This Article Abstract Full Text (PDF) All Versions of this Article: 20/8/1880    most recent Author Manuscript (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Vischer, H. F. Articles by Bogerd, J. Search for Related Content PubMed PubMed Citation Articles by Vischer, H. F. Articles by Bogerd, J. Pubmed/NCBI databases Substance via MeSH Hazardous Substances DB CHORIONIC GONADOTROPIN MENOTROPINS

Identification of Follicle-Stimulating Hormone-Selective ß-Strands in the N-Terminal Hormone-Binding Exodomain of Human Gonadotropin Receptors

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.

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Glycoprotein hormone receptors contain large N-terminal extracellular domains (ECDs) that distinguish these receptors from most other G protein-coupled receptors. Each glycoprotein hormone receptor ECD consists of a curved leucine-rich repeat domain flanked by N- and C-terminal cysteine-rich regions. Selectivity of the different glycoprotein hormone receptors for their cognate hormones is exclusively determined by their ECDs and, in particular, their leucine-rich repeat domain. To identify human (h)FSH-selective determinants we used a gain-of-function mutagenesis strategy in which ß-strands of the hLH receptor (hLH-R) were substituted with their hFSH receptor (hFSH-R) counterparts. Introduction of hFSH-R ß-strand 1 into hLH-R conferred responsiveness to hFSH, whereas hLH-R mutants harboring one of the other hFSH-R ß-strands displayed none or very limited sensitivity to hFSH. However, combined substitution of hFSH-R ß-strand 1 and some of the other hFSH-R ß-strands further increased the sensitivity of the mutant hLH-R to hFSH. The apparent contribution of multiple hFSH-R ß-strands in providing a selective hormone binding interface corresponds well with their position in relation to hFSH as recently determined in the crystal structure of hFSH in complex with part of the hFSH-R ECD.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
THE GLYCOPROTEIN HORMONES, TSH, FSH, and LH, are glycosylated heterodimeric molecules, each consisting of a noncovalent association of a common -subunit with a hormone-specific ß-subunit. The glycoprotein hormone ß-subunits as well as their cognate receptors, TSH receptor (TSH-R), FSH receptor (FSH-R), and LH receptor (LH-R), are encoded by paralogous genes that have coevolved through duplications of their common ancestral ß-subunit and glycoprotein hormone receptor genes, respectively (1, 2). In primate and equine species, a third gonadotropin, chorionic gonadotropin (CG), has evolved through additional duplications of the ancestral LH ß-subunit gene. Chorionic gonadotropin is secreted from the placenta to ensure high progesterone production by the corpus luteum during pregnancy and displays a similar receptor-binding profile as LH to the common receptor for LH and CG. In mammals, the subsequent sequence divergence of these ß-subunit and receptor genes was sufficient to confer mutual-exclusive specificity to each hormone-receptor pair with no physiological cross-reactivity between unintended hormone-receptor couples (1, 3, 4, 5, 6, 7, 8, 9). In mammalian gonads, this tight specificity, together with differential spatiotemporal expression patterns of the FSH-R and LH-R, ensures that FSH and LH bioactivities are directed to different target cells. Moreover, the intricate, complementary interplay between FSH and LH is a prerequisite for quantitative and qualitative normal gametogenesis in both sexes and, consequently, reproductive success (10).

Glycoprotein hormone receptors constitute a unique subfamily of G protein-coupled seven-transmembrane receptors by virtue of having large N-terminal extracellular domains (ECDs), which are exclusively involved in the specific recognition and high-affinity binding of their cognate glycoprotein hormones (1, 3, 4, 8, 9, 11, 12, 13). Each ECD can be divided into three structurally distinct entities: an N-terminal cysteine-rich subdomain (NCR), which is followed by a leucine-rich repeat (LRR) subdomain, consisting of nine LRR motifs, and a C-terminal cysteine-rich subdomain (CCR) (Fig. 1) (14, 15, 16, 17). LRR motifs have been recognized in a large variety of proteins (18), and crystal structure analyses of such proteins revealed that consecutive LRRs are arranged in a banana-like conformation. Each LRR motif forms a right-handed structural unit, consisting of alternating short ß-strands and helical segments, and connected by short loops, and with each ß-strand positioned nearly antiparallel to its helix. The consecutive ß-strands, arranged as a parallel ß-sheet, form the inner concave surface of the cusp-shaped ECD, to which the cognate ligand can bind using multiple contact points, whereas the helices are aligned on the convex side of the structure. Each ß-strand is composed of a conserved X₁X₂LX₃LX₄X₅ motif, in which X can be any amino acid, and L represents a hydrophobic amino acid. The side chains of the L residues are involved in maintaining the hydrophobic core of the LRR structure and are, as such, important for the stability of the protein (19, 20). The side chains of the X residues, on the other hand, are exposed to the surface of the ligand-binding site and may therefore interact with the hormone (16, 21). Consequently, these X residues were considered as promising candidates to function as ligand-selective determinants that ensure specific ligand recognition by each of the glycoprotein hormone receptor subtypes. In our previous work, we took advantage of the predicted structural conformation of the LRR domain and designed a gain-of-function mutagenesis strategy, in which we systematically exchanged the ß-strands of the human (h)FSH-R with the corresponding ß-strands of the hLH-R. Next, we determined the responsiveness of these mutant hFSH-Rs to hCG and hLH as well as to hFSH (9). In this way, we identified ß-strands 3 and 6 as hCG/hLH-selective determinants in these mutant receptors. Additional site-directed mutagenesis revealed that the hCG/hLH responsiveness of mutant hFSH-Rs depended exclusively on the identity of two amino acids, Asn¹⁰⁴, an hLH-R ß-strand 3-derived determinant, normally attracting hCG/hLH, and Lys¹⁷⁹, an hFSH-R ß-strand 6-derived determinant, normally repelling hCG/hLH. Both residues are situated on the C-terminal ends (i.e. X₅) of the ß-strands of their respective receptor proteins (7, 8).

