Ligand Selectivity of Gonadotropin Receptors
ROLE OF THE
-STRANDS OF EXTRACELLULAR LEUCINE-RICH REPEATS 3 AND 6 OF THE HUMAN LUTEINIZING HORMONE RECEPTOR*
Henry F.
Vischer
,
Joke C. M.
Granneman
,
Michiel J.
Noordam
,
Sietse
Mosselman§, and
Jan
Bogerd
¶
From the
Department of Endocrinology, Utrecht
University, 3584 CH Utrecht, and the § Department of
Pharmacology, Organon Inc., 5342 CC Oss, The Netherlands
Received for publication, January 21, 2003, and in revised form, February 20, 2003
 |
ABSTRACT |
The difference in hormone selectivity between the
human follicle-stimulating hormone receptor (hFSH-R) and human
luteinizing hormone/chorionic gonadotropin receptor (hLH-R) is
determined by their ~350 amino acid-long N-terminal receptor
exodomains that allow the mutually exclusive binding of human
follicle-stimulating hormone (hFSH) and human luteinizing hormone (hLH)
when these hormones are present in physiological concentrations. The
exodomains of each of these receptors consist of a nine-leucine-rich
repeat-containing subdomain (LRR subdomain) flanked by N- and
C-terminal cysteine-rich subdomains. Chimeric receptors, in which the
structural subdomains of the hFSH-R exodomain were substituted with
those of the hLH-R, showed a similar high responsiveness to human
chorionic gonadotropin (hCG) and hLH as long as they harbored the LRR
subdomain of the hLH-R. In addition, these chimeric receptors showed no
responsiveness to hFSH. The LRR subdomains of the gonadotropin receptor
exodomains are predicted to adopt a horseshoe-like conformation, of
which the hormone-binding concave surface is composed of nine parallel
-strands. Receptors in which individual
-strands of the hFSH-R were replaced with the corresponding hLH-R sequences revealed that hCG
and hLH selectivity is predominantly determined by hLH-R
-strands 3 and 6. A mutant receptor in which the hFSH-R
-strands 3 and 6 were
substituted simultaneously with their hLH-R counterparts displayed a
responsiveness to hCG and hLH similar to that of the wild type hLH-R.
Responsiveness to hFSH was not affected by most
-strand
substitutions, suggesting the involvement of multiple low-impact
determinants for this hormone.
 |
INTRODUCTION |
Follicle-stimulating hormone
(FSH)1 and luteinizing
hormone (LH) stimulate the FSH receptor (FSH-R) and LH receptor (LH-R), respectively, which are expressed in different target cells (1). In
some species, chorionic gonadotropin (CG) is also able to stimulate the
LH-R. The coordinated interplay between the complementary and specific
actions of FSH and LH(/CG) is required to guarantee successful
reproduction. The interaction between these gonadotropins and their
respective receptors is highly specific, and there is virtually no
cross-reactivity between hormones and heterologous receptors
(i.e. high receptor selectivity) except for LH and CG, which
both act on the LH-R (2, 3).
The FSH-R and LH-R, together with the thyroid-stimulating hormone
receptor, represent the structurally unique glycoprotein hormone
receptor (GpHR) subfamily of the G protein-coupled receptor superfamily. Characteristically, GpHRs are composed of two
approximately equally sized but functionally distinct domains: an
extracellular N-terminal half (exodomain), which is responsible for the
selective recognition and high-affinity binding of its corresponding
hormone, and a typical G protein-coupled receptor domain, consisting of seven transmembrane
-helices, which transduces the specific signal of hormone binding to the exodomain across the membrane to activate intracellular signaling pathways (4). The GpHR exodomain is subdivided
into three structural subdomains: an N-terminal cysteine-rich subdomain
(NCR subdomain) followed by a nine contiguous imperfect leucine-rich
repeat-containing subdomain (LRR subdomain) and a C-terminal
cysteine-rich subdomain (CCR subdomain; see Figs. 1 and
2A).
LLR motifs have been recognized in a large variety of proteins, with
repeats composed of 20-29 amino acids each (5). Crystal structure
analyses of some of these LRR-containing proteins, such as the porcine
ribonuclease inhibitor (6) and Listeria internalins (7),
revealed that each LRR forms a right-handed structural unit that is
composed of a short
-strand and a helical segment, which are
positioned nearly antiparallel to each other and are linked by short
loops. Tandem arrays of LRR units form a one- to three-quarter
donut-like structure with the consecutive
-strands forming a
parallel
-sheet at the concave surface, whereas the alternating
helices are aligned next to each other to form the convex side of the
structure. The concave surface of the ribonuclease inhibitor interacts
with ribonucleases using multiple contact points (6).
Based on sequence homology, the exodomains of GpHRs have been
structurally modeled using the crystal structure of porcine ribonuclease inhibitor (8-10). Consequently, the
-sheet at the inner circumference of the curved exodomain of GpHRs has been proposed
to form the main hormone-binding site, with some additional hormone
contact sites situated outside the LRR subdomain (11, 12). Each of the
LRR
-strands is composed of a highly conserved X1X2LX3LX4X5
motif (Fig. 1), in which X
indicates any amino acid and L refers to leucine,
isoleucine, or other hydrophobic residues (5). The side chains of the
conserved L residues are directed toward the helical segment
and form the hydrophobic core of the LRR structure, whereas the side
chains of the X residues are exposed to the surface of the
presumed ligand-binding site (13-15).

