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*
Yong Sang
Song
§,
Inhae
Ji
,
Jeremy
Beauchamp¶,
Neil W.
Isaacs¶, and
Tae H.
Ji
From the
Department of Chemistry, University of
Kentucky, Lexington, Kentucky 40506-0055, the § Cancer
Research Center, Seoul National University College of Medicine, Seoul
110-744, Korea, and the ¶ Department of Chemistry, University
of Glasgow, Glasgow G128QQ, United Kingdom
Received for publication, May 3, 2000, and in revised form, June 12, 2000
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ABSTRACT |
The luteinizing hormone receptor (LHR) and
follicle-stimulating hormone receptor (FSHR) have an ~350-amino
acid-long, N-terminal extracellular exodomain. This exodomain binds
hormone with high affinity and specificity and contains eight to nine
putative Leu-rich repeat (LRR) sequences. LRRs are known to assume the
horseshoe structure in ribonuclease inhibitors, and the inner lining of the horseshoe consists of the
-stranded
Leu/Ile-X-Leu/Ile motif. In the case of ribonuclease
inhibitors, these
strands interact with ribonuclease. However, it
is unclear whether the putative LRRs of LHR and FSHR play any role in
the structure and function. In this work, the
-stranded Leu/Ile
residues in all LRRs of the human LHR and FSHR were Ala-scanned and
characterized. In addition, the 23 residues around LRR2 of LHR were
Ala-scanned. The results show that
-stranded Leu and Ile residues in
all LRRs are important but not equally. These
Leu/Ile-X-Leu/Ile motifs appear to form the hydrophobic
core of the LRR loop, crucial for the LRR structure. Interestingly, the
hot spots are primarily in the upstream and downstream LRRs of the LHR
exodomain, whereas important LRRs spread throughout the FSHR exodomain.
This may explain the distinct hormone specificity despite the
structural similarity of the two receptors.
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INTRODUCTION |
The follicle-stimulating hormone receptor
(FSHR)1 and the luteinizing
hormone/chorionic gonadotropin receptor (LHR) play crucial roles in
reproduction of mammals, including humans. Human reproduction is a
major concern as we have been trying to both control and promote human
fertility. For example, humans are experiencing a dramatic population
increase, yet ~16% of families suffer from infertility problems.
FSHR is present in granulosa cells of the ovary (1) and Sertoli cells
of the testis (2). In contrast, LHR is present in luteal and granulosa
cells in the ovary and thecal cells in the testis. In the ovary, the
expression of FSHR and LHR is dependent on the developmental stage of
primordial, primary, secondary (preantral), small antral, large antral,
and preovulatory follicles during the ovulation cycle (3). Therefore, the appearance, action, and disappearance of these receptors are crucially related with the granulosa cell differentiation, follicular development, and ovulation.
LHR and FSHR belong to the structurally unique glycoprotein hormone
receptor subfamily of the G protein-coupled receptor family (4). Unlike
other receptor subfamilies, they comprise two equal halves, an
extracellular N-terminal half (exodomain) and a membrane associated
C-terminal half (endodomain) (5-9) as shown in Fig. 1A. The
exodomain is ~350 amino acids long and, alone, is capable of high
affinity hormone binding (10-13) with hormone selectivity (14-16) but
without hormone action (12, 17, 18). Hormone signal is generated in the
endodomain (19), which is structurally equivalent to the entire
molecule of many other G protein-coupled receptors such as rhodopsin
and adrenergic receptors (4). Growing evidence suggests that
glycoprotein hormones initially bind to the exodomain (4) and that the
resulting hormone-exodomain complex undergoes a conformational
change (20) and interacts with the endodomain. This secondary
interaction is thought to generate signal in the endodomain (4, 19,
21). Interestingly, the high affinity hormone binding at the exodomain
is modulated by the endodomain (22, 23). This suggests that the
exodomain, in particular the hormone contact points, and endodomain are
intimately related to each other during the initial and secondary
interactions. To understand the underlying mechanism, it is essential
to identify the exact hormone contact points in the exodomain.
Sequence comparison (Fig. 1, A and B) and
modeling suggest that the exodomain contains imperfect Leu/Ile-rich
repeats (LRRs) (5, 24-28), which are flanked by short N- and
C-terminal regions. LRRs are a structural motif in a few crystal
structures of proteins, namely ribonuclease inhibitors (29). However,
the imperfect LRR motif is present in a large family of proteins and
therefore could be a common structural motif. LRRs comprise divergent
22-29 amino acids with a short
strand consisting of two Leu and/or Ile residues in the alternate sequence (Leu/Ile-X-Leu/Ile),
which is linked by linkers to
helices in parallel to the
strand. In addition, there are four hydrophobic residues at imperfectly matching positions. Therefore, the primary homology among LRRs is the
Leu/Ile-X-Leu/Ile motif in
strands. The LRRs found in the crystal structure of ribonuclease inhibitors assume the
three-quarter donut structure with the inner lining of
strands and
the outer surface of
helices (29, 30). The inner lining of
ribonuclease inhibitor interacts with ribonuclease at multiple contact
points (31, 30). Based on the structure of ribonuclease inhibitors, the
possibility has been raised that the inner lining of LRRs in LHR and
FSHR may contact LH/CG and FSH (26-28, 32), in particular, with the
concave side of the hormones (33).
