Opposite Contribution of Two Ligand-Selective Determinants in the N-Terminal Hormone-Binding Exodomain of Human Gonadotropin Receptors
Henry F. Vischer,
Joke C. M. Granneman and
Jan Bogerd
Department of Endocrinology, Utrecht University, 3584 CH Utrecht, The Netherlands
Address all correspondence and requests for reprints to: Jan Bogerd, Utrecht University, Department of Endocrinology, Padualaan 8, 3584 CH Utrecht, The Netherlands. E-mail: J.Bogerd{at}bio.uu.nl.
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ABSTRACT
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The nine leucine-rich repeat-containing exodomains of the human FSH receptor (hFSH-R) and the human LH/chorionic gonadotropin receptor (hLH-R) harbor molecular determinants that allow the mutually exclusive binding of human FSH (hFSH) and human LH (hLH)/human chorionic gonadotropin (hCG) when these hormones are present in physiological concentrations. Previously, we have shown that the ß-strands of hLH-R leucine-rich repeats 3 and 6 can confer full hCG/hLH responsiveness and binding when simultaneously introduced into a hFSH-R background without affecting the receptors responsiveness to hFSH. In the present study, we have determined the nature of contribution of each of these two ß-strands in conferring hCG/hLH responsiveness to this mutant hFSH-R. Human LH-R ß-strand 3 appeared to function as a positive hCG/hLH determinant by increasing the hCG/hLH responsiveness of the hFSH-R. In contrast, mutagenesis of hFSH-R ß-strand 6, rather than the introduction of its corresponding hLH-R ß-strand, appeared to allow the interaction of hCG/hLH with the hFSH-R. Hence, hFSH-R ß-strand 6 functions as a negative determinant and, as such, restrains binding of hCG/hLH to the hFSH-R. Detailed mutagenic analysis revealed that the ability of the hFSH-R to interact with hCG/hLH depends primarily on the identity of two amino acids (Asn104, a positive LH-R determinant, and Lys179 a negative FSH-R determinant) that are situated on the C-terminal ends of ß-strands 3 and 6, respectively.
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INTRODUCTION
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THE THREE GLYCOPROTEIN hormone receptors, FSH receptor (FSH-R), LH receptor (LH-R), and TSH receptor (TSH-R), contain large N-terminal extracellular domains (exodomains), which are involved in the highly specific recognition as well as high-affinity binding of their respective glycoprotein hormones (1, 2, 3, 4, 5). Accordingly, glycoprotein hormone receptors have distinct regulatory functions in vertebrate physiology (6, 7). Each exodomain of these receptors consists of a nine imperfect leucine-rich repeat (LRR)-containing subdomain (LRR subdomain) that is flanked at its N and C terminus by cysteine-rich subdomains (Fig. 1
) (8, 9, 10). LRR motifs have been recognized in a large number of distinct proteins (11). The existing crystal structure of ribonuclease inhibitor (RI) showed that its LRRs are in a horseshoe-like conformation, in which each LRR is organized into a short ß-strand connected to a parallel
-helical segment (12). The consecutive ß-strands organize themselves as a parallel ß-sheet, forming a concave surface to which the ligand (i.e. RNase) binds using multiple contact points, whereas the helical segments are aligned to form the outer convex side of the RI structure.

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Fig. 1. Schematic Representation of the hFSH-R Protein Showing the N-Terminal Exodomain, the Seven-Transmembrane Helices, and the C-Terminal Intracellular Tail
The ß-strand (i.e. X1-X2-L-X3-L-X4-X5 motif) in each of the consecutive LRRs is indicated by an arrow and numbered (ß1ß9). In addition, the first and last residue of ß-strands 3 and 6 are numbered. The conserved hydrophobic (L) residues that form the core of the LRR domain are indicated by circles with a gray background. Putative N-linked carbohydrates are indicated by a Y. Cys residues present in the N- and C-terminal Cys-rich subdomains are indicated by circles with a black background. Inset, ß-strand 3 of the hLH-R.
