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Originally published In Press as doi:10.1074/jbc.M307751200 on September 8, 2003

J. Biol. Chem., Vol. 278, Issue 48, 47868-47876, November 28, 2003
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Orientation of Follicle-stimulating Hormone (FSH) Subunits Complexed with the FSH Receptor

{beta} SUBUNIT TOWARD THE N TERMINUS OF EXODOMAIN AND {alpha} SUBUNIT TO EXOLOOP 3*

Johann Sohn, HyeSook Youn, MyoungKun Jeoung, YongBum Koo{ddagger}, ChongSeoung Yi, Inhae Ji, and Tae H. Ji§

From the Departments of Chemistry and Biology, University of Kentucky, Lexington, Kentucky 40506-0055

Received for publication, July 17, 2003 , and in revised form, September 3, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Follicle-stimulating hormone (FSH) comprises an {alpha} subunit and a {beta} subunit, whereas the FSH receptor consists of two halves with distinct functions: the N-terminal extracellular exodomain and C-terminal membrane-associated endodomain. FSH initially binds to exodomain, and the resulting FSH/exodomain complex modulates the endodomain and generates signal. However, it has been difficult to determine which subunit of FSH contacts the exodomain or endodomain and in what orientation FSH interacts with them. To address these crucial issues, the receptor was Ala-scanned and the hormone subunits were probed with photoaffinity labeling with receptor peptides corresponding to the N-terminal region of the exodomain and exoloop 3 of the endodomain. Our results show that both regions of the receptors are important for hormone binding and signal generation. In addition, the FSH {beta} subunit is specifically labeled with the N-terminal peptide, whereas the {alpha} subunit is labeled with the exoloop 3 peptide. These contrasting results show that the FSH {beta} subunit is close to the N-terminal region and that the {alpha} subunit is projected toward exoloop 3 in the endodomain. The results raise the fundamental question whether the {alpha} subunit, common among the glycoprotein hormones, plays a major role in generating the hormone signal common to all glycoprotein hormones.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Follicle-stimulating hormone (FSH)1 consists of an {alpha} subunit of 92 amino acids and {beta} subunit of 111 amino acids. Both of the subunits are necessary for hormone action (1, 2). The crystal structure of human FSH (3) shows that the two subunits are tightly associated in a crescent with the C termini in the concave side and the N termini in the convex side (Fig. 1A). This is essentially the same as the human chorionic gonadotropin structure (4, 5). An exception to this intermingled subunit structure is the two polarized tips of the crescent: the {alpha} tip consisting of the {alpha} loops 1 and 3 and the {beta} tip of the {beta} loops 1 and 3.



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  		FIG. 1.
Structure of FSH and receptor. A, crystal structure of FSH, the {alpha} subunit in red and {beta} subunit in green. B, a schematic drawing of FSHR with two distinct domains, exodomain and endodomain. C, a schematic drawing of FSH interacting horizontally with LRRs of the exodomain. Note that the orientation of the {alpha} and {beta} subunits could be switched if the hormone is horizontally flipped. D, a schematic drawing of FSH interacting vertically with LRRs of the exodomain. Note that the orientation of the {alpha} and {beta} subunits could be switched if the hormone is vertically flipped. E, a hypothetical interaction of FSH {alpha} and {beta} with the LRRs of the exodomain and the exoloops of the endodomain. The FSH{beta} subunit is close to the N-terminal region of the exodomain, whereas the FSH{alpha} tip is projected toward the exoloops of the endodomain. The tilted orientation of FSH is based on this study.

 
In contrast to the tightly held hormone structure, the FSH receptor (FSHR), a G protein-coupled receptor, has two distinct domains as shown in Fig. 1B. The extracellular N-terminal exodomain comprises ~350 amino acids, and the membrane-associated C-terminal endodomain with a similar number of amino acids consists of seven transmembrane helices, three exoloops, three cytoloops, and the C-terminal cytoplasmic tail (6–8). The exodomain binds the hormone with high affinity (9–16) and selectivity (17), whereas the hormone signal is generated in the endodomain (18–22). FSH initially interacts with the exodomain, and the resulting FSH/exodomain complex modulates the endodomain to generate hormone signal. Important amino acids have been identified for the interaction of the hormone and receptor (23, 24). However, the orientation of FSH {alpha} and {beta} in the ternary complex of the hormone, exodomain, and endodomain has been a major enigma and difficult to determine.

