HUMAN MUTATION 29(1),91^98,2008
Identification of the First Germline Mutation
in the Extracellular Domain of the Follitropin
Receptor Responsible for Spontaneous Ovarian
Anne De Leener,1Gianluigi Caltabiano,2Sanly Erkan,3Mehmet Idil,3Gilbert Vassart,1,4Leonardo Pardo,2
and Sabine Costagliola1?
1Institut de Recherche Interdisciplinaire en Biologie Humaine et Mole ´culaire (I.R.I.B.H.M.), Faculte ´ de Me ´decine, Universite ´ Libre de Bruxelles
(ULB), Brussels, Belgium;2Laboratori de Medicina Computacional, Unitat de Bioestadistica, Facultat de Medicina, Universitat Autonoma de
Barcelona, Barcelona, Spain;3Department of Obstetrics and Gynaecology, Cerrahpasa Medical Faculty, Istanbul University, Istanbul, Turkey;
4Department of Genetics, Erasme Hospital, Universite ´ Libre de Bruxelles, Brussels, Belgium
Communicated by Bruce Gottlieb
The receptors for follitropin (FSHR), thyrotropin (TSHR), and lutropin/chorionic gonadotropin (LHCGR) are
the members of the glycoprotein hormone (GPH) receptors (GPHR) family. They present a bipartite structure
with a large extracellular amino-terminal domain (ECD), responsible for high-affinity hormone binding, and
a carboxyl-terminal serpentine region, implicated in transduction of the activation signal. Spontaneous ovarian
hyperstimulation syndrome (sOHSS) is a rare genetic condition in which human chorionic gonadotropin
(hCG) promiscuously stimulates the FSHR during the first trimester of pregnancy. Surprisingly, germline FSHR
mutations responsible for the disease have so far been found only in the transmembrane helices of the
serpentine region of the FSHR, outside the hormone binding domain. When tested functionally, all mutants
were abnormally sensitive to both hCG and thyrotropin (TSH) while displaying constitutive activity. This loss
of ligand specificity was attributed to the lowering of an intramolecular barrier of activation rather than to an
increase of binding affinity. Here we report the first germline mutation responsible for sOHSS (c.383C4A,
p.Ser128Tyr), located in the ECD of the FSHR. Contrary to the mutations described previously, the
p.Ser128Tyr FSHR mutant displayed increase in affinity and sensitivity toward hCG and did not show any
constitutive activity, nor promiscuous activation by TSH. Thus, sOHSS can be achieved from different
molecular mechanisms involving each functional domains of the FSHR. Based on the structure of the FSHR/
FSH complex and site-directed mutagenesis studies, we provide robust molecular models for the GPH/GPHR
complexes and we propose a molecular explanation to the binding characteristics of the p.Ser128Tyr mutant.
Hum Mutat 29(1), 91–98, 2008.
rrrr2007 Wiley-Liss, Inc.
KEY WORDS: FSHR; TSHR; GPCR; OHSS; hCG
The human follitropin (FSH) receptor (FSHR; GenBank
NM_000145.2 and MIM_136435), like the other members of
glycoprotein hormone receptors (GPHR) family, TSHR (GenBank
NM_000369.2) and LHCGR (GenBank NM_000233.2) [Ascoli
et al., 2002; Dias et al., 2002; Gether, 2000; Szkudlinski et al.,
2002], presents a bipartite structure, with a large extracellular
domain (ECD) responsible for high affinity hormone binding [Fan
and Hendrickson, 2005; Braun et al., 1991; Remy et al., 2001;
Schmidt et al., 2001; Smits et al., 2003a; Vischer et al., 2003;
Puett et al., 2007], and a carboxyl-terminal serpentine region,
shared by rhodopsin-like G-protein-coupled-receptors (GPCR),
implicated in transmission of the activation signal [Gether, 2000].
The ECD is structurally related to the family of proteins with
leucine-rich repeats (LRR) and a 2.9-A˚resolution structure of
human FSH complexed with the extracellular hormone binding
domain of its receptor was published [Fan and Hendrickson,
Published online 22 August 2007 in Wiley InterScience (www.
The Supplementary Material referred to in this article can be
accessed at http://www.interscience.wiley.com/jpages/1059-7794/
Received 7March2007; acceptedrevisedmanuscript12 June 2007.
?Correspondence to: S.Costagliola, I.R.I.B.H.M.,ULB,808 Lennik
Street, B-1070, Brussels, Belgium. E-mail: firstname.lastname@example.org
Grant sponsors: Belgian Science Policy; Belgian State; Fonds de la
Recherche Scienti¢que Me ¤ dicale (FRSM); Fonds National de la Re-
cherche Scienti¢que (FNRS); Association Recherche Biome ¤ dicale et
Diagnostic (ARBD); Fondation ERASME; Action de Recherche Con-
certe ¤ e (A.R.C) (CommunauteŁfranc -aise de Belgique); Grant sponsor:
Interuniversity Attraction Poles Programme; Grant number: P6/14;
Grant sponsor: LifeScHealth; Grant number: LSHB-CT-2003-
503337; Grant sponsor: Improving of Human Potential Programs of
the European Community;Grant number: HPRI-CT-1999-00071.
