The Amino-terminal Region of the Luteinizing Hormone/Choriogonadotropin Receptor Contacts Both Subunits of Human Choriogonadotropin
II. PHOTOAFFINITY LABELING*

Tzulip Phang, Gopal Kundu, Sohee Hong, Inhae Ji, and Tae H. JiDagger

From the Department of Molecular Biology, University of Wyoming, Laramie, Wyoming 82071-3944

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The luteinizing hormone/choriogonadotropin receptor, a seven-transmembrane receptor, is composed of two equal halves, the N-terminal extracellular exodomain and the C-terminal membrane-associated endodomain. Unlike most seven-transmembrane receptors, the exodomain alone is responsible for high affinity hormone binding, whereas signal is generated in the endodomain. These physical separations of hormone-binding and receptor activation sites are attributed to unique mechanisms for hormone binding and receptor activation of this receptor and its subfamily members. However, the precise hormone contact sites in the exodomain are unclear. In the preceding article (Hong, S., Phang, T., Ji, I., and Ji, T. H. (1998) J. Biol. Chem. 273, 13835-13840), a region immediately downstream of the N terminus of the exodomain was shown to be crucial for hormone binding. To test if the region interacts with the hormone, human choriogonadotropin (hCG) was photoaffinity-labeled with a peptide mimic corresponding to Gly18-Tyr36 of the receptor. This peptide mimic specifically photoaffinity-labeled both the alpha - and beta -subunits of hCG. Interestingly, hCGalpha was preferentially labeled. On the other hand, denatured hCG was not labeled, and a mutant analog of the peptide failed to label hCG. Furthermore, the affinity labeling was UV-dependent and saturable, indicating the specificity of the photoaffinity labeling. Our results indicate that the region of the exodomain interacts with hCG and that the contact points are near both subunits of hCG. Particularly, the alternate residues (Leu20, Cys22, and Gly24) are crucial for hCG binding. In addition, the results underscore the fact that there is a crucial hormone contact site outside of the popularly believed primary hormone-binding site that is composed of Leu-rich repeats and is located in the middle of the exodomain. Our observations are crucial for understanding the molecular mechanism through which the initial high affinity hormone binding leads to receptor activation in the endodomain.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The LH1/CG receptor belongs to a subfamily of glycoprotein hormone receptors within the seven-transmembrane receptor family. Unlike most seven-transmembrane receptors, it is composed of two equal halves, the 341-amino acid-long extracellular N-terminal exodomain and the 334-amino acid-long membrane-associated C-terminal endodomain, which includes seven transmembrane helices (1, 2). The exodomain binds the hormones with high affinity (3-7) without hormone action (5, 8). The exodomain-hCG complex is thought to make a secondary contact with the endodomain, thus generating a signal (9). Therefore, the high affinity interaction of the exodomain and hCG is the crucial first step leading to signal generation and hormone action. However, only limited information is available regarding the precise hormone contact residues and sites in the exodomain. Three peptide mimics of the exodomain, peptide-(21-38), peptide-(102-115), and peptide-(253-266), attenuated 125I-hCG binding to membranes expressing the LH/CG receptor (10). Receptors lacking the 11 amino-terminal residues or one of the leucine-rich motifs 1-6 were trapped in cells and failed to bind hCG (11).

In this work and the preceding article (12), the exodomain was examined using several independent methods, including serial truncation from the C terminus, Ala scanning, peptide mimics of the receptor, photoaffinity labeling, affinity cross-linking, and immunofluorescence. Our results show that the Leu20-Pro38 sequence contacts both the alpha - and beta -subunits of hCG. In addition, three other sequences near the junctions of exons 3-4, 6-7, and 9-10 are important for hormone binding.

    EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- The N-hydroxysuccinimide (NHS) esters of 4-azidobenzoylglycine (ABG) was synthesized as described (13). The N-hydroxysulfosuccinimide (sulfo-NHS) esters of 4-azidobenzoic acid (AB) and ethylene glycolbis(sulfosuccinimidylsuccinate) (SES) were purchased from Pierce. The hCG CR 127 and hCG alpha - and beta -subunits were supplied by the National Hormone and Pituitary Program (NIDDK, National Institutes of Health). Denatured hCG was prepared by boiling hCG in 8 M urea for 30 min. Peptide mimics including wild-type and mutant LH/CG receptor peptides, LHR18-36 (see Fig. 1), were synthesized by Biosynthesis (Lewisville, TX) and purified on a Vydac C18 HPLC 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 sequence of LHR18-36 corresponds to Gly18-Tyr36 of the LH/CG receptor. In addition, a mutant LHR18-36 was synthesized in which Leu20, Cys22, and Gly24 were substituted with Ala.

Derivatization and Radioiodination of Peptides-- NHS-ABG was freshly dissolved in dimethyl sulfoxide to a concentration of 50 mM, and sulfo-NHS-AB in 0.1 M sodium phosphate (pH 7.5) to a concentration of 20 mM. These reagent solutions were immediately used to derivatize receptor peptides. In the dark, 10 µl of NHS-ABG or sulfo-NHS-AB was added to 30 µg of LHR18-36 in 40 µl of 0.1 M sodium phosphate (pH 7.5). The mixture was incubated for 30 min for NHS-ABG or 60 min for sulfo-NHS-AB at 25 °C. The following were added to the derivatization mixture: 1 mCi of Na125I-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) (PBS). After 20 s, 7 µl of sodium metabisulfite (2.5 mg/ml) in PBS was introduced to terminate radioiodination. Derivatized and radioiodinated ABG-125I-LHR18-36 or AB-125I-LHR18-36 solution was mixed with 60 µl of 16% sucrose solution in PBS and fractionated on a Sephadex Superfine G-10 column (0.6 × 15 cm) using PBS.

Cross-linking of 125I-LHR18-36 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 of PBS), and 125I-LHR18-36 (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 SES in Me2SO 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% SDS, 100 mM dithiothreitol, and 8 M urea. The solubilized samples were electrophoresed on 8-12% polyacrylamide gradient gels. Gels were dried and exposed to Eastman Kodak 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 ABG-125I-LHR18-36 (10 ng/µl) in PBS. For labeling with AB-125I-LHR18-36, 20 µl of PBS, 10 µl of hCG (20 ng/µl) in PBS, and 10 µl of AB-125I-LHR18-36 (15 ng/µl) in PBS were mixed. 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 (13); 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, 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 the radioactive band profile 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 .

Competitive Inhibition of Photoaffinity Labeling of hCG-- Competitive inhibition experiments were carried out as described for the 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 LHR18-36.

Inhibition of 125I-hCG Binding to the LH/CG Receptor-- A human embryonic kidney 293 cell line stably expressing the rat LH/CG receptor was incubated with 100,000 cpm of 125I-hCG in the presence of increasing concentrations of nonradioactive wild-type or mutant LHR18-36 as described previously (14). After washing the cells several times, the radioactivity associated with the cells was counted to determine the Kd value.

Trichloroacetic Acid Precipitation of 125I-LHR18-36 Complexed with hCG-- 20 µl of PBS, 10 µl of 150,000 cpm of 125I-LHR18-36 (10 ng/µl) in PBS, 10 µl of hCG (10 ng/µl) in PBS, and 10 µl of increasing concentrations of unlabeled LHR18-36 in PBS were sequentially introduced to siliconized glass tubes. After incubation at 37 °C for 90 min, 5 µl of 50% trichloroacetic acid was introduced to the tubes, and the mixture was incubated at 4 °C for 10 min. The mixture was centrifuged at 2500 × g for 20 min at 4 °C, and the radioactivity of the pellet was counted.

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

In the preceding article (12), we showed that the amino acid sequence near the N terminus of the LH/CG receptor, particularly Leu20-Arg21-Cys22-Pro23-Gly24, is crucial for hCG binding. This raises the question as to whether the peptide sequence directly interacts with the hormone or indirectly influences the hormone/receptor interaction by impacting on the global structure of the receptor. To examine these possibilities, a peptide mimic corresponding to the receptor sequence from Gly18 to Tyr36, LHR18-36 (Fig. 1), was synthesized and tested for its ability to bind and to affinity label hCG. The sequence of LHR18-36 corresponds to Gly18-Tyr36 of the LH/CG receptor. In addition, a mutant LHR18-36 was synthesized in which Leu20, Cys22, and Gly24 were substituted with Ala.