View larger version (36K): [in this window] [in a new window]   Fig. 1. Amino Acid Sequence Alignment of the Exodomains of the hLH-R and hFSH-R Exodomains are subdivided into three structural subdomains: the N- and C-terminal cysteine-rich subdomains (NCR and CCR, respectively), with their conserved cysteine residues indicated by black boxes, and the LRR subdomain, consisting of nine successive LRR units. Homology modeling-predicted ß-strand motifs that form the concave surface of the cusp-shaped LRR subdomain are indicated by XXLXLXX, with the X residues pointed out by gray boxes (see text). ß-Strands present in the NCR and LRR subdomains of the crystal structure of the hFSH-R ECD are shown as arrows (13 ). The fractional solvent accessibility of each residue buried at the receptor-ligand interface in the crystal structure of hFSH bound to the hFSH-R ECD, is indicated by an open circle if greater than 0.4, a half-filled circle if it is 0.1–0.4, and a filled circle if less than 0.1. [Derived from Ref. 13 .]

In this study, we aimed to identify the determinants that allow selective molecular interactions between hFSH and its cognate receptor. To this end, we applied a gain-of-function mutagenesis strategy by systematically substituting hLH-R subdomains and/or ß-strands by their hFSH-R counterparts. While we were completing our studies, Fan and Hendrickson (13) published the crystal structure of the partially deglycosylated complex of hFSH bound to the ECD of hFSH-R, which was determined at 2.9 Å resolution. This crystal structure revealed, unexpectedly, that the C-terminal part of the hFSH-R NCR is organized as a LRR repeat, and, as such, contributed with an additional ß-strand to the hormone-binding surface. However, this additional repeat may not be conserved in the NCR of hLH-R because this region of the hLH-R NCR is nine amino acids shorter (Fig. 1). For this reason, the additional ß-strand, as identified in the crystal structure of hFSH-R and indicated as repeat 1 by Fan and Hendrickson, will be named "ß-strand 0" in our study. The gonadotropin-selective determinants identified in our present and previous (8, 9) studies are in line with the structure of this hFSH-hFSH-R ECD complex.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Gonadotropin Specificity of Receptor ECD Chimeras
Previous work by our group and others revealed that homologous substitutions of glycoprotein hormone receptor ECDs and, in particular, of the hormone-exposed X residues of their LRR ß-strands did not affect hormone binding differently from ligand-induced signaling (7, 8, 9, 19, 22). Therefore, the effect of our mutational analysis on ligand selectivity of the hLH-R/hFSH-R chimeras was analyzed by quantifying the potency of hFSH, hCG, or hLH to stimulate transiently transfected human embryonic kidney (HEK)-T 293 cells, using a cAMP-mediated reporter gene assay. Nonetheless, displacement-binding studies using [¹²⁵I]hCG were performed on wild-type hLH-R and a subset of receptor mutants to validate the receptor-signaling results.

To determine the contribution of each structural subdomain (i.e. NCR, LRR subdomain, and CCR) of the ECD in conferring hFSH selectivity, chimeric hLH-Rs were generated by substituting hLH-R subdomains with the corresponding hFSH-R sequences. Substitution of the entire hLH-R ECD with that of the hFSH-R (i.e. FFF-hLH-R) resulted in a chimeric receptor that was functionally similar to the wild-type hFSH-R, and thus displayed high responsiveness to hFSH together with physiologically negligible responsiveness to hCG/hLH (Fig. 2, B and C, and Table 1). Moreover, introducing the NCR and LRR subdomain of hFSH-R into the hLH-R (i.e. FFL-hLH-R) was already sufficient to confer full hFSH responsiveness to this chimeric hLH-R (Fig. 2C and Table 1), suggesting that the CCR is not involved in conferring gonadotropin selectivity. Remarkably, however, both FFF-hLH-R and FFL-hLH-R were consistently more responsive to hCG/hLH than the wild-type hFSH-R (Fig. 2B and Table 1), suggesting that the exoloops of the hLH-R transmembrane domain contribute to low-affinity hCG/hLH binding (23).

View larger version (68K): [in this window] [in a new window]   Fig. 2. Relative Contributions of ECD Subdomains of the hLH-R and hFSH-R in Conferring hFSH Responsiveness Schematic representation of wild-type hLH-R and hFSH-R, and chimeric hLH-R mutants (A). Chimeric hLH-R mutants FFL-hLH-R and FFF-hLH-R were generated using fusion PCR and junctions are situated between residues S248 and S253, and I364 and L362 of the hFSH-R and hLH-R, respectively. cAMP-mediated reporter gene activity in response to hCG (B) and hFSH (C) in HEK-T 293 cells expressing wild-type hLH-R or hFSH-R, or chimeric hLH-R mutants. Results are shown as mean ± SEM of triplicate observations from a single representative experiment. Mean EC50 values and receptor cell surface expression levels are presented in Table 1.

View larger version (26K): [in this window] [in a new window]   Fig. 3. cAMP-Mediated Reporter Gene Activity in Response to hCG (A and C) and hFSH (B and D) in HEK-T 293 Cells Expressing Wild-Type hLH-R or Mutant ß-strand hLH-Rs Results are shown as mean ± SEM of triplicate observations from a single representative experiment. Both hLH-R/Fß5 and hLH-R/Fß4,Fß5 receptor constructs were not expressed at the cell surface, resulting in the absence of ligand-induced signaling. Mean EC50 values and receptor cell surface expression levels are presented in Table 1.

View larger version (34K): [in this window] [in a new window]   Fig. 4. Binding of [125I]hCG to mock transfected HEK-T 293 cells or cells expressing wild-type hFSH-R, hLH-R, or mutant hLH-Rs Absolute [125I]hCG binding after overnight incubation at 4 C in the absence or presence of 3 µg/ml unlabeled hCG or hFSH (A). Percent displacement of [125I]hCG by increasing concentrations of unlabeled hCG (B) or unlabeled hFSH (C). Results are shown as mean ± SEM of duplicate observations from a single representative experiment. Mean Ki values are presented in Table 2.