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Based on the RI structure, the exodomains of glycoprotein hormone receptors have been modeled (8, 9, 10). By analogy, binding of glycoprotein hormones to their respective receptors was predicted to involve multiple contacts with the concave ß-sheet of their curved exodomains. Each of the ß-strands is composed of a conserved seven-amino acid X1-X2-L-X3-L-X4-X5 motif, in which the X residues can be any amino acid, and the L residues are Leu, Ile, or other hydrophobic residues (11). The conserved L residues are involved in maintaining the hydrophobic inner core of the LRR structure. The side chains of the X residues are exposed to the surface of the hormone-binding site and are therefore potentially involved in ligand interaction (13, 14, 15, 16).
The different glycoprotein hormone ß-subunits (i.e. FSHß, TSHß, LHß, and chorionic gonadotropin ß-subunit) as well as their cognate receptors have arisen during evolution through gene duplications of the ancestor ß-subunit and receptor genes, respectively, followed by sequence divergence (3, 17, 18). Whereas invariant X residues in the LRR domain ß-strands of FSH-R, LH-R, and TSH-R may serve as potential common contact sites for the (promiscuous) binding of the common
-subunit and/or conserved ß-subunit residues that are present in all glycoprotein hormone subtypes (13), sequence divergence in the LRR domain ß-strands between the FSH-R, LH-R, and TSH-R confers glycoprotein hormone selectivity to each of the receptor subtypes (5, 19). Our previous work revealed that simultaneous substitution of ß-strands 3 and 6 of the horseshoe-shaped hormone-binding domain of the hFSH-R with their hLH-R counterparts conferred an additive human chorionic gonadotropin (hCG)/hLH responsiveness to this mutant receptor (named hFSH-R/Lß3,Lß6) that was similar to that observed for the wild-type hLH-R and for a chimeric hFSH-R in which the entire N-terminal exodomain had been replaced with the corresponding hLH-R exodomain (5). Moreover, this mutant hFSH-R/Lß3,Lß6 receptor as well as most other mutant ß-strand hFSH-Rs displayed a hFSH responsiveness similar to that of the wild-type hFSH-R, indicating that the respective ß-strand substitutions did not alter their intrinsic capacity to induce hFSH-stimulated intracellular cAMP production. In addition, the efficacies of hFSH and hCG/hLH to stimulate these mutant ß-strand hFSH-Rs correlated well with observed ligand affinities of these receptors (5).
In the present study, we have determined in detail which molecular determinants in ß-strands 3 and 6 contribute to the increased hCG/hLH responsiveness of mutant hFSH-Rs. To this end, we initially examined the effects of substituting the unique X residues of ß-strand 3 or 6 with Ala residues on their responsiveness to these hormones. This approach revealed that each ß-strand contributed differently to the observed hCG/hLH responsiveness of hFSH-R/Lß3,Lß6. Amino acid residues crucial for hormone selectivity were then further analyzed in more detail by mutating each to a panel of amino acid residues with different physicochemical properties.
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RESULTS AND DISCUSSION
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Human LH-R ß-Strand 3 Contains Determinants that Confer hCG/hLH Responsiveness to Mutant hFSH-Rs
In contrast to an increased hCG/hLH responsiveness provoked by the introduction of hLH-R ß-strand 3 into the hFSH-R (i.e. hFSH-R/Lß3), Ala substitution of all ß-strand 3 residues, which are different between the hFSH-R and hLH-R, in the hFSH-R (i.e. hFSH-R/Alaß3) significantly impaired responsiveness to hCG/hLH (Fig. 2B
, and Table 1
). However, hFSH-R/Alaß3 also displayed a 49-fold decreased hFSH responsiveness compared with hFSH-R/Lß3 (Fig. 2A
and Table 1
). Although it can not be excluded that impaired hFSH-R/Alaß3 cell surface expression hampers hormone responsiveness to some extent (Table 1
) (5), the fact that the responsiveness to hCG/hLH and hFSH was not equally affected by the Ala substitutions indicates that the reduced hCG/hLH responsiveness is related to the removal of hCG/hLH-selective determinants that were previously introduced into hFSH-R/Lß3 rather than a disrupted overall conformation of the exodomain. In this respect, transfection of increasing amounts of the hFSH-R/Alaß3 construct (5 or 10 µg instead of 1 µg) did not affect receptor cell surface expression or ligand responsiveness (data not shown).