View larger version (7K):
[in this window]
[in a new window]
 
Fig. 1.   Sequences of LHR peptide mimics. LHR18-36 was synthesized, with its sequence corresponding to Gly18-Tyr36 of the LH/CG receptor. In addition, a mutant LHR18-36 was synthesized in which Leu20, Cys22, and Gly24 were substituted with Ala.

Cross-linking of 125I-LHR18-36 to hCG-- 125I-LHR18-36 was incubated with hCG and treated with a homobifunctional reagent, SES, which specifically reacts with amino groups to covalently cross-link two amino groups (15). Electrophoresis of the treated 125I-LHR18-36/hCG mixture showed that 125I-LHR18-36 was primarily cross-linked to hCGalpha , hCGbeta , and the hCGalpha beta dimer (Fig. 2A, lane 6). The positions of hCGalpha , hCGbeta , and the hCGalpha beta dimer were determined by comparing with the respective positions of 125I-hCGalpha , 125I-hCGbeta , and the cross-linked 125I-hCGalpha beta dimer on the autoradiograph (Fig. 2A, lanes 1-3). Without SES, ~2% of 125I-LHR18-36 remained associated with hCGalpha under reducing electrophoretic conditions. The band became conspicuous on the autoradiographs when large amounts of radioactive samples were applied and x-ray film was overexposed. This result suggests the inherent affinity of the peptide for hCG. However, cross-linking of 125I-LHR18-36 to hCG required SES, hCG, and 125I-LHR18-36 since it did not occur in the absence of any of the three. The extent of cross-linking was dependent on the SES concentration (Fig. 2, B and C), reaching the maximum level at 0.3-1 mM SES. Under this condition, ~20% of 125I-LHR18-36 was cross-linked to each hCGalpha and hCGbeta . At a higher SES concentration, e.g. 3 mM, the extent of cross-linking decreased. This decrease was due to a non-cross-linking, monofunctional reaction (only one of the two NHS groups reacting with a target amino group while leaving the other NHS group unused) of excess SES with 125I-LHR18-36, hCG, and its subunits (16). In conclusion, our results indicate that 125I-LHR18-36 was covalently cross-linked to hCGalpha and hCGbeta . Furthermore, the N-terminal amino group of 125I-LHR18-36, the only amino group of the peptide, was cross-linked to an amino group of either hCGalpha or hCGbeta . The distance between the pair of two cross-linked amino groups is expected to be <13 Å.


View larger version (76K):
[in this window]
[in a new window]
 
Fig. 2.   Autoradiography of cross-linking of 125I-LHR18-36 to hCG. After incubating 125I-LHR18-36 with hCG, the mixture was treated with increasing concentrations of SES, solubilized under reducing conditions, and electrophoresed (C). Gels were dried and autoradiographed (B) as described under "Experimental Procedures." In addition, 125I-hCGalpha , 125I-hCGbeta , and cross-linked 125I-hCGalpha beta were electrophoresed as standards (A).

Specificity of Cross-linking of LHR18-36 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-LHR18-36 while maintaining hCG at a constant concentration (Fig. 3A). Conversely, 125I-LHR18-36 and hCG were cross-linked at increasing concentrations of hCG and a constant concentration of 125I-LHR18-36 (Fig. 3B). If cross-links are specific, they should reach saturation under both conditions. The results (Fig. 3, D and E) 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. In the preceding article (12), Ala substitution for Leu20, Cys22, or Gly24 of the receptor resulted in the loss of hCG binding by the receptor. Therefore, a mutant LHR18-36 was synthesized in which Leu20, Cys22, and Gly24 were substituted with Ala (Fig. 1). As expected, mutant LHR18-36 was not cross-linked to either subunit of hCG (Fig. 3, C and F). It is not clear whether the lack of observed cross-linking of mutant LHR18-36 was caused by a lack of mutant LHR18-36 binding to hCG or by a lack of a cross-linking reaction by SES due to the putative steric hindrance even though mutant LHR18-36 successfully bound to hCG. To distinguish these possibilities, 125I-LHR18-36 was cross-linked to hCG in the presence of increasing concentrations of unlabeled wild-type LHR18-36 (Fig. 4A) and mutant LHR18-36 (Fig. 4B). Wild-type LHR18-36 attenuated the cross-linking, but mutant LHR18-36 was significantly less effective (Fig. 4C), an indication of the less efficient binding of mutant LHR18-36 to hCG.