Introduction of hFSH-R ß-strand 1 (i.e. hLH-R/Fß1) and, to a much lesser extent, the introduction of the individual hFSH-R ß-strands 7 and 8 (i.e. hLH-R/Fß7 and hLH-R/Fß8, respectively) conferred hFSH affinity and responsiveness to these mutant hLH-Rs (Fig. 3, B and D, and Table 1; Fig. 4C and Table 2). In contrast, the introduction of the other individual hFSH-R ß-strands did not confer detectable hFSH responsiveness to these mutant hLH-Rs. To determine whether hFSH-R ß-strand 1 contains hFSH-selective determinants (i.e. positive contribution) or merely replaces negative determinants normally present in this hLH-R ß-strand (i.e. residues that repel hFSH from interacting with the hLH-R), all ß-strand 1 residues that are different between hLH-R and hFSH-R were Ala substituted (i.e. hLH-R/Alaß1; see Table 1). The hLH-R/Alaß1 mutant was equally responsive to hCG/hLH as the wild-type hLH-R, but was more than 100-fold less responsive to hFSH than hLH-R/Fß1 (Fig. 5, A and B, and Table 1), suggesting the presence of negative hFSH-selective determinants in ß-strand 1 of the hLH-R.

View larger version (15K): [in this window] [in a new window]   Fig. 5. cAMP-Mediated Reporter Gene Activity in Response to hCG (A) and hFSH (B) in HEK-T 293 Cells Expressing Wild-Type, Chimeric hLH-R, or Mutant ß-Strand hLH-Rs Results are shown as mean ± SEM of triplicate observations from a single representative experiment. Mean EC50 values and receptor cell surface expression levels are presented in Table 1.

View larger version (18K): [in this window] [in a new window]   Fig. 6. cAMP-Mediated Reporter Gene Activity in Response to hCG (A) and hFSH (B) in HEK-T 293 Cells Expressing Wild-Type hLH-R or Mutant ß-Strand hLH-Rs Results are shown as mean ± SEM of triplicate observations from a single representative experiment. Mean EC50 values and receptor cell surface expression levels are presented in Table 1.

Although hFSH-R ß-strands 2, 3, 6, and 9 did not increase the hFSH responsiveness when introduced individually into the hLH-R, they may potentially contain weak hFSH-binding determinants contributing synergistically to hFSH selectivity, as suggested by receptor chimeras that were created by exchanging fragments between the rat LH-R and FSH-R using common endonuclease restriction sites (1). To identify such weak determinants, these ß-strands of the hFSH-R were stepwise introduced into the hLH-R, in combination with hFSH-R ß-strands 1, 7, and/or 8. Introducing hFSH-R ß-strand 3 into hLH-R/Fß1,Fß8 (i.e. hLH-R/Fß1,Fß3,Fß8) decreased the mutant receptor responsiveness for both hFSH and hCG/hLH (Fig. 6, A and B, and Table 1), without affecting receptor surface expression and, as a consequence, ruling out the presence of hFSH-selective determinants in this ß-strand. Although hFSH-R ß-strand 6 did not introduce additional hFSH sensitivity to the mutant hLH-R when present in combination with only hFSH-R ß-strands 1 and 8 (i.e. hLH-R/Fß1,Fß6,Fß8), exchanging ß-strand 6 together with the ß-strands 1, 7, and 8 (i.e. hLH-R/Fß1,Fß6,Fß7,Fß8) resulted in an approximately 2.4-fold gain in hFSH sensitivity compared with hLH-R/Fß1,Fß7,Fß8 (Fig. 6B and Table 1). Moreover, introducing hFSH-R ß-strand 2 together with ß-strands 1, 6, 7, and 8 increased the hFSH sensitivity of this mutant hLH-R (i.e. hLH-R/Fß1,Fß2,Fß6,Fß7,Fß8) somewhat further (Fig. 6B and Table 1). However, hLH-R/Fß1,Fß2,Fß6,Fß7,Fß8 is still 5-fold less sensitive to hFSH than FFL-hLH-R and the wild-type hFSH-R. No specific binding of [¹²⁵I]hCG was observed on hLH-R/Fß1,Fß2,Fß6,Fß7,Fß8 mutant-expressing HEK-T 293 cells (Fig. 4A), which may be related to the reduced responsiveness of this mutant to hCG in combination with reduced expression at the cell surface. The subsequent introduction of ß-strand 9 (generating the mutant receptor hLH-R/Fß1,Fß2,Fß6,Fß7,Fß8,Fß9) did not result in a further increase of the responsiveness to hFSH (Fig. 6B and Table 1).

Loss-of-(hLH/hCG Selectivity) Function of hLH-R Mutants
The substitution of individual hLH-R ß-strands with corresponding hFSH-R sequences did not significantly affect receptor responsiveness to hCG/hLH. Previous studies indicated the presence of determinants that affect hCG/hLH selectivity in ß-strands 3 and 6 of gonadotropin receptors (7, 8, 9). However, in contrast to the pronounced contribution of hLH-R ß-strands 3 or 6 in conferring hCG/hLH sensitivity to mutant hFSH-Rs [cf. hFSH-R/Lß3 and hFSH-R/Lß6 with hFSH-R (9)], the reciprocal substitutions of hLH-R ß-strands 3 or 6 (i.e. hLH-R/Fß3 and hLH-R/Fß6) resulted in an insignificant 2- to 5-fold decrease, respectively, in hCG/hLH responsiveness compared with wild-type hLH-R (Fig. 3, A and C, and Table 1). Substitution of both hLH-R ß-strands 3 and 6 with corresponding hFSH-R ß-strands (i.e. hLH-R/Fß3,Fß6) resulted in a more significant, approximately 74-fold, reduction of receptor sensitivity toward hCG/hLH compared with wild-type hLH-R (Fig. 7 and Table 1). This, however, is considerably less than the full gain in hCG/hLH responsiveness (673-fold) upon introducing hLH-R ß-strands 3 and 6 into the hFSH-R (i.e. hFSH-R/Lß3,Lß6) compared with the wild-type hFSH-R (9). The absence of detectable [¹²⁵I]hCG binding to hLH-R/Fß3,Fß6-expressing cells did not unambiguously support the proposed contribution of these ß-strands to hCG selectivity (Fig. 4A), as it may well be caused by low receptor expression at the cell surface, as indicated by ELISA (Table 1).

