Hormone Interactions to Leu-rich Repeats in the Gonadotropin Receptors

III. PHOTOAFFINITY LABELING OF HUMAN CHORIONIC GONADOTROPIN WITH RECEPTOR LEU-RICH REPEAT 4 PEPTIDE*

MyoungKun JeoungDagger , Tzulip PhangDagger , Yong Sang SongDagger §, Inhae JiDagger , and Tae H. JiDagger

From the Dagger  Department of Chemistry, University of Kentucky, Lexington, Kentucky 40506-0055 and the § Cancer Research Center, Seoul National University College of Medicine, Seoul 110-744, Korea

Received for publication, May 3, 2000, and in revised form, June 12, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Human chorionic gonadotropin (hCG) binds to the extracellular N-terminal domain, exodomain, of its receptor, and the resulting hCG-exodomain complex is thought to modulate the membrane associated domain, endodomain, of the receptor to generate hormone signal. The bulk of the exodomain is speculated to assume a crescent structure consisting of eight to nine Leu-rich repeats (LRRs), which may provide the hormone contact sites. Unfortunately, little experimental evidence is available for the precise hormone contact points in the exodomain and the endodomain. The two preceding articles (Song, Y., Ji, I., Beauchamp, J., Isaacs, N., and Ji, T. (2001) J. Biol. Chem. 276, 3426-3435; Song, Y., Ji, I., Beauchamp, J., Isaacs, N., and Ji, T. (2001) J. Biol. Chem. 276, 3436-3442) show that putative LRR2 and LRR4 are crucial for hormone binding. In particular, the N-terminal region of LRR4 assumes the hydrophobic core of the LRR4 loop, whereas the C-terminal region is crucial for signal generation. However, it is unclear whether LRR4 interacts hCG and the endodomain and how it might be involved in signal generation. In this article, our affinity labeling results present the first evidence that the N-terminal region of LRR4 interacts with hCG, preferentially the hCGalpha subunit and that the hCG/LRR4 complex interacts with exoloop 2 of the endodomain. This interaction offers a mechanism to generate hormone signal.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The luteinizing hormone/chorionic gonadotropin receptor (LHR)1 consists of an extracellular N-terminal half (exodomain) and a membrane-associated C-terminal half (endodomain) (1, 2). The ~350-amino acid-long exodomain has high affinity hormone contact sites (3-5) and shows eight to nine repeats of 22-29 amino acids with several conserved Leu/Ile residues (1, 6-10). These Leu/Ile-rich repeats (LRRs) represent a common structural motif found in a large family of proteins, which includes glycoprotein hormone receptors (11). In the crystal structure of ribonuclease inhibitors, the LRRs assume the horseshoe structure in which individual LRRs form a loop consisting of a beta  strand connected to parallel alpha  helices. The beta  strands in ribonuclease inhibitors are involved in the interaction with ribonuclease. However, it is unclear whether the putative LRR sequences of LHR and other glycoprotein hormone receptors are indeed LRRs and function as such. In the preceding articles (12, 13), we have shown that some, but not all, LRRs of LHR and the follicle-stimulating hormone receptor are crucial for hormone binding. In particular, LRR2 and LRR4 of LHR are most crucial, but it is unclear whether these LRRs make direct contacts with the hormone. In this article, the evidence is presented for the interaction of the residues around the Leu-Ser-Ile motif, the putative beta  strand, in LRR4 with hCG, in particular with the hCGalpha subunit. In addition, our data suggest the interaction of the LRR4-hCG complex with the endodomain, in particular exoloop 2, which is likely to modulate signal generation.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- The N-hydroxysuccinimide (NHS) ester of 4-azidobenzoic acid (AB) was synthesized as described previously (14). The N-hydroxysulfosuccinimide esters of ethylene glycolbis(sulfosuccinimidylsuccinate) (SES) were purchased from Pierce. The hCG CR 127 and hCG subunits were supplied by the National Hormone and Pituitary Program. Denatured hCG was prepared by boiling hCG in 8 M urea for 30 min. Receptor peptides were synthesized and N-acetylated and C-amidated by Biosynthesis (Lewisville, TX). They were purified on a Vydac C18 high performance liquid chromatography column using a solvent gradient from 100% of 0.1% trifluoroacetic acid in water to 20% of 0.1% trifluoroacetic acid in water and 80% 1-propanol. The peptide mimics include the wild type receptor peptide corresponding to the LHR sequence of Asn96-Asp115 (LHR96-115), a mutant LHR96-115 with Leu103Ala and Ile105Ala mutations (LHR96-115(L103A/I105A), a mutant LHR96-115 with the Lys101 right-arrow Ala mutation (LHR96-115(K101A)), a mutant LHR96-115 with the Lys112 right-arrow Ala mutation (LHR96-115(K112A)), a wild type peptide encompassing the sequence upstream of LHR96-115 (LHR85-104), and a wild type peptide covering the sequence downstream of LHR96-115 (LHR113-132).

