (pALTER-S9-N30, -S9-S18, -K19-R29, and -D300-F315) were then digested with EcoRI and Bsu36I to produce a small 1-kilobase pair fragment. The resultant fragment from each vector was then ligated into a transfer vector rFSHR-pVL 1392. This vector, which encodes wild-type rFSHR, was also digested with EcoRI and Bsu36I to enable ligation with the mutant fragments. Recombinant pVL plasmids were identified by PCR and sequenced. DNA preparations of the four transfer vectors pVL-S9-N30, -S9-S18, -K19-R29, and -D300-F315 were obtained with a Magic Miniprep kit (Promega Biotech). Figure 1 shows the locations of the different deletions on the rFSHR with respect to the wild-type amino acid sequence. Deletions chosen were based upon previously published data obtained from peptide challenge and immunological tests [16–19].



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  		FIG. 1. Deletions in the rFSHR created by site-directed mutagenesis: Numbering of the amino acid sequence corresponds to mature protein without a leader sequence. Shown are the locations of the four different deletions with respect to the wild-type (wt) amino acid sequence of the rFSHR. Positions of cysteines are indicated, as well as the positions of the amino acids defining and flanking the deletions. Abbreviations are according to the one-letter code for amino acids (C, cysteine; S, serine; K, lysine; R, arginine; N, asparagine; A, alanine; D, aspartic acid; F, phenylalanine)

Preparation of Recombinant Baculovirus by Homologous Recombination

The insect cell line Sf9 (Invitrogen, San Diego, CA) was maintained in TMN-FH medium, which is Grace's Insect Medium supplemented with lactalbumin hydrolysate and yeastolate with 10% fetal bovine serum (FBS). Recombinant baculovirus was produced by cotransfecting 2 x 106 Sf9 cells in a T25 flask (Becton Dickinson, Franklin Lakes, NJ) with 0.25 µg Baculogold viral DNA (PharMingen, San Diego, CA) and 2 µg recombinant pVL transfer vector using a calcium phosphate transfection protocol [21]. The culture media containing recombinant virus were harvested after 4–5 days.

A single round of virus purification was performed by agarose overlay plaque assay. Individual plaques were selected and soaked in 1 ml TMN-FH medium. Plaque extracts (0.5 ml) were used to infect 2 x 106 Sf9 cells in T25 flasks. The cells were collected after 2 days and tested for 125I-hFSH binding. If 125I-hFSH binding was not observed, the cells were tested for the presence of mutant rFSHR by Western blot analysis and flow cytometry. Media containing recombinant virus were used to generate viral stocks. A multiplicity of infection (MOI) of 1 was used for virus production. Media containing virus were collected after 2 days.

Electrophoresis and Western Blot Analysis of rFSHR

Infected Sf9 cells (10 MOI) were checked for expression of the recombinant rFSHR using denaturing SDS-PAGE in a discontinuous buffer system [22]. Two hundred thousand infected cells were solubilized for 30 min at room temperature in 150 µl Laemmli sample buffer (0.125 M Tris, pH 6.8, 4% SDS, 20% glycerol, and 0.24% bromophenol blue) containing single-strength protease inhibitor cocktail (100-strength protease inhibitor cocktail: 1.6 mg/ml benzamidine HCl, 1.0 mg/ml phenanthroline, 1.0 mg/ml aprotinin, 1.0 mg/ml leupeptin, and 1.0 mg/ml pepstatin A in 100% ethanol), 5% (v:v) 2-mercaptoethanol, and 8 M urea. SDS-PAGE was performed on ice using 10% polyacrylamide gels containing 8 M urea in the stacking and separating gels. A sample of 30 µl (40 000 cells) was loaded per lane.