View larger version (19K): [in this window] [in a new window]   Fig. 7. cAMP-Mediated Reporter Gene Activity in Response to hCG in HEK-T 293 Cells Expressing Wild-Type, Chimeric hLH-R, or Mutant ß-Strand hLH-Rs Results are shown as mean ± SEM of triplicate observations from a single representative experiment. Mean EC50 values and receptor cell surface expression levels are presented in Table 1.

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

Chimeric hLH-Rs, in which the NCR, LRR subdomain, and/or CCR were exchanged with corresponding hFSH-R sequences, revealed that key residues involved in determining hFSH selectivity are confined to the LRR subdomain of the N-terminal ECD (cf. hormone responsiveness of FFF-hLH-R and FFL-hLH-R with hLH-R, and FLL-hLH-R/Fß1 and FLL-hLH-R/Fß1,Fß8 with hLH-R/Fß1 and hLH-R/Fß1,Fß8, respectively). Likewise, introducing the LRR subdomain of the hLH-R into the hFSH-R transformed its hormone-binding profile into that of the hLH-R (9). Recent homology modeling, using the Nogo-receptor ECD crystal structure as template, suggested that the CCR contributes with one additional parallel ß-strand to the concave ß-sheet of the hormone-binding domain (17). However, sequence analysis of this so-called 10^th ß-strand revealed a high sequence similarity between the hFSH-R and hLH-R (i.e. ²⁶⁵MEASLTY²⁷¹ and ²⁶⁹LEATLTY²⁷⁵, respectively), suggesting that this ß-strand is not likely to contribute in determining hormone selectivity (cf. FFF-hLH-R and FFL-hLH-R). Moreover, both C-terminal truncation experiments of soluble hLH-R ECD (12, 26), as well as the crystal structure of the hFSH-hFSH-R complex (13), revealed a very limited contribution of the CCR to gonadotropin binding. Surprisingly, however, exchanging the CCR of rat FSH-R with that of the rat LH-R decreased hFSH binding and responsiveness (27).

In addition, the recent Nogo receptor-based homology model (17) suggested that the NCR of glycoprotein hormone receptors is integrated into the LRR structure by a short antiparallel ß-strand and a parallel ß-strand (i.e. the so-called ß-strand 0, which comprises hLH-R residues ⁴⁰GALRCPG⁴⁶ and hFSH-R residues ²⁸RVFLCQE³⁴) that are stabilized by disulfide bonds between their conserved cysteines (17). Moreover, this predicted contribution of the NCR to the concave hormone-binding domain was subsequently confirmed by the hFSH-R crystal structure (13). Although the Glu residue, present at the C terminus of hFSH-R ß-strand 0, was found to interact with Tyr¹⁰³ present in the seat belt loop of the hFSH ß-subunit, our data suggest that the NCR is not involved in determining ligand selectivity (cf. FLL-hLH-R/Fß1 and FLL-hLH-R/Fß1,Fß8 with hLH-R/Fß1 and hLH-R/Fß1,Fß8, respectively). In fact, Ala substitution of this Glu residue, together with both its adjacent residues (i.e. ³³QES³⁵ to ³³AAA³⁵), slightly increased the affinity of hFSH-R for hFSH (28).

To identify hFSH-selective determinants in the concave hormone-binding domain, we systematically substituted the five hormone-exposed X residues of each hLH-R ß-strand with their corresponding hFSH-R residues. Our mutagenesis strategy was guided by homology models of the LRR subdomain of glycoprotein hormone receptors existing at that time (14, 15, 16, 19, 21, 22). These models predicted reasonably regular repeats, in particular when considering the inner ß-sheet lining of the banana-like structure. However, the structure of the hFSH-hFSH-R complex shows that the LRRs are irregular in length and conformation (13). In addition, all ß-strands (in particular, the ß-strands 7–9) in the hFSH-R structure were found to be shorter than the X₁X₂LX₃LX₄X₅ ß-strand signature predicted by most homology models (Fig. 1). Nevertheless, all X residues that were predicted to form the inner lining of the concave ß-sheet by homology modeling were found to form the hormone-accessible binding surface of the hFSH-R structure. Our gain-of-function mutagenesis approach revealed the presence of key hFSH-selective determinant(s) in ß-strand 1 of the hFSH-R (i.e. X₁X₂LX₃LX₄X₅ = ⁴⁹IELRFVL⁵⁵), because the introduction of this ß-strand into the hLH-R yielded a significant increase in hFSH responsiveness (EC₅₀ = 11.3 ± 2.1 ng/ml). In this respect, it should be noted that the wild-type hLH-R displays no responsiveness to hFSH. The gain in hFSH responsiveness of this hLH-R/Fß1 mutant receptor resulted predominantly from the introduction of hFSH-R ß-strand 1 X residues, because Ala-substitution of the corresponding residues (i.e. hLH-R/Alaß1) resulted in a more than 100-fold decrease in hFSH-responsiveness compared with hLH-R/Fß1. However, the fact that hLH-R/Alaß1 displayed some responsiveness to hFSH, in contrast to the wild-type hLH-R, suggests that hLH-R ß-strand 1 contains one or more selective determinants that inhibit hFSH from interacting with the hLH-R ECD. The involvement of hFSH-R ß-strand 1 in determining hFSH selectivity was also indicated by the hFSH-hFSH-R crystal structure, with the side chain of Leu⁵⁵ (position X₅ of the homology model ß-strand signature, but situated in a loop in the hFSH-R structure) fitting into a shallow pocket on the hFSH surface and forming hydrophobic interactions with residues Leu⁹⁹ and Tyr¹⁰³ of the hFSH ß-subunit seat belt loop as well as with the aliphatic part of Arg⁴² in loop 2 of the -subunit (13). This shallow pocket is too small to accommodate the larger side chain of Tyr ⁵⁸, which is present in the hLH-R at the corresponding position of Leu⁵⁵ of the hFSH-R, and, as such, sterically hinders hFSH from interacting with the hLH-R. Ala substitution of the Leu⁵⁵ residue in the hFSH-R (i.e. hFSH-R/L⁵⁵A), but also other individual hFSH-R ß-strand 1 X residues, did not diminish the receptor responsiveness to hFSH, whereas substitution of hFSH-R ß-strand 1 with corresponding hLH-R sequences or Ala residues (i.e. hFSH-R/Lß1 and hFSH-R/Alaß1) disrupted receptor cell surface expression and hormone responsiveness (9). The fact that Ala substitution of Leu⁵⁵ of hFSH-R is well tolerated with respect to hFSH responsiveness suggests that hydrophobic interactions of the Leu⁵⁵ side chain contribute, to a limited extent, to the overall hFSH-hFSH-R binding energy, although this hypothesis is in contrast to the observed contribution of hFSH-R ß-strand 1 in attracting hFSH to interact with hLH-R/Fß1.