View larger version (72K):
[in this window]
[in a new window]
 
Fig. 3.   125I-LHR18-36 and hCG concentration-dependent cross-linking. A constant concentration of hCG was incubated with increasing concentrations of 125I-LHR18-36 and treated with SES (A and D). Conversely, a constant concentration of 125I-LHR18-36 was incubated with increasing concentrations of hCG and treated with SES (B and E). Wild-type (WT) and mutant (Mut) LHR18-36 were incubated with hCG and treated with SES (C and F).


View larger version (48K):
[in this window]
[in a new window]
 
Fig. 4.   Inhibition of cross-linking of 125I-LHR18-36 to hCG by unlabeled wild-type and mutant LHR18-36. 125I-LHR18-36 was incubated with hCG in the presence of increasing concentrations of wild-type (WT) LHR18-36 (A) or mutant LHR18-36 (B) and treated with SES. Samples were processed as described in the legend to Fig. 2. The intensities of the alpha  and beta  bands were plotted against increasing concentrations of unlabeled wild type (WT) and mutant peptides (C).

Affinity of LHR18-36 Binding to hCG-- To determine the binding affinity, 125I-LHR18-36 was incubated with hCG in the presence of increasing concentrations of unlabeled LHR18-36 (Fig. 5A). Unlabeled LHR18-36 inhibited 125I-LHR18-36 binding to hCG, with a Kd value of 29 µM. It is not clear whether this inhibition was caused by competitive binding of LHR18-36 and the receptor to hCG or by a putative allosteric effect of LHR18-36 binding to hCG. To examine these possibilities and the relevance of the inhibition of hCG binding to the LH/CG receptor, 125I-hCG was incubated with a 293 cell line stably expressing the receptor (14) in the presence of increasing concentrations of unlabeled LHR18-36 (Fig. 5B). Unlabeled wild-type LHR18-36, but not mutant LHR18-36, competitively attenuated binding of 125I-hCG to the receptor, with a Kd value of 25 µM. This result indicates that the interaction of LHR18-36 and hCG is specific and simulates the interaction between hCG and the receptor. Furthermore, the two independent Kd values (29 and 25 µM) are not only close, but are also similar to the value of 15 µM for the inhibition of 125I-hCG binding to the receptor by LHR21-41 (10).


View larger version (34K):
[in this window]
[in a new window]
 
Fig. 5.   Inhibition of 125I-LHR18-36 binding to hCG and of 125I-hCG binding to the LH/CG receptor. The affinity of LHR18-36 binding to hCG was determined using two independent methods. 125I-LHR18-36 was incubated with hCG in the presence of increasing concentrations of unlabeled LHR18-36 (A) as described under "Experimental Procedures." Alternatively, 125I-hCG was incubated with 293 cells stably expressing the LH/CG receptor in the presence of increasing concentrations of unlabeled LHR18-36 (B). WT, wild-type; NS, not significant.

Photoaffinity Labeling of hCG-- Despite the indication for specific cross-links between the receptor peptide and hCG, there were a series of minor cross-linked complexes larger than the complex of 125I-LHR18-36 and the hCG dimer. This suggests that a minor population of the 125I-LHR18-36·hCG dimer complex was further cross-linked to other hCG subunits or the hCG dimer. Although this is not entirely unexpected, as random collisional cross-links are possible (16), it raises concern regarding the specificity of homobifunctional cross-links between 125I-LHR18-36 and hCG. A simple way to reduce or eliminate such random collisional cross-links is photoaffinity labeling (16).

To photoaffinity label hCG with 125I-LHR18-36, the receptor peptide was derivatized with either AB or ABG (13) to produce AB-125I-LHR18-36 or ABG-125I-LHR18-36, respectively. When these peptides bind to hCG and are irradiated with UV, cross-linking will be restricted between 125I-LHR18-36 and hCGalpha or between 125I-LHR18-36 and hCGbeta . The reagent, however, will not be able to cross-link one hCG subunit to another. AB and ABG can reach and label target molecules up to 7 and 10 Å, respectively (17). These distances are considerably shorter than the maximum cross-linkable 13 Å of SES, and therefore, the labeling reaction by AB and ABG is more restricted than the cross-linking reaction by SES. As shown in Fig. 6, AB-125I-LHR18-36 and ABG-125I-LHR18-36 were capable of photoaffinity labeling either hCGalpha or hCGbeta , but not both subunits at the same time to produce the labeled hCGalpha beta complex. Interestingly, AB-125I-LHR18-36 labeled them more efficiently than ABG-125I-LHR18-36 (Fig. 6, C and D). In addition, hCGalpha was more preferentially labeled than hCGbeta . This result is consistent with the SES cross-linking results. One possible explanation is that the N terminus of the LHR18-36 derivatives is <7 Å from the hCG alpha - and beta -subunits and that the peptide derivatives are bound closer to alpha  than beta . The labeling required UV irradiation and was dependent on the irradiation time, reaching the maximum labeling after ~1 and 0.5 min of irradiation of the ABG-125I-LHR18-36·hCG and AB-125I-LHR18-36·hCG complexes, respectively. Unlike SES cross-links, the maximum levels were sustained after longer UV exposure. This UV dependence clearly indicates photoaffinity labeling. In addition, the sustained maximum levels and the preferential labeling of hCGalpha without simultaneous labeling of both subunits suggest a labeling specificity. To further examine the specificity of photoaffinity labeling, the concentration of either hCG or the peptide derivatives was changed.


View larger version (80K):
[in this window]
[in a new window]
 
Fig. 6.   UV-dependent photoaffinity labeling of hCG by AB-125I-LHR18-36 or ABG-125I-LHR18-36. A constant concentration of hCG was incubated with a constant concentration of AB-125I-LHR18-36 (B and D) or ABG-125I-LHR18-36 (A and C). The mixture was irradiated with UV for increasing time periods, solubilized, and electrophoresed as described in the legend to Fig. 2. Dried gels were analyzed on a phosphoimager, and radioactive band intensities were determined as described under "Experimental Procedures." In addition, gels were autoradiographed.

Concentration Effect of hCG and Peptide Derivatives-- When a constant amount of hCG was incubated with increasing concentrations of AB-125I-LHR18-36 or ABG-125I-LHR18-36, the intensity of labeled hCG alpha - and beta -bands gradually increased and plateaued (Fig. 7). A similar result was obtained in a converse experiment when a constant concentration of AB-125I-LHR18-36 or ABG-125I-LHR18-36 was incubated with increasing concentrations of hCG (Fig. 8). These results indicate that the photoaffinity labeling is dependent on both of 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.


View larger version (86K):
[in this window]
[in a new window]
 
Fig. 7.   Photoaffinity labeling of hCG with increasing concentrations of AB-125I-LHR18-36 or ABG-125I-LHR18-36. A constant concentration of hCG was incubated with increasing concentrations of AB-125I-LHR18-36 (B and D) or ABG-125I-LHR18-36 (A and C). The mixture was irradiated with UV, solubilized, electrophoresed, and analyzed as described in the legend to Fig. 6.


View larger version (87K):
[in this window]
[in a new window]
 
Fig. 8.   hCG concentration-dependent photoaffinity labeling. Increasing concentrations of hCG were incubated with a constant concentration of AB-125I-LHR18-36 (B and D)or ABG-125I-LHR18-36 (A and C). The mixture was irradiated with UV, solubilized, and electrophoresed as described in the legend to Fig. 6.