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Fig. 2. cAMP-Mediated Reporter Gene Activity in Response to Increasing Concentrations of hFSH (A) and hCG (B) in HEK-T 293 Cells Transiently Transfected with the Wild-Type or Mutant hFSH-Rs, and Cotransfected with a Plasmid Containing a ß-Galactosidase Gene Under Control of a Promotor Containing Five cAMP-Response Elements
hLH and hCG stimulated all constructs with a similar efficacy; for clarity, only hCG-stimulated, cAMP-mediated reporter gene activity is shown. Results are shown as the mean ± SEM of triplicate observations from a single representative experiment. Mean EC50 values and receptor cell surface expression levels are presented in Table 1 .
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Table 1. Summary of the Ligand-Induced Intracellular cAMP Production in HEK-T 293 Cells Transiently Transfected with Wild-Type and Mutant ß-Strand 3 hFSH-R Constructs
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We then used Ala scanning to determine the relative contribution of each of the hLH-R ß-strand 3 residues in conferring hCG/hLH selectivity to hFSH-R/Lß3. Each of these mutant hFSH-Rs (i.e. hFSH-R/Lß3-S98A, hFSH-R/Lß3-L101A, hFSH-R/Lß3-Q103A, and hFSH-R/Lß3-N104A) displayed a similar responsiveness to hFSH as the wild-type hFSH-R (Fig. 2A
and Table 1
). Ala substitution of Ser98 and Gln103 did not affect the hCG/hLH responsiveness of these mutant hFSH-R/Lß3 receptors (Fig. 2B
and Table 1
). In contrast, hFSH-R/Lß3-L101A displayed a significant, but only 5-fold, reduced responsiveness to hCG/hLH as compared with hFSH-R/Lß3 (Fig. 2B
and Table 1
), presumably through stabilizing a hydrophobic hormone-receptor interaction. The substitution of Asn104 of hFSH-R/Lß3 with Ala reduced its hCG/hLH responsiveness back to wild-type hFSH-R levels (Fig. 2B
and Table 1
), indicating that principally Asn104 accounted for the approximately 71-fold observed increase in hCG/hLH responsiveness of hFSH-R/Lß3 compared with the wild-type hFSH-R. Accordingly, a mutant hFSH-R, in which Lys104 was substituted with Asn (i.e. hFSH-R/K104N), was equally responsive to hCG/hLH as hFSH-R/Lß3 (Fig. 3B
and Table 1
).

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Fig. 3. cAMP-Mediated Reporter Gene Activity in Response to Increasing Concentrations of hFSH (A and C) and hCG (B and D) in HEK-T 293 Cells Transiently Transfected with the Wild-Type or Mutant hFSH-Rs and Cotransfected with a Plasmid Containing a ß-Galactosidase Gene under Control of a Promotor Containing Five cAMP-Response Elements
hLH and hCG stimulated all constructs with a similar efficacy; for clarity, only hCG-stimulated, cAMP-mediated reporter gene activity is shown. For clarity, the results from hFSH stimulation (A and C) and hCG stimulation (B and D) are divided over two separate graphs and shown as the mean ± SEM of triplicate observations from a single representative experiment. Mean EC50 values and receptor cell surface expression levels are presented in Table 1 .