The bulk of the exodomain comprises 8–9 Leu-rich repeats (LRR) (7, 25–28), which are flanked by the short upstream N-terminal region and the downstream hinge region. LRRs are thought to form a one-third of a doughnut structure (26–28). FSH appears to interact with LRRs (23, 29) and the N-terminal and hinge regions (19, 30). However, it is unclear which subunit of FSH contacts the exodomain or endodomain and in what orientation FSH interacts with them, although the concave C-terminal side of FSH appears to interact with the receptor. For example, FSH may interact horizontally or vertically with LRRs (Fig. 1, C and D) and in two directions. These are crucial pieces of information for understanding the interactions among the hormone, exodomain, and endodomain and the mechanistics of signal generation. In addition, the information will facilitate the design of agonists and antagonists and development of new therapeutics. Because of the importance, the interactions of glycoprotein hormones with their receptors have been modeled (3–5, 23, 28) based on the crystal structure of the LRRs of ribonuclease inhibitor complexed with its ligand (31). However, the evidence has been elusive.

In a step to resolve this issue, we set out to distinguish the interactions of the FSH subunits with the N-terminal region of the exodomain and exoloop 3 in the endodomain. Our results show the interaction of FSH{beta} with the N-terminal region of the exodomain and the {alpha} tip of FSH{alpha} with exoloop 3 (Fig. 1E).


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Human FSH was purchased from the National Hormone and Pituitary Program. Denatured FSH was prepared by boiling the hormone in 8 M urea for 30 min. Rabbit anti-FSH{alpha} serum, rabbit anti-FSH{beta} serum, and monoclonal anti-FSHR 106.105 antibody were kindly provided by Dr. James Dias. Anti-rabbit IgG conjugated with peroxidase was purchased from Pierce. Peptide mimics including wild type peptides corresponding to the Ser9-Lys40 sequence (FSHR9–40) and exoloop 3 and a photoactivable peptide containing benzoyl phenylalanine (Bpa) in place of Phe13 (FSHR9–40F13Bpa) were synthesized by Genemed Synthesis (San Francisco, CA) and purified on a Vydac C18 high pressure liquid chromatography column using solvent gradient from 100% of 0.1% trifluoroacetic acid in water to 20% of 0.1% trifluoroacetic acid in water and 80% 1-propanol.

Mutagenesis and Functional Expression of FSH Receptors—Mutant FSHR cDNAs were prepared in the pSELECT vector using the Altered Sites mutagenesis system (Promega), sequenced on a Beckman CEQ 2000XL capillary sequencer, subcloned into pcDNA3 (Invitrogen) as described previously (32), and sequenced again to verify mutation sequences. This procedure does not involve polymerase chain reaction and therefore does not have its infidelity problems. Wild type and mutant receptor constructs were transfected into HEK 293 cells by the calcium phosphate method as described previously (32). Stable cell lines were established in minimum essential medium containing 10% horse serum and 500 µg/ml G418. These cells were used for hormone-binding cAMP production. All of the assays were carried out in duplicate and repeated 4–5 times, and the means ± S.D. were calculated.

125I-FSH Binding and Intracellular cAMP Assay—Stable cells were assayed for 125I-FSH binding in the presence of 100,000 cpm of 125I-FSH (33) and increasing concentrations of unlabeled FSH. The Kd values were determined by Scatchard plots. For intracellular cAMP assay, cells were washed twice with Dulbecco's modified Eagle's medium and incubated in the medium containing 0.1 µg/ml isobutylmethylxanthine for 15 min. Increasing concentrations of FSH were then added, and incubation was continued for 45 min at 37 °C. After removing the medium, the cells were rinsed once with fresh medium without isobutylmethylxanthine, lysed in 70% ethanol, freeze-thawed in liquid nitrogen, and scraped. After pelleting cell debris at 16,000 x g for 10 min at 4 °C, the supernatant was collected, dried under vacuum, and resuspended in 10 µl of cAMP assay buffer (Amersham Biosciences). cAMP concentrations were determined with an 125I-cAMP assay kit (Amersham Biosciences) following the manufacturer's instructions and validated for use in our laboratory.