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Fig. 1.
Amino acid sequence alignment of the
exodomains of the hFSH-R and the hLH-R. Each exodomain was
subdivided into three structural subdomains: the N- and C-terminal
cysteine-rich subdomains (NCR subdomain and CCR
subdomain, respectively), with conserved cysteine residues
indicated by black boxes, and the LRR subdomain, consisting
of nine consecutive LRR units. Conserved amino acid residues present in
the LRRs and forming the hydrophobic core of the structural units
are depicted in bold italic letters. The -strand
X1X2LX3LX4X5
motifs (see text) that form the inner lining of the
horseshoe-shaped LRR subdomain are indicated, and the -strand amino
acid residues are underlined. The X residues (see
"Experimental Procedures"), which are directed to the surface of
the protein and are thought to interact with the respective hormones,
are indicated by gray boxes. Dashes indicate gaps introduced
for optimal alignment.
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Selective gonadotropin binding is determined exclusively by the
exodomain of the LH-R and FSH-R. Studies using chimeric receptors revealed that the sequence NCR-LRR6 of the LH-R is important to confer
LH/CG selectivity to the receptor, whereas the sequence NCR-LRR3 of the
FSH-R, in conjunction with the FSH-R sequence LRR7-CCR, is important
for FSH binding specificity (2, 3). However, these results may be
somewhat biased because the junctions of these chimeric proteins were
introduced arbitrarily, depending mainly on the presence of common
endonuclease restriction sites rather than considering the exact
structural borders within the exodomain. Taking advantage of the
current knowledge of the structural conformation of the exodomain of
GpHRs, we examined in more detail the contribution of the NCR, LRR, and
CCR subdomains of the human LH-R (hLH-R) exodomain in conferring human
chorionic gonadotropin (hCG) and/or human LH (hLH) selectivity when
placed in the context of a human FSH-R (hFSH-R) background.
Furthermore, the contribution of individual (and combinations of) hLH-R
-strands in directing hCG and hLH (hCG/hLH) selectivity to the
receptor was studied systematically with mutant receptors generated by
introducing hLH-R
-strands into the hFSH-R.
 |
EXPERIMENTAL PROCEDURES |
Glycoprotein Hormones and Antibodies--
Human recombinant FSH
(hFSH, Org32489E), human recombinant LH (hLH, 99M7019), and human
recombinant chorionic gonadotropin (hCG, 01MZ010) were kindly provided
by Dr. W. G. E. J. Schoonen (Organon Inc., Oss, The Netherlands).
The high-affinity monoclonal anti-HA antibody and goat anti-rat IgG
peroxidase conjugate were purchased from Roche Applied Science
and Sigma, respectively.
Construction of Mutant Receptor cDNAs--
The cDNAs
encoding the human FSH-R and LH-R, kindly provided by Dr. T. Minegishi
(Gunma University School of Medicine, Maebashi, Japan) and Dr.
E. Milgrom (Institut National de la Santé et de la Recherche
Médicale, Paris, France), respectively, were subcloned into the
pcDNA3.1/V5-His-TOPO expression vector (Invitrogen). To quantify
receptor cell surface expression by enzyme-linked immunosorbent assay
(ELISA), an HA epitope (Tyr-Pro-Tyr-Asp-Val-Pro-Asp-Tyr-Ala; derived
from the influenza virus hemagglutinin) was introduced between the C
termini of the signal peptide sequences and the N termini of the mature
FSH-R (i.e. between Gly17 and Cys18)
and the mature LH-R (i.e. between Leu29 and
Arg30). HA epitope tagging did not significantly
influence the ligand binding and signaling properties of the receptors
compared with their wild type counterparts (data not shown).
HA-tagged receptor cDNAs were used as templates to generate hFSH-R
and hLH-R exodomain chimeras using a fusion PCR-based method (16).
Briefly, 5'- and 3'-cDNA fragments were generated using overlapping
primers (Invitrogen and Isogen) in combination with specific primers
demarcating the cDNA insert. These PCR fragments were then fused in
a self-primed PCR taking advantage of the introduced overlapping
sequence. The fusion products were then PCR-amplified using the
specific primers demarcating the cDNA insert. All PCRs were
performed using the Advantage-HF PCR kit
(Clontech). Four different exodomain chimeras (Fig.
2A) were generated in which particular hFSH-R domains were
substituted with either the entire exodomain of the hLH-R (generating
the chimera LLL-hFSH-R), or with the LRR subdomain in combination with
either the NCR subdomain or the CCR subdomain of hLH-R (LLF-hFSH-R and
FLL-hLH-R), or with only the LRR subdomain of the hLH-R (FLF-hFSH-R).
In a similar way, a set of mutant receptors was generated in which the
residues indicated with X, and unique to the
-strand motifs
(X1X2LX3LX4X5)
of the hLH-R, were introduced to replace residues on the homologous position in the hFSH-R (see Tables II, III, and V for the
details of which residues were replaced). All cDNAs
were subcloned into the pcDNA3.1/V5-His-TOPO expression vector and
sequenced on automated ABI PRISM 310 and 377 DNA sequencers using Dye
Terminator cycle sequencing chemistry in accordance with the
manufacturer's instructions (Applied Biosystems).