Minor hFSH-selective determinants were identified in hFSH-R ß-strands 7 and 8 (i.e. X₁X₂LX₃LX₄X₅ = ¹⁹⁶DELNLSD²⁰² and ²²¹VILDISR²²⁷, respectively), with both hLH-R/Fß7 and hLH-R/Fß8 being consistently responsive to stimulation with hFSH levels of more than 1 µg/ml. Residues of the hFSH-R ß-strand signatures 7 and 8 (i.e. Asp²⁰², and Val²²¹ and Ile²²², respectively) were found to interact with residues Asp⁹⁰ (of the hFSH ß-subunit seat belt loop) and Pro⁴², Ala⁴³, Pro⁴⁵ (in loop 2 of the hFSH ß-subunit), respectively, in the crystal structure of the hFSH-hFSH-R complex (13). Substitution of all ß-strand 7 residues of hFSH-R with corresponding hLH-R- or Ala-residues (i.e. hFSH-R/Lß7 and hFSH-R/Alaß7), significantly impaired receptor cell surface expression as well as hormone responsiveness (9). However, Ala scanning mutagenesis of individual hFSH-R ß-strand X residues revealed no significant involvement in the interaction with hFSH. In line with the limited responsiveness of hLH-R/Fß8, the reciprocal substitution of hFSH-R ß-strand 8 with the corresponding hLH-R residues (i.e. hFSH-R/Lß8) resulted in a small reduction of its responsiveness to hFSH (9).

Our single ß-strand substitution strategy did not clearly indicate the involvement of the other hFSH-R ß-strands (i.e. ß-strands 2, 3, 4, 6, and 9), in conferring receptor responsiveness toward hFSH. A potential contribution of ß-strand 5 to hFSH and/or hCG/hLH selectivity is uncertain, because hLH-R ß-strand 5 substitution disrupted receptor cell surface expression as well as ligand responsiveness. However, the substitution of hFSH-R ß-strand 5 with the corresponding ß-strand of the hLH-R (i.e. hFSH-R/Lß5) did not affect the hormone selectivity profile compared with wild-type hFSH-R (9). In addition, a previous study, in which ECD receptor chimeras were created by exchanging fragments of rat LH-R and FSH-R using common endonuclease restriction sites, indicated that ß-strand 5 is not involved in determining hFSH selectivity (1).

Combining the three identified hFSH-selective determinants of hFSH-R ß-strands 1, 7, and 8 into the hormone-binding ß-sheet of the hLH-R (i.e. hLH-R/Fß1,Fß7,Fß8) synergistically increased the hFSH responsiveness of this hLH-R mutant receptor compared with the effect of introducing each of these ß-strands individually. However, hLH-R/Fß1,Fß7,Fß8 was still approximately 30-fold less responsive to hFSH than the wild-type hFSH-R and the chimeric ECD receptor FFL-hLH-R, indicating that additional hFSH-selective determinants are present in other ß-strands. The contributions of these unidentified determinants in conferring hFSH selectivity must be very weak, because the corresponding single ß-strand hLH-R mutants did not display any responsiveness to hFSH, but are predicted to act in synergy with the hFSH-selective determinants present in hFSH-R ß-strands 1, 7, and 8. Moyle et al. (1) have shown that amino acids encoded by exons 1, 2, 3, 4, 7, 8, 9, and 10 of the rat FSH-R (corresponding to the NCR, LRRs 1, 2, 3, 6, 7, 8, and 9, and the CCR) contributed to confer hFSH selectivity to a chimeric rat hLH-R. Guided by these results, we stepwise introduced hFSH-R ß-strands, in combination with ß-strands 1, 7 and/or 8 of the hFSH-R, into the hLH-R and identified small contributions of ß-strands 2 and 6 in conferring hFSH responsiveness to the mutant hLH-R. Both hFSH-R ß-strands 2 and 6 of the hFSH-R were found to interact with hFSH in the crystal structure (13): residues Lys⁷⁴ and Gln⁷⁹ of hFSH-R ß-strand 2 interact with the C terminus and loop 2, respectively, of the common -subunit of hFSH (13). Hence, the very small contribution of hFSH-R ß-strand 2 in determining hFSH selectivity is not that surprising because this ß-strand differs only in a single amino acid residue between the hFSH-R and hLH-R (i.e. X₁X₂LX₃LX₄X₅ = ⁷³EKIEISQ⁷⁹ and ⁷⁶IKIEISQ⁸², respectively). However, the substitution of Ile⁷⁶ with Glu may indirectly affect the microenvironment of hLH-R ß-strand 2, and as such apparently optimizes the interaction with the -subunit of hFSH. In this respect, it should be noted that the loops of the common -subunit display subtle conformational differences between the crystal structures of hFSH and hCG (29). The relatively small contribution of hFSH-R ß-strand 6 (i.e. X₁X₂LX₃LX₄X₅ = ¹⁷³VILWLNK¹⁷⁹) in determining hFSH selectivity, as determined by our gain-of-function strategy, may be surprising, considering the three hydrogen bonds observed in the crystal structure between Lys¹⁷⁹ of the hFSH-R and Asp⁹⁰ and Ser⁸⁹ of the hFSH ß-subunit seat belt loop (13). However, Lys¹⁷⁹ can be substituted with Ala, Gly, Met, Gln, Asn, Asp, Arg, and Trp without affecting the hFSH responsiveness of hFSH-R (8). The fact that hLH-R/Fß1,Fß2,Fß6,Fß7,Fß8 was still 5-fold less sensitive to hFSH than FFL-hLH-R may well be caused by masking interactions between neighboring hFSH-R and hLH-R ß-strands, which are apparently absent when an entire exodomain is exchanged. This may also explain why hFSH-R ß-strands 2 and 6 did not confer any hFSH responsiveness to the hLH-R when introduced individually. Such masking interactions have been described for the TSH-R, for which a naturally occurring mutant displayed increased sensitivity for hCG (6).

