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INTRODUCTION |
The glycoprotein hormone receptors comprise two equal halves; an
extracellular N-terminal half (exodomain) and a membrane-associated C-terminal half (endodomain). The exodomain is ~350 amino acids long,
which alone is capable of high affinity hormone binding (1-3) without
hormone action (3, 4). The endodomain consists of seven transmembrane
domains, three exoloops, three cytoloops, a C-terminal tail, and
a short extracellular extension connected to transmembrane domain 1. Receptor activation occurs in the endodomain (3, 5, 6), which is
structurally equivalent to the entire molecule of many other G
protein-coupled receptors (7). Existing data suggest that these two
domains are associated together before and after hormone binding (8,
9). A hormone initially binds to the exodomain, and the resulting
hormone-exodomain complex adjusts its conformation (10) and then
interacts with the endodomain (7). This change in the interaction in
the ternary complex, hCG-exodomain-endodomain, is thought to generate a
signal (6, 7, 11).
There is ample evidence that the exodomain of the luteinizing
hormone/choriogonadotropin receptor
(LH/CG-R)1 alone is capable
of binding hCG with high affinity. The truncated exodomain lacking the
endodomain binds hCG with a high affinity similar to or slightly better
than the wild type affinity (1-3). Exodomain fusion proteins are
capable of binding hCG with a high affinity (12, 13). A number of sites
in the exodomain are important for hormone binding (14-16) and hormone
specificity (17-19). LHR peptides corresponding to three regions in
the exodomain are capable of inhibiting hCG binding to the receptor
(20).
A major feature in the structure of the LHR exodomain is the Leu-rich
repeat (LRR) motif (21, 22). LRRs are found in a large number of
proteins (23). The existing crystal structures of a few LRR proteins
show up to 15 LRRs, which form a rigid 2/3 donut structure (24, 25).
Based on these LRR crystal structures, the glycoprotein hormone
receptors have been modeled (26-28). They show a 1/3 donut structure
consisting of 7-9 LRRs and suggest that the inner lining interacts
(16, 29, 30) with the concave, front side of the hormones (31, 32).
Recently, it has been shown that the putative LRRs in the gonadotropin
receptors are indeed active and that part of them interact with hCG
(33-35).
However, it is unclear whether the rigid 1/3 donut structure of the
LRRs alone interacts with hCG, adjusts its conformation, contacts with
the endodomain, and activates the endodomain or if other regions of the
exodomain are involved in the process. This agility and versatility may
come from regions other than the LRRs. For example, we have shown that
the N-terminal flanking region of the LRRs makes strong contact with
both subunits of hCG (36, 37). However, it is unknown whether the
N-terminal flanking region of the LRRs is the only exodomain region
outside of the LRRs to interact with hCG. In addition to the
insufficient knowledge on the exodomain/hCG interaction, little is
known about the contact sites between the exodomain and endodomain.
There is evidence that exoloops 2 and 3 of the endodomain are involved in the interaction with unliganded exodomain and constrain the hormone
binding at the exodomain (8, 9). It has just been reported that LRR4
(34, 35) and the C-terminal flanking (hinge) region of the LRRs (38)
modulate signal generation in the endodomain. However, there is no clue
to where the endodomain contact point is in the exodomain. In this
article, the first evidence is presented that the hinge region
interacts with hCG and that the hCG-exodomain complex makes contacts
with exoloop 2 of the endodomain.
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EXPERIMENTAL PROCEDURES |
Materials--
The N-hydroxysuccinimide of
4-azidobenzoic acid (NHS-AB) was synthesized as described previously
(39). The N-hydroxysulfosuccinimide esters of ethylene
glycolbis(sulfosuccinimidylsuccinate) (SES) were purchased from Pierce.
The hCG CR 127 and hCG subunits were supplied by the National Hormone
and Pituitary Program. Denatured hCG was prepared by boiling hCG in 8 M urea for 30 min. Receptor peptides were synthesized and
N-acetylated and C-amidated by Biosynthesis (Lewisville, TX). They were purified on a Vydac C18 high
performance liquid chromatography column using a solvent
gradient from 100% of 0.1% trifluoroacetic acid in water to 20% of
0.1% trifluoroacetic acid in water and 80% 1-propanol.
Mutagenesis and Functional Expression of LHR--
Mutant LHR
cDNAs were prepared in a pSELECT vector using the Altered Sites
Mutagenesis System (Promega), sequenced, subcloned into
pcDNA3 (Invitrogen) as described previously (40), and
sequenced again to verify mutation sequences. This procedure does not
involve polymerase chain reaction, and therefore, there are no
polymerase chain reaction-associated unintended mutations. To produce
truncated receptors, a codon was substituted with a stop codon. Mutant
and truncated LHR constructs were transfected into human embryonic kidney 293 cells by the calcium phosphate method. Stable cell lines
were established in minimum essential medium containing 10% horse
serum and 500 µg/ml of G-418. They were used for hormone binding and
cAMP production. All assays were carried out in duplicate and repeated
four to six times. Means and standard variations were calculated.
125I-hCG Binding and Intracellular cAMP
Assay--
Stable cells were assayed for 125I-hCG binding
in the presence of a constant amount of 125I-hCG (39) and
increasing concentrations of unlabeled hCG. The Kd
values were determined by Scatchard plots. For intracellular cAMP
assay, cells were washed twice with Dulbecco's modified Eagle's medium and incubated in the medium containing
isobutylmethylxanthine (0.1 µg/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, which was provided by
Amersham Pharmacia Biotech. 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. All assays for cAMP and binding to intact cells and
solubilized receptors were repeated four to six times in duplicate to
calculate means ± S.D.
125I-hCG Binding to Solubilized LHR--
Transfected
cells were washed twice with ice-cold 150 mM NaCl, 20 mM HEPES, pH 7.4 (buffer A). Cells were scraped on ice and collected in buffer A containing protease inhibitors (1 mM
phenylmethylsulfonyl fluoride, 5 mM
N-ethylmaleimide, and 10 mM EDTA) and pelleted by centrifugation at 1,300 × g for 10 min. Cells from
a 10 cm plate were resuspended in 0.6 ml of buffer A containing 1%
Nonidet P-40, 20% glycerol, and the above protease inhibitors (buffer B), incubated on ice for 15 min, and diluted with 5.4 ml of buffer A
containing 20% glycerol plus the protease inhibitors (buffer C). The
mixture was centrifuged at 100,000 × g for 60 min. The supernatant (500 µl) was mixed with 150,000 cpm of
125I-hCG and 6.5 µl of 0.9% NaCl and 10 mM
Na2HPO4 at pH 7.4 containing increasing
concentrations of unlabeled hCG. after incubation at 4 °C for
12 h, the solution was thoroughly mixed with 250 µl of buffer A
containing bovine
-globulin (5 µg/ml) and 750 µl of buffer A
containing 20% polyethylene glycol 8000. After incubation at 4 °C
for 10 min, samples were pelleted at 1300 × g for 30 min and supernatants removed. Pellets were resuspended in 1.5 ml of buffer A containing 20% polyethylene glycol 8000, centrifuged, and
counted for radioactivity.
Derivatization and Radioiodination of Peptide--
NHS-AB was
freshly dissolved in dimethyl sulfoxide to a concentration of 50 mM in 0.1 M sodium phosphate, pH 7.5, to a
concentration of 20 mM. This reagent solution was
immediately used to derivatize receptor peptides. In the dark, 10 µl
of NHS-AB was added to 30 µg of LHR246-269 in 40 µl of
0.1 M sodium phosphate, pH 7.5. The mixture was incubated for 30 min for NHS-AB or 60 min for NHS-AB at 25 °C. The following were added to the derivatization mixture: 1 mCi of Na125I
in 10 µl of 0.1 M NaOH and 7 µl of chloramine T (1 mg/ml) in 10 mM Na2HPO4 and 0.9%
NaCl, pH 7.4 (PBS). After 20 s, 7 µl of sodium metabisulfite
(2.5 mg/ml) in PBS was introduced to terminate radioiodination.
Derivatized and radioiodinated
AB-125I-LHR246-269 solution was mixed with 60 µl of 16% sucrose solution in PBS and fractionated on Sephadex
Superfine G-10 column (0.6 × 15 cm) using PBS.
Affinity Cross-linking of
125I-LHR246-269 to hCG--
Disposable glass
tubes were siliconized under dimethyldichlorosilane vapor overnight and
autoclaved. In each siliconized tube, 20 µl of PBS, hCG (70 ng in 10 µl of PBS), and 125I-LHR246-269 (100 ng in
10 µl of PBS) were mixed and incubated in 37 °C for 90 min. After
incubation, 3 µl of 0.1 mM SES in dimethyl sulfoxide was
added to each tube and further incubated at 25 °C for 20 min. The
cross-linking reaction was terminated by adding 3 µl of 5 mM Gly in PBS. The samples were boiled for 2 min in 2%
sodium dodecyl sulfate, 100 mM dithiothreitol, and 8 M urea. The solubilized samples were electrophoresed on
8-12% polyacrylamide gradient gels. Gels were dried on filter paper,
then exposed to a molecular imaging screen (Bio-Rad) overnight. The
imaging screen was scanned on a model GS-525 Molecular Imager System
Scanner (Bio-Rad), and radioactive band intensity was analyzed using
Image Analysis Systems Version 2.1 (Bio-Rad). Gels were exposed to
X-Omat x-ray film at
75 °C for ~4 days
Photoaffinity Labeling of hCG--
The following solutions were
sequentially introduced to siliconized glass tubes: 20 µl of PBS, 10 µl of hCG (10 ng/µl) in PBS, and 10 µl of
AB-125I-LHR246-269 (10 ng/µl) in PBS. The
mixtures were incubated at 37 °C for 90 min in the dark, irradiated
with Mineralight R-52 UV lamp for 3 min as described previously (41),
and solubilized in 2% SDS, 100 mM dithiothreitol, and 8 M urea. The samples were electrophoresed on 8-12%
polyacrylamide gradient gels. Gels were dried on filter paper and
processed as described above.
Competitive Inhibition of Affinity Labeling of hCG and Inhibition
of 125I-hCG Binding to LHR--
Competitive inhibition
experiments were carried out as described for the affinity
cross-linking and photoaffinity labeling experiments, except that 10 µl instead of 20 µl of PBS was introduced to each tube, and the
mixture was incubated with 10 µl of increasing concentrations of
nonradioactive wild type or mutant LHR246-269.
A human embryonic kidney 293 cell line stably expressing human LHR was
incubated with 100,000 cpm of 125I-hCG in the presence of
increasing concentrations of nonradioactive wild type or mutant
LHR246-269 as described previously (42). After washing the
cells several times, the radioactivity associated with the cells was
counted to determine the Kd value.
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RESULTS |
Truncation of the C Terminus--
LHR is encoded by 11 exons (22,
43), and the exodomain is primarily comprised of exons 1-10, whereas
the endodomain is mainly encoded in exon 11. When exons 1-10 were
expressed in mammalian cells after truncating exon 11, the exodomain
fragment was trapped within the cells and could not be detected on the
intact cell surface or in the culture medium (2, 3). However, when the cells were solubilized in nonionic detergent, the exodomain fragment was capable of binding hCG with a high affinity similar to the hormone
binding affinity of the wild type receptor (Table
I) as reported previously (1-3). When
the terminal region was further truncated by deleting both exons 10 and
11, the resulting exodomain fragment consisting of exons 1-9 was also
trapped in cells and capable of hormone binding. The binding affinity
was, however, significantly less as the Kd value was
~6-fold greater than that of the wild type receptor in detergent
solution (Table I). This result suggests the presence of a potentially
important site around the exon 9-10 boundary (36).
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Table I
Hormone binding of LHR
The wild type LHR and exodomain fragment comprised of exons 1-10 or
exons 1-9 were stably expressed in 293 cells and assayed for hCG
binding to intact cells and receptors solubilized in Nonidet P-40. NS
stands for not significant.
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Sequence Comparison and Ala Scan of Conserved Residues--
To
screen important residues in the exon 9-10 junction of LHR, the
junction sequence was aligned with the corresponding sequence of the
FSH receptor and TSH receptor (Fig. 1). A
homology sequence was found from Tyr253 to
Phe260. In addition, the sequence corresponding to LHR
Arg261-Gln268 is highly conserved within each
receptor type among species. We decided to Ala scan from
Thr250 to Gln268 except Ala259 that
was substituted with Gly. The resulting mutant receptors were stably
expressed in cells and intact cells were assayed for 125I-hCG binding and cAMP induction (Fig.
2). In addition, cells that were unable
to bind hormone were solubilized in Nonidet P-40 and assayed for
125I-hCG binding in solution.

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Fig. 1.
Sequence alignment. The amino acid
sequence around the exon 9-10 junction is aligned for the LH, FSH, and
TSH receptors. "*" represents a conserved residue.
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Fig. 2.
Ala scan of
Thr250-Gln268. Residues in the
Thr250-Gln268 sequence were individually
substituted with Ala, except the Ala259 Gly
substitution. The resulting mutants were stably expressed in 293 cells
and assayed for hCG binding to intact cells and cAMP induction as
described under "Experimental Procedures." Nonbinding cells were
solubilized in Nonidet P-40 and assayed for hCG binding. Each
experiment was repeated four to six times in duplicate and means ± S.D. were calculated. NS stands for not
significant.
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The results reveal two mutant classes: one that was capable of binding
hCG on intact cells and the other incapable of hCG binding on intact
cells. All of the latter were capable of binding hCG after
solubilization in Nonidet P-40, indicating that Ala substitutions did
not abolish the hCG binding activity and that the mutants were trapped
in cells (36). The Kd values were generally higher
than the wild type value except Cys257
Ala and
Phe260
Ala, indicating lower affinities. The lower
affinities do not appear to be due to the variations in the receptor
concentration, because they are in the range of 22,000-57,000/cell,
except for Thr252
Ala and Phe260
Ala.
At these concentrations, the receptors show normal activities (9, 33,
34, 44). It is interesting to note that all mutants in the second half
of the sequence from Ala259
Gly to Gln268
Ala were expressed on the surface, whereas only four mutants from
Thr250 to Cys258 were expressed on the surface.
This result suggests that the first half, but not the second half, of
the sequence may be important for receptor targeting to the cell surface.
Substitutions for Tyr253 Impair Surface
Expression--
To further investigate the impact on surface
expression, Tyr253 was substituted with an array of amino
acids (Table II). The substitution of Phe
did not impact the surface expression or the surface receptor
concentration. In contrast, the His substitution significantly impaired
the surface expression, and other substitutions blocked it. These
results show that Tyr253
Phe and a phenyl group at the
Tyr253 position are permissible for receptor targeting and
hormone binding. Therefore, the hydroxyl moiety of the Tyr's phenolic
group does not appear to influence the targeting and hormone binding.
The partial blockage of surface expression by the substitution of His
suggests that an imidazole at the Tyr253 position is
tolerable but not desirable. Substitutions of nonaromatic hydrophobic,
hydrophilic, neutral, and acidic amino acids for Tyr253
prevented surface expression of the receptor, although the
concentration of the mutant receptors trapped in cells was significant
and sometimes considerably higher than the total concentration of the
wild type receptor. These results suggest that a phenolic or phenyl
group is necessary at the Tyr253 position for efficient
receptor transport to the surface membrane. To test this possibility
and the essential role of Tyr253, the residue was deleted.
The resulting Tyr253 deletion mutant was not expressed on
the surface, supporting the hypothesis.
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Table II
Multiple substitutions for Tyr253
Tyr253 was substituted with an array of amino acids. Del stands
for deletion. The mutant receptors were expressed and assayed for hCG
binding to intact cells and in solution as well as cAMP induction as
described under "Experimental Procedures." NS stands for not
significant.
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One may argue that cells expressing trapped receptors might have
developed a defective mechanism for receptor transport, perhaps when
stable cell lines were being established. To test this unlikely possibility, the cells stably expressing the Tyr253
Ala
mutant were transfected again with the wild type LH/CG-R or the human
FSH receptor plasmid (45). The transfected cells were capable of
binding hCG or FSH, respectively (Table
III), indicating that the receptor
transport mechanism was functional. To exclude the possibility that the
second transfection rescued the receptor transport mechanism,
additional experiments were performed. Cells stably transfected with
the FSH receptor or wild type LH receptor were transfected again with
the Tyr253
Ala mutant plasmid. These doubly transfected
intact cells were capable of binding FSH or hCG, respectively,
indicating that the receptor-targeting mechanism was not affected by
transfection of the Tyr253
Ala mutant plasmid and
expression of the Tyr253
Ala mutant.
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Table III
Activities of cotransfected receptors
Cells that were stably expressing the Tyr253 Ala mutant
receptor in cells were transiently transfected with the wild type LHR
or wild type FSH receptor plasmid. The cells were assayed for hormone
(hCG or FSH) binding as described under "Experimental Procedures."
Conversely, cells that were stably expressing the FSH receptor on the
cell surface were transiently transfected with the Tyr253 Ala mutant receptor plasmid and assayed for hormone binding. NS stands
for not significant; WT indicates wild type.
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Constitutive Activation of cAMP Induction by Substitution for
Ser255--
The examination of cAMP induction discloses
that the mutations impaired the EC50 value, maximum
level, or both for cAMP induction (Fig. 2, A and
B). The only exception is Ser255
Ala. It
showed a similar EC50 and higher maximum cAMP as
compared with the wild type values, suggesting an improved efficacy of cAMP induction. To follow up this interesting observation,
Ser255 was replaced with several amino acids (Table
IV). The resulting mutants were
transiently surface-expressed in the ~20,000-40,000/cell range. Some
of mutations, Val and Ile, resulted in strikingly high basal cAMP
levels, despite lower receptor concentrations on the surface. This is
unusual because all activating mutations uncovered to date are found in
the endodomain (11, 46). A simple explanation is that the region may be
involved in signal generation in the endodomain. If so, it may interact
with hCG, the endodomain, or both. However, it is also possible that
the mutations might have impacted protein processing including folding and thus, the global structure, which in turn resulted in the observed
high basal cAMP level. One way to resolve this difficult issue is to
determine its interaction, which can be done by affinity labeling.
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Table IV
Multiple substitutions for Ser255
Ser255 was substituted with several amino acids. The mutant
receptors were expressed and assayed for hCG binding to intact cells
and in solution as well as cAMP induction as described under
"Experimental Procedures."
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Affinity Labeling of hCG with Receptor Peptide LHR--
We
synthesized a 24-mer peptide,
Ac-Leu246-Asn269-NH2
(LHR246-269), derivatized it with NHS-AB, and
radioiodinated it to produce
AB-125I-LHR246-269. NHS-AB couples to the Lys
residues, and Lys265 and Lys266 are present in
the peptide. To determine whether
AB-125I-LHR246-269 would bind and label hCG,
and if so, which subunit is labeled, the peptide derivative was
incubated with hCG and irradiated with UV for increasing time periods
(Fig. 3A). The samples were
solubilized in SDS under reducing conditions and electrophoresed as
described under "Experimental Procedures." The autoradiographic
phosphoimage of the gel shows that
AB-125I-LHR246-269 labeled conspicuously both
the
and
subunits in hCG as well as faintly the 
dimer.
The positions of hCG
, hCG
, and the hCG
dimer were
determined by comparing the respective positions of
125I-hCG
, 125I-hCG
, and the cross-linked
125I-hCG 
dimer on the autoradiograph (35, 37). The
labeling was dependent on UV irradiation, reaching the maximum level at 60-s exposure. The labeling of the 
dimer implies that there were
two AB groups attached to the peptide and that one was apparently closer to hCG
and labeled it, whereas the other was closer to and
labeled hCG
. This is in contrast to an LHR peptide derivatized with
an AB that is capable of labeling only one of the subunits but not both
(35, 37). It suggests the specificity of photoaffinity labeling.

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Fig. 3.
UV- and hCG-dependent
photoaffinity labeling.
AB-125I-LHR246-269 (200 ng) was incubated with
200 ng of hCG and irradiated with UV for varying time periods
(A) or incubated with increasing concentrations of hCG and
exposed to UV for 60 s (B). After electrophoresis of
the samples, gels were dried on filter paper and exposed to a molecular
imaging screen (Bio-Rad) overnight. The imaging screen was scanned on a
model GS-525 Molecular Imager System Scanner (Bio-Rad), and the
radioactive band intensity was analyzed using Image Analysis Systems
Version 2.1 (Bio-Rad). Gels were also exposed to X-Omat x-ray film at
75 °C for ~4 days. The bar graphs show the percent
radioactivity of the band and the band in a gel lane.
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Next, increasing concentrations of hCG were incubated with a constant
amount of AB-125I-LHR246-269 and irradiated
with UV for 60 s (Fig. 3B). The results show that the
hCG labeling was also dependent on the hCG concentration, reaching a
maximum labeling at and above 200 ng. In the following experiment, 200 ng of hCG was incubated with increasing concentrations of
AB-125I-LHR246-269 and UV-photolyzed for
60 s (Fig. 4A). The
result shows the dependence of the labeling on the
AB-125I-LHR246-269 concentration with the
maximum labeling at and above 200 ng of AB-125I-LHR246-269. This experiment, taken
together with the UV- and hCG-dependent experiments,
indicate that the labeling of hCG
, hCG
, and hCG
is
saturable and requires the peptide derivative, hCG, and UV photolysis.
In addition, the results indicate that hCG was covalently labeled by
AB-125I-LHR246-269 and that this labeling was
specific. To test whether the derivatization of AB impacted the
activity of the peptide, AB-125I-LHR246-269
was incubated with hCG in the presence of increasing concentrations of
nonlabeled peptide and photolyzed (Fig. 4B). Nonlabeled
peptide competitively inhibited the labeling. To further test the
labeling specificity, AB-125I-LHR246-269 was
incubated with increasing concentrations of denatured hCG and
photolyzed. No labeled bands appeared (data not included), indicating
that AB-125I-LHR246-269 labeled active, but
not denatured, hCG.

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Fig. 4.
LHR peptide-dependent
photoaffinity labeling. hCG (200 ng) was incubated with increasing
concentrations of AB-125I-LHR246-269 and
exposed to UV for 60 s (A). hCG (200 ng) was incubated
with 200 ng of AB-125I-LHR246-269 in the
presence of increasing concentrations of nonlabeled
LHR246-269 and exposed to UV for 60 s (B).
The bar graphs show the percent radioactivity of the band and the band in a gel lane.
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Affinity Cross-linking of 125I-LHR246-269
to hCG--
Despite the specificity of photoaffinity labeling, one may
raise an issue whether the AB attached to the peptide, particularly those at the contact point, might have interfered with the binding and
labeling and distorted the result. To resolve this issue, the peptide
without attached AB, 125I-LHR246-269, was
cross-linked to hCG using an amino-specific homobifunctional reagent,
SES. The results (Figs. 5 and
6) show that the peptide was cross-linked
to both hCG subunits and the hCG
dimer. The affinity
cross-linking requires SES, 125I-LHR246-269,
and hCG and is dependent on their concentrations. The maximum levels of
cross-linking reached around 15% of total peptide for hCG
and 10%
for hCG
, indicating that the labeling is saturable and specific. In
addition, hCG
was preferentially labeled compared with hCG, which is
consistent with the preferential photoaffinity labeling of the hCG
subunit. This affinity cross-linking is more effective than
photoaffinity labeling. A possible explanation is that SES can reach
further than AB can, because of the maximum cross-linkable distance,
~13 Å for SES and ~7 Å for AB. The SES cross-linking results also
show that either or both amino groups of Lys265 and
Lys266 of 125I-LHR246-269, the
only amino groups in the peptide, were cross-linked to one of seven
hCG
amines, five hCG
amines, or those in both subunits. The
SES-dependent cross-linking of
125I-LHR246-269 to the hCG subunits was
competitively inhibited by nonlabeled peptide, suggesting a
specificity. Furthermore, SES failed to cross-link
125I-LHR246-269 to denatured hCG (data not
included).

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Fig. 5.
SES- and hCG-dependent
cross-linking of 125I-LHR246-269 to hCG.
hCG was incubated with 125I-LHR246-269 and
treated with SES for 15 min. Here, the concentration of SES
(A) or hCG (B) was varied. The samples were
electrophoresed, and gels were processed as described in the legend to
Fig. 3. The bar graphs show the percent radioactivity of the
band and the band in a gel lane.
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Fig. 6.
LHR peptide-dependent
cross-linking of 125I-LHR246-269 to hCG.
hCG (200 ng) was incubated with increasing concentrations of
125I-LHR246-269 and treated with 1 mM SES (A). hCG (200 ng) was incubated with 200 ng of 125I-LHR246-269 in the presence of
increasing concentrations of nonlabeled LHR246-269 and
treated with 1 mM SES (B). The samples were
electrophoresed, and gels were processed as described in the legend to
Fig. 3. The bar graphs show the percent radioactivity of the
band and the band in a gel lane.
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Differential Effects of Exoloop Peptides on Photoaffinity Labeling
of hCG--
Our mutational analysis suggests that the
Thr250-Gln268 region may be involved in signal
generation in the endodomain, perhaps interacting with hCG, the
endodomain, or both. Such interactions, if true, might impact the
specific affinity labeling of hCG with LHR246-269. To this
end, hCG was incubated with AB-125I-LHR246-269
in the presence of an excess amount of exoloop peptides and irradiated with UV (Fig. 7A). The
labeling was inhibited most conspicuously by the exoloop 2 peptide,
whereas the inhibition by the exoloop 3 peptide or exoloop 1 peptide
was significantly less or barely noticeable, respectively (Fig. 7,
B and C).

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Fig. 7.
Effects of exoloop 1, 2, and 3 peptides on
photoaffinity labeling of hCG by
AB-125I-LHR246-269. hCG and
AB-125I-LHR246-269 were incubated with 7,000 ng of nonlabeled LHR exoloop 1, 2, or 3 peptides and exposed to UV
(A). hCG and AB-125I-LHR246-269
were incubated with increasing concentrations of nonlabeled LHR exoloop
1, 2, or 3 peptides and exposed to UV (B). The samples were
electrophoresed, and gels were processed as described in the legend to
Fig. 3. The graphs show the percent radioactivity of the band and the band in a gel lane (C).
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DISCUSSION |
The results of our mutagenesis and affinity labeling studies
indicate that the Thr250-Gln268 sequence of
the LHR exodomain interacts with both subunits of hCG, particularly
with hCG
. In previous reports, we have shown that the LRRs function
in the LHR exodomain and interact with hCG (33-35) and that the
N-flanking region of the LRRs also interacts with hCG (36, 37).
Receptor peptide mimics were useful in these studies, although
synthetic peptide mimics have limitations. They do not necessarily
mimic the function of the corresponding sequences in proteins, probably
due to structural differences. This is underscored by the fact that an
extension of either terminus or substitution of a single amino acid in
peptides mimics deprives their activity, specificity, or both (20, 35,
37). Therefore, we have carefully chosen the sequence and size of the
hinge peptide based on our mutational analysis and others' studies
(20, 36). Furthermore, short synthetic peptides can be structured for a stable tertiary structure (47) and assume a potent biological activity
(48-50).
Taken together, our observations show that the LRRs and their flanking
regions interact with hCG. This suggests an intriguing possibility that
the LRRs face hCG, probably at the front of hCG with the
seat belt
and
C terminus (31, 32), whereas the flanking regions of the LRRs
face the sides of hCG and may reach the back of hCG. This would trap
hCG in the 1/3 donut structure of the LRRs, slowing the dissociation
rate of hCG and LHR (51) and enhancing the affinity. This is consistent
with the fact that high affinities are generally ascribed to slow
dissociation rates rather than fast association rates (52). For
example, kinetic studies on interactions between hormones and
receptors, antibodies and antigens, and enzymes and substrates show
that association rate constants, kon, are in the
narrow range of 105-107
M
1 s
1,
whereas dissociation rate constants, koff, vary
widely from 1 s-1 for low affinity complexes to a half-life
of more than 3 months (10
7
s
1) (52).
An hCG contact point in the Thr250-Gln268
sequence is near or not too far from the two tandem Lys residues at 265 and 266 in the second half of the sequence. This is consistent with the
fact that most Ala substitutions for the second half residues reduced the binding affinity by up to 2.3-fold but did not noticeably impact
the surface expression and maximum cAMP induction level (Fig. 3,
A and B). In contrast to this primary role of the
second half in hCG binding, the first half of the sequence seems to be involved in the signal generation. For example, some substitutions for
Ser255 resulted in substantially high basal cAMP levels. In
particular, the cAMP level was dramatically high for Val and Ile
substitutions, indicating constitutive activation of adenylyl cyclase
(38). Taken together with the results that the interaction of the
Thr250-Gln268 sequence with hCG is inhibited
by the exoloop 2 peptide, our observations implicate the involvement of
the Thr250-Gln268 sequence in the interaction
of hCG-exodomain complex with the endodomain and the subsequent signal
generation. This is the first clue to the identification of the
endodomain contact points in the exodomain. The exoloop 2 peptide may
directly compete with LHR246-269 for the binding site or
allosterically interfere with the interaction between
LHR246-269 and hCG. On the other hand, the exoloop 3 peptide marginally inhibited the labeling of hCG by
AB-125I-LHR246-269, and the inhibition by the
exoloop 1 peptide was barely detectable. This suggests several
possibilities that these peptides bind hCG, but the affinities are
relatively low, do not share the binding site of
LHR246-269, or do not bind hCG at all. Since exoloop 3 impacts hCG binding to the exodomain, it is possible that exoloop 3 is
capable of binding the exodomain or hCG-exodomain complex, but the
affinity is low. In addition, exoloop 3 and the LHR246-269
sequence may not share a significant portion of their binding sites in
hCG and the hCG-exodomain complex.
A phenyl or phenolic group at Tyr253 is crucial for the
recognition by the surface-targeting machinery, since both Tyr and Phe are permissible. It is striking to see that substitutions of other amino acids, including other hydrophobic amino acids, completely blocked the surface expression, suggesting a high specificity. The
attenuation of surface expression by Ala substitution for Leu251-Thr252-Tyr253 and
His256-Cys257-Cys258 is specific,
and therefore, these residues may assume a particular structure. The
two groups consist of 3 contiguous amino acids, and a secondary
structure analysis suggests interesting possibilities. The
Leu251-Thr252-Tyr253 sequence is
predicted to be part of a
sheet (53), thus orienting Leu251 and Tyr253 on one side of the
sheet
and Thr252 on the other. In such a structure,
Leu251 and Tyr253 face one side, whereas
Thr252 would face the opposite side. This may explain why
Leu251 and Tyr253 were more important for
receptor transport than Thr252. Furthermore, it has been
suggested that Leu and Tyr are part of a transport determinant of
membrane proteins (54). Interestingly, the
sheet is followed by a
turn at Pro254 and Ser255 (53, 55). Therefore,
the two groups,
Leu251-Thr252-Tyr253 and
His256-Cys257-Cys258, are likely to
be in close proximity, which could allow them to form a contiguous
determinant. This is also consistent with the observation that
Pro254 and Ser255 play a role distinct from the
flanking residues, in particular the unique role in modulating signal
generation (38).
In conclusion, the results presented in this study show that the
C-terminal flanking (hinge) region (38),
Thr250-Gln268, of the LRRs in the LHR
exodomain interacts with hCG, contacting primarily hCG
. This
interaction is specifically inhibited by exoloop 2 but not by exoloops
1 and 3 of the endodomain, suggesting an intimate relationship in the
ternary complex of Thr250-Gln268, exoloop 2 and hCG. Taken together, our observations in this and previous articles
(33-35) suggest a new paradigm that the LRRs face the front of
hCG, while both of the flanking regions of the LRRs interact with the
sides of hCG and possibly reach the back of hCG as well. This would
trap hCG in the 1/3 donut structure of the LRRs, increasing the binding
affinity. In addition, mutations of Ser255 in the exodomain
sequence can constitutively activate the receptor. This result, along
with the ternary complex, provides a new clue for the identity of the
enigmatic signal modulator in the exodomain.