125I-FSH Binding to Solubilized FSHR—Transfected cells were washed twice with ice-cold 150 mM NaCl, 20 mM HEPES, pH 7.4 (buffer A). Cells were scraped on ice, collected in buffer A containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 5 mM N-ethylmaleimide, and 10 mM EDTA), and pelleted by centrifugation at 1300 x g for 10 min. Cells were resuspended in 0.6 ml of buffer A containing 1% Nonidet P-40, 20% glycerol, and the above protease inhibitors (buffer B), incubated on ice for 15 min, and diluted with 5.4 ml of buffer A containing 20% glycerol plus the protease inhibitors (buffer C). The mixture was centrifuged at 100,000 x g for 60 min. The supernatant (500 µl) was mixed with 100,000 cpm of 125I-FSH and 6.5 µl of 0.9% NaCl and 10 mM Na2HPO4 at pH 7.4 containing increasing concentrations of unlabeled FSH. After incubation for 12 h at 4 °C, the solution was thoroughly mixed with 250 µl of buffer A containing bovine {gamma}-globulin (5 µg/ml) and 750 µl of buffer A containing 20% polyethylene glycol 8000. After incubation for 10 min at 4 °C, samples were pelleted at 1,300 x g for 30 min and supernatants removed. Pellets were resuspended in 1.5 ml of buffer A containing 20% polyethylene glycol 8000, centrifuged, and counted for radioactivity. Monoclonal anti-FSHR antibodies were radioiodinated and used for binding to nonbinding mutant FSHRs expressed on the intact cell surface as described previously (34).

Derivatization and Radioiodination of Peptides—In the dark, 30 µg of receptor peptides in 40 µl of 0.1 M sodium phosphate, pH 7.5, was mixed with 1 mCi of Na-[125I]iodine in 10 µl of 0.1 M NaOH and 7 µl of chloramine-T (1 mg/ml) in 10 mM Na2HPO4, pH 7.4. After 20 s, 7 µl of sodium metabisulfite (2.5 mg/ml) in 10 mM Na2HPO4, pH 7.4, was introduced to terminate radioiodination. Radioiodinated peptides were mixed with 60 µl of 16% sucrose solution in PBS and fractionated on Sephadex Superfine G-10 column (0.6 x 15 cm) using PBS. Peptides were derivatized with 4-azidobenzoyl glycine (ABG) and radioiodinated as described previously (35).

Photoaffinity Labeling of FSH—The following solutions were sequentially introduced to siliconized glass tubes: 20 µl of 0.9% NaCl and 10 mM Na2HPO4, pH 7.4, in PBS, 10 µl of FSH in PBS, and 10 µl of 125I-FSHR9–40F13Bpa in PBS. Competitive inhibition experiments were carried out as described for the photoaffinity-labeling experiments with the exception that 10 µl instead of 20 µl of PBS was introduced to each tube and the mixture was incubated with 10 µl of increasing concentrations of nonradioactive receptor peptides. The mixtures were incubated at 37 °C for 90 min in the dark, irradiated with a Mineralight R-52 UV lamp for 3 min as described previously (36), and solubilized in 2% SDS, 100 mM dithiothreitol, and 8 M urea. The samples were electrophoresed on 8–12% polyacrylamide gradient gels. Gels were dried on filter paper and exposed to an imaging screen overnight, which was scanned on a PhosphorImager (Molecular Dynamics).

Deglycosylation—The FSH {alpha} and {beta} subunits co-migrate on SDS-PAGE. To separate them on the gel, FSH was photoaffinity-labeled and deglycosylated with PNGase F. Enzymatic cleavage was done by incubation of the labeled FSH complex with 20 or 50 units of PNGase F (New England BioLabs) in 40 µl for 18 h at 37 °C. The samples were solubilized in SDS under the reducing condition and electrophoresed on 15% gel containing 9 M urea.