After SDS-PAGE, proteins were stained with Coomassie blue or transferred to nitrocellulose membranes by electroblotting [23]. The nitrocellulose membranes were blocked in protein images blocking solution (USB, Cleveland, OH) overnight at room temperature. Primary antibody W970 (anti-hFSHR 150–183) was prepared by immunizing a rabbit against a synthetic peptide corresponding to amino acids 150–183 of hFSHR coupled to keyhole limpet cyanin (KLH) [20]. The peptide (18 mg) was coupled to KLH (15 mg) in 4.5 ml of PBS using water-soluble carbodiimide (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide [EDAC]). Antiserum W970 was diluted in protein images salt buffer (USB) with 1% nonfat dry milk and incubated with the nitrocellulose blots for 1–4 h at room temperature. The blots were washed once with salt buffer/1% milk and incubated with goat-anti-rabbit immunoglobulin G conjugated to alkaline phosphatase (IgG; Fisher, Pittsburgh, PA) for 1 h at room temperature. The membranes were washed extensively with salt buffer before the addition of Western Blue stabilized substrate for alkaline phosphatase (Promega Biotech). The blot was developed until sufficient color was evident.

Radioreceptor Assay (RRA)

The ability of mutant rFSHR to bind FSH was determined in an equilibrium displacement binding isotherm assay using purified hFSH prepared in this laboratory as both radiolabeled ligand and reference preparation. hFSH was purified as previously described [24, 25], except that zone electrophoresis was omitted and final purification was performed by hFSH monoclonal antibody (46.3H6.B7, anti-hFSH) affinity chromatography. Infected intact Sf9 cells (10 MOI) were resuspended in cold buffer (25 mM MgCl2, 50 mM Tris-HCl pH 7.5) 2 days after infection. An amount of 200 000 cells was added to the reaction buffer (25 mM MgCl2, 50 mM Tris-HCl pH 7.5, 25 mM sucrose, 0.1% BSA) in a glass tube (Falcon, Bedford, NJ). For competitive displacement, 150 000 cpm 125I-hFSH (1–2 ng) was added to each tube. The specific activity of the label was approximately 50 µCi/µg [26]. The maximum bindable fraction was 30%.

Preliminary tests for FSH binding were performed with 300 ng of hFSH as competitor. If binding was observed, full displacement curves were generated with increasing concentrations of unlabeled pure hFSH (0–2500 ng). The RRA was carried out in a reaction volume of 400 µl with shaking for 16 h at room temperature. After the addition of 1 ml cold 50 mM Tris-HCl (pH 7.5) to each tube, the cells were pelleted using an HG-4L-rotor (Sorvall, Waltham, MA) at 3000 rpm (2323 x g) for 1 h. The supernatants were aspirated, and the pellets were counted in a gamma-counter (Wallac, Gaithersburg, MD). The data were analyzed using the LIGAND program [27]. Hormone binding specificity was evaluated by incubating 200 000 Sf9 cells with 300 ng unlabeled pure hCG (CR 127) under the same conditions as described above. Displacement curves were generated using increasing concentrations of hCG (0–30 µg) as competitor for 125I-hFSH.

Measurement of Adenylyl Cyclase Activation by FSH

Sf9 cells were harvested 2 days after infection (10 MOI) and washed once with Sf 900 II serum-free medium (Gibco, Grand Island, NY). To determine a dose-response relationship, 200 000 cells were resuspended in 100 µl of the above medium containing 1 mM of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine and shaken with various concentrations (0–100 ng) of hFSH for 1.5 h at room temperature in a final volume of 200 µl. To end the reaction, the cells were put on dry ice, as previously described. After the samples were boiled for 5 min, 800 µl 0.05 M sodium acetate was added to each tube. The suspension was spun for 10 min in a microcentrifuge at 12 000 rpm, and the supernatants were either frozen or assayed. At the same time, a radioreceptor assay was conducted (see above) to evaluate FSH binding and to determine FSH receptor concentration. Total cAMP production of 20 000 cells was measured by RIA as previously described [20], using an antiserum kindly provided by Dr. F. Labrie (Laval University, PQ, Canada).