Derivatization and Radioiodination of Peptides-- NHS-AB was freshly dissolved in dimethyl sulfoxide to a concentration of 50 mM in 0.1 M sodium phosphate (pH 7.5) to a concentration of 20 mM. This reagent solution was immediately used to derivatize receptor peptides. In the dark, 10 µl of NHS-AB was added to 30 µg of LHR96-115 in 40 µl of 0.1 M sodium phosphate (pH 7.5). The mixture was incubated for 30 min for NHS-AB or 60 min for NHS-AB at 25 °C. The following were added to the derivatization mixture: 1 mCi of Na125I in 10 µl of 0.1 M NaOH and 7 µl of chloramine T (1 mg/ml) in 10 mM Na2HPO4 and 0.9% NaCl (pH 7.4) (PBS). After 20 s, 7 µl of sodium metabisulfite (2.5 mg/ml) in PBS was introduced to terminate radioiodination. Derivatized and radioiodinated AB-125I-LHR96-115 solution was mixed with 60 µl of 16% sucrose solution in PBS and fractionated on Sephadex Superfine G-10 column (0.6 × 15 cm) using PBS.

Affinity Cross-linking of 125I-LHR96-115 to hCG-- Disposable glass tubes were siliconized under dimethyldichlorosilane vapor overnight and autoclaved. In each siliconized tube, 20 µl of PBS, hCG (70 ng in 10 µl PBS), and 125I-LHR96-115 (100 ng in 10 µl of PBS) were mixed and incubated in 37 °C for 90 min. After incubation, 3 µl of 0.1 mM of SES in dimethyl sulfoxide was added to each tube and further incubated at 25 °C for 20 min. The cross-linking reaction was terminated by adding 3 µl of 5 mM Gly in PBS. The samples were boiled for 2 min in 2% sodium dodecyl sulfate, 100 mM dithiothreitol, and 8 M urea. The solubilized samples were electrophoresed on 8-12% polyacrylamide gradient gels. Gels were dried on filter paper, which was exposed to a molecular imaging screen (Bio-Rad) overnight. The imaging screen was scanned on a model GS-525 Molecular Imager System Scanner (Bio-Rad), and radioactive band intensity was analyzed using Image Analysis Systems, Version 2.1 (Bio-Rad). Gels were exposed to X-Omat x-ray film at -75 °C for ~4 days.

Photoaffinity Labeling of hCG-- The following solutions were sequentially introduced to siliconized glass tubes: 20 µl of PBS, 10 µl of hCG (10 ng/µl) in PBS, and 10 µl of AB-125I-LHR96-115 (10 ng/µl) in PBS. 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 (14), 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 processed as described above.

Competitive Inhibition of Affinity Labeling of hCG-- Competitive inhibition experiments were carried out as described for the affinity cross-linking and photoaffinity labeling experiments, except 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 wild type or mutant LHR96-115.

Inhibition of 125I-hCG binding to LHR-- A human embryonic kidney 293 cell line stably expressing human LHR was incubated with 100,000 cpm of 125I-hCG in the presence of increasing concentrations of nonradioactive wild type or mutant LHR96-115 peptides as described previously (15). After several times washing the cells, the radioactivity associated with the cells was counted, and percent bound 125I-hCG was plotted against the nonradioactive receptor peptides. The results were converted to Scatchard plot by plotting bound/free peptide versus bound peptide. The plot was used to calculate the Kd value following the Scatchard equation (16).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In the preceding articles (12, 13), we showed the crucial roles of LRRs of LHR in hormone binding, particularly LRR4. This raises the question as to whether the LRRs directly interact with the hormone or indirectly influence the hormone/receptor interaction by impacting the global structure of the receptor exodomain. To examine these possibilities a peptide mimic corresponding to the receptor sequence encompassing the beta -stranded Leu103-Ile105, LHR96-115, was synthesized and tested for its ability to bind and affinity label hCG. For affinity labeling, we employed two complementary affinity labeling methods. In the first approach, 125I-LHR96-115 incubated with hCG, and the resulting 125I-LHR96-115-hCG complexes were cross-linked using SES, a homobifunctional reagent that is capable of cross-linking two amino groups up to 13 Å apart (17). In the second approach, 125I-LHR96-115 was derivatized with AB, an UV-activable reagent, to produce AB-125I-LHR96-115 and incubated with hCG. The resulting 125I-LHR96-115-hCG complex was irradiated with UV to photoaffinity label hCG with AB-125I-LHR96-115. The advantages and disadvantages of both methods will be discussed later.