Immunoblot of FSH Subunits—Separated proteins were blotted onto 0.2-µm nitrocellulose membrane as described previously (37). Membranes were treated for 1 h with 5% blocking buffer (25 mM Tris-HCl, 1.4 M NaCl, 5% nonfat dry milk, 0.2% sodium azide, 1% Nonidet P-40, pH 7.4) and incubated with polyclonal anti-FSH and anti-FSH {alpha} and {beta} antibodies (dilution 1:2000 and 1:3500 each in blocking buffer) for 1 h at room temperature. Membranes were washed three times (5 min each) with the blocking buffer and incubated with anti-rabbit peroxidase-conjugated IgG (dilution 1:5000 in the blocking buffer) for 1 h at room temperature. Membranes were washed three times (5 min each) with the blocking buffer and twice (5 min each) with 25 mM Tris-HCl, pH 7.4. Membranes were incubated in staining solution (0.05% 3,3'-diaminobenzidine, 0.02% CoCl2, 0.03% H2O2) until bands became visible.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Activities of the N-terminal Region—In a first step to check the importance of the N-terminal region of human FSHR, each amino acid of the 8SNRVFLCQESKVTEIPSDLPRNAIE33 sequence was individually substituted with Ala. This sequence is diverse among the glycoprotein hormone receptors (Fig. 2), although these receptors share a high overall homology and structural similarity (38). In contrast, the FSHR sequence is highly conserved among species, implicating its importance. HEK 293 cells were stably transfected with mutant receptor plasmids and selected for stably expressing individual mutant receptors. These cells were assayed for 125I-FSH binding and FSH-dependent cAMP induction. The results show that some of the Ala substitutions significantly impacted the hormone binding and/or cAMP induction.



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  		FIG. 2.
Comparison of the primary sequence of the first 34 residues of the glycoprotein hormone receptors. The FSH receptor sequences of various species were compared with the corresponding sequences of the human LH receptor and TSH receptor. Cys15, Ser18, Pro24, Asp26, and Leu27 of FSHR are conserved among the species.

 
Ala substitutions for Ser9, Asn10, Arg11, Val12, Phe13, and Leu14 improved FSH binding (Fig. 3, A and B), FSH-dependent cAMP induction (Fig. 3C), or both. The Kd values of FSHRN10A, FSHRR11A, and FSHRL14A were lower than the wild type value as were the EC50 values of FSHRS9A, FSHRN10A, FSHRV12A, FSHRF13A, and FSHRL14A (see tables in Fig. 3). Ala substitutions for Gln16, Glu17, Lys19, and Val20 did not impact the EC50 values and maximal cAMP induction (Fig. 3F), and Kd values of the mutants were similar to or somewhat higher than the wild type value (Fig. 3, D and E). In contrast, the S18A substitution resulted in a considerably lower EC50 value despite a higher Kd value. These results show an improved cAMP induction despite a lower hormone binding affinity and suggest an interesting and potentially crucial role of Ser18 in modulating signal generation.



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  		FIG. 3.
Ala substitutions for Ser9-Val20. A–F, residues from Ser9 to Val20 of the FSH receptor were individually substituted with Ala, and the resulting mutant receptors were stably expressed in HEK 293 cells. Intact cells were used for 125I-FSH binding in the presence of increasing concentrations of unlabeled FSH (A and D) and for cAMP production (C and F). The competitive inhibition data (A and D) were converted to Scatchard plot (B and E). Experiments were repeated 4–6 times in duplicate, and the means ± S.D. were calculated. NS, not significant.

 
Ala substitution for Thr21, Glu22, or Ser25 did not significantly impact hormone binding or cAMP induction (Fig. 4). On the other hand, the I23A substitution partially impaired the cAMP induction with a 23-fold higher EC50 value and 2.6-fold lower maximal cAMP level. The P24A, D26A, and L27A substitutions completely abrogated hormone binding and therefore cAMP induction, suggesting the importance of these residues and this region. The P28A, R29A, N30A, A31G, I32A, and E33A substitutions did not dramatically impact the Kd and EC50 values or the maximal cAMP production (Fig. 4). The nonbinding mutants were either incapable of binding the hormone or trapped in cells, and these possibilities have successfully been tested by assaying hormone binding after solubilization of cells in nonionic detergents (34, 39, 40). The binding assay for receptors solubilized in Nonidet P-40 showed that FSH did not bind to any of the C15A, P24A, D26A, and L27A mutants (Fig. 5A), indicating that they are incapable of hormone binding. However, the result does not necessarily prove the surface expression of the nonbinding mutants and nonbinding on the surface. Therefore, the cells expressing them were probed with 125I-anti-FSHR antibody. The antibody bound to the cells (Fig. 5B), indicating the surface expression of the mutants and their inability to bind the hormone on the cell surface. These results, taken together, show several distinct effects of Ala substitutions as shown in the summary bar graph (Fig. 6). C15A, P24A, D26A, and L27A abolished hormone binding. In contrast to these nonbinding mutations, N10A, R11A, and L14A improved hormone binding. On the other hand, I23A impaired cAMP induction by dramatically increasing the EC50 value. Remarkably, S9A, V12A, F13A, S18A, and I32A reduced the EC50 value by 2–3-fold while maintaining or slightly enhancing the maximum cAMP induction level. These results suggested the importance of this region of the receptor in hormone binding and cAMP induction and raised a question as to whether this region directly interacts with the hormone or indirectly impacts the global structure of the receptor.