Flow Cytometry Analysis of Cell Surface Expression of rFSHR

One million Sf9 cells per 35-mm tissue culture dish were infected with virus at 10 MOI and incubated at 27°C for 2 days. To prevent cell lysis and inadvertent accessibility of intracellular protein, the following steps were performed. Media were aspirated, and the cells were washed once with 1 ml insect saline (170 mM NaCl, 6 mM KCl, 2 mM NaHCO3, 17 mM glucose, 6 mM NaH2PO4·H2O, 2 mM CaCl2·2H2O, 4 mM MgCl2·6H2O, pH 7.0). Then 0.3 ml insect saline with 10% FBS and normal rabbit serum (NRS) or anti-peptide antibody X179 (rabbit anti-hFSHR 265–296) [20] was added to each plate. The plates were gently rocked for 2 h at 4°C. Fluid was aspirated, and the cells were washed once with 1 ml insect saline, transferred to 12 x 75-mm tubes (Falcon), and pelleted at 3000 rpm (2060 x g; Beckman, Fullerton, CA).

In an attempt to permeabilize cells without detergent/fixation, 2 x 107 Sf9 cells in a T175 flask (Becton-Dickinson) were infected with virus and manipulated as follows. After 2 days, the cells were counted and 1 x 106 cells were washed with insect saline and transferred to 12 x 75 tubes (Falcon). To each tube was added 0.3 ml insect saline with 10% FBS and NRS or anti-peptide antibody X179 (anti-hFSHR 265–296) [20]. The tubes were gently shaken for 2 h at 4°C to shear the cells and make them permeable to antibody. For incubation with the secondary antibody, all cells were resuspended in 0.2 ml PBSA (phosphate buffered saline with 0.02% sodium azide) containing 2 µl fluorescein isothiocyanate-labeled anti-rabbit IgG (Vector, Burlingame, CA) and shaken for 1 h at 4°C. After the incubation, the cells were washed twice with 1 ml PBSA and resuspended in a final volume of 0.5 ml PBSA.

Fluorescence intensity of individual cells was determined using a Becton-Dickinson PC-LYSYS system counting 5000 events. Background staining and autofluorescence were controlled using cells treated with NRS. Any event with higher fluorescence intensity than the background was counted as positive.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Evaluation of Mutant rFSHR Expression by Western Blot

To determine whether mutant receptors {Delta}rFSHRS9-N30, {Delta}rFSHRS9-S18, {Delta}rFSHRK19-R29, and {Delta}rFSHRD300-F315 were expressed, whole-cell extracts were analyzed by SDS PAGE and Western blotting (Fig. 2). It was possible to solubilize the insect cells in conventional Laemmli buffer, but resolution of bands on Coomassie Blue-stained protein gels was poor. To alleviate protein band blurring and increase Western blot quality, 8 M urea was used in the sample buffer and gel systems, yielding reasonably resolved protein bands on 10% SDS-PAGE gels (Fig. 2, top panel).



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  		FIG. 2. A) Coomassie staining of the SDS-polyacrylamide gel of insect cells infected with wild-type (wt) and recombinant baculovirus. Cells were harvested 48 h postinfection and resuspended in Laemmli sample buffer containing 8 M urea. Amounts of 40 000 cells per lane were loaded on 8% SDS-polyacrylamide gels supplemented with 8 M urea. B) Wild-type and mutant rFSHR protein expressed in insect cells visualized by Western blot analysis. Western blots were probed with antibody W970 (anti-rFSHR 150–183) [20] for 1 h and developed for approximately 30 sec. The left arrows indicate the molecular weight markers. The top and bottom right arrows indicate the two forms of rFSHR detected. The middle right arrow indicates a nonspecific staining band, which, although not visible in this figure, is present also in vector controls