Anne De Leener and Gianluigi Caltabiano contributed equally to
rrrr2007 WILEY-LISS, INC.
2005]. In this crystal, the hormone is bound in a hand-clasp
fashion to an elongated and curved FSHR ectodomain and the
authors suggested that all glycoprotein hormones (GPH) would
bind to their receptors in this mode. Retrospectively, these
structural data validate functional studies generated by extensive
site-directed mutagenesis of the thyrotropin (TSH) and FSH
receptors, where exchange of specific residues in the LRR by their
LHCGR counterparts could switch hormonal specificity [Smits
et al., 2003a].
The GPH are heterodimeric proteins made of an alpha (a)
subunit common to all four proteins and hormone-specific beta
(b) subunits encoded by paralogous genes [Li and Ford, 1998] and
sharing about 40% sequence identity as do the ECD of the
corresponding receptors. Despite similarity between these para-
logous hormones and receptors, coevolution of the hormone-
receptor couples resulted in the establishment of tight specificity
barriers, preventing promiscuous activation under normal physio-
logical conditions. These barriers can be overruled in diseases, as
in severe hypothyroidism, where very high concentrations of
TSH have been made responsible for cases of ovarian hypersti-
mulation due to spillover of TSH activity on the FSH receptor
[Anasti et al., 1995; Nappi et al., 1998]. The emergence of
chorionic gonadotropin (CG) in primates, which achieves very
high concentration during early pregnancy and is 85% identical to
lutropin (LH), constituted an evolutionary challenge to the
specificity barrier in GPHR couples. In humans, in particular,
during the first trimester of pregnancy, human CG (hCG) reaches
concentrations at which it displays some thyrotropic activity,
bringing most pregnant women to the fringe of hyperthyroidism
[Glinoer, 1997]. Overproduction of hCG in molar or twin
pregnancies may result in overt gestational hyperthyroidism
(MIM_603373) [Hershman, 1999].
However, specific mutations affecting the hormone binding
surface of the TSHR or FSHR would be expected to lower the
specificity barrier, and cause gestational hyperthyroidism or
MIM# 608115), even in the presence of normal levels of hCG.
One case of gestational hyperthyroidism was reported with a
mutation (p.Lys183Arg, K183R) [Rodien et al., 1998] in the ECD
of the human TSHR, but so far and quite unexpectedly, all cases of
sOHSS were linked to mutations in the serpentine domain of the
FSHR [Smits et al., 2003b; Vasseur et al., 2003; Montanelli et al.,
2004a, 2004b; De Leener et al., 2006]. In these reports, a relation
was established between constitutive activity and lowering
of specificity in the FSHR mutants, suggesting that the gain
of sensitivity of the mutants to hCG would be due to lowering of
an intramolecular barrier to activation rather than to increase
in binding affinity [Vassart et al., 2004].
In the present study, we report the first mutation responsible of
sOHSS located in the ECD of the human FSHR (c.383C4A,
p.Ser128Tyr, S128Y), in the vicinity of residues implicated in
hormone binding. When tested functionally, the p.Ser128Tyr
mutant displayed promiscuous activation by hCG but, in contrast
with the previous cases, it did not show any constitutive activity or
promiscuous activation by TSH. As predicted from its location in
the ECD, the p.Ser128Tyr mutant presented an increase of binding
affinity for hCG. This finding demonstrates that in sOHSS,
promiscuous activation of the FSHR by hCG can be achieved by
different molecular mechanisms involving each of the two
functional domains of the FSHR.
Additional site-directed mutagenesis demonstrates that some
substitutions at this position were less selective than p.Ser128Tyr
and could, in addition, open the specificity toward TSH. Serine
128 is located in the vicinity of residues implicated in hormone
binding in the crystal structure of the FSHR/FSH [Fan and
Hendrickson, 2005]. Molecular models of the illegitimate FSHR/
hCG and FSHR/TSH complexes were built by homology with the
FSHR/FSH crystal structure. They provided a molecular explana-
tion for the observed increase of affinity of FSHR mutants to hCG,
and, for some of them, to TSH.