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  		FIG. 4.
Ala substitutions for Thr21-Glu33. A–F, residues from Thr21 to Glu33 of the FSH receptor were individually substituted with Ala, and the resulting mutant receptors were expressed in HEK 293 cells and assayed as described in the legend to Fig. 3.

 



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  		FIG. 5.
Hormone binding in solution and anti-FSHR antibody binding to intact cells. A, cells individually transfected with the C15A, P24A, D26A, or L27A mutant receptor were solubilized in Nonidet P-40 and assayed for 125I-FSH binding as described under "Experimental Procedures." B, intact cells were also probed with 125I-labeled monoclonal anti-FSHR 106.105 antibody for the surface expression of the nonbinding mutants.

 



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  		FIG. 6.
Comparison of Ala substitution mutations. To easily compare the activities of the wild type and mutant receptors, the ratios of Kd wild type mutant, EC50 wild type mutant, and maximum cAMP mutant wild type were presented in a bar graph.

 
Photoaffinity Labeling of FSH—To examine the two general possibilities, a peptide mimic corresponding to the receptor sequence of 8SNRVFLCQESKVTEIPSDLPRNAIELRFVLTK40 was synthesized, FSHR9–40 (Fig. 7A). A Tyr residue was attached to the N terminus for radioiodination, and the N terminus was acetylated while the C terminus amidated. Phe13 was substituted with Bpa for photoaffinity labeling (41). The ketone moiety of the Bpa group can be activated with UV at >350 nm and is capable of reacting with unreactive {alpha}-CH bonds of amino acids (41, 42, 44). To determine whether the resulting peptide 125I-FSHR9–40F13Bpa could bind and photoaffinity-label FSH, it was incubated with FSH and irradiated with UV for increasing time periods. Samples were solubilized in SDS under reducing conditions and then electrophoresed. The autoradiographic phosphorimage of the gel (Fig. 7B) revealed labeling of the FSH band. The autoradiograph suggests that the two subunits of human FSH comigrated. The band was not labeled when the sample was not irradiated with UV, indicating the requirement for UV irradiation. The extent of the labeling was dependent on the irradiation time, reaching maximum labeling after 30-s irradiation. The result shows that the labeling is saturable. The hormone was labeled next with increasing concentrations of 125I-FSHR9–40F13Bpa while maintaining FSH at a constant concentration (Fig. 7C). Conversely, increasing concentrations of FSH were labeled with a constant concentration of 125I-FSHR9–40F13Bpa (Fig. 7D). If the labeling was specific, the concentrations should reach a plateau under both conditions. Indeed, the labeling plateaued under both conditions, indicating saturable and specific labeling of a substantial portion (>50%) of FSH. Furthermore, the labeling should be inhibited by nonradioactive peptide and unmodified wild type peptide. Therefore, FSH was incubated with 125I-FSHR9–40F13Bpa in the presence of increasing concentrations of wild type peptide and nonradioactive FSHR9–40F13Bpa (Fig. 7E). The peptides inhibited the photoaffinity labeling in a dose-dependent manner and eventually blocked the labeling. Peptides corresponding to exoloops 1–3, FSHRexo1, FSHRexo2, and FSHRexo3, were also tested. FSHRexo2 inhibited the labeling as FSHR9–40 did. In contrast, FSHRexo1 inhibited the labeling with a ~30-fold less potency. On the other hand, FSHRexo3 failed to block the labeling.