Transient Expression of Wild Type, Chimeric, and Mutant
Receptors--
Human embryonic kidney (HEK-T 293) cells were cultured
at 37 °C under 5% CO2 in culture medium (Dulbecco's
modified Eagle's medium containing 2 mM glutamine, 10%
fetal bovine serum, and 1× antibiotic/antimycotic; all from
Invitrogen). Transient transfections were performed in a 10-cm dish
containing ~3.5 × 106 cells, with 1 µg of (wild
type, chimeric, or mutant) HA-tagged receptor expression vector
construct in combination with 10 µg of a pCRE/
-gal plasmid, using
the modified bovine serum transfection method according to the
instructions of the manufacturer (Stratagene) and as described
previously (17). The pCRE/
-gal plasmid consists of a
-galactosidase gene under the control of a human vasoactive intestinal peptide promoter containing five cAMP-response
elements (18). "Empty" pcDNA3.1/V5-His vector was used for mock
transfections. The next day, cells were collected and split into
96-well tissue culture plates (~2 × 105 cells/well)
for ligand stimulation studies. In addition, duplicate aliquots
(~4.5 × 105 cells/well) were transferred to
poly-D-lysine (Sigma)-coated 24-well tissue culture plates
for ELISA detection of receptor cell surface expression.
Detection of Ligand-induced cAMP Production--
The
receptor-mediated stimulation of cAMP-induced reporter gene activity
was assayed according to Chen et al. (18) with minor
modifications as described previously (17). Briefly, 2 days after
transfection the cells were stimulated for 6 h with various
concentrations of hFSH, hCG, and hLH in 25 µl of Hepes-modified Dulbecco's modified Eagle's medium containing 0.1% bovine serum albumin and 0.1 mM 3-isobutyl-1-methylxanthine (all from
Sigma). Ligand-induced changes in
-galactosidase activity
(conversion of
o-nitrophenyl-
-D-galactopyranoside into
o-nitrophenol) were measured at 405 nm and related to the
forskolin-induced changes (10 µM) in each 96-well plate.
Therefore, the results are expressed as arbitrary units related to the
forskolin-induced cAMP-mediated reporter gene activation. 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 or chimeric) receptors. Two days after transfection,
HEK-T 293 cells were rinsed with Dulbecco's phosphate-buffered saline
(Sigma), subsequently harvested in ice-cold Tris buffer (10 mM Tris-HCl, 5 mM MgCl2·6 H2O, pH 7.4), and centrifuged at 200 × 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 × g at 4 °C for 30 min. The
pellet was resuspended in Tris buffer. Cell membranes (~50 µg of
protein) were incubated for 18-20 h in 300 µl of Tris buffer
supplemented with 0.1% bovine serum albumin at room temperature with
10,000 cpm of [125I]hFSH (i.e. NEX 173 with a
specific activity of 163 µCi/µg; purchased from PerkinElmer Life
Sciences) in the presence of increasing concentrations of unlabeled hCG
or hFSH. The reaction was terminated by adding 500 µl of ice-cold
Tris buffer supplemented with 0.1% bovine serum albumin and
subsequently centrifuged at 15,000 × g at room
temperature for 5 min. The supernatant was aspirated, and the
radioactivity in the membrane pellet was determined in an LKB
-counter (PerkinElmer Life Sciences). All binding studies were
performed in triplicate in two independent experiments.
ELISA Detection of HA-tagged Receptors on the Cell
Surface--
Aliquots of the cells used for signal transduction
experiments were used for cell surface receptor quantification by
ELISA, essentially as described previously (19). Briefly, 2 days after transfection, cells were fixed using 4% paraformaldehyde in
phosphate-buffered saline 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 (a 1:200 dilution in
Tris-buffered saline containing 0.1% bovine serum albumin) overnight
at 4 °C. The next day, the samples were incubated with
peroxidase-conjugated goat anti-rat IgG (1:500 dilution in 1% nonfat
dried milk in 0.1 M NaHCO3) at room temperature
for 2 h. The peroxidase activity was then visualized using the
3,3',5,5'-tetramethylbenzidine liquid substrate system (Sigma) for
~10 min. Absorbance values (at 450 nm) of mock transfected cells were
subtracted, and mutant or chimeric receptor expression values were
expressed as the percentage of the wild type hFSH-R expression. All
experiments were repeated at least three times using cells from
independent transfections, each performed in duplicate.
Data Analysis--
The ligand concentrations that induce
half-maximal stimulation (i.e. EC50 values) were
calculated by fitting the cAMP data to sigmoidal dose-response curves
using GraphPad PRISM3 (GraphPad Software, Inc.). Ligand binding
affinities (Ki) were calculated from one-site
competition curves using GraphPad PRISM3. All data shown in Tables I,
II, IV, and V are presented as the calculated mean ± S.E.
based on observations derived from at least three independent
experiments. Statistical comparisons were performed on the
log(EC50) or log(Ki) values using
one-way analysis of variance followed by the Bonferroni test using
GraphPad PRISM3.