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Responsiveness to hCG/hLH of Mutant hFSH-Rs Is Dependent on the Presence of an Asn Residue at the C-Terminal Position of ß-Strand 3
The specific contribution of the Asn side chain at position 104 of hFSH-R/Lß3 in conferring receptor responsiveness to hCG/hLH was studied by detailed mutagenic analysis. This revealed that substitutions of this residue with Lys, Gln, or Thr impaired the hCG/hLH responsiveness (Fig. 3
, B and D, and Table 1
). In fact, only the substitution of Asn with Asp was fairly tolerated with respect to hCG/hLH responsiveness. Nonetheless, hFSH-R/Lß3-N104D was approximately 12-fold less sensitive to hCG/hLH than hFSH-R/Lß3 (Fig. 3D
and Table 1
). Intriguingly, however, this apparent key determinant for hCG/hLH responsiveness is not exclusively found in LH-Rs, but is also conserved in ß-strand 3 of all TSH-Rs. However, human TSH-R (hTSH-R) is almost devoid of responsiveness to hCG, as more than an approximately 1000-fold higher concentration of hCG as compared with hTSH is required to promiscuously bind/activate the hTSH-R (19, 20, 21). A potential hCG/hLH-selective role of this Asn in the hTSH-R is apparently neutralized by other key selecting amino acid residues, present in the ligand-binding domain of this receptor type. For example, residues neighboring the position that corresponds to Asn104 were more similar between hFSH-R and hLH-R, but were considerably different in the hTSH-R, in particular with respect to charge and polarity.
Lysine179 of hFSH-R ß-Strand 6 Restrains hCG/hLH from Interacting with the hFSH-R
Ala substitution of all ß-strand 6 residues, which are different between the hFSH-R and hLH-R, in the hFSH-R (i.e. hFSH-R/Alaß6) did not affect the responsiveness to hFSH (Fig. 4A
and Table 2
). More importantly, analysis of the mutant hFSH-R/Alaß6 receptor revealed that not the presence of hLH-R-specific residues, but rather the absence of hFSH-R-specific residues, conferred hCG/hLH responsiveness to this mutant receptor (Fig. 4B
and Table 2
). Subsequent Ala scanning of the individual hFSH-R ß-strand 6 residues (i.e. Ile174, Trp176, Asn178, or Lys179; see Table 2
) identified Lys179 as the predominant determinant that restrains hCG/hLH from interacting with the hFSH-R, and Asn178 as a minor hCG/hLH-repelling determinant (Fig. 4B
and Table 2
). In fact, hFSH-R/K179A displayed a similar responsiveness to hCG/hLH as hFSH-R/Lß6. Similar to hFSH-R/K179A, the hLH-R also has a small, nonpolar amino acid residue (Gly) at the corresponding position of Lys179 in the hFSH-R, suggesting that size and/or charge of Lys179 may sterically hinder and/or electrostatically repulse the interaction of hCG/hLH with the hFSH-R.

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Fig. 4. cAMP-Mediated Reporter Gene Activity in Response to Increasing Concentrations of hFSH (A) and hCG (B) in HEK-T 293 Cells Transiently Transfected with the Wild-Type or Mutant hFSH-Rs and Cotransfected with a Plasmid Containing a ß-Galactosidase Gene under Control of a Promotor Containing Five cAMP-Response Elements
hLH and hCG stimulated all constructs with a similar efficacy; for clarity, only hCG-stimulated, cAMP-mediated reporter gene activity is shown. Results are shown as the mean ± SEM of triplicate observations from a single representative experiment. Mean EC50 values and receptor cell surface expression levels are presented in Table 2 .
