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

The luteinizing hormone/choriogonadotropin receptor is a seven-transmembrane receptor. Unlike most seven-transmembrane receptors, it is composed of two halves of equal size, the N-terminal extracellular exodomain and the C-terminal membrane-associated endodomain. The exodomain is exclusively responsible for high affinity hormone binding, whereas receptor activation occurs only in the endodomain. This mutually exclusive physical separation of the two functional domains sets the lutropin receptor and its subfamily of receptors apart from all other seven-transmembrane receptors. The mechanisms of hormone binding and receptor activation also appear to be different from those of other receptors in that binding occurs in at least two steps. However, the precise hormone contact sites in the exodomain are unknown. To determine the hormone/receptor contact sites, we have examined the receptor using progressive truncation from the C terminus, Ala scanning, immunofluorescence microscopy, and antibody binding. Progressive truncation from the C terminus of the receptor indicates several discrete regions that impact hormone binding. These regions are around the boundaries of exons 1–2, 4–5, 6–7, and 9–10. Ala scanning of the Asp17–Arg26 region near the exon 1–2 junction uncovered three alternating residues (Leu20, Cys22, and Gly24) crucial for hormone binding. Ala substitution for any one of these residues abolished hormone binding, although the resulting mutant receptors were successfully expressed on the cell surface. In contrast, Ala substitution for their flanking and intervening residues did not impair hormone binding. These results and the data in the accompanying article (Phang, T., Kundu, G., Hong, S., Ji, I., and Ji, T. (1998)J. Biol. Chem. 273, 13841–13847) indicate that this region directly contacts the hormone and suggest a novel mode of embracing the hormone.

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 extracellu-lar 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 hormone 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. Despite the importance, only limited information is available concerning the precise hormone contact residues and sites in the exodomain. Roche et al. (10) found that 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. More recently, Segaloff and co-workers (11) observed that receptors lacking any of several discrete exodomain sequences, the 11 N-terminal residues and leucine-rich motifs 1-6, were trapped in cells and failed to bind hCG.
In this work and the accompanying 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 microscopy. Our results show that the Leu 20 -Pro 38 sequence near the exon 1-2 junction contacts both the ␣and ␤-subunits of hCG. In addition, another three sequences near the junctions of exons 4 -5, 6 -7, and 9 -10 influence hormone binding.

Mutagenesis and Functional Expression of LH/CG Receptors-Mu-
tant LH/CG receptor cDNAs were prepared in the pSELECT vector using the Altered Sites Mutagenesis system (Promega), sequenced, subcloned into pcDNA3 (Invitrogen) as described (13), and sequenced again to verify mutation sequences. This procedure does not involve polymerase chain reaction. To produce truncated receptors, a stop codon was introduced at the 3Ј-end of the exons. Mutant and truncated LH/CG receptor constructs were transfected into human embryonic kidney 293 cells by the calcium phosphate method. Stable cell lines were established in minimal essential medium containing 10% horse serum and 500 g/ml G418. These cells were used for hormone binding, cAMP production, antibody binding, and fluorescence microscopy. All assays were carried out in duplicate and repeated four to five times. Means Ϯ S.D. were calculated. 125 I-hCG Binding and Intracellular cAMP Assay-Stable cells were assayed for 125 I-hCG binding in the presence of 150,000 cpm of 125 I-hCG (14) and increasing concentrations of unlabeled hCG. The K d values were determined by Scatchard plots. hCG (batch CR 127) was supplied by the National Hormone and Pituitary Program. For intracellular cAMP assay, cells were washed twice with Dulbecco's modified Eagle's medium and incubated in the medium containing isobutylmethylxanthine (0.1 g/ml) for 15 min. Increasing concentrations of hCG were then added, and incubation was continued for 45 min at 37°C. After removing the medium, the cells were rinsed once with fresh medium without isobutylmethylxanthine, lysed in 70% ethanol, freeze-thawed in liquid nitrogen, and scraped. After pelleting cell debris at 16,000 ϫ g for 10 min at 4°C, the supernatant was collected, dried under vacuum, and resuspended in 10 l of cAMP assay buffer (Amersham Pharmacia Biotech). cAMP concentrations were determined with a 125 I-cAMP assay kit (Amersham Pharmacia Biotech) following the manufacturer's instructions and validated for use in our laboratory.
125 I-hCG Binding to Solubilized LH/CG Receptor-Transfected cells were washed twice with ice-cold 150 mM NaCl and 20 mM HEPES, pH 7.4 (buffer A). Cells were scraped on ice, collected in buffer A containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 5 mM N-ethylmaleimide, and 10 mM EDTA), and pelleted by centrifugation at 1300 ϫ g for 10 min. Cells from a 10-cm plate were resuspended in 0.6 ml of buffer A containing 1% Nonidet P-40, 20% glycerol, and the above protease inhibitors; incubated on ice for 15 min; and diluted with 5.4 ml of buffer A containing 20% glycerol plus the protease inhibitors. The mixture was centrifuged at 100,000 ϫ g for 60 min. The supernatant (500 l) was mixed with 150,000 cpm of 125 I-hCG and 6.5 l of 0.9% NaCl and 10 mM Na 2 HPO 4 , pH 7.4, containing increasing concentrations of unlabeled hCG. After incubation at 4°C for 12 h, the solution was thoroughly mixed with 250 l of buffer A containing bovine ␥-globulin (5 g/ml) and 750 l of buffer A containing 20% polyethylene glycol 8000. After incubation at 4°C for 10 min, samples were pelleted at 1300 ϫ g for 30 min, and supernatants were removed. Pellets were resuspended in 1.5 ml of buffer A containing 20% polyethylene glycol 8000, centrifuged, and counted for radioactivity.
Immunofluorescence Microscopy-For fluorescence labeling of LH/CG receptors, the Flag epitope (15), Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (5Ј-GAC TAC AAG GAC GAT GAC GAT AAG-3Ј), was inserted between the C terminus (Ser 26 ) of the signal sequence and the N terminus (Arg 27 ) of mature receptors. The Flag epitope (16) has successfully been used as a marker to identify, trace, and purify recombinant proteins carrying the tag without significantly impairing their biological activities (17,18). For labeling intact cells, cells were cultured on coverslips in 6-well plates for 2 days and fixed with 4% formaldehyde in PBS for 10 min at 25°C. For labeling permeabilized cells, cells were fixed with 4% formaldehyde in PBS for 5 min at 4°C and treated with 0.1% Triton X-100 in PBS for 5 min at 4°C. Fixed intact or permeabilized cells were washed five times with PBS for 2.5 min each at 25°C. They were sequentially treated with 0.4% type IV gelatin isolated from calf skin (Sigma) in modified Eagle's medium free of phenol red for 10 min at 25°C and then with 5% goat serum and 1% fetal calf serum in the same medium for 20 min at 25°C. The treated cells were incubated with 500 l/well primary antibody solution (25 g of mouse anti-Flag antibody in 1 ml of modified Eagle's medium containing 5% goat serum and 1% fetal calf serum) for 2 h at 37°C. The cells were washed three times with PBS for 2.5 min each at 25°C and treated with a 400-fold dilution of Texas Red-conjugated goat anti-mouse IgG (Molecular Probes, Inc.). Finally, the cells were washed with PBS six times for 2.5 min each at 25°C. The coverslip containing processed cells was mounted on slide glass using 50% glycerol in PBS and sealed using nail polish. Specimens were examined under a Leica TCS-4D laser scanning confocal microscope equipped with Scanware analysis software. Entire experiments were completed in 1 day to prevent increasing background fluorescence.
Radioimmunoassay for Flag-LH/CG Receptors-Mouse anti-Flag monoclonal antibody M2 (Eastman Kodak Co.) was iodinated with 125 I according to the published procedure for radioiodination of hCG (14), and 125 I-anti-Flag antibodies were purified on a Sephadex G-150 column. Binding of 125 I-anti-Flag antibodies to 293 cells expressing Flag-LH receptors was carried out according to the 125 I-hCG binding assay described above.
Interestingly, K d values for the fragments increased stepwise in groups rather than continuously as the C terminus was progressively truncated (Fig. 1C). Truncation of exon 11 to produce the exon 1-10 fragment slightly reduced the K d value compared with that of the wild type. In contrast, the exon 1-9 fragment, resulting from truncation of exons 10 and 11, showed FIG. 1. hCG binding to exodomain fragments. The 11 exons of the LH/CG receptor were progressively truncated from the C terminus to produce receptor fragments consisting of exons 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, and 1 (D). These exon fragments were stably expressed in 293 cells, solubilized in Nonidet P-40, and assayed for 125 I-hCG binding in the presence of increasing concentrations of unlabeled hCG. The results are presented as displacement of 125 I-hCG binding (A) and in a Scatchard plot (B). Experiments were repeated four to five times in duplicate, and means Ϯ S.D. were calculated (C). Nontransfected cells did not show specific binding of hCG. WT, wild type; NS, not significant. Arrows indicate regions that influence hormone binding. a ϳ20-fold higher K d value. As the C terminus was successively truncated, the K d values increased in several discrete steps. This result suggests that the ultimate effects of the truncated exons on hCG binding are not equal. There are four potentially important regions around the junctions between exons 1 and 2, 4 and 5, 6 and 7, and 9 and 10 ( Fig. 1D). Among the four, the region near the exon 1-2 junction appears to be most crucial for hCG binding. Therefore, the region was further investigated by Ala scanning.
Ala Scanning of the Asp 17 -Arg 26 Sequence-As a first step to identify important residues near the exon 1-2 junction, Asp 17 -Gly 18 -Ala 19 -Leu 20 -Arg 21 -Cys 22 -Pro 23 -Gly 24 -Pro 25 -Arg 26 was Ala-scanned (Fig. 2). Ala substitution for Arg 21 , Pro 23 , Pro 25 , or Arg 26 did not impair hCG binding to intact cells but increased the K d values approximately 5-6-fold (Fig. 2, A and B). All of the mutant receptors that bound hCG on intact cells were capable of inducing cAMP production (Fig. 2C). Their EC 50 values for cAMP induction were similar to the wild type value except, the R26A mutant, which has a 2.4-fold higher EC 50 value (Fig. 2, table). In contrast, Ala substitution for Asp 17 , Gly 18 , Leu 20 , Cys 22 , or Gly 24 impaired hCG binding to intact cells. EC 50 values for cAMP production are generally lower than K d values of the corresponding receptors. This is thought to be due to the fact that receptors are activated before they are fully occupied by hormone. Sometimes, it happens when Ͻ5% of receptors are occupied. Furthermore, the maximum receptor activation is reached long before receptors are fully occupied.
Hormone Binding of Solubilized Receptors-To determine whether the non-binding mutants are defective in hCG binding or trapped in the cytoplasm, cells expressing the mutants were solubilized in Nonidet P-40 and assayed for hCG binding (Fig.  3). hCG bound to the D17A and G18A mutants. Their K d values are comparable to those of the R21A, P23A, P25A, and R26A mutants, but ϳ5-8-fold higher than the wild type value. This result suggests that the D17A and G18A mutants are trapped in cells. In contrast to these binding-competent mutants, the L20A, C22A, and G24A mutants failed to bind hCG. Therefore, the L20A, C22A, and G24A mutants either were not synthesized or were synthesized but incapable of binding hormone. Even if they were expressed, our data shown in Figs. 2 and 3 could not pinpoint whether the mutants were located either on the cell surface or within the cells. To distinguish these possibilities, we utilized two independent immunological methods, immunofluorescence microscopy and 125 I-antibody that should bind to receptors expressed on the cell surface and in cells.
Immunofluorescence Microscopy-For immunological studies, the Flag epitope (16) was inserted between the C terminus of the signal sequence and the N terminus of mature receptors. The resulting receptors are the Flag-wild type LH/CG receptor, Flag-LH/CG-R D17A , Flag-LH/CG-R G18A , Flag-LH/CG-R L20A , Flag-LH/CG-R C22A , and Flag-LH/CG-R G24A . Cells were transfected with the plasmids encoding receptors carrying the Flag epitope. They were either examined intact or after treatment  19 , of the LH/CG receptor were individually substituted with Ala, and the resulting mutant receptors were stably expressed in 293 cells. Intact cells were used for 125 I-hCG binding in the presence of increasing concentrations of unlabeled hCG (A and B) and for cAMP production (C). Experiments were repeated four to five times in duplicate as described in the legend to Fig. 1. As a control to verify intended mutations and to identify unintended changes, if any, in the cDNA sequence, mutant receptor cDNAs were reverted to the wild type, and their sequences were confirmed. The revertant cDNAs were used to transfect cells and were assayed for hormone binding and cAMP induction. All revertants behaved the same as the wild type (data not shown). NS, not significant.