Competitive Inhibition of Photoaffinity Labeling by Nonderivatized Peptides-- A nonderivatized peptide is expected to displace specific labeling. Therefore, hCG was incubated with AB-125I-LHR18-36 or ABG-125I-LHR18-36 in the presence of increasing concentrations of nonderivatized peptide (Fig. 9, A and D). Increasing concentrations of LHR18-36 inhibited photoaffinity labeling in a dose-dependent manner and eventually completely blocked it. These results indicate the specificity of LHR18-36 for the photoaffinity labeling. However, this specific labeling should not be blocked by a peptide that does not recognize hCG. Increasing concentrations of mutant LHR18-36 failed to significantly block the photoaffinity labeling of hCG by AB-125I-LHR18-36 or ABG-125I-LHR18-36 (Fig. 9, B and E). Only at the highest concentrations of the mutant peptide was labeling by AB-125I-LHR18-36 slightly reduced. Although these results indicate the labeling specificity of AB-125I-LHR18-36 and ABG-125I-LHR18-36, the futile inhibition could be interpreted as the mutant peptide binding to a site in hCG different from the AB-125I-LHR18-36- or ABG-125I-LHR18-36-binding site. To test this hypothesis, mutant LHR18-36 was derivatized and radioiodinated to prepare AB-125I-mutant LHR18-36 and ABG-125I-mutant LHR18-36.


View larger version (82K):
[in this window]
[in a new window]
 
Fig. 9.   Inhibition of photoaffinity labeling by nonderivatized wild-type or mutant LHR18-36. hCG was photoaffinity-labeled with AB-125I-LHR18-36 (D-F) or ABG-125I-LHR18-36 (A-C) in the presence of increasing concentrations of nonderivatized wild-type (WT) or mutant LHR18-36. Samples were processed as described in the legend to Fig. 6.

Photoaffinity Labeling by Mutant LHR18-36-- As shown in Fig. 10, AB-125I-mutant LHR18-36 and ABG-125I-mutant LHR18-36 failed to conspicuously label the hCG subunits. Only trace amounts of labeling were detected, indicating that the labeling affinities were significantly low. These results are consistent with the observation that the highest concentrations of nonderivatized mutant LHR18-36 slightly attenuated the labeling by AB-125I-LHR18-36. In addition, AB-125I-LHR18-36 and ABG-125I-LHR18-36 did not photoaffinity label denatured hCG that was boiled in M urea and did not bind to the receptor (data not shown).


View larger version (78K):
[in this window]
[in a new window]
 
Fig. 10.   Photoaffinity labeling by wild-type and mutant AB-125I-LHR18-36 or ABG-125I-LHR18-36. hCG was incubated with wild-type (WT) and mutant (Mut) AB-125I-LHR18-36 (B and D) or ABG-125I-LHR18-36 (A and C) and photoaffinity-labeled as described in the legend to Fig. 6.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Our results show that AB-125I-LHR18-36 and ABG-125I-LHR18-36 photoaffinity label hCG. The alpha -subunit is preferentially labeled. Ample evidence was presented to support the specificity of the photoaffinity labeling of hCG. The labeling is saturable and dependent on the hCG concentration, derivatized 125I-LHR18-36 concentration, and UV exposure. AB-125I-LHR18-36 and ABG-125I-LHR18-36 photoaffinity label bioactive hCG, but not denatured hCG. This labeling is blocked by nonderivatized wild-type LHR18-36, but not by nonderivatized mutant LHR18-36. Furthermore, AB-125I-mutant LHR18-36 and ABG-125I-mutant LHR18-36 do not photoaffinity label bioactive hCG and denatured hCG.

Both subunits of hCG are labeled, indicating that the UV-activable group coupled to LHR18-36 can reach them. This is consistent with other studies (18-20) and not surprising since the two subunits are closely intertwined in the crystal structure (21, 22). Interestingly, hCGalpha was preferentially labeled. Since only one photosensitive group is attached to the N terminus of each derivatized peptide, AB-125I-LHR18-36 and ABG-125I-LHR18-36 bound to hCG can photoaffinity label only one, but not both, of the subunits. Obviously, the reagent more readily reaches and labels the alpha -subunit than the beta -subunit. Since the maximum labeling distances of AB and ABG are 7 and 10 Å, respectively (17), and AB-125I-LHR18-36 labels hCG more efficiently than ABG-125I-LHR18-36, the N terminus of the LHR18-36 derivatives is <7 Å from both subunits. Therefore, both subunits of hCG are likely to contact the LHR18-36 derivatives. Our results are not consistent with the unlikely possibility that the peptide associates with hCG at sites other than the receptor contact site, impacts on the global structure of hCG, and interferes with the hormone/receptor interaction. Clearly, LHR18-36 interacts with hCG at or near a contact site of hCG and the LH/CG receptor.

The recent crystallization of Leu-rich repeats (23, 24) and their presence in the middle of the exodomain of all glycoprotein hormone receptors (1) generated a deluge of the popular and probable thoughts (11, 25-27) that eight to nine Leu-rich repeats compose the primary contact site for the ligand. They compose the bulk of the exodomain at its center and are computer-modeled to show a crescent structure (Fig. 11). The inner surface of the crescent consists of beta -sheets of the repeats and is thought to be the ligand contact site (24-26), perhaps interacting with the putative receptor-binding C-terminal and seat belt side of hCG (21). Our results in this work and the preceding article (12) indicate that there is a crucial hormone contact site outside of this Leu-rich crescent. It will be interesting to see if the Gly18-Tyr36 sequence of the receptor reaches the opposite face of the seat belt side (Fig. 11). In the Gly18-Tyr36 sequence, the alternate Leu20, Cys22, and Gly24 residues are crucial for hormone binding (12). These three residues appear to be at one side of a beta -like structure and could face the hormone and provide a direct contact site.


View larger version (33K):
[in this window]
[in a new window]
 
Fig. 11.   Schematic model for the interaction of the LH/CG receptor exodomain with hCG. To facilitate the visualization of the interaction of the exodomain with hCG, a schematic model was prepared. hCG interacts with the inner face of the crescent structure of the receptor as suggested by computer modeling (25, 28, 29).

Our results are consistent with the observations of others indicating multiple contact sites for the hCG/receptor interaction (10, 11, 28, 29). Each contact site is likely to contribute to the overall interaction and affinity. For complete understanding of the interaction, it is necessary to know whether the multiple contact sites are independent or related and whether they interact with hCG simultaneously or sequentially. This information will be useful for designing agonists and antagonists of hCG.

    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.

Dagger To whom correspondence should be addressed. Tel.: 307-766-6272; Fax: 307-766-5098; E-mail: ji{at}uwyo.edu.

1 The abbreviations used are: LH, luteinizing hormone; LHR, LH receptor; CG, choriogonadotropin; hCG, human CG; NHS, N-hydroxysuccinimide; sulfo-NHS, N-hydroxysulfosuccinimide; ABG, 4-azidobenzoylglycine; AB, 4-azidobenzoic acid; SES, ethylene glycolbis(sulfosuccinimidylsuccinate); PBS, phosphate-buffered saline.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

  1. McFarland, K., Sprengel, R., Phillips, H., Kohler, M., Rosemblit, N., Nikolics, K., Segaloff, D., and Seeburg, P. (1989) Science 245, 494-499[Medline] [Order article via Infotrieve]
  2. Loosfelt, H., Misrahi, M., Atger, M., Salesse, R., Thi, M., Jolivet, A., Guiochon-Mantel, A., Sar, S., Jallal, B., Garnier, J., and Milgrom, E. (1989) Science 245, 525-528[Medline] [Order article via Infotrieve]
  3. Tsai-Morris, C. H., Buczko, E., Wang, W., and Dufau, M. L. (1990) J. Biol. Chem. 265, 19385-19388[Abstract/Free Full Text]
  4. Xie, Y. B., Wang, H., and Segaloff, D. L. (1990) J. Biol. Chem. 265, 21411-21414[Abstract/Free Full Text]
  5. Ji, I., and Ji, T. H. (1991) Endocrinology 128, 2648-2650[Abstract]
  6. Seetharamaiah, G. S., Kurosky, A., Desai, R. K., Dallas, J. S., and Prabhakar, B. S. (1994) Endocrinology 134, 549-554[Abstract]
  7. Davis, D., Liu, X., and Segaloff, D. (1995) Mol. Endocrinol. 9, 159-170[Abstract]
  8. Remy, J. J., Bozon, V., Couture, L., Goxe, B., Salesse, R., and Garnier, J. (1993) Biochem. Biophys. Res. Commun. 193, 1023-1030[CrossRef][Medline] [Order article via Infotrieve]
  9. Ji, T. H., Murdoch, W., and Ji, I. (1995) Endocrine 3, 187-194T. H.
  10. Roche, P. C., Ryan, R. J., and McCormick, D. J. (1992) Endocrinology 131, 268-274[Abstract]
  11. Thomas, D., Rozell, T., Liu, X., and Segaloff, D. (1996) Mol. Endocrinol. 10, 760-768[Abstract]
  12. Hong, S., Phang, T., Ji, I., and Ji, T. H. (1998) J. Biol. Chem. 273, 13835-13840[Abstract/Free Full Text]
  13. Ji, I., and Ji, T. H. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 7167-7170[Abstract]
  14. Ryu, K.-S., Gilchrist, R. L., Ji, I., Kim, S.-J., and Ji, T. H. (1996) J. Biol. Chem. 271, 7301-7304[Abstract/Free Full Text]
  15. Ji, I., Bock, J., and Ji, T. H. (1985) J. Biol. Chem. 260, 12815-12821[Abstract/Free Full Text]
  16. Ji, T. H. (1979) Biochim. Biophys. Acta 559, 39-69[Medline] [Order article via Infotrieve]
  17. Ji, I., and Ji, T. H. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 5465-5469[Abstract]
  18. Morbeck, D. E., Roche, P. C., Keutmann, H. T., and McCormick, D. J. (1993) Mol. Cell. Endocrinol. 97, 173-181[Medline] [Order article via Infotrieve]
  19. Liu, C., Roth, K. E., Shepard, B. A., Shaffer, J. B., and Dias, J. A. (1993) J. Biol. Chem. 268, 21613-21617[Abstract/Free Full Text]
  20. Cosowsky, L., Lin, W., Han, Y., Bernard, M. P., Campbell, R. K., and Moyle, W. R. (1997) J. Biol. Chem. 272, 3309-3314[Abstract/Free Full Text]
  21. Lapthorn, J. P., Harris, D. C., Littlejohn, A., Lustbader, J. W., Canfield, R. E., Machin, K. J., Morgan, F. J., and Isaacs, N. W. (1994) Nature 369, 455-461[CrossRef][Medline] [Order article via Infotrieve]
  22. Wu, H., Lustabader, J. W., Liu, Y., Canfield, R. E., and Hendrickson, W. A. (1994) Structure 2, 545-558[Medline] [Order article via Infotrieve]
  23. Kobe, B., and Deisenhofer, J. (1994) Trends Biochem. Sci. 19, 415-421[CrossRef][Medline] [Order article via Infotrieve]
  24. Kobe, B., and Deisenhofer, J. (1995) Nature 374, 183-186[CrossRef][Medline] [Order article via Infotrieve]
  25. Jiang, X., Dreno, M., Buckler, D., Cheng, S., Ythier, A., Wu, H., Hendrickson, W., and Tayar, N. (1995) Structure 3, 1341-1353[Medline] [Order article via Infotrieve]
  26. Remy, J. J., Couture, L., Pantel, J., Haertle, T., Rabesona, H., Bozon, V., Pajot-Augy, E., Robert, P., Troalen, F., Salesse, R., and Bidart, J. M. (1996) Mol. Cell. Endocrinol. 125, 79-91[CrossRef][Medline] [Order article via Infotrieve]
  27. Puett, D., Bhowmick, N., Fernandez, L. M., Huang, J., Wu, C., and Narayan, P. (1996) Mol. Cell. Endocrinol. 125, 55-64[CrossRef][Medline] [Order article via Infotrieve]
  28. Couture, L., Naharisoa, H., Grebert, D., Remy, J. J., Pajot-Augy, E., Bozon, V., Haertle, T., and Salesse, R. (1996) J. Mol. Endocrinol. 16, 15-25[Abstract]
  29. Bhowmick, N., Huang, J., Puett, D., Isaacs, N. W., and Lapthorn, A. J. (1996) Mol. Endocrinol. 10, 1147-1159[Abstract]


Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.