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Table 2. Summary of the Ligand-Induced Intracellular cAMP Production in HEK-T 293 Cells Transiently Transfected with Wild-Type and Mutant ß-Strand 6 hFSH-R Constructs
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Relative Contribution of Substituting Lys179 with Different Amino Acids to the hCG/hLH Responsiveness of Mutant hFSH-Rs
To further investigate this presumed Lys179-mediated hCG/hLH repulsion, Lys179 was replaced with a panel of amino acids with different physicochemical properties. Substitution with the somewhat larger cationic Arg resulted in a significant approximately 3-fold increase in hCG/hLH responsiveness as compared with the wild-type hFSH-R (Fig. 5D
and Table 2
). Nevertheless, the relative low responsiveness of both the wild-type hFSH-R (Lys179) and hFSH-R/K179R to hCG/hLH suggests that this specific determinant is involved in an electrostatic repulsion of the net positively charged determinant loop of the seat belt regions of hCG and hLH ß-subunits, which has been shown to be essential for receptor recognition (3, 22, 23). However, the inability of a chimeric hCG harboring the net negatively charged determinant loop of hFSH to bind to the hFSH-R (3, 22), as well as the ability of a chimeric hFSH that contains the positively charged determinant loop of hCG to bind to the hFSH-R with a similar affinity as wild type hFSH (23), contradicts such an electrostatic repulsion between the seat belt loop of hCG/hLH and Lys179. The substitution of Lys179 with Glu or Asp increased the hCG/hLH responsiveness only up to approximately 12-fold as compared with wild-type hFSH-R (Fig. 5D
and Table 2
), suggesting that ionic interactions are indeed not the main driving force behind hCG/hLH recognition by this receptor determinant. This was further confirmed by the fact that hFSH-R/K179Q and hFSH-R/K179N displayed a similar responsiveness to hCG/hLH as their Glu- and Asp-substituted counterparts (Fig. 5D
and Table 2
). It should be noted that the approximately 4-fold difference in ligand responsiveness (both to hFSH and hCG/hLH) of hFSH-R/K179E compared with hFSH-R/K179D, hFSH-R/K179Q, and hFSH-R/K179N is probably related to its impaired cell surface expression levels (Table 2
), as discussed previously (5). Transfection of more hFSH-R/K179E construct did not enhance the receptor cell surface expression (data not shown). In contrast to our results, the release of a Glu residue (situated at the X3 position of TSH-R ß-strand 5) from an ionic interaction with a neighboring Lys (situated at the X3 position of TSH-R ß-strand 6) as a result of a naturally occurring mutation of this Lys, was found to significantly increase the sensitivity of TSH-R for hCG leading to gestational hyperthyroidism during pregnancy (19). Moreover, additional mutagenic experiments by Smits and co-workers (19) revealed the importance of an anionic side chain at or nearby position X3 in ß-strand 5 of the TSH-R, suggesting that electrostatic attraction allows hCG to interact with the TSH-R. The approximately 37-fold increase in hCG/hLH responsiveness of hFSH-R/K179M as compared with wild-type hFSH-R revealed that the presence of the hydrophobic side chain of Met allows hCG/hLH to interact with this mutant hFSH-R, rather than the size of this side chain (Fig. 5B
and Table 2
). However, substitution of K179 with the more bulky Trp had only minor effects (
5-fold increase) on the hCG/hLH responsiveness as compared with wild-type hFSH-R, whereas the presence of Ile on this position presumably disrupted the LRR conformation as hFSH-R/K179I was not detectable at the cell surface and displayed significantly reduced responsiveness to hCG/hLH and hFSH (Table 2
).

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Fig. 5. cAMP-Mediated Reporter Gene Activity in Response to Increasing Concentrations of hFSH (A and C) and hCG (B and D) in HEK-T 293 Cells Transiently Transfected with the Wild-Type or Mutant hFSH-Rs, and Cotransfected with a Plasmid Containing a ß-Galactosidase Gene Under Control of a Promotor Containing Five cAMP-Response Elements
hLH and hCG stimulated all constructs with a similar efficacy; for clarity, only hCG-stimulated, cAMP-mediated reporter gene activity is shown. For clarity, the results from hFSH stimulation (A and C) and hCG stimulation (B and D) are divided over two separate graphs and shown as the mean ± SEM of triplicate observations from a single representative experiment. Mean EC50 values and receptor cell surface expression levels are presented in Table 2 .