Plasmid pBluescript SK1 and Pfu turbo polymerase were
obtained from Stratagene (La Jolla, CA, USA) and plasmid pSVL
was obtained from Amersham Pharmacia Biotech (Roosendaal,
The Netherlands). Restriction enzymes were obtained from
Invitrogen (Merelbeke, Belgium) and New England Biolabs
(Beverly, MA). Mouse monoclonal antibody 5B2 was obtained
by genetic immunization with the cDNA encoding the human
FSHR [Costagliola et al., 1998]. Recombinant human FSH
(rhFSH) was from Organon Belge (Brussels, Belgium), recombi-
nant hCG (rhCG) from Sigma Chemical (St. Louis, MO) and
recombinant human TSH (rhTSH) from Genzyme (Cambridge,
MA). Polyethylenimine (PEI), linear, molecular weight (MW)
?25,000 was from Polysciences, Inc. (Warrington, PA) [Boussif
et al., 1995]. [125I]hFSH was from Perkin Elmer (Rodgau-
Ju ¨gesheim, Germany).
The clinical characteristics of the sOHSS patient have been
described previously [Cepni et al., 2006]. The proband was
a 21-year-old Turkish woman, gravida 1, para 0, presenting with
severe sOHSS at week 11 of her first pregnancy. The b-hCG level
was normal for a 3-month singleton pregnancy. The TSH level was
normal. Symptoms regressed during the second and third trimester
with a conservative medical treatment and resolved in postpartum.
Her medical and family history was unremarkable.
DNA Sequencing and Mutation Identi¢cation
Human DNA was extracted from peripheral blood leukocytes
and the sequences of all the exons of the human FSHR gene
(GenBank NM_000145.2), together with intron–exon boundaries,
were determined as previously described [Smits et al., 2003b]. The
patient gave informed consent to participation in this study, which
has been approved by the Ethical Committee of Erasme Hospital,
Brussels, Belgium. The patient is heterozygote for a C to A
transversion (c.383C4A) that substitutes a tyrosine (Tyr, Y) for
serine (Ser, S) at position 128 (p.Ser128Tyr, S128Y). The DNA
mutation numbering is based on cDNA sequence where 11
corresponds to the A of the ATG initiation codon. The sequence
of the segment harboring this mutation has been determined from
the product of two independent polymerase chain reactions
(PCRs). The presence of the mutation has been confirmed on a
second blood sample. The same region was sequenced from
96 control blood samples from the same ethnical origin and the
same substitution was not found.
Construction of Human FSHR Mutants
Mutations were introduced in the human wild-type (wt) FSHR
by site mutagenesis as previously described [Vlaeminck-Guillem
et al., 2002]. The appropriate mutated portions of SK1FSHR
mutants were subcloned in the pSVL-FSHR cDNA using natural
restriction sites. The constructs were verified by sequencing on
92HUMAN MUTATION 29(1),91^98,2008
Human Mutation DOI 10.1002/humu
COS-7 cells were used for all the transient expression
experiments. The cells were transfected with PEI [Boussif et al.,
1995] (stock 1mg/ml, pH 7.2, in water, conserved at ?801C) with
modified protocol. Briefly, 6mg of DNA were mixed with 18mg PEI
in NaCl (150mM, 1,400ml final volume), then added on 106cells
in suspension (in 1,200ml of culture medium) after 30 minutes of
incubation at room temperature. After addition of 6ml of fresh
culture medium, cells were distributed in 48 wells (250ml per well)
for stimulation with hormones or for flow immunocytometry
(FACS), 24 wells (500ml per well) for binding experiments, or
six wells (1.5ml per well) for basal cyclic adenosine monophosphate
(cAMP) accumulation. The medium was changed the day after
transfection and cells were used 2 days after transfection for
Quanti¢cation of Cell Surface Expression by FACS
Expression on the cell surface was assessed by flow immuno-
cytometry (FACScan flow cytofluorometer; Becton Dickinson,
Erembodegem, Belgium) with the 5B2 mouse monoclonal antibody
as previously described [Costagliola et al., 1998]. Cells transfected
by pSVL alone (empty vector) and by pSVL-FSHR wt were always
used as negative and positive controls, respectively.
Determination of cAMP Production
At 48hr after transfection the intracellular accumulation of
cAMP was measured by radioimmunoassay (RIA) as described
previously [Smits et al., 2003a]. cAMP concentrations were
determined in duplicate on extracts from duplicate transfection
dishes or wells. Results are expressed as picomoles cAMP per
milliliter (pmol/ml), or percentage of maximal cAMP response.
The Prism computer program (GraphPad Software, Inc., San
Diego, CA) was used for curve fitting and for EC50determination.
Ligand binding was measured on COS-7 cells transfected with
various constructs with PEI as previously described [Smits et al.,
2003a], with minor modifications, when volumes were adjusted to
work in 24-well culture dishes. Briefly, 48hr after transfection,
cells were washed twice with 400ml modified Krebs-Ringer-HEPES
buffer (without NaCl, isotonicity maintained with 280mM
sucrose). Thereafter, cells were incubated overnight at room
temperature with 200ml of the same buffer supplemented with 5%
low fat milk, [125I]hFSH (100,000 counts per minute [cpm] per
well) and graded concentration of cold rhCG or rhFSH. There-
after, the cells were washed twice with 400ml of the same ice-cold
buffer and solubilized with 200ml of 1N NaOH. Radioactivity was
measured in a g counter. All experiments were carried out at least
in duplicate and results are expressed as cpm [125I]hFSH bound.