Antibody staining of a high-molecular-weight broad band at the top of the gel (Fig. 2A, right top arrow) was evident and was specific to cells expressing the rFSHR, since it was not observed in cells infected with the wild-type pVL virus (negative control). Surprisingly, despite clear resolution of protein bands using urea gels, the persistence of high-molecular-weight bands of the rFSHR (Fig. 2B, right top arrow) suggests that these are forms of rFSHR resistant even to denaturant. Similar results have been observed for other G-protein coupled receptors, and the significance of these higher-molecular-weight forms is currently being studied [28, 29]. Nonspecific staining at approximately 100 kDa was observed in vector controls as well (Fig. 2B, middle right arrow). There appeared to be some differences between the ratio of protein loading and immunostaining intensity. This probably reflects differences in the efficacy of production and of stability of different molecular forms of rFSHR.

The results demonstrate that {Delta}rFSHRK19-R29 and {Delta}rFSHRS9-S18 stain at approximately 75 kDa, the same molecular size as the wild-type rFSHR (Fig. 2B, right bottom arrow). The appearance of a doublet is possibly due to glycosylation differences. {Delta}rFSHRD300-F315 appears to run slightly lower than the wild-type rFSHR. The deletion is located close to a glycosylation site at residue 276 and may have affected glycosylation. {Delta}rFSHRS9-N30 was found to migrate at around 70 kDa, possibly reflecting the size of the deletion. In summary, the Western blot analyses revealed that all mutants were expressed in Sf9 cells.

FSH Binding Properties of Deletion Mutants of rFSHR

The regions of primary sequence deleted from the RFSHR in the present study were previously suggested to be involved in hormone binding and ligand specificity [16, 17, 19, 30]. Therefore, it was important to test the ability of each of the mutants to bind FSH selectively by an RRA using whole-cell preparations. {Delta}rFSHRK19-R29 and {Delta}rFSHRD300-F315 bound hFSH with high affinity (Fig. 3). Cells expressing {Delta}rFSHRS9-N30 and {Delta}rFSHRS9-S18 mutations were unable to bind hFSH (Fig. 3A).



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  		FIG. 3. A) Specific FSH binding by insect cells expressing wild-type (wt) rFSHR or FSHR deletion mutants. B) Comparison of the FSH binding properties of {Delta}K19-R29 and {Delta}D300-F315 with wild-type rFSHR by 125I-hFSH ligand binding isotherm assay. Each tube contained 200 000 cells in a final volume of 400 µl. The number of receptors per cell and the molar binding constants (Kd) were calculated using the LIGAND program [27]. Data are representative of eight independent experiments, and each point was determined in triplicate

When detailed dose-response curves were compared, it was found that the hormone binding activity of {Delta}rFSHRK19-R29 and {Delta}rFSHRD300-F315 were similar to that of wild-type rFSHR (Fig. 3B). Analysis of duplicate assays was done using the computer program LIGAND [27]. Data sets were analyzed as a cohort; then the hypothesis was tested that all affinity constants were similar. There was no significant difference between fits (P = 0.24), revealing that dissociation constants (Kd) for {Delta}rFSHRK19-R29, {Delta}rFSHRD300-F315, and wild-type rFSHR were similar (~10-10 M). It was important to know if the number of {Delta}rFSHRK19-R29 cell surface receptors was comparable to that of wild-type rFSHR. Whole-cell preparations were used in the binding assays, and analysis of the ligand binding data using the program LIGAND did not show any difference in {Delta}rFSHRK19-R29 receptors per cell compared to wild-type rFSHR (P = 0.08). These considerations become more important when analyzing the deletion mutation for cAMP production as described below, since an interpretation can be made only if matching numbers of receptors per cell are present. Regions K19-R29 and D300-F315 of the rFSHR appear to be unimportant or nonessential for hormone binding since deletion of these regions had no effect on the ability of the rFSHR to bind to its cognate ligand in comparison with the wild-type receptor.