with Triton X-100 to permeabilize the plasma membrane and to allow the antibody to enter the cytosol. The cells were treated sequentially with mouse anti-Flag and Texas Red-conjugated goat anti-mouse IgG monoclonal antibodies. Confocal laser fluorescence microscopy showed bright fluorescence of the Flag-wild type receptor on intact cells and in permeabilized cells (Fig. 4). Cells expressing the wild type receptor lacking the Flag tag did not show fluorescence, regardless of permeabilization. In addition, the cells expressing the Flag-wild type receptor did not show fluorescence when treated for fluorescence labeling without anti-Flag antibody. These controls demonstrate that the fluorescence staining is specific for the Flag epitope and that the Flag-wild type receptor is expressed both on the cell surface and within cells. Flag-LH/CG-R L20A , Flag-LH/CG-R C22A , and Flag-LH/CG-R G24A were also observed on intact and permeabilized cells, indicating they were expressed on the cell surface and within cells. On the other hand, Flag-LH/CG-R D17A and Flag-LH/CG-R G18A were observed in permeabilized cells, but not intact cells, indicating that they were not transported to the cell surface.
125 I-Anti-Flag Antibody Binding to Intact Cells-125 I-Anti-Flag antibody was used to verify the surface expression of receptors. Fig. 5 shows that 125 I-anti-Flag antibody bound to cells expressing the Flag-wild type receptor, Flag-LH/CG-R L20A , Flag-LH/CG-R C22A , or Flag-LH/CG-R G24A , but not to those expressing Flag-LH/CG-R D17A or Flag-LH/CG-R G18A . The K d values were 29, 96, 106, and 135 nM for the Flag-wild type receptor, Flag-LH/CG-R L20A , Flag-LH/CG-R C22A , and Flag-LH/CG-R G24A , respectively. The numbers of binding sites on the cell surface (27,000 and 187,000 per cell) are significant, and therefore, these bindings are likely to be specific. Taken together, these and the fluorescence microscopy results demonstrate that the Flag-wild type receptor, Flag-LH/CG-R L20A , Flag-LH/CG-R C22A , and Flag-LH/CG-R G24A were expressed on the cell surface, whereas Flag-LH/CG-R D17A and Flag-LH/CG-R G18A were trapped in the cells.
Activities of Flag-tagged Receptors-To test whether the Flag epitope might have interfered with normal processing so that the mutant receptors carrying the Flag epitope were fortu-itously expressed on the cell surface, the activities of the Flag-LH/CG receptors (hCG binding, and cAMP induction) were determined. The Flag-wild type receptor and the wild type receptor on intact cells bound hCG with the same affinity (Fig.  6, A and B). In addition, the Flag-wild type receptor was capable of hCG-dependent cAMP induction, although the EC 50 value for cAMP induction was ϳ3-fold higher than the value of the wild type receptor (Fig. 6C). These data show that the Flag-LH/CG receptors are active, although their potency is somewhat different compared with LH/CG receptors lacking the Flag epitope. With this in mind, we examined Flag-LH/CG-R L20A , Flag-LH/CG-R C22A , and Flag-LH/CG-R G24A . They did not bind hCG or induce cAMP production (data not shown). These results, along with the results obtained from the non-Flag mutants, demonstrate that LH/CG-R L20A , LH/CG-R C22A , and LH/CG-R G24A are expressed on the surface of intact cells, but are defective in hCG binding. In contrast, LH/CG-R D17A , LH/CG-R G18A , LH/CG-R R21A , LH/CG-R G24A , LH/CG-R P25A , and LH/CG-R R26A are capable of binding hCG.

DISCUSSION
Truncation at several discrete regions of the exodomain, near the boundaries of exons 1-2, 4 -5, 6 -7, and 9 -10, noticeably influences hormone binding. It is unclear whether these regions represent hormone contact sites or whether their truncation has allosteric effects on the global structure of the exodomain and thus indirectly impacts hormone binding. In the sequence near the exon 1-2 junction, three alternate residues (Leu 20 , Cys 22 , and Gly 24 ) are important for hormone binding. It has been speculated that a primary hCG-binding site is the putative crescent structure (21)(22)(23) that is composed of Leurich motifs (24). However, the three residues are upstream (but not part) of the Leu-rich motifs and crescent structure of the exodomain. Therefore, this upstream region near the N terminus of the receptor appears to be at least equally important for hormone binding as the crescent structure. It will be interesting to see whether this upstream sequence is a hormone contact site and, if so, how it interacts with the hormone. Role of Leu 20 , Cys 22 , and Gly 24 -The data in this work suggest two general possible roles for the region covering the three residues. It may contact hCG or be important for the exodomain to assume a structure necessary for hormone binding without directly interacting with the hormone. The latter possibility could be a result of misfolding of the L20A, C22A, or G24A mutant. However, the results of our specific photoaffinity labeling of both hCG ␣and ␤-subunits by a peptide mimic of the receptor region indicate the direct interaction of the receptor region with hCG (12). Besides, several observations described in this study are consistent with the interaction of the receptor region with hCG. The effect of Ala substitutions for the three alternate residues on hormone binding are remarkably consistent with, yet strikingly different from, the effects of Ala substitutions for the intervening and flanking residues. For example, Ala substitutions for the flanking and intervening residues of Leu 20 , Cys 22 , and Gly 24 reduced the hormone binding affinity approximately 5-8-fold, but never abrogated hCG binding. Therefore, the flanking and intervening residues appear to be important for hormone binding, but are not as crucial as Leu 20 , Cys 22 , and Gly 24 . The three residues are 20 amino acids from the N terminus, and the Flag epitope was recognized in the Flag-wild type receptor as well as in Flag-LH/CG-R L20A , Flag-LH/CG-R C22A , and Flag-LH/CG-R G24A . These results suggest that the structure of the N-terminal region including Leu 20 , Cys 22 , and Gly 24 is similar regardless of Ala substitutions for the three residues. The alternate sequence of Leu 20 , Cys 22 , and Gly 24 suggests a ␤-like structure, orienting the three residues on one side where hCG might contact. Our results are consistent with other reports that the presence of the peptide mimic of receptor Arg 21 -Pro 38 (10), substitution for Cys 22 (25), or deletion of exon 1 (11) interferes with hormone binding.
In addition to the three residues, our data and the peptide mimic (10) and deletion (11) studies predict the importance of the boundaries of exons 1-2 and 4 -5 in hormone binding. On the other hand, our data and the peptide mimic study (10) suggest the potential role of the exon 9 -10 boundary in hormone binding. In contrast, the deletion of residues 212-341 covering the exon 9 -10 boundary from the receptor increased the K d value merely by ϳ2-fold (11). Our study and the deletion study (11), but not the peptide mimic study (10), suggest a role of the exon 6 -7 boundary in hormone binding. These results suggest the usefulness and limitations of individual methods by themselves.
Some of the mutant receptors such as R21A, P23A, P25A, Flag-L20A, Flag-C22A, and Flag-G24A were expressed at significantly higher levels compared with expression of the wild type receptor. It is unclear whether the higher expression levels are caused by better plasmid preparation, better transfection, more efficient translation and/or processing, or a reduced degradation rate. We have experienced that the expression level is dependent on the transfection efficiency, which, in turn, is affected by the quality and amount of plasmid preparation as well as the cell condition. If the transfection efficiency is equal, it is possible that mutations could impact on the expression level. Similarly, substitutions for a crucial amino acid can diversely influence the machinery for the surface expression depending on the side chain of the amino acid (26).
Importance of Asp 17 and Gly 18 in Targeting-When either Asp 17 and Gly 18 was substituted with Ala, the corresponding mutant receptors were trapped within cells and could not be detected on the cell surface. This total lack of their surface expression implies the importance of these residues in targeting the receptor to the plasma membrane. Furthermore, the targeting machinery is extremely sensitive to a change in the structure of this sequence. At least in the case of the D17A and G18A substitutions, the targeting mechanism appears to be more sensitive than hormone binding is. Therefore, targeting to the cell surface could be used as an indicator of structural changes of the receptor including mutant receptors.
FIG. 6. Activities of Flag-tagged receptors. Hormone binding and cAMP induction by Flag-tagged receptors were determined as described in the legend to Fig. 2 and under "Experimental Procedures." NS, not significant.