The Prism computer program was used for curve fitting.
Construction of Homology Models for wt and Mutant
Glycoprotein Hormone Receptors in
The crystallographic structure of the extracellular domain of the
FSHR in complex with FSH (Protein Databank [PDB] code 1XWD)
[Fan and Hendrickson, 2005] has been employed to build homology
models of the LHCGR-hCG and TSHR-TSH complexes, and
mutants FSHR in complex with hCG and TSH. SCWRL 3.0 was
employed to add the side chains of the nonconserved residues based
on a backbone-dependent rotamer library [Canutescu et al., 2003].
The resulting structures were placed in a rectangular box (?101A˚
? 97A˚? 108A˚in size) containing ?27,000 Monte Carlo–equili-
brated TIP3P water molecules. Initially, the system was subjected to
500 iterations of energy minimization and then heated to 3001K in
15ps. Structures were collected every 10ps during 1ns (100
structures per simulation). During the molecular dynamics simula-
tions of mutant FSHR in complex with hCG and TSH, a positional
restraint of 1kcal mol?1A˚?2was applied to the Caatoms of the
FIGURE 1. Presentation of the mutation. Left: Locations of the mutations identi¢ed to date in the FSHR (GenBankNM_000145.2) of
patientspresentingwith sOHSS.Thenumberingof amino acids begins atATG initiatingcodon. Right: Representationof thenucleo-
tide sequence around codon128 (exon 5) of theFSHR in the new sOHSS patient.The patient is heterozygote for a c.383C4A muta-
tionencodingap.Ser128Tyrproteinchange.TheDNA mutationnumberingisbasedoncDNAsequencewhere 11correspondstothe
Aof theATG initiationcodon.
HUMAN MUTATION 29(1),91^98,2008 93
Human Mutation DOI 10.1002/humu
receptor structure. Langevin temperature regulation was used at
constant pressure using the particle mesh Ewald method to evaluate
electrostatic interactions. The molecular dynamics simulations were
run with the Sander module of AMBER 9 [Case et al., 2005], the
all-atom force field [Ponder and Case, 2003], SHAKE bond
constraints in all bonds, a 2-fs integration time step, and constant
temperature of 3001K coupled to a heat bath.
Functional Characterization of the S128Y FSHR
Mutant Responsible for sOHSS
We describe here a mutation resulting in the replacement of a
Serine (Ser, S) by a Tyrosine (Tyr, Y) at codon 128 (p.Ser128Tyr,
c.383C4A) in the FSHR of a patient presenting with sOHSS
(see case report in Materials and Methods [Cepni et al., 2006]
and Fig. 1). Serine 128 is located in position X4 of the fifth
LRR (LRR5) in the FSHR ECD. The mutant was analyzed
functionally by transient expression in COS-7 cells. Despite
reduction of expression at the cell surface (36% of the wt FSHR;
Table 1), the p.Ser128Tyr mutant showed a clear lowering of its
specificity toward hCG with an increase of sensitivity (Fig. 2A;
Table 1) and affinity (Fig. 2B) toward this hormone. In
contrast with the results obtained with previously described
sOHSS mutations [Smits et al., 2003b; Vasseur et al., 2003;
Montanelli et al., 2004a, 2004b; De Leener et al., 2006], neither
the basal activity (Fig. 2) nor the sensitivity to TSH (Table 1;
Fig. 3) were affected by the mutation (see Supplementary
TABLE 1. Functional Characterization ofAll the Ser128 Mutantsy
FACS % expression of wt rhCG screening 300UI/ml rhTSH screening100mUI/ml
Mutation MeanSEM Mean RangerhCG?100/FACS MeanRange
yColumn 1:The FSHR mutants characterized in this study. Column 2: Expression of wt or mutant receptors as measured by FACS (expressed in % of the wt FSHR; GenBank
NM_000145.2). Sensitivity of individual mutants to rhCG (columns 3 and 4), or rhTSH (column 5). Results are expressed as mean7standard error of the mean (SEM) pmol/ml
of cAMP. Since maximal stimulation could not be achieved (see Fig. 2), sensitivity to rhCG or rhTSH could not be measured as EC50. Hence, cAMP accumulation over basal
(empty vector transfected) for a ¢xed concentration of hormone was used as an index of sensitivity (normalized to surface expression [for hCG, column 4], or not [columns 3
aTwo measurementswere performed. Results areexpressed asmean7range of duplicate.