Signal Transduction by Deletion Mutants of rFSHR That Bind FSH

Functional coupling between the rFSHR and adenylate cyclase in insect cells has been demonstrated [20]. Therefore, we were able to examine whether FSH activates {Delta}rFSHRK19-R29 or {Delta}rFSHRD300-F315 and signal transduction, measurable as elevation in cAMP levels. Sf9 cells expressing the mutant and wild-type receptors were exposed to increasing doses of pure pituitary hFSH for a period of 90 min. Measurements of resultant total cAMP levels indicated that the amounts of cAMP generated by {Delta}rFSHRD300-F315 were comparable to those generated by the wild type (Fig. 4). Nonlinear regression analysis determined that maximal cAMP production was 5.3 ± 0.4 and 5.4 ± 0.4 pmol cAMP/20 000 cells, for wild type and D300-R315, respectively. At the ED50 (approximately 10 ng/200 µl hFSH; ~1.5 nM), both produced about 4 pmol cAMP. Thus, the removal of D300-F315 had no effect on the FSH-induced synthesis of cAMP, and we conclude that this region is unimportant for signal transduction. In contrast, deleting K19-R29 resulted in impaired (2- to 3-fold) FSH-stimulated cAMP production. Maximal cAMP production was 1.84 ± 0.1 pmol cAMP/20 000 cells. At the ED50, only about 1.5 pmol cAMP could be detected. Therefore, amino acids within K19-R29 are likely to play a role in signal transduction.



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  		FIG. 4. Signal transduction properties of rFSHR{Delta}K19-R29 and rFSHR{Delta}D300-F315 in comparison with wild-type (wt) rFSHR. Data are represented as total cAMP production per 20 000 Sf9 cells in a final volume of 200 µl. Data are representative of three independent experiments and were calculated using the program NIH-RIA [48]. Each point was determined in sets of three triplicates per hormone concentration

Determination of hCG Binding to Deletion Mutants rFSHR

We reasoned that just as the determinant loop guides the specificity of pituitary glycoprotein hormone binding [31–33], the receptor may also harbor a reciprocating specificity determinant. Therefore, we tested whether the deletion of variant regions K19-R29 or D300-F315 broadens rFSHR binding specificity by using unlabeled hCG instead of hFSH as a competitor against 125I-hFSH. Indicative of an expected very low-affinity interaction, 1–30 µg hCG competed against hFSH for wild-type rFSHR with an ED50 of 5.6 µg hCG (Fig. 5). However, no difference in binding affinity to hCG could be found between {Delta}rFSHRK19-R29, {Delta}rFSHRD300-F315, and wild-type rFSHR. Importantly, deletion of the two regions of primary sequence did not increase the affinity of hCG for {Delta}rFSHR. Saturation analysis with radioiodinated hCG of cells expressing these deletion mutants failed to detect any hCG binding (not shown). Thus, regions K19-R29 and D300-F315 do not appear to be specificity determinants that allow FSH binding but preclude LH and TSH binding.



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  		FIG. 5. Comparison of the hormone binding specificity of {Delta}K19-R29 and {Delta}D300-F315 with wild-type rFSHR by displacement of 125I-hFSH by hCG. Each tube contained 200 000 cells in a final volume of 400 µl. All data points were determined in triplicate

Flow Cytometric Analysis of Surface Expression of Deletion Mutants of rFSHR

The {Delta}rFSHRS9-N30 and {Delta}rFSHRS9-S18 forms of rFSHR did not bind hFSH. It was conceivable that this was due either to the loss of a binding site or to trafficking problems. A question remained, therefore, as to whether these forms of rFSHR were trapped in cells in addition to being unable to bind hormone. To address this question, cells infected with {Delta}rFSHRS9-N30 or {Delta}rFSHRS9-S18 were analyzed by flow cytometry. A comparison was made between cells whose membranes had either been disrupted by shearing or left attached to the cell culture plates during the immunostaining procedure. For cells incubated with antibody while still attached to plates, an antibody to the rFSHR can only bind to its antigen on the cell surface and will thus indicate correct receptor trafficking. An antiserum generated against the hFSHR peptide sequence 265–296, which is well away from the site of mutagenesis, was used for this study [20]. For cells whose membranes had been sheared before incubation with the primary antibody, rFSHR trapped on the inside of the cell could be detected.