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RESULTS |
Hormone Specificity of Chimeric Gonadotropin Receptors--
To
exactly determine which parts of the exodomains confer ligand
selectivity to the gonadotropin receptors, we divided their exodomains
into three structural subdomains (i.e. NCR, LRR, and CCR
subdomains). Next, we substituted individual or combinations of the
hFSH-R subdomains with their homologous hLH-R subdomains (Fig.
2A). We then tested these
chimeric receptors for their ability to mediate hCG- and hLH-induced
cAMP production in transiently transfected HEK-T 293 cells.
Substitution of the entire exodomain or the NCR subdomain in
conjunction with the LRR domain of the hFSH-R with the corresponding
hLH-R sequences (i.e. Fig. 2A,
LLL-hFSH-R and LLF-hFSH-R, respectively) resulted
in chimeric receptors that were readily expressed at the cell surface
(130 and 148% of wild type hFSH-R cell surface expression,
respectively; Fig. 2B). Moreover, these chimeric receptors
were functionally similar to wild type hLH-R (Fig. 2, C and
D, and Table I) in their
responsiveness to hFSH, as well as to hCG and hLH. An equal
responsiveness to hCG and hLH is indicated by hCG/hLH. Chimeric
hFSH-Rs, in which only the hLH-R LRR subdomain or the combination of
the hLH-R LRR and CCR subdomain was introduced (i.e. Fig.
2A, FLF-hFSH-R and FLL-hFSH-R,
respectively), were severely hampered in cell surface expression (both
<1% of the wild type hFSH-R cell surface expression; see Fig.
2B). Nevertheless, both FLF-hFSH-R and FLL-hFSH-R responded significantly better to hCG and hLH than the wild type hFSH-R (p < 0.001), although they were significantly less
efficient than wild type hLH-R, LLL-hFSH-R, and LLF-hFSH-R (Fig.
2C and Table I). Similar to the hLH-R, all chimeric hFSH-R
constructs were devoid of responsiveness to hFSH (Fig. 2D
and Table I).

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Fig. 2.
Cell surface expression and ligand-induced
cAMP production of wild type hFSH-R, wild type hLH-R, and chimeric
hFSH-Rs transiently expressed in HEK-T 293 cells. A,
schematic representation of the chimeric receptors (see
"Experimental Procedures"). Human FSH-R amino acids are
indicated by open circles and hLH-R amino acids by
filled circles. Arrows indicate the nine -strands in each
receptor. B, cell surface expression of wild type or
chimeric receptors as determined by HA tag ELISA. C
and D, human CG- and hFSH-stimulated, cAMP-mediated reporter
gene activity in HEK-T 293 cells transiently transfected with wild type
or chimeric receptor constructs. The variable levels of basal cAMP
signaling are related to different levels of receptor expression. Human
LH and hCG stimulated the various (chimeric) receptors with a similar
efficacy; for clarity, only the hCG-induced, cAMP-mediated reporter
gene activity is shown. Results are shown as the mean ± S.E. of
triplicate observations from a single representative experiment. Mean
EC50 values are presented in Table I.
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Table I
Summary of the ligand-induced intracellular cAMP production in HEK-T
293 cells transiently transfected with wild type hFSH-R, wild type
hLH-R, and chimeric hFSH-R constructs
Cyclic AMP production upon stimulation with hCG, hLH, and hFSH was
measured in HEK-T 293 cells, transiently cotransfected with various
receptor constructs and a plasmid (pCRE/ -gal) containing a
-galactosidase gene under control of a promoter containing five
cAMP-response elements. The identity of the exodomain subdomains
present in the chimeric hFSH-Rs is indicated by capital F or L for each
of the three subdomains if they were derived from the hFSH-R or the
hLH-R, respectively (see text). The EC50 values presented are
the calculated mean ± S.E. of EC50 values derived from at
least three independent experiments. Human LH and hCG yielded a similar
efficacy as determined for every construct in at least two independent
assays; for clarity, only the hCG data are shown.
EC50wt/mut values were calculated by dividing the
EC50 for the wild type hFSH-R with the wild type hLH-R or
chimeric receptor EC50 values. ND, not detectable.
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Hormone Selectivity of Mutant
-Strand hFSH Receptors--
To
identify which of the nine
-strands of the hLH-R confers hCG/hLH
selectivity to its LRR subdomain, all X residues of
individual hFSH-R
-strand motifs (with the consensus sequence
X1X2LX3LX4X5)
were mutated to their corresponding hLH-R residues (see Table II). Next, each of the mutant
-strand
hFSH-Rs was transiently expressed in HEK-T 293 cells and analyzed for
cell surface expression as well as hCG-, hLH-, and
hFSH-dependent cAMP production. Alternatively, this
strategy may also lead to the identification of
-strands of the
hFSH-R that usually are involved in repelling hCG and/or hLH from its
LRR subdomain. Most of the mutant
-strand hFSH-Rs were expressed at
the cell surface, with levels ranging from 21 to 147% of wild type
hFSH-R expression (Fig. 3, A
and D). However, hFSH-R/L
4 and hFSH-R/L
5 were
expressed at lower levels (4 and 15%, respectively), whereas
hFSH-R/L
1 and hFSH-R/L
7 were undetectable in the anti-HA tag
ELISA (Fig. 3, A and D).