FIGURE 2. Functional characterization of the p.Ser128Tyr FSHR mutant.Various amount of DNA of the wt FSHR were transfected
in COS-7 cells and the conditions were selected giving equal expression of the wt FSHR and the Ser128Tyr mutant (see inset
panel B). A: Stimulation of the cells expressing wt FSHR or the Ser128Tyr mutant with rhCG. Results are expressed as mean7
standarderrorofthemean (SEM) pmol/mlofcAMP. B:Competitionbetweenbindingof[125I]hFSHandcoldrhCGtothewtFSHRand
the Ser128Tyr mutant. Results are expressed as mean7SEM cpm bound [125I]hFSH. Each graph is representative of at least three
94HUMAN MUTATION 29(1),91^98,2008
Human Mutation DOI 10.1002/humu
com/jpages/1059-7794/suppmat; which compares in vitro pheno-
types of mutant p.Ser128Tyr and sOHSS mutant p.Asp567Asn
previously described). The sensitivity to FSH remained unchanged
(data not shown).
S1;available online athttp://www.interscience.wiley.
Phenotype of Other Ser128 Mutants
To explore the molecular mechanisms of the gain of function
caused by substitutions at position 128, a panel of mutants was
engineered in which Ser128 was replaced by the 19 other amino
acids. None of the expressed mutants presented detectable
constitutive activity (data not shown). Differences were observed
in the level of expression at the cell surface of individual mutants,
ranging from 0% (Ser128Pro) to 150% (Ser128Lys) of the wt
FSHR (Table 1). The sensitivity to hCG remained unchanged for
mutants Ser128Ala/His/Lys/Pro/Arg/Trp and to a lesser extent
mutants Ser128Cys/Met (Table 1). Interestingly the substitution of
Ser128 by Cys or Phe (residues located at this position in the ECD
of LHCGR and TSHR, respectively) was with minor effect.
Despite weak expression (38% of the wt), mutant Ser128Ile was
FIGURE 3. rh TSH response of the Ser128 mutants. COS-7 cells
transiently transfectedwith Ser128Tyr(Y), Ser128Ile(I), Ser128-
Glu(E), Ser128Gln(Q), andSer128Val(V) mutantreceptorswere
stimulated with increasingconcentration of rhTSH,expressed in
mIU/ml. Intracellular cAMP accumulation was determined by
RIA. This graph is representative of at least two independent
FIGURE 4. Molecular models of GPHR in complex with hormones. A: General view of the FSHR-FSH crystal structure (PDB code
1XWD) [Fan and Hendrickson,2005].The position of LRR2^9and the seat-belt portion of the b-subunit are shown in blue and dark
red. B: StructureofarepresentativeLRRshowing thepositionsofX1X2LX3LX4X5.C^E: Detailedview (LRR2^9) of thecrystalFSHR-
FSH (C) structureandcomputermodelsofLHCGR-hCG (D) andTSHR-TSH (E) complexes. SchematicrepresentationoftheX1^5side
seat-belt portionof the b-subunit ofGPH.
HUMAN MUTATION 29(1),91^98,2008 95
Human Mutation DOI 10.1002/humu
particularly sensitive to hCG (see Table 1) and in addition,
together with Ser128Gln and Ser128Val mutants, it displayed also
a strong increase of sensitivity to TSH (Table 1; Fig. 3). The
Ser128Glu mutants was sensitive to hCG (like Ser128Asp) but
without effect regarding TSH (see Table 1).
The only structural data available is from the crystal of the
complex between FSHR ECD and FSH [Fan and Hendrickson,
2005]. Therefore, in order to interpret in molecular terms
the increase in sensitivity of individual Ser128 mutants to
hCG (and/or TSH), we used homology modeling techniques
(see Materials and Methods) to build models of LHCGR-hCG
and TSHR-TSH complexes (accessible at http://gris.ulb.ac.be),
employing the FSHR-FSH crystal structure as a template (Fig. 4A)
and the ECD gain of function mutants available in the literature.
Speci¢city of Recognition of GPHR
Selectivity of FSHR toward hCG.
the different GPH is guided by the ‘‘seat-belt’’ portion of the
b-subunit [Dias et al., 1994; Moyle et al., 1994; Grossmann et al.,
1997]. Interestingly, the seat-belt of b-hCG (CGB; GenBank
NM_000737.2) has two basic residues (Arg94 [R94b] and Arg95
[R95b]), while b-FSH (FSHB; GenBank NM_000510.2) has
acidic residues in this part of the hormone (D88b and D90b)
(Fig. 4). Appropriately, X5of LRR7,8,9contain G183, E206, and
S231 in LHCGR (negative electrostatics in this area of the
The specificity for
receptor) and K179, D202, and R227 in FSHR (positive
electrostatics), respectively. Thus, similarly to the interaction
between D90b of FSHB and K179 of FSHR (Fig. 4C), we propose
that the CGB-specific R94b forms a salt bridge with E206 of
LHCGR while R95b interacts with both E154 and E203 (Fig. 4D).