The results of this experiment are shown in Figure 6. Antibody binding is expressed as the mean peak fluorescence. As a control, cells were carried through the staining procedures using NRS instead of X179 antiserum. For cells infected with the wild-type rFSHR, there was a greater mean peak fluorescence observed in intact cells than in cells with disrupted membranes. This difference is believed to be due to staining of previously inaccessible additional rFSHR inside the cell. In intact cells infected with either one of the mutants, no staining could be detected unless cells were sheared during the immunostaining process. In that case, cells evidenced staining for {Delta}rFSHRS9-N30 and for {Delta}rFSHRS9-S18. This result demonstrated that {Delta}rFSHRS9-S18 is produced at the levels comparable to those of wild-type rFSHR but is not detectable at the cell membrane. However, these results should not be construed as quantitative since this staining does not discriminate between intact and degraded protein. The larger deletion, {Delta}RFSHRS9-N30, seemed to be produced at a lower level than {Delta}RFSHRS9-S18, and also was not detectable at the cell surface. It is worth noting that under the conditions of the RRA, with shaking at room temperature for 16 h, intracellular receptor is likely to be accessible to radiolabeled hormone, just as it is accessible to antibody. It therefore seems reasonable to conclude that these deletion mutants of rFSHR cannot bind hFSH.



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  		FIG. 6. Cells expressing {Delta}S9-N30, {Delta}S9-S18, and wild-type rFSHR were examined by flow cytometry using rabbit antipeptide antisera X179 and NRS as a control. Antibody binding to the rFSHR is expressed as the mean peak fluorescence. Data are representative of three independent experiments, with 5000 cells analyzed per sample. A) Staining of rFSHR expressed on insect cells still attached to plates during the staining procedure. B) Antigen staining when cells were sheared by detachment and shaking during incubation to allow the antibody access to antigen trapped inside the cell


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Only the large ECD of rFSHR is necessary for high-affinity FSH binding because truncated forms of rFSHR bind their cognate ligand with high affinity [10–13]. Likewise, the large ECD of the gonadotropin receptors can be exchanged, conferring hormone-specific signaling upon homologous transmembrane domains [34]. Shorter fragments of the ECD also have hormone binding properties. The first four exons of ovine rFSHR (encoding 117 amino acids), when expressed in Escherichia coli, bind the glycoprotein hormones [35]. Similarly, the first 125 amino acids of the rFSHR expressed in E. coli were capable of inhibiting binding of radiolabeled FSH and LH to their receptors, but only recombinant rFSHR peptide 201–319 inhibited FSH binding in a specific manner [36].

Additional information about specific linear sequences of gonadotropin receptors that inhibit hormone binding have been identified by peptide challenge tests. In the case of LH/CG-R, ECD sequences have been identified that can inhibit hormone binding to receptor, three of which are located in the N-terminal half of the receptor (amino acids 21–38, 102–115, 253–272) [37]. In the case of rFSHR, a synthetic peptide corresponding to rFSHR residues S9-N30 inhibited binding of 125I-hFSH to its receptor in a concentration-dependent manner, albeit at millimolar levels [16, 17, 30]. Similarly, 125I-hFSH could bind to immobilized rFSHRS9-N30 peptide, and this interaction was specifically inhibited by FSH. The present data support the idea that determinants in rFSHR 9–18 contain hormone binding regions. The {Delta}rFSHRS9-S18 mutant did not bind hFSH, but in accord with studies with the LH receptor, the present work indicates that this region is extremely sensitive to amino acid substitution, often leading to trapping or binding defects [38].