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Table II
Summary of the ligand-induced intracellular cAMP production in HEK-T
293 cells transiently transfected with wild type hFSH-R, the
chimeric LLL-hFSH-R (see Table I), and mutant hFSH-R constructs
Cyclic AMP production upon stimulation with hCG, hLH, and hFSH was
measured in HEK-T 293 cells, transiently cotransfected with various
receptor constructs and a plasmid containing a -galactosidase gene
under control of a promoter containing five cAMP-response elements
(pCRE/ -gal). Residues that were introduced into the hFSH-R
-strands are underlined. The EC50 values presented are the
calculated mean ± S.E. of EC50 values derived from at
least three independent experiments. Human LH and hCG yielded a similar
efficacy as determined for every construct in at least two independent
assays; for clarity, only the hCG data are shown.
EC50wt/mut values were calculated by dividing the
EC50 for the wild type hFSH-R with the mutant receptor
EC50 values. ND, not detectable.
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Fig. 3.
Cell surface expression and ligand-induced
cAMP production of wild type, chimeric, or mutant hFSH-Rs transiently
expressed in HEK-T 293 cells. A, D, and G,
cell surface expression of wild type, chimeric, or mutant -strand
hFSH-Rs as determined by HA tag ELISA. B, E, and
H, human CG-stimulated, cAMP-mediated reporter gene activity
in HEK-T 293 cells transiently transfected with wild type, chimeric, or
mutant -strand hFSH-R constructs. C, F, and I,
human FSH-stimulated, cAMP-mediated reporter gene activity in HEK-T 293 cells transiently transfected with wild type, chimeric, or mutant
-strand hFSH-R constructs. Human LH and hCG stimulated all
constructs with a similar efficacy; for clarity, only the hCG-induced,
cAMP-mediated reporter gene activity is shown. Results are shown as the
mean ± S.E. of triplicate observations from a single
representative experiment. Mean EC50 values are presented
in Table II. ND, not detectable.
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The hFSH-R/L
2, hFSH-R/L
3, hFSH-R/L
5, hFSH-R/L
6, and
hFSH-R/L
9 constructs responded to hFSH with EC50 values
(ranging from 0.064 to 0.232 ng/ml; p > 0.05) similar
to that of wild type hFSH-R (Fig. 3, C and F, and
Table II). This finding indicates that these hFSH-R
-strands, when
replaced by hLH-R
-strands, are not crucial in conferring hFSH
selectivity to the hFSH-R exodomain. In contrast, the responsiveness to
hFSH was decreased significantly in hFSH-R/L
8 (5-fold;
p < 0.05), hFSH-R/L
4 (14-fold; p < 0.001), hFSH-R/L
7 (130-fold; p < 0.001), and
hFSH-R/L
1 (> 2600-fold; p < 0.001), indicating
that these
-strands contain important molecular determinants
necessary for specific hFSH recognition and binding (Fig. 3,
C and F, and Table II). However, the reduced ligand responsiveness of some of these receptor mutants may also be
related to their impaired cell surface expression. However, the
transfection of increasing amounts of these receptor constructs (5 or
10 µg instead of 1 µg) did not affect receptor cell surface expression or ligand responsiveness (data not shown).
Introducing hLH-R-specific X residues into
-strands 3 and
6 of the hFSH-R (hFSH-R/L
3 and hFSH-R/L
6, respectively) resulted in a significant 60- and 100-fold (p < 0.001) increase
in responsiveness to hCG/hLH, respectively, as compared with wild type
hFSH-R (Fig. 3, B and E, and Table II). In
addition, hFSH-R/L
9 displayed a less significant increase in
responsiveness to hCG/hLH (~2.5-fold; p < 0.05),
suggesting a hCG/hLH-specific interaction site with lower affinity in
this
-strand. In contrast, all other chimeric
-strand hFSH-Rs
displayed either similar (hFSH-R/L
2 and hFSH-R/L
8) or decreased
(hFSH-R/L
1, hFSH-R/L
4, hFSH-R/L
5, and hFSH-R/L
7) responsiveness to hCG/hLH compared with the wild type hFSH-R (Fig. 3,
B and E, and Table II).
Although both hFSH-R/L
3 and hFSH-R/L
6 approached the
responsiveness of the LLL-hFSH-R to hCG and hLH, their EC50
values were still significantly higher (p < 0.001;
Table II). To determine a potential synergistic contribution of hLH-R
-strands 3 and 6 in conferring hCG/hLH selectivity to the "host"
hFSH-R, an hFSH-R/hLH-R
3,
6 double mutant was generated
(i.e. Table II, hFSH-R/L
3,L
6). Unexpectedly, hFSH-R/L
3,L
6 was hardly expressed at the cell surface (~1% of wild type hFSH-R; see Fig. 3D). Nevertheless,
hFSH-R/L
3,L
6 displayed significantly improved responsiveness to
hCG/hLH compared with the individual
3 and
6 mutant receptors
(p < 0.001), with EC50 values similar to
that of LLL-hFSH-R (Fig. 3E and Table II). As expected,
hFSH-R/L
3,L
6 responded to hFSH with an efficacy similar to that
of the wild type hFSH-R (Fig. 3F and Table II).
