Hormone interactions to Leu-rich repeats in the gonadotropin receptors. I. Analysis of Leu-rich repeats of human luteinizing hormone/chorionic gonadotropin receptor and follicle-stimulating hormone receptor.

The luteinizing hormone receptor (LHR) and follicle-stimulating hormone receptor (FSHR) have an approximately 350-amino acid-long, N-terminal extracellular exodomain. This exodomain binds hormone with high affinity and specificity and contains eight to nine putative Leu-rich repeat (LRR) sequences. LRRs are known to assume the horseshoe structure in ribonuclease inhibitors, and the inner lining of the horseshoe consists of the beta-stranded Leu/Ile-X-Leu/Ile motif. In the case of ribonuclease inhibitors, these beta strands interact with ribonuclease. However, it is unclear whether the putative LRRs of LHR and FSHR play any role in the structure and function. In this work, the beta-stranded Leu/Ile residues in all LRRs of the human LHR and FSHR were Ala-scanned and characterized. In addition, the 23 residues around LRR2 of LHR were Ala-scanned. The results show that beta-stranded Leu and Ile residues in all LRRs are important but not equally. These Leu/Ile-X-Leu/Ile motifs appear to form the hydrophobic core of the LRR loop, crucial for the LRR structure. Interestingly, the hot spots are primarily in the upstream and downstream LRRs of the LHR exodomain, whereas important LRRs spread throughout the FSHR exodomain. This may explain the distinct hormone specificity despite the structural similarity of the two receptors.

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 ϳ350-amino acid-long exodomain has high affinity hormone contact sites (3)(4)(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 ␤ strand connected to parallel ␣ helices. The ␤ 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 ␤ strand, in LRR4 with hCG, in particular with the hCG␣ 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.
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 l of NHS-AB was added to 30 g of LHR 96 -115 in 40 l 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 Na 125 I in 10 l of 0.1 M NaOH and 7 l of chloramine T (1 mg/ml) in 10 mM Na 2 HPO 4 and 0.9% NaCl (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 AB-125 I-LHR 96 -115 solution was mixed with 60 l of 16% sucrose solution in PBS and fractionated on Sephadex Superfine G-10 column (0.6 ϫ 15 cm) using PBS.
Affinity Cross-linking of 125 I-LHR 96 -115 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 PBS), and 125 I-LHR 96 -115 (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 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 l 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 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 AB-125 I-LHR 96 -115 (10 ng/l) 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 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 96 -115 .
Inhibition of 125 I-hCG binding to LHR-A human embryonic kidney 293 cell line stably expressing human LHR was incubated with 100,000 cpm of 125 I-hCG in the presence of increasing concentrations of nonradioactive wild type or mutant LHR 96 -115 peptides as described previously (15). After several times washing the cells, the radioactivity associated with the cells was counted, and percent bound 125 I-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 K d value following the Scatchard equation (16).

RESULTS
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 ␤-stranded Leu 103 -Ile 105 , LHR 96 -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, 125 I-LHR 96 -115 incubated with hCG, and the resulting 125 I-LHR 96 -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, 125 I-LHR 96 -115 was derivatized with AB, an UV-activable reagent, to produce AB-125 I-LHR 96 -115 and incubated with hCG. The resulting 125 I-LHR 96 -115 -hCG complex was irradiated with UV to photoaffinity label hCG with AB-125 I-LHR 96 -115 . The advantages and disadvantages of both methods will be discussed later.
To determine whether AB-125 I-LHR 96 -115 and 125 I-LHR 96 -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-125 I-LHR 96 -115 and 125 I-LHR 96 -115 labeled both the ␣ and ␤ subunits in hCG (Fig. 1). In addition, the hCG ␣␤ dimer was cross-linked and labeled with 125 I-LHR 96 -115 when the 125 I-LHR 96 -115 -hCG complex was treated with SES. The positions of hCG␣, hCG␤, and the hCG␣␤ dimer were determined by comparing the respective positions of 125 I-hCG␣, 125 I-hCG␤, and the cross-linked 125 I-hCG ␣␤ dimer on the autoradiograph (Fig. 1, lanes 1 and 5).