A synthetic peptide spanning another unique region of the rFSHR, D300-F315, bound to immobilized FSH and also to thyroid-stimulating hormone (TSH) and LH, suggesting a nonspecific interaction [17]. However, immunoneutralization studies with antisera generated against hFSHR E299-D326 demonstrated that hFSH action could be blocked by this antisera [19]. We have previously shown that antibodies raised against hFSHR residues 265–296 inhibit FSH binding to receptor [20]. Moreover, deletion of regions 299–301 and 387–395 of TSH receptor (TSHR) rendered the TSHR unable to bind TSH. In fact, those data suggested that these two distal sites (9–30, 300–315) were involved in hFSH binding and thus formed the basis for the hypothesis tested in this study [39]. The present results demonstrate that D300-F315 is not involved in hormone binding or in hormone binding specificity.

Although in the present work hFSH bound to {Delta}rFSHRK19–29 and to {Delta}rFSHRD300-F315, previous studies demonstrated that antiserum against either of the synthetic peptides rFSHRK19-R29, rFSHRS9-N30, or hFSHR 299–326 inhibited binding of FSH to membrane-bound receptors in a concentration-dependent manner [16,18] or blocked FSH action [19]. Those results are not discordant with the present findings; rather, it is possible that the antiserum generated against these FSHR regions contains antibodies that, when they bind to the receptor, offer considerable steric hindrance to the binding site.

Sensitivity to mutational analysis has hampered determination of the activation site and the mechanism of the LHR activation of signal transduction because the receptors are often trapped in cells, precluding analysis of signal transduction capabilities [38, 40]. In addition, in cases in which decreased signal transduction has been measured, it is usually commensurate with a decrease in receptor number or affinity. Information from studies with other gonadotropin receptors helps in the interpretation of the present findings, yet differences between the gonadotropin receptors are expected, since gain of function mutations in LHR frequently do not translate to similar phenotypes when expressed in rFSHR [13]. In the present study, measurements of FSH-induced cAMP production demonstrated a role for residues within region rFSHRK19-R29 in signal transduction. It is unlikely that the failure to signal maximally is due to perturbation of receptor conformation, since FSH binding was normal. In this regard, the precedent in the literature is that loss of signal transduction is commensurate with loss of binding. An exception is the case of transmembrane domain mutations, in which gain or loss of function can occur in the absence of a change in binding characteristics. Indeed, trafficking defects appear even more sensitive than binding defects [38, 40], and there was no trafficking defect observed with this mutant. Thus, when exposed to different amounts of hFSH, cells expressing the wild-type rFSHR or {Delta}rFSHR D300-F315 consistently produced a 2- to 3-fold higher level of total cAMP than cells displaying {Delta}rFSHRK19-R29. This loss of function by the rFSHR{Delta}K19-R29 mutant is on the order of magnitude of rFSHR loss of function mutations in humans [1, 41]. In contrast, deletion of rFSHR regions S9-S18 and S9-N30 renders cells expressing the {Delta}rFSHR undetectable at the cell surface. In comparison, deletion of LHR amino acids 12–33 also resulted in intracellular trapping and loss of binding activity [42]. Similarly, mutants {Delta}rFSHRS9-N30 and {Delta}rFSHRS9-S18, which should be accessible to radiolabeled hFSH during the course of the assay, still did not exhibit any affinity for FSH. Although it is reasonable to assume that this mutant rFSHR does not bind FSH because a binding determinant has been removed, it is equally plausible that a local conformational change precluded binding. By example, rLHR does not localize to the cell surface if alanine is substituted for rLHR amino acids D17 or G18 and does not bind hCG if alanine is substituted for G24, L20, and C22 [38]. Thus, deletion of either S9-N30 or S9-S18 causes the loss of cysteine 15, which is part of a cysteine cluster of the rFSHR. Cysteine clusters like this are conserved in the TSHR, LHR, and proteins as different as CD 14, fibromodulin, and biglycan [43], which may point to a generalized role in protein processing. The importance of cysteines in protein folding is well established, and a number of reports underscore their role in hormone binding. Substitution of C22 in rLHR [38] and replacement of all four cysteines from exon 1 in rLHR [44] results in a loss of hCG binding. Despite these considerations, because the leucine-rich repeat (LRR) domains of the G-protein-coupled receptors are successive beta-sheet-alpha helix building blocks, the ends of the rFSHR are likely to have considerable elasticity as in the ribonuclease inhibitor [45] as modeled for the rFSHR [46]. In this light, a local rather than global effect of deletions seems reasonable to posit, again suggesting that this domain is a hormone binding domain.

