The Amino-terminal Region of the Luteinizing Hormone/Choriogonadotropin Receptor Contacts Both Subunits of Human Choriogonadotropin

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 α- and β-subunits of hCG. Interestingly, hCGα 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.

The LH 1 /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 125 I-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 crosslinking, and immunofluorescence. Our results show that the Leu 20 -Pro 38 sequence contacts both the ␣and ␤-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
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 ␣and ␤-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, LHR 18 -36 (see Fig. 1), were synthesized by Biosynthesis (Lewisville, TX) and purified on a Vydac C 18 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 LHR 18 -36 corresponds to Gly 18 -Tyr 36 of the LH/CG receptor. In addition, a mutant LHR 18 -36 was synthesized in which Leu 20 , Cys 22 , and Gly 24 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 l of NHS-ABG or sulfo-NHS-AB was added to 30 g of LHR 18 -36 in 40 l 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 Na 125 I-iodine in 10 l of 0.1 M NaOH and 7 l of chloramine T (1 mg/ml) in 10 mM Na 2 HPO 4 (pH 7.4) (PBS). After 20 s, 7 l of sodium metabisulfite (2.5 mg/ml) in PBS was introduced to terminate radioiodination. Derivatized and radioiodinated ABG-125 I-LHR 18 -36 or AB-125 I-LHR 18 -36 solution was mixed with 60 l of 16% sucrose solution in PBS and fractionated on a Sephadex Superfine G-10 column (0.6 ϫ 15 cm) using PBS.
Cross-linking of 125 I-LHR 18 -36 to hCG-Disposable glass tubes were siliconized under dimethyldichlorosilane vapor overnight and autoclaved. In each siliconized tube, 20 l of PBS, hCG (70 ng in 10 l of PBS), and 125 I-LHR 18 -36 (100 ng in 10 l of PBS) were mixed and incubated in 37°C for 90 min. After incubation, 3 l of 0.1 mM SES in Me 2 SO was added to each tube and further incubated at 25°C for 20 min. The cross-linking reaction was terminated by adding 3 l 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 Ϫ75°C for ϳ4 days .
Photoaffinity Labeling of hCG-The following solutions were sequentially introduced to siliconized glass tubes: 20 l of PBS, 10 l of hCG (10 ng/l) in PBS, and 10 l of ABG-125 I-LHR 18 -36 (10 ng/l) in PBS. For labeling with AB-125 I-LHR 18 -36 , 20 l of PBS, 10 l of hCG (20 ng/l) in PBS, and 10 l of AB-125 I-LHR 18 -36 (15 ng/l) 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 Ϫ75°C for ϳ4 days .
Competitive Inhibition of Photoaffinity Labeling of hCG-Competitive inhibition experiments were carried out as described for the photoaffinity labeling experiments, except that 10 l instead of 20 l of PBS was introduced to each tube, and the mixture was incubated with 10 l of increasing concentrations of nonradioactive wild-type or mutant LHR 18 -36 .
Inhibition of 125 I-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 125 I-hCG in the presence of increasing concentrations of nonradioactive wild-type or mutant LHR 18 -36 as described previously (14). After washing the cells several times, the radioactivity associated with the cells was counted to determine the K d value.
Trichloroacetic Acid Precipitation of 125 I-LHR 18 -36 Complexed with hCG-20 l of PBS, 10 l of 150,000 cpm of 125 I-LHR 18 -36 (10 ng/l) in PBS, 10 l of hCG (10 ng/l) in PBS, and 10 l of increasing concentrations of unlabeled LHR 18 -36 in PBS were sequentially introduced to siliconized glass tubes. After incubation at 37°C for 90 min, 5 l 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 ϫ g for 20 min at 4°C, and the radioactivity of the pellet was counted.
FIG. 1. Sequences of LHR peptide mimics. LHR 18 -36 was synthesized, with its sequence corresponding to Gly 18 -Tyr 36 of the LH/CG receptor. In addition, a mutant LHR 18 -36 was synthesized in which Leu 20 , Cys 22 , and Gly 24 were substituted with Ala.

RESULTS
In the preceding article (12), we showed that the amino acid sequence near the N terminus of the LH/CG receptor, particularly Leu 20 -Arg 21 -Cys 22 -Pro 23 -Gly 24 , 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 Gly 18 to Tyr 36 , LHR 18 -36 (Fig. 1), was synthesized and tested for its ability to bind and to affinity label hCG. The sequence of LHR 18 -36 corresponds to Gly 18 -Tyr 36 of the LH/CG receptor. In addition, a mutant LHR 18 -36 was synthesized in which Leu 20 , Cys 22 , and Gly 24 were substituted with Ala.
Cross-linking of 125 I-LHR 18 -36 to hCG-125 I-LHR 18 -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 125 I-LHR 18 -36 /hCG mixture showed that 125 I-LHR 18 -36 was primarily cross-linked to hCG␣, hCG␤, and the hCG␣␤ dimer ( Fig. 2A, lane 6). The positions of hCG␣, hCG␤, and the hCG␣␤ dimer were determined by comparing with the respective positions of 125 I-hCG␣, 125 I-hCG␤, and the crosslinked 125 I-hCG␣␤ dimer on the autoradiograph ( Fig. 2A, lanes  1-3). Without SES, ϳ2% of 125 I-LHR 18 -36 remained associated with hCG␣ 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 125 I-LHR 18 -36 to hCG required SES, hCG, and 125 I-LHR 18 -36 since it did not occur in the absence of any of the three. The extent of crosslinking was dependent on the SES concentration (Fig. 2, B and C), reaching the maximum level at 0.3-1 mM SES. Under this condition, ϳ20% of 125 I-LHR 18 -36 was cross-linked to each hCG␣ and hCG␤. 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 125 I-LHR 18 -36, hCG, and its subunits (16). In conclusion, our results indicate that 125 I-LHR 18 -36 was covalently cross-linked to hCG␣ and hCG␤. Furthermore, the N-terminal amino group of 125 I-LHR 18 -36 , the only amino group of the peptide, was crosslinked to an amino group of either hCG␣ or hCG␤. The distance between the pair of two cross-linked amino groups is expected to be Ͻ13 Å.
Specificity of Cross-linking of LHR 18 -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 125 I-LHR 18 -36 while maintaining hCG at a constant concentration (Fig. 3A). Conversely, 125 I-LHR 18 -36 and hCG were cross-linked at increasing concentrations of hCG and a constant concentration of 125 I-LHR 18 -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 Leu 20 , Cys 22 , or Gly 24 of the receptor resulted in the loss of hCG binding by the receptor.
Therefore, a mutant LHR 18 -36 was synthesized in which Leu 20 , Cys 22 , and Gly 24 were substituted with Ala (Fig. 1). As expected, mutant LHR 18 -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 LHR 18 -36 was caused by a lack of mutant LHR 18 -36 binding to hCG or by a lack of a cross-linking reaction by SES due to the putative steric hindrance even though mutant LHR 18 -36 successfully bound to hCG. To distinguish these possibilities, 125 I-LHR 18 -36 was cross-linked to hCG in the presence of increasing concentrations of unlabeled wild-type LHR 18 -36 (Fig. 4A) and mutant LHR 18 -36 (Fig. 4B). Wild-type LHR 18 -36 attenuated the crosslinking, but mutant LHR 18 -36 was significantly less effective (Fig. 4C), an indication of the less efficient binding of mutant LHR 18 -36 to hCG.
Affinity of LHR 18 -36 Binding to hCG-To determine the binding affinity, 125 I-LHR 18 -36 was incubated with hCG in the presence of increasing concentrations of unlabeled LHR 18 -36 (Fig. 5A). Unlabeled LHR 18 -36 inhibited 125 I-LHR 18 -36 binding to hCG, with a K d value of 29 M. It is not clear whether this inhibition was caused by competitive binding of LHR 18 -36 and the receptor to hCG or by a putative allosteric effect of LHR 18 -36 binding to hCG. To examine these possibilities and the relevance of the inhibition of hCG binding to the LH/CG receptor, 125 I-hCG was incubated with a 293 cell line stably expressing the receptor (14) in the presence of increasing concentrations of unlabeled LHR 18 -36 (Fig. 5B). Unlabeled wildtype LHR 18 -36 , but not mutant LHR 18 -36 , competitively attenuated binding of 125 I-hCG to the receptor, with a K d value of 25 M. This result indicates that the interaction of LHR 18 -36 and hCG is specific and simulates the interaction between hCG and the receptor. Furthermore, the two independent K d values (29 and 25 M) are not only close, but are also similar to the value of 15 M for the inhibition of 125 I-hCG binding to the receptor by LHR   (10).
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 125 I-LHR 18 -36 and the hCG dimer. This suggests that a minor population of the 125 I-LHR 18 -36 ⅐hCG 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 125 I-LHR 18 -36 and hCG. A simple way to reduce or eliminate such random collisional cross-links is photoaffinity labeling (16).
To photoaffinity label hCG with 125 I-LHR 18 -36 , the receptor peptide was derivatized with either AB or ABG (13) to produce AB-125 I-LHR 18 -36 or ABG-125 I-LHR 18 -36 , respectively. When these peptides bind to hCG and are irradiated with UV, crosslinking will be restricted between 125 I-LHR 18 -36 and hCG␣ or between 125 I-LHR 18 -36 and hCG␤. 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-125 I-LHR 18 -36 and ABG-125 I-LHR 18 -36 were capable of photoaffinity labeling either hCG␣ or hCG␤, but not both subunits at the same time to produce the labeled hCG␣␤ complex. Interestingly, AB-125 I-LHR 18 -36 labeled them more efficiently than ABG-125 I-LHR 18 -36 (Fig. 6, C and D). In addition, hCG␣ was more preferentially labeled than hCG␤. This result is consistent with the SES cross-linking results. One possible explanation is that the N terminus of the LHR 18 -36 derivatives is Ͻ7 Å from the hCG ␣and ␤-subunits and that the peptide deriva-tives are bound closer to ␣ than ␤. The labeling required UV irradiation and was dependent on the irradiation time, reaching the maximum labeling after ϳ1 and 0.5 min of irradiation of the ABG-125 I-LHR 18 -36 ⅐hCG and AB-125 I-LHR 18 -36 ⅐hCG 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 hCG␣ 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.