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  		FIG. 7.
Photoaffinity labeling of FSH with photoactivable FSHR9–40F13Bpa. The FSH receptor peptide corresponding to the sequence Ser9-Lys40 (FSHR9–40) was synthesized with a Tyr at the N terminus for radioiodination and Bpa at the position of Phe13 for photoaffinity labeling (A). The peptide was radioiodinated, and the resulting 125I-FSHR9–40F13Bpa was incubated with FSH and irradiated with UV. B, the sample was irradiated with UV for increasing time periods from 0 to 150 s, solubilized in SDS under the reducing condition, and electrophoresed on polyacrylamide gel. After drying gels, they were exposed to a phosphorimaging screen and scanned on a PhosphorImager. The peptide appeared as the lower band, and the FSH {alpha} and FSH {beta} subunits comigrated and appeared in the upper band. The intensity of each band in a gel lane was measured, and the percentage of the labeled FSH band in a gel lane was calculated based on the total intensity of a gel lane and presented in the bar graph above the autoradiograph. C, increasing amounts of 125I-FSHR9–40F13Bpa from 0 to 3.7 µM were incubated with a constant amount (0.1 µM) of FSH and photolyzed for 60 s. The samples were processed as described above. D, increasing amounts of FSH from 0 to 0.2 µM were incubated with a constant amount (3.1 µM) of 125I-FSHR9–40F13Bpa. E, FSH (80 nM) was incubated with 3 µM 125I-FSHR9–40F13Bpa in the presence of increasing concentrations of unlabeled FSHR peptides, FSHR9–40, FSHR9–40F13Bpa, exoloop 1 peptide (FSHRexo1), exoloop 2 peptide (FSHRexo2), and exoloop 3 peptide (FSHRexo3). The samples were irradiated and processed as described in B.

 
Labeling Specificity—Although the photoaffinity labeling was specific for FSH, our data do not show the biological specificity of the affinity labeling. To address this concern, a constant amount of denatured FSH was incubated with increasing concentrations of 125I-FSHR9–40F13Bpa and treated with UV. Denatured FSH was not labeled at all despite high concentrations of the peptide (Fig. 8A). Denatured FSH was not labeled when increasing concentrations of denatured FSH were incubated with a constant amount of 125I-FSHR9–40F13Bpa and treated with UV (Fig. 8B). When FSH was denatured by boiling in 8 M urea for 30 min, it did not bind to FSHR and induce cAMP production. To test whether the denatured FSH remained in solution, the mixture of radioactively labeled FSH and unlabeled FSH was denatured and varying volumes of the mixture were transferred to other tubes and the radioactivity was counted. The transfer was quantitative with 99–100% efficiency, indicating that denatured FSH was present in the photoaffinity-labeling tube. These results indicate the specificity of the affinity labeling for biologically active FSH. To determine the labeling specificity, luteinizing hormone (LH), thyroid-stimulating hormone (TSH), growth hormone, phospholipase A, and urokinase were subjected to photoaffinity labeling with 125I-FSHR9–40F13Bpa (Fig. 8C). None of them was labeled. If 125I-FSHR9–40Bpa13 specifically binds to FSH and labels it as indicated by the results, the peptide is expected to inhibit the in vivo binding of FSH to the receptor on intact cells. Indeed, FSHR9–40Bpa13 and FSHR9–40 inhibited 125I-FSH binding to the receptor (Fig. 8D).



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  		FIG. 8.
Photoaffinity labeling of denatured FSH and other glycoproteins. A, increasing concentrations of 125I-FSHR9–40F13Bpa were incubated with 80 nM denatured FSH, irradiated with UV, and processed as described in the legend to Fig. 7. FSH was denatured by boiling in 8 M urea for 30 min. B, a constant amount of 125I-FSHR9–40F13Bpa was incubated with increasing concentrations of denatured FSH, treated with UV, and processed. C, a constant amount of 125I-FSHR9–40F13Bpa was incubated with 5 nM each of FSH, phospholipase A (PLA), urokinase, growth hormone (GH), LH, or TSH, treated with UV, and processed as described in the legend to Fig. 7. D, cells stably expressing FSHR were incubated with 125I-FSH and washed as described in Fig. 3 but in the presence of increasing concentrations of FSHR9–40 or FSHR9–40F13Bpa. Cells were washed three times and counted for the bound radioactivity.