To confirm that the ligand responsiveness of a receptor represents its
affinity for that ligand, displacement studies were performed for a
selected number of mutant receptors that displayed a responsiveness to
hFSH similar to that of wild type hFSH-R. Ki values
similar to those obtained for the wild type hFSH-R were found for
hFSH-R/L
3, hFSH-R/L
6, and hFSH-R/L
3,L
6 (Fig.
4B and Table III). The mutant
receptors hFSH-R/L
3 and hFSH-R/L
6 displayed a significantly
increased affinity for hCG compared with the wild type hFSH-R
(p < 0.001), which is in
accordance with the functional assay results. Likewise, the
simultaneous introduction of hLH-R
-strands 3 and 6 into hFSH-R
(hFSH-R/L
3,L
6) resulted in a further synergistic increase of the
receptor affinity for hCG (p < 0.001; Fig.
4A and Table III).

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Fig. 4.
Ligand binding to membranes prepared from
HEK-T 293 cells transiently transfected with wild type or mutant
-strand hFSH-R constructs. Displacement of
[125I]hFSH binding to membranes prepared from HEK-T 293 cells transiently transfected with wild type or mutant -strand
hFSH-Rs by various concentrations of unlabeled hCG (A) or
unlabeled hFSH (B). Results are shown as the mean ± S.E. of triplicate observations from a single representative
experiment. Mean Ki values are presented in Table
III.
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Table III
Summary of the ligand-binding properties of membranes of HEK-T 293 cells transiently transfected with wild type and mutant hFSH-R
constructs
Ki values were obtained from competitive binding
experiments of [125I]hFSH to membranes prepared from HEK-T
293 cells, transiently expressing wild type and mutant hFSH receptors,
in the presence of various concentrations of unlabeled hCG or hFSH. The
Ki values presented are the calculated mean ± S.E. of Ki values derived from two independent
assays. Kiwt/mut values were calculated by
dividing the Ki for the wild type receptor with the
mutant receptor Ki values.
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Cys Residues in hLH-R
-Strands 4 and 5 Are Important for hLH-R
Cell Surface Expression--
In contrast to the situation in the
hFSH-R, Cys residues are present on position X4
of
-strands 4 and 5 of the hLH-R (Fig. 1). These Cys residues form a
putative disulfide bridge (10) and have been shown to be involved in
LH-R cell surface expression (20). Because the "unpaired" Cys
residues in hFSH-R/L
4 and hFSH-R/L
5 may have caused reduced
receptor cell surface expression (Fig. 3A) by inappropriate receptor folding and/or intracellular trafficking, each individual Cys
was substituted with a Ser residue (hFSH-R/L
4-C133S and
hFSH-R/L
5-C158S, respectively; Table II). In addition, the adjacent
Cys pair was restored by substituting hFSH-R
4- and
5-strands in
conjunction with their corresponding hLH-R
-strands (Table II,
hFSH-R/L
4,L
5). The hFSH-R/L
4-C133S and hFSH-R/L
5-C158S
receptors were equally well expressed at the cell surface as the wild
type hFSH-R. The hFSH-R/L
4,L
5 receptor was expressed at ~56%
of wild type hFSH-R levels (Fig. 3G) but at higher
expression levels than hFSH-R/L
4 or hFSH-R/L
5 (Fig.
3A). All three mutant receptor constructs (hFSH-R/L
4-C133S, hFSH-R/L
5-C158S, and hFSH-R/L
4,L
5)
displayed a similar responsiveness to hFSH, hCG, and hLH as the wild
type hFSH-R (Fig. 3, H and I, and Table II).
Cell Surface Expression of the hFSH-R Is Sensitive to Mutations in
-Strands 1 and 7--
The mutant hFSH-R/L
1 and hFSH-R/L
7
receptors displayed completely impaired cell surface expression (Fig.
3, A and D), together with a 2600- and 130-fold
decreased hFSH responsiveness, respectively (Fig 3, C and
F, and Table II), and no response to hCG or hLH (Fig. 3,
B and E, and Table II). To test whether the
observed reduction in responsiveness is related directly to reduced
cell surface receptor expression, HEK-T 293 cells were transfected with
10, 100, and 1000 ng of wild type HA-tagged hFSH-R construct. Although
these amounts of transfected construct appeared to be expressed at
different levels (3 and 23% and set at 100%, respectively) on the
cell surface, the production of cAMP in response to hFSH, hCG, and hLH
stimulation appeared to be only slightly, although significantly,
affected by the different receptor numbers (Fig. 5 and Table IV); a 33-fold reduction in
cell surface receptor expression was
accompanied by a less than 5-fold reduction in ligand responsiveness.
To determine whether replacing hFSH-R
-strands 1 or 7 with
the corresponding hLH-R
-strands either affected hFSH binding
directly or severely disrupted the exodomain conformation, the
hLH-R-specific residues in hFSH-R/L
1 and hFSH-R/L
7 were mutated.
An Ala substitution of all X residues in
-strands 1 or 7 (i.e. hFSH-R/Ala
1 and hFSH-R/Ala
7 in Table
V) impaired receptor surface expression
as well as ligand responsiveness in a similar way as found for
hFSH-R/L
1 and hFSH-R/L
7 (Fig. 6 and Table V). In contrast, an Ala substitution of individual X
residues in
-strands 1 and 7 did not affect the receptor
responsiveness to hFSH, hCG, and hLH (Fig. 6 and Table V), whereas
their cell surface expression levels varied from 2 to 140% of wild
type hFSH-R.