Concentration Effect of hCG and Peptide Derivatives-When a constant amount of hCG was incubated with increasing concentrations of AB-125 I-LHR 18 -36 or ABG-125 I-LHR 18 -36 , the intensity of labeled hCG ␣and ␤-bands gradually increased and plateaued (Fig. 7). A similar result was obtained in a converse experiment when a constant concentration of AB-125 I-LHR 18 -36 or ABG-125 I-LHR 18 -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 hCG␣ more than hCG␤, an indication of a labeling specificity.
Competitive Inhibition of Photoaffinity Labeling by Nonderivatized Peptides-A nonderivatized peptide is expected to displace specific labeling. Therefore, hCG was incubated with AB-125 I-LHR 18 -36 or ABG-125 I-LHR 18 -36 in the presence of increasing concentrations of nonderivatized peptide (Fig. 9, A  and D). Increasing concentrations of LHR 18 -36 inhibited photoaffinity labeling in a dose-dependent manner and eventually completely blocked it. These results indicate the specificity of LHR 18 -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 LHR 18 -36 failed to significantly block the photoaffinity labeling of hCG by AB-125 I-LHR 18 -36 or ABG-125 I-LHR 18 -36 (Fig. 9, B and E). Only at the highest concentrations of the mutant peptide was label- ing by AB-125 I-LHR 18 -36 slightly reduced. Although these results indicate the labeling specificity of AB-125 I-LHR 18 -36 and ABG-125 I-LHR 18 -36 , the futile inhibition could be interpreted as the mutant peptide binding to a site in hCG different from the AB-125 I-LHR 18 -36 -or ABG-125 I-LHR 18 -36 -binding site. To test this hypothesis, mutant LHR 18 -36 was derivatized and radioiodinated to prepare AB-125 I-mutant LHR 18 -36 and ABG-125 I-mutant LHR 18 -36 .
Photoaffinity Labeling by Mutant LHR 18 -36 -As shown in Fig. 10, AB-125 I-mutant LHR 18 -36 and ABG-125 I-mutant LHR 18 -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 LHR 18 -36 slightly attenuated the labeling by AB-125 I-LHR 18 -36 . In addition, AB-125 I-LHR 18 -36 and ABG-125 I-LHR 18 -36 did not photoaffinity label denatured hCG that was boiled in 8 M urea and did not bind to the receptor (data not shown). DISCUSSION Our results show that AB-125 I-LHR 18 -36 and ABG-125 I-LHR 18 -36 photoaffinity label hCG. The ␣-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 125 I-LHR 18 -36 concentration, and UV exposure. AB-125 I-LHR 18 -36 and ABG-125 I-LHR 18 -36 photoaffinity label bioactive hCG, but not denatured hCG. This labeling is blocked by nonderivatized wild-type LHR 18 -36 , but not by nonderivatized mutant LHR 18 -36 . Furthermore, AB-125 I-mutant LHR 18 -36 and ABG-125 I-mutant LHR 18 -36 do not photoaffinity label bioactive hCG and denatured hCG.
Both subunits of hCG are labeled, indicating that the UVactivable group coupled to LHR 18 -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, hCG␣ was preferentially labeled. Since only one photosensitive group is attached to the N terminus of each derivatized peptide, AB-125 I-LHR 18 -36 and ABG-125 I-LHR 18 -36 bound to hCG can photoaffinity label only one, but not both, of the subunits. Obviously, the reagent more readily reaches and labels the ␣-subunit than the ␤-subunit.
Since the maximum labeling distances of AB and ABG are 7 and 10 Å, respectively (17), and AB-125 I-LHR 18 -36 labels hCG more efficiently than ABG-125 I-LHR 18 -36 , the N terminus of the LHR 18 -36 derivatives is Ͻ7 Å from both subunits. Therefore, both subunits of hCG are likely to contact the LHR 18 -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, LHR 18 -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)(26)(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 ␤-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 Gly 18 -Tyr 36 sequence of the receptor reaches the opposite face of the seat belt side (Fig. 11). In the Gly 18 -Tyr 36 sequence, the alternate Leu 20 , Cys 22 , and Gly 24 residues are crucial for hormone binding (12). These three residues appear to be at one side of a ␤-like structure and could face the hormone and provide a direct contact site.
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.