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Hormone-Binding Characteristics of Mutant ß-Strand 3 and/or 6 hFSH-Rs
Most mutant ß-strand hFSH-Rs displayed a similar hFSH responsiveness as the wild-type hFSH-R [present study, and as observed in a previous study (5)], indicating that the substitutions introduced did not alter their intrinsic capacity to induce hFSH-stimulated intracellular cAMP production. To confirm that the observed ligand responsiveness of these mutant receptors represents their affinity for that ligand, displacement studies were performed for a selected number of mutant receptors. In contrast to the observed hFSH and hCG responsiveness of human embryonic kidney (HEK)-T 293 cells, expressing hFSH-R/Lß3-N104A, almost no [125I]hFSH was bound specifically to membrane preparations of these cells (Fig. 6A
), which is most likely related to the low hFSH-R/Lß3-N104A cell surface expression levels (
5% of wild-type hFSH-R; see Table 1
). These low receptor cell surface expression levels could not be increased by transfecting more (5 or 10 µg instead of 1 µg) hFSH-R/Lß3-N104A construct into the HEK-T 293 cells. Consequently, no unambiguous binding parameters could be determined for this mutant receptor (Fig. 6
, B and C, and Table 3
), as revealed by the relatively high SE of the estimated log (IC50) values (i.e. between 0.2 to 0.9 log units). As expected from the low degree of hCG responsiveness of the wild-type hFSH-R, hCG could not fully displace the cognate binding of [125I]hFSH to this receptor (Fig. 6
, A, C, and E, and Table 3
). Except for the slightly increased affinity of hFSH-R/K179A for hFSH, all other mutant receptors that were tested displayed a similar affinity to hFSH as the wild-type hFSH-R (Fig. 6
, B and D, and Table 3
). The mutant receptors hFSH-R/Alaß6 and hFSH-R/K179A displayed similar ligand-binding affinity (Ki) values as hFSH-R/Lß6, and thus bound hCG with a significant 119- to 288-fold higher affinity than the wild-type hFSH-R. These binding data are comparable to the corresponding responsiveness upon stimulation with the respective hormones. This indicated that the responsiveness to hCG of these mutant receptors represents their affinity for hCG, provided that their sensitivity to hFSH is similar to that of the wild-type hFSH-R.

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Fig. 6. Binding of [125I]-hFSH to Membranes Prepared from HEK-T 293 Cells Expressing Wild-Type or Mutant hFSH-Rs
Absolute [125I]hFSH binding in the absence or presence of 3 µg/ml unlabeled hFSH or hCG (A). Percent displacement of [125I]hFSH by increasing concentrations of unlabeled hFSH (B and D) or unlabeled hCG (C and E). Results are shown as the mean ± SEM of triplicate observations from a single representative experiment. Mean Ki values are presented in Table 3 .
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Table 3. Summary of the Ligand-Binding Properties of Membranes of HEK-T 293 Cells Transiently Transfected with Wild-Type and Mutant hFSH-R Constructs
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Roles of Asn 104 and Lys179 in the Hormone Selectivity of Human Gonadotropin Receptors
Hitherto, there is no general consensus about the spatial orientation of glycoprotein hormones docking into the presumed horseshoe-shaped binding site of the exodomain of their cognate receptors. Notably, both amino acids on positions 104 and 179 of the hFSH-R are situated at the C-terminal ends (i.e. amino acid residue X5) of its X1-X2-L-X3-L-X4-X5 motif of ß-strand 3 and ß-strand 6, respectively, suggesting a predominant role of this rim of the curved LRR domain in conferring ligand selectivity (Fig. 1
). With an estimated interatomic distance of 1314 Å between the
-carbon positions of amino acids 104 and 179 (8, 9), these determinants may interact with different contact points of the glycoprotein hormones. Mutagenesis, epitope mapping, and modeling experiments suggested that the central area of these hormones, which consists of the C termini and the proximal part of loop 2 of both subunits and the seat belt loop of the ß-subunit interacts with the receptor (23, 24). However, other studies suggested that the groove formed by loop 2 of the
-subunit and the proximal regions of ß-subunit loop 1 and 3 of the glycoprotein hormones faces the horseshoe-shaped binding site of the receptors (25, 26). The identification of the exact contact sites between the two determinants identified in this study (i.e. the amino acids on positions 104 and 179) in the receptor and hCG/hLH ultimately requires the three-dimensional crystal structure of a soluble gonadotropin-receptor complex. This may be expected in the near future with the recent developments in overexpressing such complexes (27, 28).