Cross-linking of 125 I-LHR 96 -115 to hCG-As a first step to determine the specificity of affinity labeling, 125 I-LHR 96 -115 was incubated with hCG and treated with increasing concentrations of SES. Electrophoresis of the treated hCG/ 125 I-LHR 96 -115 mixture ( Fig. 2A) shows that 125 I-LHR 96 -115 was cross-linked to hCG␣, hCG␤, and the hCG␣␤ 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, ϳ20% of 125 I-LHR 96 -115 was cross-linked to hCG␣ and ϳ10% to hCG␤. 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 125 I-LHR 96 -115 , hCG, and its subunits (18). In conclusion, our results indicate that 125 I-LHR 96 -115 was covalently crosslinked to hCG␣ and hCG␤. Furthermore, either or both amino groups of Lys 101 and Lys 112 of 125 I-LHR 96 -115 , the only amino groups of the peptide, were cross-linked to an amino group(s) of either hCG␣ or hCG␤. The distance between the pair of two cross-linked amino groups is expected to be Ͻ13 Å.
Saturable Cross-linking of 125 I-LHR 96 -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 125 I-LHR 96 -115 while maintaining hCG at a constant concentration (Fig. 2B). Conversely, 125 I-LHR 96 -115 and hCG were cross-linked at increasing concentrations of hCG and a constant concentration of 125 I-LHR 96 -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 125 I-LHR 96 -115 and the hCG dimer. They suggest that a minor population of the 125 I-LHR 96 -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 crosslinks between 125 I-LHR 96 -115 and hCG. A simple way to reduce or eliminate such random collisional cross-links is photoaffinity labeling (18). To photoaffinity label hCG with 125 I-LHR 96 -115 , the receptor peptide was derivatized with AB to produce AB-125 I-LHR 96 -115 . When the derivatized peptide binds to hCG and is irradiated with UV, the cross-link will be restricted between 125 I-LHR 96 -115 and hCG␣ or between 125 I-LHR 96 -115 and hCG␤. 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 crosslinking 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-125 I-LHR 96 -115 was capable of photoaffinity labeling either hCG␣ or hCG␤ but not both subunits at the same time. The labeling is generally confined to hCG␣ with the labeling of hCG␤ being faint. This result is consistent with the SES cross-linking results. One possible explanation is that the peptide is bound closer to ␣ than ␤. The labeling required UV irradiation and was dependent on the irradiation time, reaching the maximum labeling after 30ϳ60-s irradiation. This UV dependence clearly indicates photoaffinity labeling. In addition, the preferential labeling of hCG␣ 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-125 I-LHR 96 -115 was incubated with increasing concentrations of hCG, the intensity of labeled hCG␣ and ␤ 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-125 I-LHR 96 -115 (Fig. 2F). These results indicate that the photoaf-finity labeling is dependent on both 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.
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 Leu 103 3 Ala or Ile 105 3 Ala substitution in LHR abrogated hormone binding. Therefore, Leu 103 and Ile 105 were substituted with Ala in LHR 96 -115 to produce a mutant peptide, LHR 96 -115(L103A/I105A) . To test whether the wild type and mutant LHR peptides could inhibit affinity labeling, hCG was incubated with AB-125 I-LHR 96 -115 in the presence of increasing concentrations of nonderivatized wild type peptide (Fig. 3A) and nonderivatized mutant peptide (Fig. 3C). Increasing concentrations of LHR 96 -115 inhibited photoaffinity labeling in a dose-dependent manner and eventually, completely blocked it. These results indicate the specificity of LHR 96 -115 for the photoaffinity labeling. In contrast, the inhibition by mutant LHR 96 -115(L103A/I105A) was significantly less effective (Fig. 3C). Similar results were obtained with affinity cross-linking of 125 I-LHR 96 -115 to hCG (Fig. 3, B, D, and F). 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 Ϫ75°C for ϳ4 days. The bar graphs show the percent radioactivity of the ␣ band and the ␤ band in a gel lane. finities were significantly low. These results are consistent with the observation that the highest concentrations of nonderivatized LHR 96 -115(L103A/I105A) slightly attenuated the labeling by AB-125 I-LHR 96 -115 and 125 I-LHR 96 -115 .
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 125 I-hCG binding to the receptor on intact cells. For the first test, denatured hCG was incubated with increasing concentrations of AB-125 I-LHR 96 -115 or 125 I-LHR 96 -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 125 I-LHR 96 -115 to denatured hCG, 125 I-LHR 96 -115 appears to have a difficulty to recognize denatured hCG. To test this possibility, 125 I-hCG was incubated with intact cells expressing LHR in the presence of increasing concentrations of the wild type or mutant peptide, LHR 96 -115 or LHR 96 -115(L103A/I105A) (Fig. 6). The wild type LHR 96 -115 inhibited 125 I-hCG binding to the receptor with a K d value of 43.4 M, suggesting its binding to the receptor with a reasonable affinity for a peptide (20,21). In contrast, the K d value of mutant LHR 96 -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 LHR 96 -115 to hCG. Furthermore, the results show that the interaction between hCG and LHR 96 -115 simulates the interaction between hCG and the receptor.
Affinity Labeling Site-LHR 96 -115 has two Lys residues, Lys 101 and Lys 112 , 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 LHR 96 -115 to produce LHR 96 -115(K101A) and LHR 96 -115(K112A) . These two peptides were capable of inhibiting 125 I-hCG binding to the receptor on intact cells, but their K d values were 12-15-fold higher than the corresponding K d value of the wild type LHR 96 -115 (Fig. 6). The result is consistent with the effect of Ala substitution for Lys 101 or Lys 112 in intact receptor on hCG binding. The K d value for hCG binding of LHR increased by 2.6 -3.4-fold when Lys 101 or Lys 112 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-125 I-LHR 96 -115 and affinity cross-linking of 125 I-LHR 96 -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-125 I-LHR 96 -115(K101A) and AB-125 I-LHR 96 -115(K112A) (Fig. 9). AB-125 I-LHR 96 -115(K112A) photoaffinity labeled hCG similar to the photoaffinity labeling of hCG by AB-125 I-LHR 96 -115 , whereas the labeling of hCG with AB-125 I-LHR 96 -115(K101A) was less (Fig. 9A). This result indicates that the photoaffinity labeling is significantly more effective when AB is attached to Lys 101 than to Lys 112 .
If this is true, one would expect the same trend with affinity cross-linking using the two peptides. Indeed, 125 I-   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. LHR 96 -115(K112A) was cross-linked to hCG with SES significantly better than 125 I-LHR 96 -115(K101A) (Fig. 9B). However, neither of the derivatized peptides labeled denatured hCG, indicating a specificity of affinity labeling of hCG by AB-125 I-LHR 96 -115(K112A) and 125 I-LHR 96 -115(K112A) (data not shown). Taken together, these results indicate that Lys 101 is more suitable for affinity labeling hCG than Lys 112 is. They also suggest that Lys 101 is at or near the hCG contact point as suggested by the computer model that the short ␤ strand is a ligand contact site, and the Lys 101 is projected toward ligand (Fig. 7). In contrast, Lys 112 is located near the ␣ helix as part of the outer lining of the donut structure, at the opposite side from the ligand binding site. Since Lys 101 is in the N-terminal area of LHR 96 -115 , whereas Lys 112 is in the C-terminal region, one way to verify the conclusion is to use peptide mimics covering the sequences upstream and downstream of LHR 96 -115 . To this end, we synthesized two peptide mimics, LHR 84 -104 and LHR 113-132 , and tested them for their ability to inhibit photoaffinity labeling of hCG by AB-125 I-LHR 96 -115 and affinity crosslinking of 125 I-LHR 96 -115 to hCG (Fig. 10). LHR 84 -104 and LHR 113-132 inhibited the affinity labeling of hCG, but their potency was less than that of LHR 96 -115 . LHR 84 -104 was more effective in inhibiting hCG␣ than LHR 113-132 was. On the other hand, LHR85-104 was similar to LHR 113-132 in inhibiting the labeling of hCG␤.
Interaction of LHR 96 -115 -hCG Complex with Exoloops-In the preceding article (13), we pointed out the absolute homology in the 8 residues (boldface) in the Leu 98 -Pro-Gly-Leu-Lys-Tyr-Leu-Ser-Ile-Cys-Asn-Thr-Gly 109 sequence among cloned LHR, follicle-stimulating hormone receptor, and thyroid-stim- ulating hormone receptor of various species. Furthermore, we showed that the tandem three conserved residues, Asn 107 -Thr-Gly 109 , 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)(23)(24)(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 (LHR exo1 , LHR exo2 , and LHR exo3 ) and tested whether they could inhibit the photoaffinity labeling of hCG by AB-125 I-LHR 96 -115 (Fig. 11). LHR exo2 effectively inhibited the photoaffinity labeling, whereas the inhibition by LHR exo1 was less. In contrast, LHR exo3 did not inhibit the labeling. These differential effects suggest the specificity of the inhibition. DISCUSSION Our results show that AB-125 I-LHR 96 -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 125 I-LHR 96 -115 concentration, and UV activation. AB-125 I-LHR 96 -115 photoaffinity labels bioactive hCG but not denatured hCG. This labeling is blocked by nonderivatized wild type LHR 96 -115 but not by nonderivatized mutant LHR 96 -115(L103A/I105A) . The same Ala mutations in LHR abolish the hCG binding activity of LHR. Furthermore, AB-125 I-LHR 96 -115(L103A/I105A) does not photoaffinity label bioactive hCG and denatured hCG. LHR 96 -115 inhibits 125 I-hCG binding to the receptor expressed on intact cells but LHR 96 -115(L103A/I105A) is not capable of inhibiting 125 I-hCG binding to the receptor. To avoid the potential interference of the photoactivable group on binding of AB-125 I-LHR 96 -115 to the receptor and the subsequent labeling, 125 I-LHR 96 -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 UVactivable group coupled to AB-125 I-LHR 96 -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, hCG␣ was preferentially labeled. Obviously, the reagent more readily reaches and labels the ␣ subunit than the ␤ subunit. Since the maximum labeling distances of AB is 7 Å (19), hCG␣ is likely to contact AB-125 I-LHR 96 -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  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 hCG␣ (A) and hCG␤ (B) and affinity cross-linking to hCG␣ (C) and hCG␤ (D). In addition, the inhibition of unlabeled wild type LHR 96 -115 was presented for comparison. LHR 96 -115 inhibits hCG binding to the receptor, AB-125 I-LHR 96 -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 hCG␣␤ subunits, but not both, is labeled, although two AB could be attached to the two Lys residues of LHR 96 -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 Lys 101 is capable of labeling hCG, whereas the AB attached to Lys 112 is less effective. This is strong evidence to support the orientation of Lys 101 and Lys 112 in the LRR4 loop model (Fig. 7) and implicates the N-terminal region of LHR 96 -113 , including the putative ␤ 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)(33)(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 ␤ sheets of the repeats and is thought to be the ligand contact site (8,31,32), perhaps interacting with the putative receptor binding ␣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 ␤ 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 FIG. 10. Roles of peptides flanking LHR 96 -115 on affinity labeling. hCG was photoaffinity-labeled with AB-125 I-LHR 96 -115 and affinity-cross-linked to 125 I-LHR 96 -115 with SES in the presence of increasing concentrations of LHR 96 -115 , LHR 85-104 , or LHR 113-132 as described in the legend to Fig. 3. After processing the samples, the percent labeling of hCG␣ and hCG␤ was determined. The results are presented for the inhibition of photoaffinity labeling of hCG␣ (A) and hCG␤ (B) and the inhibition of affinity crosslinking of hCG␣ (C) and hCG␤ (D).