To determine whether AB-125I-LHR96-115 and 125I-LHR96-115 would bind and label hCG, they were incubated with hCG and treated with UV or SES, respectively. The samples were solubilized in SDS under the reducing condition and electrophoresed, as described under "Experimental Procedures." The autoradiographic phosphoimage of the gel shows that both AB-125I-LHR96-115 and 125I-LHR96-115 labeled both the alpha  and beta  subunits in hCG (Fig. 1). In addition, the hCG alpha beta dimer was cross-linked and labeled with 125I-LHR96-115 when the 125I-LHR96-115-hCG complex was treated with SES. The positions of hCGalpha , hCGbeta , and the hCGalpha beta dimer were determined by comparing the respective positions of 125I-hCGalpha , 125I-hCGbeta , and the cross-linked 125I-hCG alpha beta dimer on the autoradiograph (Fig. 1, lanes 1 and 5).



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Fig. 1.   Autoradiograph of affinity-labeled hCG subunits. hCG was incubated with AB-125I-LHR96-115 (lanes 2 and 3) or 125I-LHR96-115 (lanes 4 and 5) and treated with UV for 1 min (lane 3) or 0.3 mM SES (lane 5), respectively, as described under "Experimental Procedures." After electrophoresis of the samples, the gel was dried and autoradiographed using PhosphoImager. Lane 1, 125I-hCG showing radiolabeled alpha  and beta  subunits as standards.

Cross-linking of 125I-LHR96-115 to hCG-- As a first step to determine the specificity of affinity labeling, 125I-LHR96-115 was incubated with hCG and treated with increasing concentrations of SES. Electrophoresis of the treated hCG/125I-LHR96-115 mixture (Fig. 2A) shows that 125I-LHR96-115 was cross-linked to hCGalpha , hCGbeta , and the hCGalpha beta dimer. The extent of cross-linking was dependent on the SES concentration, reaching the maximum level at 0.3-1 mM SES. Under this condition, ~20% of 125I-LHR96-115 was cross-linked to hCGalpha and ~10% to hCGbeta . At higher SES concentrations, for example 10 mM, the extent of cross-linking decreased. This decrease was due to noncross-linking, monofunctional reactions (only one of the two NHS groups reacting with a target amino group while the other NHS group undergoing hydrolysis) of excess SES with 125I-LHR96-115, hCG, and its subunits (18). In conclusion, our results indicate that 125I-LHR96-115 was covalently cross-linked to hCGalpha and hCGbeta . Furthermore, either or both amino groups of Lys101 and Lys112 of 125I-LHR96-115, the only amino groups of the peptide, were cross-linked to an amino group(s) of either hCGalpha or hCGbeta . The distance between the pair of two cross-linked amino groups is expected to be <13 Å.



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Fig. 2.   Affinity labeling is saturable. hCG was incubated with 125I-LHR96-115 and treated with SES (A-C). In this series of experiments, the increasing concentrations of SES (A), 25I-LHR96-115 (B), or hCG (C) were applied, while the other two conditions were kept constant. In D-F, hCG was incubated with AB-125I-LHR96-115 and irradiated with UV. In this series, the UV irradiation time (D), AB-125I-LHR96-115 concentration (E), or hCG concentration (F) was varied, while the other two factors were kept constant. After electrophoresis of the samples, gels were dried on filter paper and exposed to a molecular imaging screen (Bio-Rad) overnight. The imaging screen was scanned on a model GS-525 Molecular Imager System Scanner (Bio-Rad), and the radioactive band intensity was analyzed using Image Analysis Systems Version 2.1 (Bio-Rad). Gels were also exposed to X-Omat x-ray film at -75 °C for ~4 days. The bar graphs show the percent radioactivity of the alpha  band and the beta  band in a gel lane.

Saturable Cross-linking of 125I-LHR96-115 to hCG-- To determine whether the cross-links are specific between the receptor peptide and hCG, cross-linking was performed under increasing concentrations of 125I-LHR96-115 while maintaining hCG at a constant concentration (Fig. 2B). Conversely, 125I-LHR96-115 and hCG were cross-linked at increasing concentrations of hCG and a constant concentration of 125I-LHR96-115 (Fig. 2C). If cross-links are specific, they should reach saturation under both conditions. The results indeed show plateaus under both conditions, an indication of saturable and specific cross-linking. This specific cross-linking is not expected to occur with peptides that do not recognize hCG.

Photoaffinity Labeling of hCG-- Despite the indication for saturable and specific cross-links between the receptor peptide and hCG, there were a series of minor cross-linked complexes larger than the complex of 125I-LHR96-115 and the hCG dimer. They suggest that a minor population of the 125I-LHR96-115-hCG dimer complex may be further cross-linked to another hCG subunit or hCG dimer. Although this is not entirely unexpected, as random collisional cross-links are possible (18), it raises a concern on the specificity of homobifunctional cross-links between 125I-LHR96-115 and hCG. A simple way to reduce or eliminate such random collisional cross-links is photoaffinity labeling (18). To photoaffinity label hCG with 125I-LHR96-115, the receptor peptide was derivatized with AB to produce AB-125I-LHR96-115. When the derivatized peptide binds to hCG and is irradiated with UV, the cross-link will be restricted between 125I-LHR96-115 and hCGalpha or between 125I-LHR96-115 and hCGbeta . The reagent, however, will not be able to cross-link an hCG subunit to another. AB can reach and label target molecules up to 7 Å (19). The distance is considerably shorter than the maximum cross-linkable 13 Å of SES and therefore, the labeling reaction by AB is more restricted than the cross-linking reaction by SES. On the other hand, cross-linking with SES can be useful when AB attached at the contact point might interfere with the interaction.

As shown in Fig. 2, D-F, AB-125I-LHR96-115 was capable of photoaffinity labeling either hCGalpha or hCGbeta but not both subunits at the same time. The labeling is generally confined to hCGalpha with the labeling of hCGbeta being faint. This result is consistent with the SES cross-linking results. One possible explanation is that the peptide is bound closer to alpha  than beta . The labeling required UV irradiation and was dependent on the irradiation time, reaching the maximum labeling after 30~60-s irradiation. This UV dependence clearly indicates photoaffinity labeling. In addition, the preferential labeling of hCGalpha without simultaneously labeling of both subunits suggests a labeling specificity. To further examine the specificity of photoaffinity labeling, the concentration of either hCG or the peptide derivatives was changed. When a constant concentration of AB-125I-LHR96-115 was incubated with increasing concentrations of hCG, the intensity of labeled hCGalpha and beta  bands gradually increased and plateaued (Fig. 2E). A similar result was obtained in a converse experiment when a constant amount of hCG was incubated with increasing concentrations of AB-125I-LHR96-115 (Fig. 2F). These results indicate that the photoaffinity labeling is dependent on both the derivatized peptides and hCG as they are limiting factors. In both cases, the derivatized peptides labeled hCGalpha more than hCGbeta , an indication of a labeling specificity.

Labeling Specificity-- Specific labeling should be displaced by wild type peptide but not by a peptide that could not bind hCG. We have shown in the previous reports (12), (13) that the Leu103 right-arrow Ala or Ile105 right-arrow Ala substitution in LHR abrogated hormone binding. Therefore, Leu103 and Ile105 were substituted with Ala in LHR96-115 to produce a mutant peptide, LHR96-115(L103A/I105A). To test whether the wild type and mutant LHR peptides could inhibit affinity labeling, hCG was incubated with AB-125I-LHR96-115 in the presence of increasing concentrations of nonderivatized wild type peptide (Fig. 3A) and nonderivatized mutant peptide (Fig. 3C). Increasing concentrations of LHR96-115 inhibited photoaffinity labeling in a dose-dependent manner and eventually, completely blocked it. These results indicate the specificity of LHR96-115 for the photoaffinity labeling. In contrast, the inhibition by mutant LHR96-115(L103A/I105A) was significantly less effective (Fig. 3C). Similar results were obtained with affinity cross-linking of 125I-LHR96-115 to hCG (Fig. 3, B, D, and F). Although these results indicate the labeling specificity of AB-125I-LHR96-115 and 125I-LHR96-115, the futile inhibition could be interpreted as the mutant peptide binding to a site in hCG different from the AB-125I-LHR96-115 binding site. To test this hypothesis and test whether the mutant peptide could label hCG, LHR96-115(L103A/I105A) was radioiodinated or derivatized and then radioiodinated to prepare 125I-LHR96-115(L103A/I105A) or AB-125I-LHR96-115(L103A/I105A), respectively. As shown in Fig. 4, AB-125I-LHR96-115(L103A/I105A) and 125I-LHR96-115(L103A/I105A) labeled the hCG subunits significantly less. Only trace amounts of labeling were detected, indicating the labeling affinities were significantly low. These results are consistent with the observation that the highest concentrations of nonderivatized LHR96-115(L103A/I105A) slightly attenuated the labeling by AB-125I-LHR96-115 and 125I-LHR96-115.



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Fig. 3.   Competitive inhibition of affinity labeling by unlabeled wild type peptide and mutant peptide. AB-125I-LHR96-115 was incubated with hCG in the presence of increasing concentrations of wild type LHR96-115 (A) or mutant LHR96-115(L103A/I105A) (B) and irradiated with UV for 30 s. Samples were processed as described in the legend to Fig. 2. In addition, 125I-LHR96-115 was incubated with hCG in the presence of increasing concentrations of wild type LHR96-115 (C) or mutant LHR96-115(L103A/I105A) (D) and treated with 0.3 mM SES. The samples were electrophoresed and processed to determine the percent labeling of the hCG alpha  and beta  subunits. The percent intensities of the labeled alpha  and beta  bands were determined and plotted against increasing concentrations of unlabeled wild type and mutant peptide (E plotted with the percent labeling data from A and C, and F plotted with the percent labeling data from B and D).



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Fig. 4.   Futile affinity labeling of hCG by mutant LHR96-115. hCG was incubated with mutant AB-125I-LHR96-115(L103A/I105A) (lanes 3 and 4) or 125I-LHR96-115(L103A/I105A) (lanes 7 and 8) and treated with UV for 1 min (lane 4) or 0.3 mM SES (lane 8), respectively. These samples were processed as described in the legend to Fig. 1. The autoradiograph shows no affinity labeling of hCG as compared with successful labeling of hCG by wild type LHR96-115 (lanes 2 and 6). Lanes 1 and 5 show the control hCG samples that were incubated with the wild type peptide but without UV or SES treatment.

Biological Specificity of Affinity Labeling-- Although the affinity labeling is specific, our data do not show the biological significance of the affinity labeling. To test this concern, two different experiments were performed. In the first test, denatured hCG was tested for affinity labeling, and in the second the peptides were examined whether they could inhibit 125I-hCG binding to the receptor on intact cells. For the first test, denatured hCG was incubated with increasing concentrations of AB-125I-LHR96-115or 125I-LHR96-115 and treated with UV or SES, respectively (Fig. 5). Denatured hCG was not labeled at all by either of the LHR peptide derivatives, despite high concentrations of the peptide probes. The results suggest the specificity of the affinity labeling for biologically active hCG. Since SES failed to cross-link 125I-LHR96-115 to denatured hCG, 125I-LHR96-115 appears to have a difficulty to recognize denatured hCG. To test this possibility, 125I-hCG was incubated with intact cells expressing LHR in the presence of increasing concentrations of the wild type or mutant peptide, LHR96-115 or LHR96-115(L103A/I105A) (Fig. 6). The wild type LHR96-115 inhibited 125I-hCG binding to the receptor with a Kd value of 43.4 µM, suggesting its binding to the receptor with a reasonable affinity for a peptide (20, 21). In contrast, the Kd value of mutant LHR96-115(L103A/I105A) was 5 mM, which is insignificant. This result, taken together with the futile labeling of denatured hCG (Fig. 5), shows the biological specificity of the binding and labeling of LHR96-115 to hCG. Furthermore, the results show that the interaction between hCG and LHR96-115 simulates the interaction between hCG and the receptor.



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Fig. 5.   Denatured hCG is not affinity-labeled. Denatured hCG (200 ng) was incubated with increasing concentrations of AB-125I-LHR96-115 (A) or 125I-LHR96-115 and treated with UV for 60 s or 0.3 mM SES (B). The samples were processed as described in the legend to Fig. 1.



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Fig. 6.   Inhibition of 125I-hCG binding to the receptor by LHR peptides. 125I-hCG was incubated with intact 293 cells expressing LHR in the presence of increasing concentrations of unlabeled wild type and mutant LHR96-115 peptides. After washing cells several times to remove unbound 125I-hCG, cells were counted for the bound 125I-hCG as described under "Experimental Procedures." The results were plotted against the concentrations of unlabeled peptides (left panel) and converted to Scatchard plots (right panel). The Kd values of individual peptides in the table were determined with standard deviations based on the bound/free and bound peptide values as described previously (16).

Affinity Labeling Site-- LHR96-115 has two Lys residues, Lys101 and Lys112, which are derivatized with AB or reacted with SES. Since these two Lys are 11 amino acids apart and located in the opposite side of the LRR4 (Fig. 7), it is important to know whether both or only one of them is involved in the affinity labeling. The information will be crucial for defining the orientation and hormone interacting phase of LRR4. To determine the labeling activity of the two Lys residues, one of them was substituted with Ala in LHR96-115 to produce LHR96-115(K101A) and LHR96-115(K112A). These two peptides were capable of inhibiting 125I-hCG binding to the receptor on intact cells, but their Kd values were 12-15-fold higher than the corresponding Kd value of the wild type LHR96-115 (Fig. 6). The result is consistent with the effect of Ala substitution for Lys101 or Lys112 in intact receptor on hCG binding. The Kd value for hCG binding of LHR increased by 2.6-3.4-fold when Lys101 or Lys112 was substituted with Ala (13). Since they were capable of binding hCG, we examined whether the two mutant peptides were also capable of inhibiting photoaffinity labeling of hCG by AB-125I-LHR96-115 and affinity cross-linking of 125I-LHR96-115 to hCG (Fig. 8). The results show their ability to inhibit the labeling, but the potency was noticeably less than the inhibition potency of the wild type peptide. Again, this result is consistent with the lower affinity of the two mutant peptides to hCG as compared with the affinity of the wild type peptide to hCG. All of these results show their specific interaction with hCG. Finally, we attempted to photoaffinity label hCG with AB-125I-LHR96-115(K101A) and AB-125I-LHR96-115(K112A) (Fig. 9). AB-125I-LHR96-115(K112A) photoaffinity labeled hCG similar to the photoaffinity labeling of hCG by AB-125I-LHR96-115, whereas the labeling of hCG with AB-125I-LHR96-115(K101A) was less (Fig. 9A). This result indicates that the photoaffinity labeling is significantly more effective when AB is attached to Lys101 than to Lys112.



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Fig. 7.   Model of LRR4. Lys101 and Lys112 in LRR4 are projected on the opposite side of LRR4 (13).



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Fig. 8.   Inhibition of affinity labeling by Lys to Ala mutant peptides. hCG was affinity-labeled with AB-125I-LHR96-115 and 125I-LHR96-115 in the presence of increasing concentrations of LHR96-115(K101A) or LHR96-115(K112A) as described in the legend to Fig. 3. After processing the samples, the phosphoimage of the gel was analyzed to determine the percent labeling the hCG subunits. The results were plotted against the concentration of peptides for the inhibition of photoaffinity labeling hCGalpha (A) and hCGbeta (B) and affinity cross-linking to hCGalpha (C) and hCGbeta (D). In addition, the inhibition of unlabeled wild type LHR96-115 was presented for comparison.



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Fig. 9.   Affinity labeling of hCG with Lys to Ala mutant peptides. A, hCG was photoaffinity-labeled with AB-125I-LHR96-115, AB-125I-LHR96-115(K101A), or AB-125I-LHR96-115(K101A) as described in the legend to Fig. 3. B, likewise, hCG was affinity-cross-linked to 125I-LHR96-115, 125I-LHR96-115(K101A), or 215I-LHR96-115(K101A) as described in the legend to Fig. 3. "+" and "-" indicate the treatment with or without UV or SES.

If this is true, one would expect the same trend with affinity cross-linking using the two peptides. Indeed, 125ILHR96-115(K112A) was cross-linked to hCG with SES significantly better than 125I-LHR96-115(K101A) (Fig. 9B). However, neither of the derivatized peptides labeled denatured hCG, indicating a specificity of affinity labeling of hCG by AB-125I-LHR96-115(K112A) and 125I-LHR96-115(K112A) (data not shown). Taken together, these results indicate that Lys101 is more suitable for affinity labeling hCG than Lys112 is. They also suggest that Lys101 is at or near the hCG contact point as suggested by the computer model that the short beta  strand is a ligand contact site, and the Lys101 is projected toward ligand (Fig. 7). In contrast, Lys112 is located near the alpha  helix as part of the outer lining of the donut structure, at the opposite side from the ligand binding site. Since Lys101 is in the N-terminal area of LHR96-115, whereas Lys112 is in the C-terminal region, one way to verify the conclusion is to use peptide mimics covering the sequences upstream and downstream of LHR96-115. To this end, we synthesized two peptide mimics, LHR84-104 and LHR113-132, and tested them for their ability to inhibit photoaffinity labeling of hCG by AB-125I-LHR96-115 and affinity cross-linking of 125I-LHR96-115 to hCG (Fig. 10). LHR84-104 and LHR113-132 inhibited the affinity labeling of hCG, but their potency was less than that of LHR96-115. LHR84-104 was more effective in inhibiting hCGalpha than LHR113-132 was. On the other hand, LHR85-104 was similar to LHR113-132 in inhibiting the labeling of hCGbeta .