K179 of FSHR is thus a key determinant of the specificity against
hCG because it is repulsive with R94b of CGB [Smits et al.,
Selectivity ofFSHR towardTSH.
4C) and TSHR (K209; Fig. 4E) form at the bottom of LRR7,8an
ionic interaction with a negatively-charged side chain of FSHB
(D90b) or b-TSH (TSHB; GenBank NM_000549.3) (D111b),
respectively. The specificity of the FSHR against TSHB resides at
LRR2–4. TSHR contains K58 and R109, conferring a positive
electrostatic surface to this domain, which forms a salt bridge with
the TSHB-specific E118b (Fig. 4E). In contrast, E76 confers to
FSHR a negative electrostatic region that interacts with the
FSHB-specific R97b (Fig. 5A).
FSHR toward hCG.
Mutation of Ser128 to Gln(Q), Glu(E),
or Tyr(Y) permits FSHR to hydrogen bond the CGB-specific
side chain of R95b, increasing the binding of the mutant FSHR
toward hCG (Fig. 5). In particular, the side chain of Q128 bridges
R95b and D99b of hCG (Fig. 5D); E128 forms attractive
D99b (Fig. 5E); and Y128 forms a hydrogen bond with R95b of
Both FSHR (K179; Fig.
FIGURE 5. Detailed view of LRR3^6of GPHR in complex with hormones.The crystal FSHR-FSH (A) structure, computer models of
LHCGR-hCG (B) andTSHR-TSH (C) complexes, computer models of Ser128Gln(Q)/Glu(E)/Tyr(Y) mutant FSHR in complex with
hCG (D^F), and computer models of Ser128Ile(I) mutant FSHR in complex with hCG (G) andTSH (H).The Ser128Gln(Q)/Glu(E)/
Tyr(Y) mutationpermitsFSHR tohydrogenbondthe bhCG-speci¢csidechainofR95b(D^F), increasingthesensitivityofthemutant
FSHR toward hCG.The bulky and b-branched side chain of Ile(I)128 modi¢es the conformation of the nearby Glu(E)103 side chain
anditsionicArg(R)101partner, allowing Lys(K)104b of hCG tointeract withGlu(E)103 ofFSHR (G) andGlu(E)118b ofTSH tointer-
act withArg(R)101ofFSHR (H).
96HUMAN MUTATION 29(1),91^98,2008
Human Mutation DOI 10.1002/humu
hCG (Fig. 5F). The repulsive interaction between E128 and D99b
explains the lower effect in hCG binding of the S128E mutation
relative to S128Q.
Sensitivity ofSer128Ile(I)/Val(V) mutantsFSHRtoward
hCG and TSH.
The Ser128Ile(I)/Val(V) mutation in FSHR
adds a bulky and b-branched side chain, which the Cg moiety
modifies the conformation of the nearby E103 side chain and its
ionic R101 partner in the wt receptor (Fig. 5A). Thus, S128I/V
mutants force R101 and E103 to point toward LLR3and allow
K104b of hCG to interact with E103 of FSHR (Fig. 5G) and
E118b of TSH to interact with R101 of FSHR (Fig. 5H),
increasing the sensitivity of the mutant FSHR toward hCG and
TSH. Importantly, non-b-branched, more flexible, hydrophobic
residues do not elicit this effect due to the possibility of their side
chain adopting a conformation distant from E103.
In opposition to the five previously described mutations found
in sOHSS cases [Smits et al., 2003b; Vasseur et al., 2003;
Montanelli et al., 2004a, 2004b; De Leener et al., 2006] (see
Introduction), the location of the mutation in the extracellular
binding region of the FSHR and the functional characterization of
the p.Ser128Tyr mutant clearly suggests that the increase of
sensitivity toward hCG observed here is the consequence of an
increase of affinity toward this hormone. Interestingly, the
p.Ser128Tyr substitution is selective because no increase of
sensitivity to TSH was measured. In addition, the absence of
constitutive activity of this p.Ser128Tyr mutant demonstrates that
this is not a prerequisite condition in vivo to develop OHSS,
contrary to what previous natural mutations found in sOHSS had
suggested before [Delbaere et al., 2005].