In the structure of the hFSH-hFSH-R complex, ß-strand 3 appeared to substantially contribute to the hormone-binding interface, with multiple residues in ß-strand 3 and its adjacent C-terminal loop interacting with both subunits of hFSH (13). Residues Arg¹⁰¹ (corresponding to Leu¹⁰⁴ in hLH-R) and Lys¹⁰⁴ (corresponding to Asn¹⁰⁷ in hLH-R) were identified to be involved in hormone selectivity, because of their interaction with hFSH-specific residues in the seat belt loop of the hFSH ß-subunit. However, introducing ß-strand 3 of hFSH-R, in combination with hFSH-R ß-strands 1 and 8 into the hLH-R, surprisingly diminished the responsiveness of this mutant hLH-R to both hFSH and hCG/hLH (see discussion below).

Full hCG/hLH responsiveness can be conferred to the hFSH-R by substituting two amino acid residues with their corresponding hLH-R residues [i.e. Lys¹⁰⁴ in ß-strand 3, and Lys¹⁷⁹ in ß-strand 6; (7, 8, 9)]. Detailed mutagenic analysis revealed that these two determinants have an opposite contribution in hCG/hLH-selectivity. An Asn residue should be present at the C-terminal X₅ position (i.e. position 104) of hFSH-R ß-strand 3, whereas Lys¹⁷⁹ (normally repelling hLH and hCG from binding to the receptor) should be absent at the C-terminal X₅ position of ß-strand 6, in the hormone-binding domain of hFSH-R to gain affinity for hCG/hLH (8). However, in contrast to this so-called gain-of-function hFSH-R mutations, with respect to hCG/hLH selectivity, the reciprocal loss-of-function hLH-R mutations (i.e. hLH-R/Fß3, hLH-R/Fß6, and hLH-R/Fß3,Fß6) on hCG/hLH responsiveness were significantly less effective. The puzzling difference in level of hCG/hLH selectivity between the gain-of-function and loss-of-function mutants of these hCG/hLH-selective determinants in ß-strands 3 and 6 may be related to differences in side chain accessibility on the hormone-binding surface, which can be influenced by interactions with residues of adjacent ß-strands. Interactions between residues in neighboring ß-strands have been shown to underlie gestational hyperthyroidism resulting from an increased hCG sensitivity, which is caused by a single point mutation (K¹⁸³R) in the hTSH-R (6). The K¹⁸³R mutation disrupts a salt bridge between Lys¹⁸³ in ß-strand 6 and Glu¹⁵⁷ in ß-strand 5, resulting in the surface exposure and subsequent interaction of Glu¹⁵⁷ with hCG. However, inspection of the amino acids constituting the microenvironment of hCG/hLH-selective determinants in ß-strands 3 and 6 revealed no striking differences in physicochemical side chain properties between the ß-strands 3 and 6 mutants of hFSH-R (9) and hLH-R (Fig. 1). Alternatively, the difference in potency of the hCG/hLH-selective determinants in ß-strands 3 and 6 between the hFSH-R and hLH-R may indicate subtle differences in orientation, in which hCG/hLH binds to the hormone-binding domain of hFSH-R and hLH-R. To determine whether hCG/hLH interacts in a similar orientation with the promiscuous hFSH-R mutant and the wild-type hLH-R, we are currently investigating the effects of Ala-scanning of conserved ß-strand X residues on hCG/hLH responsiveness between these two receptors.

In conclusion, this study was primarily undertaken to identify hFSH-selective determinants that ensure the highly specific interaction of this hormone with its cognate receptor. Previous loss-of-function mutational analysis of the hFSH-R did not persuasively identify hFSH-selective determinants (9). For this reason, we have applied our gain-of-function mutational strategy (9) by introducing hFSH responsiveness to the hLH-R through the stepwise substitution of hLH-R domains with corresponding hFSH-R sequences. This study was initiated and performed before the crystal structure of hFSH in complex with its hormone-binding domain of the hFSH-R was solved. Hence, our mutants were rationally designed, based on the homology models of LRR domain available at that time. However, post hoc comparison of the designed mutants with the crystal structure of the hFSH-R ECD revealed that our mutants correspond well to the structural subdomains (i.e. ß-strands that make up the parallel ß-sheet) that make up the hormone-binding domain (13). Our results revealed the presence of predominant hFSH-selective determinant(s) in hFSH-R ß-strand 1, with minor contributions of the hFSH-R ß-strands 2, 6, 7, and 8. These findings are a substantial refinement of the crude analysis of hFSH selectivity using chimeric rat gonadotropin receptors (1). Moreover, our results are in accordance with the proposed selective interaction sites between hFSH-R and hFSH in the crystal structure (13). In addition, mutational analysis of the hLH-R ß-strands allowed the evaluation of previously identified hCG/hLH-selective determinants in a loss-of-function setting. The differential potency, in which similar hCG/hLH-selective determinants act between the hFSH-R and hLH-R, suggests that the different gonadotropins interact in a somewhat different orientation with each of the ECDs of their receptors. The latter is currently under investigation using mutational analysis. However, the recent success in resolving the three-dimensional structure of hFSH bound to a large part of the N-terminal ECD of hFSH-R requires similar analyses of hCG in complex with the hLH-R as well as with the promiscuous hFSH-R mutant (7, 8). Moreover, the recently initiated postcrystal structure era in the field of gonadotropin receptor research should finally resolve the questions of how these unique members of the G protein-coupled receptor family coevolved with their ligands into highly selective ligand-receptor pairs as well as how hormone binding triggers the subsequent steps of receptor activation in the transmembrane domain.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Construction of the hLH-R Mutants
Mutant receptor cDNA pcDNA3.1/V5-His-TOPO vector (Invitrogen, San Diego, CA) constructs were generated by PCR-based mutagenesis using hLH-R cDNA templates, containing an HA-epitope (derived from the influenza virus hemagglutinin) sequence inserted between the C terminus of the signal peptide and the N terminus of the mature hLH-R protein (9).