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Fig. 10.   Roles of peptides flanking LHR96-115 on affinity labeling. hCG was photoaffinity-labeled with AB-125I-LHR96-115 and affinity-cross-linked to 125I-LHR96-115 with SES in the presence of increasing concentrations of LHR96-115, LHR85-104, or LHR113-132 as described in the legend to Fig. 3. After processing the samples, the percent labeling of hCGalpha and hCGbeta was determined. The results are presented for the inhibition of photoaffinity labeling of hCGalpha (A) and hCGbeta (B) and the inhibition of affinity cross-linking of hCGalpha (C) and hCGbeta (D).

Interaction of LHR96-115-hCG Complex with Exoloops-- In the preceding article (13), we pointed out the absolute homology in the 8 residues (boldface) in the Leu98-Pro-Gly-Leu-Lys-Tyr-Leu-Ser-Ile-Cys-Asn-Thr-Gly109 sequence among cloned LHR, follicle-stimulating hormone receptor, and thyroid-stimulating hormone receptor of various species. Furthermore, we showed that the tandem three conserved residues, Asn107-Thr-Gly109, were more important for cAMP induction than hormone binding. This is unique because the exodomain is responsible of high affinity hormone binding and mutations in the exodomain impact hormone binding, which in turn affected cAMP induction, not the other way around. Therefore, we have raised the possibility that this region may be involved in the interaction with the endodomain and, thus, in signal generation. This is a crucial issue, because the exodomain and endodomain are known to interact (22-25), and this interaction regulates the generation of hormone signals (22), (23). However, the exact contact points in the exodomain and endodomain are unknown. Since the three exoloops in the endodomain are a logical candidate for the exodomain/endodomain interaction, we have synthesized peptide mimics for the exoloops 1, 2, and 3 of LHR (LHRexo1, LHRexo2, and LHRexo3) and tested whether they could inhibit the photoaffinity labeling of hCG by AB-125I-LHR96-115 (Fig. 11). LHRexo2 effectively inhibited the photoaffinity labeling, whereas the inhibition by LHRexo1 was less. In contrast, LHRexo3 did not inhibit the labeling. These differential effects suggest the specificity of the inhibition.



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Fig. 11.   Roles of exoloop peptides on affinity labeling. hCG was photoaffinity-labeled with AB-125I-LHR96-115 in the presence of increasing concentrations of LHR peptide mimics corresponding to the sequences of exoloops 1, 2, and 3 (LHRexo1, LHRexo2, and LHRexo3) as described in the legend to Fig. 3. Exoloop 1 connects transmembrane domains 2 and 3, exoloop 2 connects TMs 4 and 5, and exoloop 3 connects transmembrane domains 6 and 7. The LHRexo1 sequence is Asp-Ser-Gln-Thr-Lys-Gly-Gln-Tyr-Tyr-Asn-His-Ala-Ile-Asp-Trp-Gln-Thr-Gly-Ser-Gly-Cys-Ser-Thr. The LHRexo2 sequence is Ser-Asn-Tyr-Met-Lys-Val-Ser-Ile-Cys-Phe-Pro-Met-Asp-Val-Glu-Thr-Thr-Leu-Ser-Gln. LHRexo3 comprises Lys-Val-Pro-Leu-Ile-Thr-Val-Thr-Asn-Ser-Lys. Labeled hCG was processed, and the percent labeling of hCGalpha and hCGbeta was determined as described in the legend to Fig. 3.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Our results show that AB-125I-LHR96-115 photoaffinity labels hCG. Ample evidence is presented to support the specificity of the photoaffinity labeling under rigorous conditions. The labeling is saturable and dependent on the hCG concentration, derivatized 125I-LHR96-115 concentration, and UV activation. AB-125I-LHR96-115 photoaffinity labels bioactive hCG but not denatured hCG. This labeling is blocked by nonderivatized wild type LHR96-115 but not by nonderivatized mutant LHR96-115(L103A/I105A). The same Ala mutations in LHR abolish the hCG binding activity of LHR. Furthermore, AB-125I-LHR96-115(L103A/I105A) does not photoaffinity label bioactive hCG and denatured hCG. LHR96-115 inhibits 125IhCG binding to the receptor expressed on intact cells but LHR96-115(L103A/I105A) is not capable of inhibiting 125I-hCG binding to the receptor. To avoid the potential interference of the photoactivable group on binding of AB-125I-LHR96-115 to the receptor and the subsequent labeling, 125I-LHR96-115 was affinity-cross-linked to hCG with SES. This affinity labeling is equally successful with similar specificity.

Both subunits of hCG are labeled, indicating that the UV-activable group coupled to AB-125I-LHR96-115 can reach them. This is consistent with other studies (26-28) and not surprising, since the two subunits are closely intertwined in the crystal structure (29, 30). Interestingly, hCGalpha was preferentially labeled. Obviously, the reagent more readily reaches and labels the alpha  subunit than the beta  subunit. Since the maximum labeling distances of AB is 7 Å (19), hCGalpha is likely to contact AB-125I-LHR96-115. Our results are inconsistent with the unlikely possibility that the peptide associates with hCG at sites other than the receptor contact site, impacts the global structure of hCG, and interferes with the hormone/receptor interaction. Since LHR96-115 inhibits hCG binding to the receptor, AB-125I-LHR96-115 interacts with hCG at or near a contact site of hCG and the LH/CG receptor.

It is significant that only one of the hCGalpha beta subunits, but not both, is labeled, although two AB could be attached to the two Lys residues of LHR96-115. This suggests that only one of the Lys residues is close to hCG. Indeed, photoaffinity labeling using mutant peptides lacking one of the Lys residues shows that the AB coupled to Lys101 is capable of labeling hCG, whereas the AB attached to Lys112 is less effective. This is strong evidence to support the orientation of Lys101 and Lys112 in the LRR4 loop model (Fig. 7) and implicates the N-terminal region of LHR96-113, including the putative beta  strand of LRR4, in the interaction with hCG.

The crystallization of Leu-rich repeats (11, 31) and their presence in the middle of the exodomain of all glycoprotein hormone receptors (1) generated much speculation (8, 32-34) that the eight to nine LRRs provide the primary contact site for the cognate ligands, LH/CG, FSH, and TSH. They comprise the bulk of the exodomain at its center and are computer-modeled to show a crescent structure. The inner surface of the crescent consists of beta  sheets of the repeats and is thought to be the ligand contact site (8, 31, 32), perhaps interacting with the putative receptor binding alpha C terminus and seat belt side of hCG (29). However, little experimental evidence has been available to support these popular views. Our results of this and the preceding articles (12, 13) are the first experimental evidence supporting the LRR structure of LHR and the direct interaction of the LRR4 beta  strand with hCG. Our studies have laid the ground work to determine the contact residues of the receptor and the hormone.

It has been known that LHR interacts hCG initially at the exodomain, and the exodomain-hCG complex impacts the endodomain. This secondary contact is thought to generate the hormone signals (22, 23). There is evidence that the exodomain and endodomain are intimately associated before and after hormone binding (24, 25). This association is crucial because it affects the hormone binding affinity and provides a mechanism for the signal generation (24, 25). Unfortunately, there are few clues to the site of the interaction between the exodomain and endodomain except the recent reports implicating exoloops 2 and 3 (24, 25). The observations described in this and preceding articles (12, 13) show the involvement of LRR4 in the signal generation, implicating exoloop 2 and, perhaps, exoloop 1 as contact points of the exodomain/hCG complex. In fact, our computer modeling shows that the exoloop 2 projects straight up from the connecting the transmembranes 4 and 5, like a hairpin, toward the exodomain. It will be interesting to see whether the hairpin structure of exoloop 2 interacts with the crescent LRR structure of the exodomain, in particular LRR4. Such an exodomain/endodomain interaction could provide a mechanism for the mutual modulation of the two distinct domains (24, 25) and signal generation.


    FOOTNOTES

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

To whom all correspondence should be addressed: Dept. of Chemistry, University of Kentucky, Lexington, KY 40506-0055. Tel.: 859-257-3163; Fax: 859-527-3229; E-mail: tji@pop.uky.edu.

Published, JBC Papers in Press, July 3, 2000, DOI 10.1074/jbc.M003774200


    ABBREVIATIONS

The abbreviations used are: LH, luteinizing hormone; LHR, LH receptor; CG, choriogonadotropin; h, human; LRR, Leu-rich repeat; AB, 4-azidobenzoyl; NHS, N-hydroxysuccinimide; SES, ethylene glycolbis(sulfosuccinimidylsuccinate); PBS, phosphate-buffered saline.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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