 
Labeling of FSH {beta} Subunit—Because the two subunits of purified human FSH appeared to comigrate on SDS-PAGE, it was unclear which of the subunits was labeled. To resolve the subunits, FSH was deglycosylated with PNGase F and electrophoresed (Fig. 9A, lane 2). The two subunits were clearly separated into two distinct bands. Because the {beta} subunit is larger than the {alpha} subunit, the upper band is probably the {beta} subunit. To conclusively determine the identity of the upper band, deglycosylated FSH was electrophoresed, the gel was blotted on nitrocellulose membrane, and the membrane was probed with anti-FSH{alpha} and anti-FSH{beta} antibodies. Anti-FSH{alpha} antibody conspicuously labeled the lower band, whereas the anti-FSH{beta} antibody recognized primarily the upper band and faintly the lower band (Fig. 9, A and B). These results show that the lower band represents the FSH{alpha} subunit, whereas the upper band is the FSH{beta} subunit, indicating that FSH{beta} was labeled. To compare this labeling of FSH{beta} with the N-terminal peptide, the exoloop 3 peptide, FSHRexo3, was used for labeling FSH. The peptide was derivatized with a UV-activable reagent, ABG, and radioiodinated. The resulting 125I-ABG-FSHRexo3 was incubated with FSH, irradiated with UV, deglycosylated, and electrophoresed. The labeled FSH subunit appeared in the lower band, indicating the labeling of the FSH{alpha} subunit (Fig. 9C) as reported previously (35).



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  		FIG. 9.
Immunoblot of FSH {alpha} and {beta} subunit bands. A, FSH was treated with PNGase F, solubilized in SDS under the reducing condition, and electrophoresed along with nondeglycosylated FSH. Gel lanes were either stained with Coomassie Brilliant Blue (CBB) or blotted and stained with anti-FSH, anti-FSH{alpha}, or anti-FSH{beta} antibodies. B, FSH was incubated with increasing concentrations of 125I-FSHR9–40F13Bpa, treated with UV, deglycosylated with PNGase F, solubilized, and electrophoresed along with 125I-FSH. C, the peptide corresponding to exoloop 3 (FSHRexo3) was derivatized with a UV-activable reagent, ABG, and radioiodinated. The resulting 125I-ABG-FSHRexo3 was incubated with FSH, treated with UV, deglycosylated with PNGase F, solubilized, and electrophoresed (E3). E3 was compared with FSH labeled with 125I-FSHR9–40F13Bpa (N).

 
The labeling of FSH{beta} by 125I-FSHR9–40F13Bpa contrasts the labeling of the FSH{alpha} subunit by the FSHR exoloop 3 peptide. If this contrasting labeling is specific and reflects the true interaction between FSH and the receptor, some of the peptides representing parts of the {alpha} subunit sequence might inhibit the labeling. Particularly, if some of the {alpha} peptides block the labeling of the {alpha} subunit but not the {beta} subunit, the result would support the selective labeling results and show labeling specificity. Four {alpha} peptides, {alpha}1–15, {alpha}26–46, {alpha}61–75, and {alpha}81–92 were tested, and none of them inhibited the labeling of the FSH{beta} subunit by 125I-FSHR9–40F13Bpa (Fig. 10A). In contrast, the labeling of the FSH{alpha} subunit by 125I-ABG-FSHRexo3 was blocked by {alpha}26–46 and somewhat by {alpha}61–75 (Fig. 10B). {alpha}1–15 and {alpha}81–92 Peptides failed to block the labeling. These results support the differential labeling and its selectivity and validity.



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  		FIG. 10.
Inhibition of photoaffinity labeling of FSH. Peptides corresponding to the {alpha} subunit sequences, 1–15, 26–46, 61–75 and 81–92, were synthesized. FSH was incubated with either 125I-FSHR9–40F13Bpa or 125I-ABG-FSHRexo3 in the presence of an excess amount of {alpha}1–15, {alpha}26–46, {alpha}61–75, or {alpha}81–92. The samples were irradiated with UV and processed, and the percentage of the labeled FSH was calculated above the autoradiographs as described in Fig. 7.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Our Ala-scanning results indicate that the Ser9-Glu33 sequence of the FSH receptor is important for surface expression, hormone binding, and signal generation. The photoaffinity-labeling results show that FSHR9–40F13Bpa photoaffinity labels FSH but not LH, TSH, growth hormone, phospholipase A, and urokinase. The labeling is saturable and dependent on the concentrations of FSH and derivatized 125I-FSHR9–40, UV activation, and UV exposure time. 125I-FSHR9–40F13Bpa photoaffinity labels bioactive FSH but not denatured hormone, and the labeling is blocked by nonderivatized wild type peptide and nonradioactive FSHR9–40F13Bpa. The labeling specificity is further underscored by the fact that it labeled the {beta} subunit but not the {alpha} subunit. These results suggest that the N-terminal region of the FSH receptor is in close proximity to FSH, probably interacting with the hormone. This conclusion is consistent with the previous reports that some residues of the region are important for hormone binding and receptor trafficking (45–47) and that the similar region of the LH receptor interacts with human chorionic gonadotropin (34, 48).