In conclusion, while several common determinants allow the (promiscuous) binding of related glycoprotein hormones to the ligand-binding domain of gonadotropin receptors, ligand selectivity of these receptors is determined by a relatively small number of positive (i.e. attracting) and negative (i.e. repelling) key determinants. In this way, promiscuous human gonadotropin receptors can be generated by introducing hLH-R ß-strand 3 amino acid sequences (in particular Asn on position 104) into the hFSH-R, while simultaneously replacing the hFSH-R-specific Lys179 in ß-strand 6. Identification of hFSH-selective determinants by systematically introducing hFSH-R ß-strands into the hLH-R is currently under investigation. In addition, this approach will also allow verification of the selective potential of the hCG/hLH determinants identified in this study, when situated in a hLH-R background.
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MATERIALS AND METHODS
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Construction of the hFSH-R Mutants
Mutant receptor cDNA pcDNA3.1/V5-His-TOPO vector (Invitrogen, San Diego, CA) constructs (see Tables 1
and 2
) were generated by PCR-based mutagenesis using hFSH-R cDNA templates containing an HA epitope (derived from the influenza virus hemagglutinin) sequence insertion between the C terminus of the signal peptide and the N terminus of the mature hFSH-R protein (5).
Transfection Experiments
HEK-T cells (
3.5 x 106 cells) were transiently transfected with 1 µg (wild type or mutant) HA-tagged hFSH-R expression vector construct in combination with 10 µg of a pCRE/ß-gal plasmid using a modified bovine serum transfection method (5). The pCRE/ß-gal plasmid consists of a ß-galactosidase gene under the control of a human vasoactive intestinal peptide promotor containing five cAMP-response elements (29). Empty pcDNA3.1/V5-His vector was used for mock transfections. The next day, cells were trypsinized and seeded into poly-D-lysine (Sigma Chemical Co., St. Louis, MO)-coated 24-well (Costar) plates (
4.5 x 105 cells per well) for ELISA detection of receptor cell surface expression, and 96-well (Costar) plates (
2 x 105 cells per well) for ligand-stimulation studies.
ELISA Detection of HA-Tagged Receptors on the Cell Surface
HA-tagged receptor cell surface expression was quantified by ELISA as previously described (5). Briefly, 2 d after transfection, cells were fixed using 4% paraformaldehyde in PBS at room temperature for 30 min. Next, the samples were blocked with 1% nonfat dried milk in 0.1 M NaHCO3 at room temperature for 4 h and subsequently incubated with anti-HA high-affinity antibodies (Roche Applied Science, Almere, The Netherlands; 1:200 dilution in Tris-buffered saline containing 0.1% BSA) overnight at 4 C. The next day, the samples were washed and incubated with peroxidase-conjugated goat antirat IgG (Sigma; 1:500 dilution in 1% nonfat dried milk in 0.1 M NaHCO3) at room temperature for 2 h. Peroxidase activity was visualized using the 3,3', 5,5'-tetramethylbenzidine liquid substrate system (Sigma) for approximately 10 min. Absorbance values (450 nm) of mock transfected cells were subtracted, and mutant hFSH-R expression values were expressed as the percentage of wild-type hFSH-R expression. All experiments were repeated at least three times using cells from independent transfections, each performed in duplicate.
Detection of Ligand-Induced cAMP Production
Receptor-mediated stimulation of cAMP-induced reporter gene activity was assayed as previously described (5). Briefly, 2 d after transfection, cells were stimulated for 6 h with various concentrations of human recombinant FSH (hFSH, Org32489E), LH (hLH, 99M7019), and CG (hCG, 01MZ010) in 25 µl HEPES-modified DMEM containing 0.1% BSA and 0.1 mM 3-isobutyl-1-methylxanthine (all from Sigma). All human recombinant gonadotropins were kindly provided by Dr. W. G. E. J. Schoonen (NV Organon, Oss, The Netherlands). Ligand-induced changes in ß-galactosidase activity (conversion of o-nitrophenyl-ß-D-galactopyranoside into o-nitrophenol) were measured at 405 nm, and related to 10 µM forskolin-induced changes (Sigma) in each 96-well plate. Hence, results are expressed as arbitrary units. Ligand concentrations that induce half-maximal stimulation (i.e. EC50 values) were determined by fitting the cAMP-related reporter gene activity to sigmoidal dose-response curves using GraphPad PRISM3 (GraphPad Software, Inc., San Diego, CA). All experiments were repeated at least three times using cells from independent transfections, each performed in triplicate.
Receptor Binding Assay
Competition ligand-binding assays were carried out on purified cell membranes from HEK-T 293 cells expressing mutant receptors. Two days after transfection, HEK-T 293 cells were rinsed with Dulbeccos PBS (Sigma), subsequently harvested in ice-cold Tris buffer [10 mM Tris-HCl, 5 mM MgCl2·6 H2O (pH 7.4)] and centrifuged at 200 x g at 4 C for 10 min. The pellet was resuspended in ice-cold Tris buffer containing 250 mM sucrose and homogenized by 40 strokes in a Dounce homogenizer at 4 C, and the cell suspension was centrifuged at 15,000 x g at 4 C for 30 min. The pellet was resuspended in Tris buffer. Cell membranes (
50 µg protein) were incubated for 1820 h in 300 µl Tris buffer supplemented with 0.1% BSA at room temperature with 10,000 cpm [125I]hFSH (i.e. NEX 173 with a specific activity of 163 µCi/µg; purchased from Perkin Elmer Life Sciences, Norwalk, CT) in the presence of increasing concentrations of unlabeled hCG or hFSH. The reaction was terminated by adding 500 µl ice-cold Tris buffer supplemented with 0.1% BSA and subsequently centrifuged at 15,000 x g at room temperature for 5 min. The supernatant was aspirated and the radioactivity in the membrane pellet was determined in a LKB
-counter (Perkin Elmer Life Sciences). Ligand binding affinities (Ki) were calculated from one-site competition curves using GraphPad PRISM3. All binding studies were performed in triplicate in two independent experiments.
Data Analysis
All data are presented as mean ± SEM. Statistical comparisons were performed on log(EC50) or log(Ki) values using one-way ANOVA, followed by the Bonferroni test, using GraphPad PRISM3. P < 0.05 was considered to be significant.
Note Added in Proof
After submission of this manuscript, similar results were published by Smits et al. (30).
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ACKNOWLEDGMENTS
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The authors thank Dr. S. Mosselman and R. H. L. van de Wetering (NV Organon, Oss, The Netherlands) for valuable assistance with performing receptor binding assays; M. J. Noordam (Utrecht University, The Netherlands) for assistance with generating mutant receptor constructs; and Dr. M. L. C. E. Kouwijzer (NV Organon, Oss, The Netherlands) for helpful discussions.
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FOOTNOTES
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Present address for H.F.V.: Leiden/Amsterdam Center for Drug Research, Division of Medicinal Chemistry, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.
Abbreviations: CG, Chorionic gonadotropin; FSH-R, FSH receptor; HA, hemagglutinin; HEK, human embryonic kidney; LH-R, LH receptor; LRR, leucine-rich repeat; RI, ribonuclease inhibitor; TSH-R, TSH receptor.
Received for publication May 12, 2003.
Accepted for publication July 7, 2003.
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