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Fig. 5.
Effect of different amounts of hFSH-R
construct, transiently transfected into HEK-T 293 cells on receptor
cell surface expression and ligand responsiveness. HEK-T 293 cells
were transiently transfected with 0.01, 0.1, and 1 µg of expression
vector containing the wild type hFSH-R cDNA insert. A,
cell surface expression of hFSH-R as determined by HA tag ELISA.
B and C, human CG- and hFSH-stimulated,
cAMP-mediated reporter gene activity in HEK-T 293 cells transiently
transfected with different amounts of hFSH-R. Human LH and hCG
stimulated all constructs with a similar efficacy; for clarity, only
the hCG-induced, cAMP-mediated reporter gene activity is shown. Results
are shown as the mean ± S.E. of triplicate observations from a
single representative experiment. Mean EC50 values are
presented in Table IV.
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Table IV
Summary of the ligand-induced intracellular cAMP production in HEK-T
293 cells transiently transfected with different quantities of wild
type hFSH-R construct
Cyclic AMP production upon stimulation with hCG, hLH, and hFSH was
measured in HEK-T 293 cells, transiently cotransfected with various
amounts of hFSH-R construct and a plasmid containing a
-galactosidase gene under control of a promoter containing five
cAMP-response elements (pCRE/ -gal). The EC50 values
presented are the calculated mean ± S.E. of EC50 values
derived from at least three independent experiments. Human LH and hCG
yielded a similar efficacy for all constructs as determined for every
construct in at least two independent assays; for clarity, only the hCG
data are shown. EC50wt/red values were calculated by
dividing the EC50 of 1 µg of transfected hFSH-R with the
EC50 values of the 0.1 and 0.01 µg of hFSH-R transfections.
red, reduced amount.
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Table V
Summary of the ligand-induced intracellular cAMP production in HEK-T
293 cells transiently transfected with wild type and mutant hFSH-R
constructs
Cyclic AMP production upon stimulation with hCG, hLH, and hFSH was
measured in HEK-T 293 cells, transiently cotransfected with various
receptor constructs and a plasmid containing a -galactosidase gene
under control of a promoter containing five cAMP-response elements
(pCRE/ -gal). Residues that were introduced into the hFSH-R
-strands are underlined. The EC50 values presented are the
calculated mean ± S.E. of EC50 values derived from at
least three independent experiments. Human LH and hCG yielded a similar
efficacy for all constructs as determined for every construct in at
least two independent assays; for clarity, only the hCG data are shown.
EC50wt/mut values were calculated by dividing the
EC50 for the wild type hFSH-R with the mutant receptor
EC50 values. ND, not detectable.
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Fig. 6.
Cell surface expression and ligand-induced
cAMP production of wild type or -strand 1 or 7 mutant hFSH-Rs transiently expressed in HEK-T 293 cells.
A and D, cell surface expression of wild type or
-strand 1 or 7 mutant hFSH-Rs as determined by HA tag ELISA.
B and E, human CG-stimulated, cAMP-mediated
reporter gene activity in HEK-T 293 cells transiently transfected with
wild type or mutant -strand hFSH-R constructs. C and
F, human FSH-stimulated, cAMP-mediated reporter gene
activity in HEK-T 293 cells transiently transfected with wild type or
mutant -strand hFSH-R constructs. Human LH and hCG stimulated all
constructs with a similar efficacy; for clarity, only the hCG-induced
cAMP-mediated reporter gene activity is shown. Results shown are the
mean ± S.E. of triplicate observations from a single
representative experiment. Mean EC50 values are presented
in Table V. ND, not detectable.
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 |
DISCUSSION |
Mammalian glycoprotein hormones are bound with high affinity and
high selectivity by the exodomains, which are highly related and are
thought to share a similar structural conformation of their
corresponding receptors. Nevertheless, sequences in the exodomains of
these receptors have diverged sufficiently to generate the above
mentioned selectivity toward their respective glycoprotein hormones.
Taking advantage of their structural similarity, the hormone
selectivity of the hLH-R was studied in the present study by
substituting hFSH-R-specific sequences with their corresponding hLH-R
sequences. In contrast to previous studies (2, 3, 21), chimeric
junctions were designed to coincide with the presumed structural
borders of the extracellular subdomains (i.e. the NCR, LRR,
and CCR subdomains, as well as the
-strands within the LRR subdomain).
Receptor chimeras in which the extracellular NCR, LRR, and/or CCR
subdomains of the hFSH-R were substituted by their homologous hLH-R
counterparts indicated that the determinants involved in ligand
selectivity are confined mainly to the LRR subdomain (cf. LLL-hFSH-R, LLF-hFSH-R, and hLH-R). Similar to other extracellular LRR-containing proteins, it is thought that the hydrophobic core of the
LRR subdomain of gonadotropin receptors is protected from the
solvent at both ends by its flanking NCR and CCR subdomains (5).
Despite the conservation of four almost equally spaced Cys residues,
which are presumably disulfide-bonded and essential for correct folding
(10, 20), the NCR subdomain of the hFSH-R was not compatible with the
LRR subdomain of hLH-R as revealed by the severely impaired cell
surface expression of the FLL-hFSH-R and FLF-hFSH-R chimeras.
The current knowledge of the putative spatial arrangement of the LRR
subdomain of GpHRs (13, 14) was used to systematically exchange only
those residues that were expected to have their side chains directed
toward the presumed hormone-binding site (i.e. the
X amino acid residues of the nine
-strand motifs arranged in parallel, each with the consensus sequence
X1X2LX3LX4X5).
Human FSH, hCG, and hLH binding selectivity is very likely associated
with the ~73% divergence of these X residues between hFSH-R and hLH-R. Identical X residues (e.g. the
highly conserved Asp at position X5 in
-strand 5) may be involved in common hormone-receptor contacts (10).
Substitution of individual
-strands of the hFSH-R with the
corresponding hLH-R
-strands allowed the identification of
-strands 3 and 6 as modules containing hCG- and hLH-selective determinants, as these mutant receptors mediate a 60 (
-strand 3)- to
100-fold (
-strand 6), respectively, enhanced responsiveness to hCG
as well as to hLH. Moreover, both
-strands appeared to act
synergistically, as a mutant receptor (i.e.
hFSH-R/L
3,L
6) displayed a responsiveness to hCG and hLH similar
to that of wild type hLH-R. Hence, hLH-R selectivity toward hCG and hLH
can be confined mainly to just these two
-strands. In fact,
none of the other
-strands appeared to confer additional hCG/hLH
selectivity to a major extent. These results are consistent with
previous studies (2, 3) in which hCG-selective determinants were predicted to be localized within the NCR-LRR6 sequence of the LH-R.
Most of the mutant hFSH-R constructs that harbored hLH-R
-strands
did not exhibit severely impaired hFSH responsiveness, indicating that
no essential hFSH-selective determinants are present in the
corresponding hFSH-R
-strands. Moreover, this means that mutations
in these
-strands did not alter the ligand-stimulated capacity of
the receptors to induce cAMP production. Hence, the observed efficacy
of hFSH, hCG, and hLH to stimulate receptor-mediated signaling
correlates with the respective hormone binding affinities of these
receptors as confirmed by ligand binding studies. Although some mutant
receptors that were expressed at low levels (e.g. hFSH-R/L
3,L
6 and hFSH-R/L
5) exhibited efficient responsiveness to ligand stimulation, reduced responsiveness to hFSH stimulation was
often related to severely impaired surface expression of mutant
-strand hFSH-Rs (e.g. hFSH-R/L
1, hFSH-R/L
4, and
hFSH-R/L
7). This was likely because of disrupted protein
folding/conformation, because reduced numbers (i.e.
transfecting different concentrations of wild type hFSH-R constructs)
of correctly folded receptors on the cell surface had only minor
effects (<5-fold) on the level of ligand responsiveness. Also
hFSH-R/Ala
1 and hFSH-R/Ala
7 exhibited disrupted cell surface
expression and, as a consequence, reduced ligand responsiveness.
However, Ala substitution of the individual residues in
-strands 1 and 7 revealed that these
-strands are not involved the binding of
hFSH, hCG, or hLH.
In conclusion, the primary objective of this study was to identify
hCG/hLH selective determinants in the extracellular N terminus of the
hLH-R by generating potential gain-of-function mutant receptors in the
context of an hFSH-R background. Using this strategy, hCG/hLH selectivity was tracked down to hLH-R
-strands 3 and 6. Alternatively, the results observed may also be explained by the loss
of hCG/hLH repulsion in these mutant hFSH-Rs. Surprisingly, hFSH
signaling (and binding) was hardly affected by the introduction of most hLH-R
-strands, suggesting that selective hFSH responsiveness, in
contrast to the responsiveness to hCG and hLH, appears to be determined
by numerous hFSH-R
-strands, each, however, with a relatively low
contribution. The identification of the amino acid residues in
-strands 3 and 6 that are critically involved in the molecular
mechanism determining hCG/hLH selectivity is currently under investigation.
 |
ACKNOWLEDGEMENTS |
We thank R. H. L. van der Wetering (Organon
Inc., Oss, The Netherlands) for valuable assistance in performing
receptor binding assays, and Dr. M. L. C. E. Kouwijzer (Organon
Inc.) for helpful discussions.
 |
FOOTNOTES |
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: Utrecht
University, Faculty of Biology, Dept. of Endocrinology, Padualaan 8, NL-3584 CH Utrecht, The Netherlands. Tel.: 31-30-2534177; Fax:
31-30-2532837; E-mail: J.Bogerd@bio.uu.nl.
Published, JBC Papers in Press, February 21, 2003, DOI 10.1074/jbc.M300634200
 |
ABBREVIATIONS |
The abbreviations used are:
FSH, follicle-stimulating hormone;
FSH-R, FSH receptor;
hFSH, human FSH;
LH, luteinizing hormone;
hLH, human LH;
LH-R, LH receptor;
LRR, leucine-rich repeat;
CG, chorionic gonadotropin;
hCG, human CG;
GpHR, glycoprotein hormone receptor;
NCR, N-terminal cysteine-rich;
CCR, C-terminal cysteine-rich;
ELISA, enzyme-linked immunosorbent assay;
HA, hemagglutinin;
HEK-T, human embryonic kidney T cells.
 |
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Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.