Primary sequence D300-F315 is part of LRR 13 of the rFSHR and lies outside the region necessary for receptor function (LRR 1–11) [34]. Even though D300-F315 is located close to a number of conserved cysteines (C258, 259, 275, 320, and 328), changing the distance between the first three and the last two cysteines does not seem to impair receptor function. Unique sites in the hormone receptors have been presumed to be involved in hormone selectivity, and it has been demonstrated that residues encoded by exons 2–4 and 7–9 of the LHR and exons 5–6 of the rFSHR confer binding specificity [14]. As region K19-R29 lies in exon 1 and D300-F315 in exon 10 [9], our results support the notion that unique regions of the rFSHR are not necessarily involved in hormone selectivity [14]. A cDNA encoding the first eight exons of the sheep FSH receptor along with a carboxy-terminal extension that contributed a hypothetical single transmembrane domain has been cloned. This cDNA, which represented the first eight exons of the FSHR gene and which lacked the conventional seven-transmembrane motif of the full-length 695-residue wild-type receptor protein, was also efficiently expressed on the cell surface and exhibited high affinity and specificity for FSH binding [47]. These data further support the present findings.

In summary, the present results indicate a previously unreported role in signal transduction for rFSHR region K19-R29 and an involvement in hormone binding or proper folding for region S9-S18. The possibility also exists, however, that the K19-R29 domain is involved not in signaling but in reorientation of the FSHR complex. Additional low-affinity FSH binding has also been localized to amino acids 140–283 [34] and 265–296 [20], thus underscoring the possibility of a discontinuous binding region comprising residues from different parts of the receptor.

Current three-dimensional models of gonadotropin-gonadotropin receptor suggest that the hormone binding site is centrally located at the beta-sheet face [46]. Localization of critical residues on the gonadotropins to a discrete and common site support this consideration [7]. However, signal transduction defects in hormone mutations can usually be overcome by increasing the mass of hormone to accommodate decreases in binding affinity caused by the mutation. This phenomenon suggests that it is the receptor that contains critical signal transduction residues. On the basis of the present results, and literature cited within, a key question for future studies is how the ECD is oriented towards the transmembrane domains, and whether discrete domains in the ECD such as the exon-one encoded domain make key contacts with the transmembrane domains, thereby effecting signal transduction. One might envision several mechanisms underlying such an activating effect, with stabilization of the active state of the receptor the most likely.


   ACKNOWLEDGMENTS
 
Oligonucleotides were prepared by the Wadsworth Center Molecular Genetics core facility. Flow cytometry analysis was performed by Mr. Renjie Song in the Wadsworth Center Molecular Immunology core facility. Purified hCG was generously provided by Dr. Steven Birken of Columbia University, New York. Dr. Cheryl Nechamen and Mr. Howard Brumberg assisted in collecting some data.


   FOOTNOTES
 
First decision: 4 January 1999.

1 Supported by NIH HD-18407. This study was conducted in partial fulfillment of the requirements for a master's degree at the University at Albany, Albany, NY (K.I.M.). Back

2 Correspondence: James A. Dias, Wadsworth Center, David Axelrod Institute for Public Health, New York State Department of Health, 120 New Scotland Avenue, Albany, NY 12208. FAX: 518 474 5978; james.dias{at}wadsworth.org Back

Accepted: December 13, 1999.

Received: November 30, 1998.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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