In contrast to the labeling of the {beta} subunit by the N-terminal peptide, the exoloop 3 peptide labeled the {alpha} subunit. The significance of these contrasting results is 2-fold. First, it supports the validity and specificity of the photoaffinity labeling. Second, it provides the crucial information on the overall arrangement of the ternary complex involving the exodomain, FSH, and endodomain. The results suggest that the {beta} subunit is near the N-terminal region of the exodomain, whereas the {alpha} subunit is close to the exoloops of the endodomain. These are consistent with the previous reports that only the {alpha}{beta} dimer is capable of high affinity binding to receptors and inducing biological responses (49, 50). Interestingly, all glycoprotein hormones utilize similar if not identical signal pathways consisting of adenylyl cyclase and phospholipase C{beta}. Therefore, the {alpha} subunit has been implicated in the signal generation (49).

Based on these results, it is now possible to project the hormone interacting with both the exodomain and endodomain. Furthermore, they suggest that the hormone is probably in a vertically tilted position with respect to LRRs of the exodomain and the endodomain. To help visualize the arrangement and facilitate modeling of the ternary complex, one of several possible models is presented in Fig. 1E. The model suggests that parts of the {alpha} subunit might interact with LRRs and, conversely, that some parts of the {beta} subunit may be close to the exoloops. Such interactions could be probed by strategically attaching a photoactivable group at appropriate positions of the hormone subunits and/or using a reagent that can reach farther than ABG and Bpa. These two reagents can reach 10 and 7 Å, respectively. Other additional information will also be necessary to more precisely define the ternary structure. For example, the crystal structure of FSH shown in the model does not likely represent its structure in the ternary complex, because the gonadotropin undergoes conformational changes, particularly the interaction between the two subunits upon the initial interaction with the receptor (51). These conclusions are consistent with observations that the original quaternary structure of unbound hormone dimers is not essential for hormone action (1, 52).

The results that Ala substitution for some N-terminal residues improved hormone binding, cAMP induction or both suggest an interesting possibility that this region is involved in modulating not only hormone binding but also signal generation. The most dramatic improvement is seen in the S18A substitution, which improved the EC50 value of cAMP induction by 3-fold as compared with the wild type value. Additionally, the maximum level of cAMP production only slightly increased. These observations indicate that the affinity and maximum level of cAMP induction are distinctly regulated. They suggest that FSH activates FSHRS18A more effectively than the wild type receptor does, which in turn results in better activation of the G protein. It will be interesting to see whether the number of activated G protein molecules is the limiting factor. The improved EC50 is not related to the hormone binding affinity because the binding affinity of the mutant is somewhat less than the wild type affinity. These novel observations suggest an intriguing possibility that FSHRS18A is more sensitive to hormone binding and is capable of activating the G protein with higher affinity without significantly impacting the level of activation. Because the exodomain is likely to modulate the endodomain to generate hormone signals at the exoloops (18–20, 38, 43), a simple possibility is that the affinity of the modulation at the interface between the exodomain and exoloops is improved in FSHRS18A. Several other Ala substitutions, S9A, V12A, and F13A, also showed similar yet less dramatic results.

In conclusion, the evidence is presented that in the ternary exodomain/FSH/endodomain complex, FSH is vertically oriented with the {beta} subunit close to the N-terminal region and the {alpha} tip projecting toward the exoloop 3.


   FOOTNOTES
 
* This work was supported by Grants DK-51469 and HD-18702 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Currently on leave from InJe University (GhimHe, Korea). Back

§ To whom correspondence should be addressed: Dept. of Chemistry, University of Kentucky, Lexington, KY 40506-0055. Tel.: 859-257-3163; Fax: 859-257-3229; E-mail: tji{at}uky.edu.

1 The abbreviations used are: FSH, follicle-stimulating hormone; FSHR, FSH receptor; LRR, leucine-rich repeat; PBS, phosphate-buffered saline; HEK, human embryonic kidney; ABG, 4-azidobenzoyl glycine; LH, luteinizing hormone; TSH, thyroid-stimulating hormone; Bpa, benzoyl phenylalanine; PNGase F, peptide N-glycosidase F. Back



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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