Finally, extensive directed mutagenesis at the position 128,
demonstrates that the increase of sensitivity toward hCG is not
the consequence of the loss of the Serine at position 128 because
some substitutions were neutral (e.g., p.Ser128Ala and p.Ser128-
His; Table 1). Also, some substitutions were poorly selective,
increasing the sensitivity to both hCG and TSH (e.g., p.Ser128Ile
Previously, one case of gestational hyperthyroidism was reported
with a mutation (p.Lys183Arg, K183R) [Rodien et al., 1998] in
the ECD of the human TSHR. In vitro characterization
demonstrated that mutation of K183 (X3of LRR7) in the TSHR
to any amino acid [Smits et al., 2002] or adding a negative charge
in X3of LRR8(the Y206E mutation) [Smits et al., 2003a] increase
the sensitivity of TSHR for hCG. Analysis of this mutant in the
light of the molecular models provided here, confirms the
suggestion that the increase of sensitivity for hCG was due to
the release of neighboring residue E157 from a neutralizing
interaction with K183 [Smits et al., 2002], permitting the access of
R95b of hCG to the acidic E157 of TSHR. Thus, both S128Y of
FSHR and K183R of TSHR, the two unique natural mutations in
the ECD of a GPHR increasing its sensitivity toward hCG, cause
their irregular function by interacting with R95b of hCG.
Together with the crystal structure of the FSH-FSHR complex
[Fan and Hendrickson, 2005], natural [Smits et al., 2003b;
Vasseur et al., 2003; Montanelli et al., 2004a, 2004b; De Leener
et al., 2006; Rodien et al., 1998], or experimental [Smits et al.,
2003a] gain of function mutants of the GPHR showing an increase
of sensitivity regarding hCG allowed us to build robust models of
both legitimate and illegitimate interactions between these
families of paralogous gene products. With the increasing
availability of animal genome and GPH-GPHR sequences, these
models will be of great help to decipher the evolution of ligand
binding specificity during the expansion of the GPH-GPHR family.
We thank G. Smits for fruitful discussions and comments on the
manuscript and V . Janssens for technical assistance. A.D.L. is Research
Fellow at the Fonds National de la Recherche Scientifique (FNRS).
Anasti JN, Flack MR, Froehlich J, Nelson LM, Nisula BC. 1995. A
potential novel mechanism for precocious puberty in juvenile hypothyr-
oidism. J Clin Endocrinol Metab 80:276–279.
Ascoli M, Fanelli F, Segaloff DL. 2002. The lutropin/choriogonadotropin
receptor, a 2002 perspective. Endocr Rev 23:141–174.
Boussif O, Lezoualc’h F, Zanta MA, Mergny MD, Scherman D, Demeneix
B, Behr JP . 1995. A versatile vector for gene and oligonucleotide transfer
into cells in culture and in vivo: polyethylenimine. Proc Natl Acad Sci
Braun T, Schofield PR, Sprengel R. 1991. Amino-terminal leucine-rich
repeats in gonadotropin receptors determine hormone selectivity. EMBO
Canutescu AA, Shelenkov AA, Dunbrack RL Jr. 2003. A graph-theory
algorithm for rapid protein side-chain prediction. Protein Sci 12:
Case DA, Cheatham TE, III, Darden T, Gohlke H, Luo R, Merz KM Jr,
Onufriev A, Simmerling C, Wang B, Woods RJ. 2005. The Amber
biomolecular simulation programs. J Comput Chem 26:1668–1688.
Cepni I, Erkan S, Ocal P , Ozturk E. 2006. Spontaneous ovarian
hyperstimulation syndrome presenting with acute abdomen. J Postgrad
Costagliola S, Rodien P , Many MC, Ludgate M, Vassart G. 1998. Genetic
immunization against the human thyrotropin receptor causes thyroiditis
and allows production of monoclonal antibodies recognizing the native
receptor. J Immunol 160:1458–1465.
De Leener A, Montanelli L, Van Durme J, Chae H, Smits G, Vassart G,
Costagliola S. 2006. Presence and absence of follicle-stimulating
hormone receptor mutations provide some insights into spontaneous
ovarian hyperstimulation syndrome physiopathology. J Clin Endocrinol
Delbaere A, Smits G, De Leener A, Costagliola S, Vassart G. 2005.
Understanding ovarian hyperstimulation syndrome. Endocrine 26:
Dias JA, Zhang Y, Liu X. 1994. Receptor binding and functional properties
of chimeric human follitropin prepared by an exchange between a small
hydrophilic intercysteine loop of human follitropin and human lutropin.
J Biol Chem 269:25289–25294.
Dias JA, Cohen BD, Lindau-Shepard B, Nechamen CA, Peterson AJ,
Schmidt A. 2002. Molecular, structural, and cellular biology of follitropin
and follitropin receptor. Vitam Horm 64:249–322.
Fan QR, Hendrickson WA. 2005. Structure of human follicle-stimulating
hormone in complex with its receptor. Nature 433:269–277.
Gether U. 2000. Uncovering molecular mechanisms involved in activation
of G protein-coupled receptors. Endocr Rev 21:90–113.
Glinoer D. 1997. The regulation of thyroid function in pregnancy:
pathways of endocrine adaptation from physiology to pathology. Endocr
Grossmann M, Szkudlinski MW, Wong R, Dias JA, Ji TH, Weintraub BD.
1997. Substitution of the seat-belt region of the thyroid-stimulating
hormone (TSH) beta-subunit with the corresponding regions of
choriogonadotropin or follitropin confers luteotropic but not follitropic
activity to chimeric TSH. J Biol Chem 272:15532–15540.
Hershman JM. 1999. Human chorionic gonadotropin and the thyroid:
hyperemesis gravidarum and trophoblastic tumors. Thyroid 9:653–657.
Li MD, Ford JJ. 1998. A comprehensive evolutionary analysis based on
nucleotide and amino acid sequences of the alpha- and beta-subunits of
glycoprotein hormone gene family. J Endocrinol 156:529–542.
HUMAN MUTATION 29(1),91^98,2008 97
Human Mutation DOI 10.1002/humu
Montanelli L, Delbaere A, Di Carlo C, Nappi C, Smits G, Vassart G,
Costagliola S. 2004a. A mutation in the follicle-stimulating hormone
receptor as a cause of familial spontaneous ovarian hyperstimulation
syndrome. J Clin Endocrinol Metab 89:1255–1258.
Montanelli L, Van Durme JJ, Smits G, Bonomi M, Rodien P , Devor EJ,
Moffat-Wilson K, Pardo L, Vassart G, Costagliola S. 2004b. Modulation
of ligand selectivity associated with activation of the transmembrane
region of the human follitropin receptor. Mol Endocrinol 18:2061–2073.
Moyle WR, Campbell RK, Myers RV, Bernard MP , Han Y, Wang X. 1994.
Co-evolution of ligand-receptor pairs. Nature 368:251–255.
Nappi RG, Di Naro E, D’Aries AP , Nappi L. 1998. Natural pregnancy in
hypothyroid woman complicated by spontaneous ovarian hyperstimula-
tion syndrome. Am J Obstet Gynecol 178:610–611.
Ponder JW, Case DA. 2003. Force fields for protein simulations. Adv
Protein Chem 66:27–85.
Puett D, Li Y, DeMars G, Angelova K, Fanelli F. 2007. A functional
transmembrane complex: the luteinizing hormone receptor with bound
ligand and G protein. Mol Cell Endocrinol 260–262:126–136.
Remy JJ, Nespoulous C, Grosclaude J, Grebert D, Couture L, Pajot E,
Salesse R. 2001. Purification and structural analysis of a soluble human
chorionogonadotropin hormone-receptor complex. J Biol Chem 276:
Rodien P , Bremont C, Sanson ML, Parma J, Van Sande J, Costagliola S,
Luton JP , Vassart G, Duprez L. 1998. Familial gestational hyperthyroid-
ism caused by a mutant thyrotropin receptor hypersensitive to human
chorionic gonadotropin. N Engl J Med 339:1823–1826.
Schmidt A, MacColl R, Lindau-Shepard B, Buckler DR, Dias JA. 2001.
Hormone-induced conformational change of the purified soluble
hormone binding domain of follitropin receptor complexed with single
chain follitropin. J Biol Chem 276:23373–23381.
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.
Smits G, Campillo M, Govaerts C, Janssens V, Richter C, Vassart G, Pardo
L, Costagliola S. 2003a. Glycoprotein hormone receptors: determinants
in leucine-rich repeats responsible for ligand specificity. EMBO J 22:
Smits G, Olatunbosun O, Delbaere A, Pierson R, Vassart G, Costagliola S.
2003b. Ovarian hyperstimulation syndrome due to a mutation in the
follicle-stimulating hormone receptor. N Engl J Med 349:760–766.
Szkudlinski MW, Fremont V, Ronin C, Weintraub BD. 2002. Thyroid-
stimulating hormone and thyroid-stimulating hormone receptor struc-
ture-function relationships. Physiol Rev 82:473–502.
Vassart G, Pardo L, Costagliola S. 2004. A molecular dissection of the
glycoprotein hormone receptors. Trends Biochem Sci 29:119–126.
Vasseur C, Rodien P , Beau I, Desroches A, Gerard C, de Poncheville L,
Chaplot S, Savagner F, Croue A, Mathieu E, Lahlou N, Descamps P ,
Misrahi M. 2003. A chorionic gonadotropin-sensitive mutation in the
follicle-stimulating hormone receptor as a cause of familial gestational
spontaneous ovarian hyperstimulation syndrome. N Engl J Med 349:
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:
Vlaeminck-Guillem V, Ho SC, Rodien P , Vassart G, Costagliola S. 2002.
Activation of the cAMP pathway by the TSH receptor involves
switching of the ectodomain from a tethered inverse agonist to an
agonist. Mol Endocrinol 16:736–746.
98 HUMAN MUTATION 29(1),91^98,2008
Human Mutation DOI 10.1002/humu