Unfortunately, there is no experimental evidence for the existence of
the LRR structure in LHR and FSHR and the LRRs' interaction with
hormone. Mutational and photoaffinity analyses of LHR show that a
number of regions and amino acids in the exodomain, particularly in the
N-terminal half of the exodomain, either interact with hormone (34, 35)
or are important for hormone binding (36-39). Interestingly, some of
these hormone contact sites are outside of the LRRs (34, 35), whereas
certain regions and ionic residues in the LRR region are directly or
indirectly important for hormone binding (37, 39). Furthermore, the
presence of Leu versus Ile in the eight
Leu/Ile-X-Leu/Ile motifs is conserved except one residue
between LHR and FSHR. This underscores the potential importance of LRRs
in these receptors. These results, however, do not address whether
putative LRRs and
strand Leu/Ile-X-Leu/Ile motifs are
indeed involved in hormone binding. It is also enigmatic how the two
receptors produce totally distinct specificities with the 94%
homologous Leu//Ile-X-Leu/Ile motifs.
In this study, we examined the conserved
strand Leu/Ile residues of
LRRs in the human LHR and FSHR. The results show that
strand Leu
and Ile residues in all LRRs are important, suggesting multiple contact
sites and crucial roles of LRRs in the receptors. Furthermore, some
LRRs are more crucial than others and the crucial LRRs are primarily
present in the upstream half LRRs in the LHR exodomain, in contrast to
their presence in the C-terminal half LRRs of the FSHR exodomain. This
suggests the distinct hormone binding specificity of the two receptors
resides at spatially separate sites. In addition, the Ala scan of the
sequence around LRR2 in the LHR shows that not only the
Ile-X-Ile sequence in the
strand but also other
conserved hydrophobic residues are indispensable for hormone binding.
These results are compatible with the LRR model structure proposed by
Bhowmick et al. (28) and show for the first time that
LRRs in the gonadotropin receptors, particularly the
Leu/Ile-X-Leu/Ile motifs, play a crucial role.
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EXPERIMENTAL PROCEDURES |
Mutagenesis and Functional Expression of Receptors--
Each
mutant human LHR and FSHR cDNA was prepared in a pSELECT vector
using the non-polymerase chain reaction-based Altered Sites
Mutagenesis System (Promega), sequenced, and subcloned into pcDNA3 (Invitrogen) as described previously (40). After subcloning pcDNA3, the mutant cDNAs were sequenced again. Plasmids were
transfected into human embryonic kidney (HEK) 293 cells by the calcium
phosphate method. Stable cell lines were established in minimum
essential medium containing 10% horse serum and 500 µg/ml
G-418 and then used for hormone binding and cAMP assays. In addition to
stable cell lines expressing receptors, some receptors were transiently expressed using varying amounts of plasmid to express different concentrations of mutant receptors. All assays were carried out in
duplicate and repeated four to six times. Means and standard variations
were calculated.
Hormone Binding and Intracellular cAMP Assay--
hCG and human
FSH were provided by the National Hormone and Pituitary Program and
radioiodinated as described previously (41). Cells were assayed for
125I-hormone binding in the presence of increasing
concentrations of nonradioactive hormone. Scatchard plots determined
the Kd values. For intracellular cAMP assay, cells
were washed twice with Dulbecco's modified Eagle's medium and
incubated in the medium containing isobutylmethylxanthine (0.1 mg/ml)
for 15 min. Increasing concentrations of hCG were then added, and the
incubation was continued for 45 min at 37 °C. After removing the
medium, the cells were rinsed once with fresh medium
without isobutylmethylxanthine, lysed in 70% ethanol, freeze-thawed in
liquid nitrogen, and scraped. After pelleting cell debris at
16,000 × g for 10 min at 4 °C, the supernatant was
collected, dried under vacuum, and resuspended in 10 µl of the cAMP
assay buffer that was provided by the manufacturer. cAMP concentrations
were determined with an 125I-cAMP assay kit (Amersham
Pharmacia Biotech) following the manufacturer's instruction and
validated for use in our laboratory.
Radioimmunoassay for FLAG-G Receptor--
FLAG-LHR was
prepared by inserting the FLAG epitope (42),
Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (5'-GAC TAC AAG GAC GAT GAC GAT AAG-3'), between the C terminus (Ser26) of the signal
sequence and the N terminus (Arg27) of mature receptors. We
have shown that the FLAG tag did not significantly impair the activity
of wild type and mutant LH/CG-Rs (34). Also, the FLAG epitope (43) has
successfully been used as a marker to identify, trace, and purify
recombinant proteins carrying the tag without significantly impairing
their biological activities of other molecules (44, 45). Mouse
anti-FLAG monoclonal antibody M2 (Eastman Kodak Co.) was iodinated with
125I (41), and 125I-anti-FLAG antibodies were
purified on a Sephadex G-150 column. Binding of
125I-anti-FLAG to HEK 293 cells expressing FLAG-LH
receptors was carried out as described previously (34).
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RESULTS |
The eight LRRs of LHR and FSHR contain the
Leu/Ile-X-Leu/Ile motif, which constitutes the core of the
putative
strands present in all LRRs of the glycoprotein hormone
receptors and other LRR proteins (29). In fact, the presence of Leu
versus Ile in the eight Leu/Ile-X-Leu/Ile motifs
is conserved except one residue between LHR and FSHR. The exception is
that the LRR1 motif is Leu-X-Leu in LHR and
Leu-X-Phe in FSHR (Fig. 1,
A and B). This 94% homology is striking,
particularly in light of the less than 50% homology in the exodomain
sequences (46) and the distinct hormone binding specificities of LHR
and FSHR. In addition to the eight LRRs, there is putative LRR9 in the
LHR (28), but it lacks the Leu/Ile-X-Leu/Ile motif and the
Ala-Phe sequence. Therefore, we focused on LRRs 1-8 in this study.

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Fig. 1.
Structure and sequence of the gonadotropin
receptors. The LHR and FSHR consist of a ~350-amino
acid-long extracellular domain (exodomain) and a membrane-associated
domain (endodomain) of equal size. The exodomain is thought to include
eight to nine LRRs, each consisting of a strand and an helix.
The endodomain consists of three exoloops, three cytoloops, seven
transmembrane domains, and the C-terminal tail. A, the
exodomain sequence of human LHR is aligned based on the LRR motif with
conserved residues in bold letters. B, likewise,
the LRR sequences of human FSHR are aligned. C, a schematic
model of the LRRs in the exodomain and the seven transmembrane domains
in the endodomain.
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Leu/Ile-X-Leu/Ile Motifs in LHR LRRs--
To examine the
importance of the Leu and Ile residues in the putative
strands of
LHR, they were individually substituted with Ala. Each of the Ala
substitution mutants was stably expressed in HEK 293 cells and
assayed for 125I-hCG binding on intact cells or after
solubilization in Triton X-100 and for hCG-dependent cAMP
induction. For example, the cells transfected with the vector carrying
the Leu29
Ala mutant cDNA were incubated
with a constant amount of 125I-hCG in the presence of
increasing concentrations of unlabeled hCG. The resulting displacement
data (Fig. 2A) did not show
hCG binding (Fig. 2B). To test whether the Leu29
Ala mutant was capable of binding hCG but trapped in cells, the
cells transfected with the Leu29
Ala mutant vector were
solubilized in Triton X-100 and assayed for 125I-hCG
binding (Fig. 2, C and D). Again,
125I-hCG binding was not observed. Since the cAMP assay is
significantly more sensitive than the 125I binding assay,
we tested if the cells produce cAMP in response to hCG. cAMP production
was insignificant up to 10 nM hCG treatment (Fig.
2E). Although the cells produced cAMP, 2-5% of the maximum cAMP level of the wild type receptor, when treated with 100 nM, we considered it insignificant. This result, however,
suggests the surface expression of the mutant receptor. We and others
(28, 34, 47) have shown that mutant receptors that do not bind 125I-hCG on intact cells and in solution are more likely to
be defective in hormone binding than unexpressed. Therefore, mutant
receptors that do not bind hCG on intact cells and in solution are
generally considered to be incapable of binding hormone. However, to
demonstrate their expression on the cell surface, antibodies were
utilized. Because most of anti-LHR antibodies lack high affinity and
specificity, the FLAG tag (42) was attached to the mutant receptor. The
FLAG tag can be detected by monoclonal anti-FLAG antibody band,
and it does not interfere with the expression and activity of
receptors (34, 48). The FLAG-Leu29
Ala mutant was
expressed and assayed for binding monoclonal 125I-anti-FLAG
antibody (34). The antibody bound to the intact cells expressing the
FLAG-Leu29
Ala mutant as well as the FLAG-wild type
receptor, indicating the surface expression of the
FLAG-Leu29
Ala mutant (Table
I). On the other hand, the antibody did not bind to the cells expressing the wild type LH/CG-R lacking the FLAG
tag nor the FLAG-Asp17
Ala that is known to be trapped
in cells but not expressed on the cell surface (34). These results show
that the Leu29
Ala mutant was expressed on the surface
but was defective in binding hCG. The fact that anti-FLAG
antibody recognized the mutant indicates that the mutant was folded to
a conformation similar to that of the wild type FLAG receptor, at least
in the FLAG region.

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Fig. 2.
Analysis of Leu and Ile in the
strands of LRR1-LRR4. Leu and Ile of the
putative strands in LRR1 and LRR2 of LHR were individually
substituted with Ala, and the resulting mutant receptors were stably
expressed on HEK 293 cells. Cells were assayed for
125I-hCG binding in the presence of increasing
concentrations of nonradioactive hCG (A), and Scatchard
analysis (B) was plotted against specific binding. Cells
were also solubilized in Triton X-100 and assayed for
125I-hCG binding in the presence of unlabeled hCG
(C), and the results were converted to Scatchard plots
(D). In addition, intact cells were treated with increasing
concentrations of unlabeled hCG, and intracellular cAMP was measured
(E) as described under "Experimental Procedures."
Experiments were repeated four to six times in duplicate. NS
stands for not significant. F-J, the same as
A-E for Leu/Ile residues in LRR3 and LRR4.
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Table I
Anti-FLAG antibody binding to intact cells
Mutant LHRs with Ala substitution transiently were expressed on intact
cells and assayed for increasing concentrations of
125I-anti-FLAG monoclonal antibody binding as previously
described (34).
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Next, the Leu31
Ala mutant was examined.
125I-hCG bound to intact cells expressing the
Leu31
Ala mutant with a Kd value of
690 pM as compared with the wild type value of 600 pM (Fig. 2, A and B). The mutant
solubilized in Triton X-100 was also capable of binding the hormone
with the affinity similar to the wild type affinity. It also produced
cAMP in response to hCG, although the affinity and maximum cAMP level were significantly lower than the wild type receptor. These results show that these two Leu residues of LRR1 responded dissimilarly to Ala
substitution. In contrast to the dissimilar sensitivity to Ala
substitution for the two Leu residues of LRR1, Ala substitution for the
two Ile residues in LRR2 abolished hCG binding on intact cells and in
solution as well as cAMP induction, although the two mutants were
detected on the cell surface by anti-FLAG antibody (Table I).
In LRR3, the Leu/Ile pair comprises Ile78 and
Ile80. The Ile78
Ala mutant was capable of
binding 125I-hCG and inducing cAMP (Fig. 2,
F-J), but the affinities were lower than the wild type
affinities. On the other hand, the Ile80
Ala mutant was
incapable of hormone binding and cAMP induction, despite its expression
on the cell surface (Table I). In LRR4, both of the Leu103
Ala and Ile105
Ala mutants lost the receptor
activities as did the Ile53
Ala and Ile55
Ala mutants of LRR2.
In LRR5 and LRR6, individual Ala substitutions for Leu128,
Ile130, Leu154, and Leu156
partially impaired, but did not abolish, the hormone binding and cAMP
induction (Fig. 3, A-E).
Interestingly, the affinities of hormone binding to intact cells of
these mutants were similar, approximately 2-fold lower than the wild
type affinity. This result indicates similar impacts of the Ala
substitutions. In addition, the binding affinity does not appear to be
dependent on the receptor concentration on the cell surface. To test
the relationship between the receptor concentration and the binding
affinity, cells were transfected with varying concentrations of
plasmids carrying the wild type receptor and Leu128
Ala
mutant. Cells expressing varying surface concentrations of the
receptors showed similar Kd values (Table
II). Ala substitutions for the LRR7 and
LRR8 residues showed split results (Fig. 3). In LRR7,
Leu177
Ala partially impaired hormone binding and cAMP
induction, whereas Leu179
Ala entirely abolished them.
The Leu202
Ala mutant of LRR8 completely lost the
ability to bind the hormone and induce cAMP, but the effect was only
partial for the Ile204
Ala substitution.

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Fig. 3.
Analysis of Leu and Ile in the
strands of LRR5-LRR8. Leu and Ile of the strands of LRR5-LRR8 were individually Ala-scanned and analyzed as
described in the legend to Fig. 2.
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Table II
Receptor concentration and binding affinity
To determine the effect of receptor concentrations on the binding
affinity, varying concentrations of receptors were transiently
expressed on intact cells and assayed for hormone binding.
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Overall, the results show three distinct LRR groups. In LRR2 and LRR4,
both residues of the Leu/Ile-X-Leu/Ile motif are sensitive to Ala substitution; only one residue is sensitive in LRR1, LRR3, LRR7,
and LRR8; and neither is sensitive in LRR5 and LRR6. To readily compare
various binding affinities of the mutant receptors, the wild type
Kd value was divided by each mutant's
Kd value (Fig. 4). The
lower the
Kdwt/Kdmut
ratio implies the lower hormone binding affinity of the mutant. All of
the mutants have a Kd ratio less than the unity, consistent with their lower binding affinities than the wild type affinity. Most of the Kd ratios are in the range of
0.2-0.5 except those incapable of hormone binding. This clearly
indicates the significant impact of Ala substitution for the conserved
Leu/Ile-X-Leu/Ile motif of the putative
strand of LRRs
in LHR. It also suggests the importance of the motif in most of LRRs,
in particular those in the region of LRR2, LRR3, and LRR4 where all Ala
substitutions except Ile78
Ala completely abolished
hormone binding. The results also indicate that the partial and
complete losses of cAMP induction by the Ala mutants were due to
impaired hormone binding rather than defective cAMP induction,
per se. There was no mutant receptor that was capable of
binding hormone but incapable of inducing cAMP. This suggests that the
Leu/Ile-X-Leu/Ile motifs enable hormone binding, not
inducing cAMP.

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Fig. 4.
Comparative binding affinities of LRR
mutants: comparative binding affinities of LRR mutants. The wild
type Kd value was divided with the
Kd value of each mutant. Solid bars
represent Kd values of receptors on intact cells and
open bars for solubilized receptors.
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Fidelity of Mutagenesis and Other Controls--
Our site-directed
mutagenesis utilizes synthetic oligonucleotides containing a mutant
sequence for each mutation and, furthermore, does not involve
polymerase chain reaction. After mutagenesis, the mutant and flanking
sequences are verified by sequencing. In addition, the same sequence is
confirmed once more after a mutant cDNA is subcloned into the
expression vector. Therefore, it is highly unlikely that a mutant
cDNA might have undergone an unintended mutation(s) during the
mutagenesis and subcloning. To confirm this mutation, cDNAs were
reverted to the wild type cDNA, which, in turn, was used to
transfect cells. The revertants behaved the same as the wild type
receptor in surface expression, hormone binding, and cAMP induction
(data not included), indicating that there was no mutation other than
the intended substitutions. In addition, intact or solubilized HEK 293 cells that were transfected with the vector containing no receptor
cDNA or that were not transfected did not bind hCG and FSH (data
not included).
Sequence around Ile53-X-Ile55 in
LRR2--
As a first step to examine crucial LRR2 and LRR4, amino acid
residues (Val38-Arg63) flanking
Ile53-Glu54-Ile55 of LRR2 were
Ala-scanned. Ala substitution for each of Val38,
Pro40, Ser41, and Gln42 did not
abolish hormone binding as did Ala substitution for Ile39
(Fig. 5, A-D), although the
affinities for hormone binding to Ala substituents on intact cells were
similarly ~3-fold lower than the wild type affinity. In addition, the
mutants were capable of inducing cAMP in response to hCG binding,
indicating that the Ala substitutions were tolerable. In contrast, the
Ile39
Ala mutant failed to bind hCG both on intact
cells and even after solubilization in Triton X-100, showing the
mutant's inability of hCG binding. The next five Ala substituents
behaved similarly to the first five Ala substituents. Arg45
Ala, Gly46
Ala, Leu47
Ala, and
Asn48
Ala were tolerable for hCG binding with 2-4-fold
lower affinities and cAMP induction, whereas Phe44
Ala
abrogated hCG binding and cAMP induction (Fig. 5, F-J). Among the next five Ala substituents, Glu49
Ala,
Val50
Ala, Ile51
Ala, Lys52
Ala, and Ile53
Ala (Fig.
6, A-E), Ile53
Ala was intolerable as Ile53 was part of the
Leu/Ile-X-Leu/Ile motif. On the other hand, the others were
all tolerable. The results were the same for the next five Ala
substitutions for Glu54, Ile55,
Ser56, Gln57, and Ile58 (Fig. 6,
F-J). Ile55
Ala was intolerable, whereas
the other Ala substitutions reduced the affinities for hormone binding
and cAMP induction. Ala substitutions for the last five residues
produced different results. Asp59
Ala and
Arg63
Ala were tolerable, whereas the Ala substitutions
for the three tandem residues, Ser60, Leu61,
and Glu62, abolished hCG binding and cAMP induction. The
Ala substitution mutants were again reverted to the wild type, and the
revertants behaved the same as the wild type receptor.

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Fig. 5.
Ala scan and characterization of residues
around LRR2 of LHR. Amino acids from Val38 to
Asn48 around LRR2 were individually substituted with Ala,
and the resulting mutants were assayed as described in the legend to
Fig. 2.
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Fig. 6.
Ala scan and characterization of residues
around LRR2 of LHR. Amino acids from Glu49 to
Arg63 around LRR2 were individually substituted with Ala,
and the resulting mutants were assayed as described in the legend to
Fig. 2.
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The comparative
Kdwt/Kdmut
ratios (Fig. 7) show that all Ala
substitutions resulted in the reduction of hormone binding affinity by
2-fold or more. In particular, Ala substitution for Ile39,
Phe44, Ile53, Ile55,
Ser60, Leu61, and Glu62 abolished
hormone binding, although they were expressed on the cell surface
according to the anti-FLAG antibody binding assay (data not shown).
Interestingly, Ile39, Phe44, Ile53,
Ile55, and Leu61 are conserved among all LRRs
(Fig. 1B), whereas Ser60 and Glu62
are not, an indication of the importance of the conserved hydrophobic residues. The result is consistent with the importance of the conserved
residues and, therefore, the LRRs in the LHR. The fact that the
conserved residues are located in the putative
strand and
helix
signifies their structural role.

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Fig. 7.
Comparative binding affinities of LRR2
mutants. The wild type Kd value was divided
with the Kd value of each mutant described in the
legends to Figs. 5 and 6. Solid bars represent
Kd values of receptors on intact cells and
open bars for solubilized receptors.
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Leu/Ile-X-Leu/Ile Motifs in LRRs of FSHR--
The
Leu/Ile-X-Leu/Ile sequences of FSHR are exactly the same as
those of LHR except LRR1. In contrast to this near perfect sequence,
the sequence homology of the exodomains of the two receptors is less
than 50% (46), the selectivity for LH/CG and FSH is nearly 100%, and
the affinity is extremely high with the Kd value of
~500 pM. To understand these seemingly contrasting
results and determine whether the nearly identical
Leu/Ile-X-Leu/Ile sequences of the two receptors possibly
contribute distinguish the two hormones, we examined the LRRs of FSHR.
In LRR1, the Leu32
Ala mutant on intact cells and after
solubilization was capable of binding 125I-FSH (Fig.
8, A-D). Interestingly, the
Kd value was 1,010 pM as compared with
the wild type Kd value of 2,420 pM,
indicating a 2-fold improvement in the hormone binding affinity. However, cAMP induction was marginal (Fig. 8E). The Ala
substitution for Phe34 abolished FSH binding and cAMP
induction (Fig. 8). The cells transfected with the mutant cDNA
plasmid did not bind 125I-FSH nor those solubilized in
Triton X-100, although the FLAG-Phe34
Ala mutant was
detected by 125I-anti-FLAG antibody on the cell surface
(data not shown).

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Fig. 8.
Ala scan and characterization of the
strand residues of FSHR. Leu, Ile, and their
equivalent residues of the strands of LRR1-LRR8 of FSHR were
individually Ala-scanned and analyzed as described for LRR residues of
LHR in the legend to Fig. 2.
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In LRR2, the Ile58
Ala mutant, but not the
Ile60
Ala mutant, was capable of binding hormone and
inducing cAMP (Fig. 8, A-E). The binding affinity of the
Ile58
Ala mutant was nearly 3-fold better than the wild
type receptor, but the cAMP induction was marginal, as was the
Leu32
Ala mutant of LRR1. The Ala substitution of the
second residue of LRR2, Ile60
Ala, abolished hormone
binding and cAMP induction, as did the Ala substitution for the second
residue of LRR1, Phe34
Ala.
In LRR3 the Ile83
Ala and Ile85
Ala
mutants failed to bind the hormone and induce cAMP. In LRR4 the result
of the Ala substitutions was the same as the result of Ala
substitutions for LRR1 andLRR2 residues. For example, the
Leu108
Ala mutant bound FSH and induced cAMP, whereas
the Ile110
Ala did not bind the hormone nor induced
cAMP. In LRR5 the two Ala substitution mutants were capable of binding
the hormone and cAMP induction (Fig. 8, F-J). However,
their binding affinities and cAMP induction were quite different. The
Ala substitutions for Leu158 and Leu160 of LRR6
had split results. The Leu158
Ala mutant was incapable
of binding the hormone and inducing cAMP, whereas the Leu60
Ala mutant bound FSH and induced cAMP. In LRR7 and LRR8 none of the
Ala mutants were capable of binding FSH and inducing cAMP.
Taken together, Ala substitutions for the Leu/Ile-X-Leu/Ile
motifs in LRRs significantly impact the hormone binding and/or cAMP
induction of FSHR. Interestingly, some of the Ala substitutions noticeably improved the hormone binding affinity. However, the ability
to induce cAMP by the mutants was consistently impaired. The
Kd ratios of the wild type receptor and individual mutants (Fig. 4B) show that LRR5, LRR6, LRR7, and LRR8 in
the C-terminal region of the exodomain were more severely impaired than
those LRRs in the N-terminal regions of the exodomain were.
 |
DISCUSSION |
In this study, we set out to determine whether putative LRRs and
strand Leu/Ile-X-Leu/Ile motifs are indeed required for hormone binding. In addition, we hoped to learn how the 94% homologous Leu//Ile-X-Leu/Ile motifs of LHR and FSHR might contribute
to their distinct hormone specificities.
Ala substitutions for all Leu and Ile residues, except
Leu31, in the LRR
strands impaired the hormone binding
activity of LHR, although the Ala mutants were expressed on the cell
surface. Some Ala substitutions abolished hormone binding, whereas
others had less significant effects as shown in the computer models
(Fig. 9, A and B).
These results suggest that the Leu and Ile residues are important,
although in varying degree. Since the conserved and important residues
are spread around an LRR (Fig. 1A), some of them are
probably necessary for the structural integrity of LRRs. The
examination of the residues around the Ile-X-Ile sequence of
LRR2 reveals the importance of the two alternate Ile residues, since
the Ala substitutions for the intervening and flanking residues had
less dramatic or marginal effects (Fig. 9C). In addition to the Ile-X-Ile sequence, Ala substitutions for
Ile39, Phe44, Ile53,
Ile55, and Leu61, which are conserved among
LRRs, resulted in the complete loss of the hormone binding without
exception. These residues form the hydrophobic core of the LRR2. In
contrast, the substitution effects of other residues were less
significant. These results support the LRR structure in LHR and its
significant role in hormone binding.

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|
Fig. 9.
Exodomain models. For models
A for LHR, B for FSHR, and C for LRR2
of LHR, the receptor exodomain was homology-modeled, as described
previously (28), based on the coordinates of ribonuclease inhibitor
from the Brookhaven data base as the template with the O and Frodo
software packages (51) using an Indigo 2-Extreme (Silicon Graphics) and
easv10 (Evans and Sutherland). They were modified based on the
experimental data presented in this study. In each case,
yellow indicates an natural residue, red
indicates Ala substitutions with a maximum effect on hormone binding
(Kd of hormone binding to cells) and/or expression,
blue indicates a minimum effect on hormone binding, and
colors in between (red > crimson > magenta > blue) indicate the effects
between these extremes. Note that in this scheme the wild type FSH
receptor would be a crimson color. FSH apparently binds
better to the mutant receptors, so these are going to be
bluer than the wild type. LRR 2 of LHR is shown with the
green strand and red helix in C.
|
|
The next question is whether or not the crucial LRRs interact with the
hormone. The results of this study could not provide a direct answer.
The LRR2 study, however, shed some light on the issue. The residues
that are most sensitive to Ala substitution are hydrophobic and appear
to form the hydrophobic core of LRR2. Since an LRR assumes an oblong
loop (Fig. 9), it is unlikely that all sides of an LRR contact with the
hormone. In particular, the hydrophobic core residues, including the
conserved Ile53 and Ile55 in the
strand,
are unlikely to interact with the hormone. If an LRR interacts with
hormone at all, the residues involved will be present in the inner
lining of the LRR horseshoe or on the linkers. In fact, our model
predicts that Lys52, Glu54, Ser56,
and Gln57 are to face the hormone. However, the Ala
substitutions for these residues did not completely impair the hormone
binding capacity. Therefore, they do not appear to provide the major
force to the hormone-receptor interaction.
One may raise an issue whether the Ala substitutional effects were due
to changes in the global structure of LHR rather than local structure
around the substituted residue. This is a general and contentious issue
concerning the interpretation of the result from a substitutional
study. Although it is difficult to test for an unequivocal answer,
there is evidence supporting local conformational changes induced by
substitution of a single amino acid. For example, crystal structures of
other proteins show that single amino acid substitutions generally
cause subtle and local conformational alterations (49, 50). In our
hand, mutations did not impact the recognition of the FLAG
epitope at the exodomain N terminus by anti-FlAG antibodies (34, 48).
The previous studies also suggest that the targeting machinery of LHR
is capable of detecting some conformational changes, which hormone
binding could not. This indicates that the machinery is more sensitive to such conformational changes of LHR than the hormone binding could
recognize. However, in the absence of foolproof evidence and
conformational antibodies, it is premature to dismiss the potential
interference of misfolding on the local and global conformation of
the mutants.
Despite the 94% homology in the Leu/Ile-X-Leu/Ile
sequences between the two receptors, LHR and FSHR are capable of
recognizing their cognate ligands, LH/CG and FSH, respectively, with a
high affinity and no cross-reactivity. Therefore, it is necessary to know how the homologous Leu/Ile-X-Leu/Ile sequences in the
putative hormone contact sites contribute to the discrete hormone
specificity. The Kd ratios in Fig. 4 provide some
answers, at least in part. For example, the location of the important
LRRs is distinct. The upstream LRRs of LHR are more sensitive to Ala
substitution than the downstream LRRs, whereas the downstream LRRs of
FSHR are more sensitive. This suggests different contact points in LRRs
of the two receptors. The role of LRRs may also differ as Ala
substitutions improved the binding affinity of some FSHR mutants, whereas Ala substitutions attenuated the binding affinity of LHR. This
result suggests a clear difference in the structural role of LRRs in
the two receptors, which could explain their dramatically distinct
hormone binding specificities. Despite the higher binding affinities of
the FSHR mutants and the lower binding affinities of the LHR mutants,
the EC50 values for cAMP induction increased for both
receptors. Therefore, both receptors appear to share some common
mechanism to generate the cAMP signal.
 |
FOOTNOTES |
*
This work was supported by Grants HD-18702 and DK-51469 from
the National Institutes of Health.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 all correspondence should be addressed: Dept. of
Chemistry, University of Kentucky, Lexington, KY 40506-0055. Tel.: 859-257-3163; Fax: 859-527-3229; E-mail tji@pop.uky.edu.
Published, JBC Papers in Press, July 3, 2000, DOI 10.1074/jbc.M003772200
 |
ABBREVIATIONS |
The abbreviations used are:
FSH, follicle-stimulating hormone;
FSHR, FSH receptor;
h, human;
CG, chorionic gonadotropin;
LH, luteinizing hormone;
LHR, LH receptor;
LRR, Leu-rich repeat;
HEK, human embryonic kidney.
 |
REFERENCES |
1.
|
Hsueh, A. J.,
Jones, P. B.,
Adashi, E. Y.,
Wang, C.,
Zhuang, L. Z.,
and Welsh, T. H., Jr.
(1983)
J. Reprod. Fertil.
69,
325-342[Abstract]
|
2.
|
Griswold, M. D.
(1993)
in
The Sertoli Cells
(Russell, L. D.
, and Griswold, M. D., eds)
, pp. 496-508, Cache River Press, Clearwater, FL
|
3.
|
Adashi, E. Y.
(1996)
in
Reproductive Endocrinology, Surgery, and Technology
(Adashi, E. Y.
, Rock, J. A.
, and Rosenwaks, Z., eds), Vol. 1
, pp. 17-40, Lippincott-Raven, Philadelphia
|
4.
|
Ji, T. H.,
Grossmann, M.,
and Ji, I.
(1998)
J. Biol. Chem.
273,
17299-17302[Free Full Text]
|
5.
|
McFarland, K.,
Sprengel, R.,
Phillips, H.,
Kohler, M.,
Rosemblit, N.,
Nikolics, K.,
Segaloff, D.,
and Seeburg, P.
(1989)
Science
245,
494-499[Medline]
[Order article via Infotrieve]
|
6.
|
Loosfelt, H.,
Misrahi, M.,
Atger, M.,
Salesse, R.,
Thi, M.,
Jolivet, A.,
Guiochon-Mantel, A.,
Sar, S.,
Jallal, B.,
Garnier, J.,
and Milgrom, E.
(1989)
Science
245,
525-528[Medline]
[Order article via Infotrieve]
|
7.
|
Nagayama, Y.,
Kaufman, K. D.,
Seto, P.,
and Rapoport, B.
(1989)
Biochem. Biophys. Res. Commun.
165,
1184-1190[Medline]
[Order article via Infotrieve]
|
8.
|
Sprengel, R.,
Braun, T.,
Nikolics, K.,
Segaloff, D. L.,
and Seeburg, P. H.
(1990)
Mol. Endocrinol.
4,
525-530[Abstract]
|
9.
|
Tilly, J. L.,
Aihara, T.,
Nishimori, K.,
Jia, X. C.,
Billig, H.,
Kowalski, K. I.,
Perlas, E. A.,
and Hsueh, A. J.
(1992)
Endocrinology
131,
799-806[Abstract]
|
10.
|
Tsai-Morris, C. H.,
Buczko, E.,
Wang, W.,
and Dufau, M. L.
(1990)
J. Biol. Chem.
265,
19385-19388[Abstract/Free Full Text]
|
11.
|
Xie, Y. B.,
Wang, H.,
and Segaloff, D. L.
(1990)
J. Biol. Chem.
265,
21411-21414[Abstract/Free Full Text]
|
12.
|
Ji, I.,
and Ji, T. H.
(1991)
Endocrinology
128,
2648-2650[Abstract]
|
13.
|
Davis, D.,
Liu, X.,
and Segaloff, D.
(1995)
Mol. Endocrinol.
9,
159-170[Abstract]
|
14.
|
Braun, T.,
Schofield, P. R.,
and Sprengel, R.
(1991)
EMBO J.
10,
1885-1890[Abstract]
|
15.
|
Moyle, W. R.,
Campbell, R. K.,
Myers, R. V.,
Bernard, M. P.,
Han, Y.,
and Wang, X.
(1994)
Nature
368,
251-255[CrossRef][Medline]
[Order article via Infotrieve]
|
16.
|
Liu, X.,
DePasquale, J. A.,
Griswold, M. D.,
and Dias, J. A.
(1994)
Endocrinology
135,
682-691[Abstract]
|
17.
|
Remy, J. J.,
Bozon, V.,
Couture, L.,
Goxe, B.,
Salesse, R.,
and Garnier, J.
(1993)
Biochem. Biophys. Res. Commun.
193,
1023-1030[CrossRef][Medline]
[Order article via Infotrieve]
|
18.
|
Osuga, Y.,
Hayashi, M.,
Kudo, M.,
Conti, M.,
Kobilka, B.,
and Hsueh, A.
(1997)
J. Biol. Chem.
272,
25006-25012[Abstract/Free Full Text]
|
19.
|
Ji, T. H.,
Murdoch, W. J.,
and Ji, I.
(1995)
Endocrine
3,
187-194
|
20.
|
Ji, I.,
Pan, Y.-N.,
Lee, Y.-M.,
Phang, T.,
and Ji, T. H.
(1995)
Endocrine
3,
907-911
|
21.
|
Dufau, M. L.
(1998)
Annu. Rev. Physiol.
60,
461-496[CrossRef][Medline]
[Order article via Infotrieve]
|
22.
|
Ryu, K.,
Lee, H.,
Kim, S.,
Beauchamp, J.,
Tung, C.,
Isaacs, N. W.,
Ji, I.,
and Ji, T. H.
(1998)
J. Biol. Chem.
273,
6285-6291[Abstract/Free Full Text]
|
23.
|
Ryu, K.,
Gilchrist, R. L.,
Tung, C.,
Ji, I.,
and Ji, T. H.
(1998)
J. Biol. Chem.
273,
28953-28958[Abstract/Free Full Text]
|
24.
|
Koo, Y. B.,
Ji, I.,
Slaughter, R. G.,
and Ji, T. H.
(1991)
Endocrinology
128,
2297-2308[Abstract]
|
25.
|
Moyle, W.,
Campbell, R.,
Rao, S.,
Ayad, N.,
Bernard, M.,
Han, Y.,
and Wang, Y.
(1995)
J. Biol. Chem.
270,
20020-20031[Abstract/Free Full Text]
|
26.
|
Jiang, X.,
Dreno, M.,
Buckler, D.,
Cheng, S.,
Ythier, A.,
Wu, H.,
Hendrickson, W.,
and Tayar, N.
(1995)
Structure (Lond.)
3,
1341-1353[Medline]
[Order article via Infotrieve]
|
27.
|
Couture, L.,
Naharisoa, H.,
Grebert, D.,
Remy, J. J.,
Pajot-Augy, E.,
Bozon, V.,
Haertle, T.,
and Salesse, R.
(1996)
J. Mol. Endocrinol.
16,
15-25[Abstract]
|
28.
|
Bhowmick, N.,
Huang, J.,
Puett, D.,
Isaacs, N. W.,
and Lapthorn, A. J.
(1996)
Mol. Endocrinol.
10,
1147-1159[Abstract]
|
29.
|
Kobe, B.,
and Deisenhofer, J.
(1994)
Trends Biochem. Sci.
19,
415-421[CrossRef][Medline]
[Order article via Infotrieve]
|
30.
|
Papageorgiou, A. C.,
Shapiro, R.,
and Acharya, K. R.
(1997)
EMBO J.
16,
5162-5177[Abstract/Free Full Text]
|
31.
|
Kobe, B.,
and Deisenhofer, J.
(1995)
Nature
374,
183-186[CrossRef][Medline]
[Order article via Infotrieve]
|
32.
|
Dias, J. A.,
Lindau-Shepard, B.,
Hauer, C.,
and Auger, I.
(1998)
Biol. Reprod.
58,
1331-1336[Medline]
[Order article via Infotrieve]
|
33.
|
Lapthorn, J. P.,
Harris, D. C.,
Littlejohn, A.,
Lustbader, J. W.,
Canfield, R. E.,
Machin, K. J.,
Morgan, F. J.,
and Isaacs, N. W.
(1994)
Nature
369,
455-461[CrossRef][Medline]
[Order article via Infotrieve]
|
34.
|
Hong, S.,
Phang, T.,
Ji, I.,
and Ji, T. H.
(1998)
J. Biol. Chem.
273,
13835-13840[Abstract/Free Full Text]
|
35.
|
Phang, T.,
Kundu, G.,
Hong, S.,
Ji, I.,
and Ji, T. H.
(1998)
J. Biol. Chem.
273,
13841-13847[Abstract/Free Full Text]
|
36.
|
Huang, J.,
and Puett, D.
(1995)
J. Biol. Chem.
270,
30023-30028[Abstract/Free Full Text]
|
37.
|
Thomas, D.,
Rozell, T.,
Liu, X.,
and Segaloff, D.
(1996)
Mol. Endocrinol.
10,
760-768[Abstract]
|
38.
|
Zhang, R.,
Buczko, E.,
and Dufau, M.
(1996)
J. Biol. Chem.
271,
5755-5760[Abstract/Free Full Text]
|
39.
|
Bhowmick, N.,
Narayan, P.,
and Puett, D.
(1999)
Endocrinology
140,
4558-4563[Abstract/Free Full Text]
|
40.
|
Ji, I.,
and Ji, T. H.
(1995)
J. Biol. Chem.
270,
15970-15973[Abstract/Free Full Text]
|
41.
|
Ji, I.,
and Ji, T.
(1981)
Proc. Natl. Acad. Sci. U. S. A.
78,
5465-5469[Abstract]
|
42.
|
Prikett, K.,
Amberg, D.,
and Hopp, T.
(1989)
BioTechniques
7,
580-589[Medline]
[Order article via Infotrieve]
|
43.
|
Hopp, T.,
Prikett, K.,
Price, V.,
Libby, R.,
March, C.,
Cerretti, P.,
Urdal, D.,
and Conlon, P.
(1988)
Bio/Technology
6,
1205-1210
|
44.
|
Guan, Z.-M.,
Kobilka, T.,
and Kobilka, B.
(1992)
J. Biol. Chem.
267,
21995-21998[Abstract/Free Full Text]
|
45.
|
Chiang, C.,
and Roeder, R.
(1993)
Pept. Res.
6,
62-64[Medline]
[Order article via Infotrieve]
|
46.
|
Ji, T.,
Ji, I.,
and Kim, S.
(1996)
in
Gonadotropin Receptors. Reproductive Endocrinology, Surgery, and Technology
(Adashi, E.
, Rock, J.
, and Rosenwaks, Z., eds)
, pp. 21-26, Lippincott-Raven, Philadelphia
|
47.
|
Abell, A.,
Liu, X.,
and Segaloff, D. L.
(1996)
J. Biol. Chem.
271,
4518-4527[Abstract/Free Full Text]
|
48.
|
Hong, S.,
Ryu, K.-S.,
Oh, M.-O.,
Ji, I.,
and Ji, T. H.
(1997)
J. Biol. Chem.
272,
4166-4171[Abstract/Free Full Text]
|
49.
|
Sundström, M.,
Lundqvist, T.,
Rödin, J.,
Giebel, L. B.,
Milligan, D.,
and Norstedt, G.
(1996)
J. Biol. Chem.
271,
32197-32203[Abstract/Free Full Text]
|
50.
|
Hamm, H.
(1998)
J. Biol. Chem.
273,
669-673[Free Full Text]
|
51.
|
Jones, T.,
Zou, J.,
Cowan, S.,
and Kjeldaard, M.
(1991)
Acta Crystallogr. Sect. A
47,
110-119[CrossRef][Medline]
[Order article via Infotrieve]
|
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.