Transfection Experiments
HEK-T 293 cells (3.5 x 10⁶ cells) were transiently transfected with 1 µg HA-tagged hLH-R expression vector construct in combination with 10 µg pCRE/ß-gal plasmid using a modified bovine serum transfection method (9). The pCRE/ß-gal plasmid consists of a ß-galactosidase gene under the control of a human vasoactive intestinal peptide promoter containing five cAMP-response elements (30). Empty pcDNA3.1/V5-His vector was used for mock transfections. For receptor binding experiments, HEK-T 293 cells were transfected with HA-tagged receptor expression vector construct using 25-kDa linear polyethylenimine (PEI; Polysciences, Inc., Niles, IL). Briefly, three million HEK-T 293 cells were seeded in a 10-cm dish the day before transfection. DNA (10 µg) DNA and PEI (30 µg) were separately diluted in 250 µl NaCl solution (150 mM). Next, the PEI solution was added to the DNA solution, vortexed, and incubated for 15 min at room temperature. Culture medium was replaced by 6 ml DMEM, and the DNA/PEI solution was added dropwise to the cells. Cells were incubated at 37 C and after 5 h the transfection medium was replaced by culture medium. The next day, cells were trypsinized and seeded into poly-D-lysine (Sigma Chemical Co., St. Louis, MO) coated 24-well (Costar Corp., Cambridge, MA) 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 and -binding studies.

ELISA Detection of HA-Tagged Receptors on the Cell Surface
HA-tagged receptor cell surface expression was quantified by ELISA as previously described (9). 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, Indianapolis, IN; 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 hLH-R expression values were expressed as the percentage of wild-type hLH-R expression. All experiments were repeated at least three times using cells from independent transfections.

Detection of Ligand-Induced cAMP Production
Receptor-mediated stimulation of cAMP-induced reporter gene activity was assayed as described previously (9). 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 (AU). 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 PRISM4 (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
Displacement binding experiments were performed 48 h after transfection on whole cells. Cells were incubated with 0.7 ng/ml (10,000 cpm) [¹²⁵I]hCG (i.e. NEX106 with a specific activity of 105 µCi/µg; purchased from PerkinElmer Life Sciences, Inc., Boston, MA) in the absence or presence of increasing concentrations of unlabeled hCG or hFSH in binding buffer (10 mM Tris, 5 mM MgCl₂ · 6 H₂O, 200 mM sucrose, 0.1% BSA, pH 7.5). After an overnight incubation at 4 C, cells were washed twice with ice-cold binding buffer. Cells were collected in lysis buffer (0.5% Nonidet P-40, 0.1% sodium dodecyl sulfate, 0.5% deoxycholic acid), and bound radioactivity was quantified in a -counter. One-site competition curves were fitted using GraphPad PRISM4, and binding affinities (K_i) were calculated from the IC₅₀ values. All binding studies were performed in duplicate in at least three independent experiments.

Data Analysis
All data are presented as mean ± SEM. Statistical comparisons were performed on log (EC₅₀) values using one-way ANOVA, followed by the Bonferroni test, using GraphPad PRISM4. P < 0.05 was considered to be significant.

	ACKNOWLEDGMENTS

 
We thank T. M. Hulscher and L. Bosch (Medicinal Chemistry, Vrije Universiteit Amsterdam, The Netherlands) for technical assistance with receptor binding assays.

	FOOTNOTES

 
Present address for H.F.V: Leiden/Amsterdam Center for Drug Research, Division of Medicinal Chemistry, Faculty of Sciences, Vrije Universiteit Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.

This work was supported by National Institutes of Health Grant DK 69711 (to J.B.).

First Published Online March 30, 2006

Abbreviations: AU, arbitrary units; CCR, C-terminal cysteine-rich subdomain; CG, chorionic gonadotropin; ECD, extracellular domain; FSH-R, FSH receptor; HA, hemagglutinin; HEK, human embryonic kidney; LH-R, LH receptor; LRR, leucine-rich repeat; NCR, N-terminal cysteine-rich subdomain; PEI, polyethylenimine; TSH-R, TSH receptor.

Received for publication May 20, 2005. Accepted for publication March 22, 2006.

	REFERENCES

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 

1. Moyle WR, Campbell RK, Myers RV, Bernard MP, Han Y, Wang X 1994 Co-evolution of ligand-receptor pairs. Nature 368:251–255[CrossRef][Medline]
2. Li MD, Ford JJ 1998 A comprehensive evolutionary analysis based on nucleotide and amino acid sequences of the - and ß-subunits of glycoprotein hormone gene family. J Endocrinol 156:529–542[Abstract]
3. Braun T, Schofield PR, Sprengel R 1991 Amino-terminal leucine-rich repeats in gonadotropin receptors determine hormone selectivity. EMBO J 10:1885–1890[Medline]
4. Nagayama Y, Wadsworth HL, Chazenbalk GD, Russo D, Seto P, Rapoport B 1991 Thyrotropin-luteinizing hormone/chorionic gonadotropin receptor extracellular domain chimeras as probes for thyrotropin receptor function. Proc Natl Acad Sci USA 88:902–905[Abstract/Free Full Text]
5. Combarnous Y 1992 Molecular basis of the specificity of binding of glycoprotein hormones to their receptors. Endocr Rev 13:670–691[CrossRef][Medline]
6. Smits G, Govaerts C, Nubourgh I, Pardo L, Vassart G, Costagliola S 2002 Lysine 183 and glutamic acid 157 of the TSH receptor: two interacting residues with a key role in determining specificity toward TSH and human CG. Mol Endocrinol 16:722–735[Abstract/Free Full Text]
7. Smits G, Campillo M, Govaerts C, Janssens V, Richter C, Vassart G, Pardo L, Costagliola S 2003 Glycoprotein hormone receptors: determinants in leucine-rich repeats responsible for ligand specificity. EMBO J 22:2692–2703[CrossRef][Medline]
8. Vischer HF, Granneman JC, Bogerd J 2003 Opposite contribution of two ligand-selective determinants in the N-terminal hormone-binding exodomain of human gonadotropin receptors. Mol Endocrinol 17:1972–1981[Abstract/Free Full Text]
9. Vischer HF, Granneman JC, Noordam MJ, Mosselman S, Bogerd J 2003 Ligand selectivity of gonadotropin receptors. Role of the ß-strands of extracellular leucine-rich repeats 3 and 6 of the human luteinizing hormone receptor. J Biol Chem 278:15505–15513[Abstract/Free Full Text]
10. Themmen APN, Huhtaniemi IT 2000 Mutations of gonadotropins and gonadotropin receptors: elucidating the physiology and pathophysiology of pituitary-gonadal function. Endocr Rev 21:551–583[Abstract/Free Full Text]
11. Osuga Y, Kudo M, Kaipia A, Kobilka B, Hsueh AJ 1997 Derivation of functional antagonists using N-terminal extracellular domain of gonadotropin and thyrotropin receptors. Mol Endocrinol 11:1659–1668[Abstract/Free Full Text]
12. Hong S, Phang T, Ji I, Ji TH 1998 The amino-terminal region of the luteinizing hormone/choriogonadotropin receptor contacts both subunits of human choriogonadotropin. I. Mutational analysis. J Biol Chem 273:13835–13840[Abstract/Free Full Text]
13. Fan QR, Hendrickson WA 2005 Structure of human follicle-stimulating hormone in complex with its receptor. Nature 433:269–277[CrossRef][Medline]
14. Kajava AV, Vassart G, Wodak SJ 1995 Modeling of the three-dimensional structure of proteins with the typical leucine-rich repeats. Structure 3:867–877[Medline]
15. Jiang X, Dreano M, Buckler DR, Cheng S, Ythier A, Wu H, Hendrickson WA, el Tayar N 1995 Structural predictions for the ligand-binding region of glycoprotein hormone receptors and the nature of hormone-receptor interactions. Structure 3:1341–1353[Medline]
16. Bhowmick N, Huang J, Puett D, Isaacs NW, Lapthorn AJ 1996 Determination of residues important in hormone binding to the extracellular domain of the luteinizing hormone/chorionic gonadotropin receptor by site-directed mutagenesis and modeling. Mol Endocrinol 10:1147–1159[Abstract]
17. Kleinau G, Jaschke H, Neumann S, Lattig J, Paschke R, Krause G 2004 Identification of a novel epitope in the thyroid-stimulating hormone receptor ectodomain acting as intramolecular signaling interface. J Biol Chem 279:51590–51600[Abstract/Free Full Text]
18. Kobe B, Kajava AV 2001 The leucine-rich repeat as a protein recognition motif. Curr Opin Struct Biol 11:725–732[CrossRef][Medline]
19. Song YS, Ji I, Beauchamp J, Isaacs NW, Ji TH 2001 Hormone interactions to Leu-rich repeats in the gonadotropin receptors. II. Analysis of Leu-rich repeat 4 of human luteinizing hormone/chorionic gonadotropin receptor. J Biol Chem 276:3436–3442[Abstract/Free Full Text]
20. Song BH, Lee GC, Moon MS, Cho YH, Lee CH 2001 Human cytomegalovirus binding to heparan sulfate proteoglycans on the cell surface and/or entry stimulates the expression of human leukocyte antigen class I. J Gen Virol 82:2405–2413[Abstract/Free Full Text]
21. Jeoung M, Phang T, Song YS, Ji I, Ji TH 2001 Hormone interactions to Leu-rich repeats in the gonadotropin receptors. III. Photoaffinity labeling of human chorionic gonadotropin with receptor Leu-rich repeat 4 peptide. J Biol Chem 276:3443–3450[Abstract/Free Full Text]
22. Song YS, Ji I, Beauchamp J, Isaacs NW, Ji TH 2001 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. J Biol Chem 276:3426–3435[Abstract/Free Full Text]
23. Ji IH, Ji TH 1991 Human choriogonadotropin binds to a lutropin receptor with essentially no N-terminal extension and stimulates cAMP synthesis. J Biol Chem 266:13076–13079[Abstract/Free Full Text]
24. Zhang R, Buczko E, Dufau ML 1996 Requirement of cysteine residues in exons 1–6 of the extracellular domain of the luteinizing hormone receptor for gonadotropin binding. J Biol Chem 271:5755–5760[Abstract/Free Full Text]
25. Sohn J, Youn H, Jeoung M, Koo Y, Yi C, Ji I, Ji TH 2003 Orientation of follicle-stimulating hormone (FSH) subunits complexed with the FSH receptor ß-subunit toward the N terminus of exodomain and subunit to exoloop 3. J Biol Chem 278:47868–47876[Abstract/Free Full Text]
26. Zeng H, Phang T, Song YS, Ji I, Ji TH 2001 The role of the hinge region of the luteinizing hormone receptor in hormone interaction and signal generation. J Biol Chem 276:3451–3458[Abstract/Free Full Text]
27. Moyle WR, Xing Y, Lin W, Cao D, Myers RV, Kerrigan JE, Bernard MP 2004 Model of glycoprotein hormone receptor ligand binding and signaling. J Biol Chem 279:44442–44459[Abstract/Free Full Text]
28. Nechamen CA, Dias JA 2000 Human follicle stimulating hormone receptor trafficking and hormone binding sites in the amino terminus. Mol Cell Endocrinol 166:101–110[CrossRef][Medline]
29. Dias JA, Van Roey P 2001 Structural biology of human follitropin and its receptor. Arch Med Res 32:510–519[CrossRef][Medline]
30. Chen WB, Shields TS, Stork PJS, Cone RD 1995 A colorimetric assay for measuring activation of G(s)- and G(q)-coupled signaling pathways. Anal Biochem 226:349–354[CrossRef][Medline]

This article has been cited by other articles:

				M. Y.-K. Leung, P. J. Steinbach, D. Bear, V. Baxendale, P. Y. Fechner, O. M. Rennert, and W.-Y. Chan Biological Effect of a Novel Mutation in the Third Leucine-Rich Repeat of Human Luteinizing Hormone Receptor Mol. Endocrinol., October 1, 2006; 20(10): 2493 - 2503. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 20/8/1880    most recent Author Manuscript (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire