From the Department of Molecular Biology, University of Wyoming,
Laramie, Wyoming 82071-3944
The luteinizing hormone/choriogonadotropin
receptor, a seven-transmembrane receptor, is composed of two equal
halves, the N-terminal extracellular exodomain and the C-terminal
membrane-associated endodomain. Unlike most seven-transmembrane
receptors, the exodomain alone is responsible for high affinity hormone
binding, whereas signal is generated in the endodomain. These physical
separations of hormone-binding and receptor activation sites are
attributed to unique mechanisms for hormone binding and receptor
activation of this receptor and its subfamily members. However, the
precise hormone contact sites in the exodomain are unclear. In the
preceding article (Hong, S., Phang, T., Ji, I., and Ji, T. H. (1998) J. Biol. Chem. 273, 13835-13840), a region
immediately downstream of the N terminus of the exodomain was shown to
be crucial for hormone binding. To test if the region interacts with
the hormone, human choriogonadotropin (hCG) was photoaffinity-labeled
with a peptide mimic corresponding to
Gly18-Tyr36 of the receptor. This peptide
mimic specifically photoaffinity-labeled both the
- and
-subunits
of hCG. Interestingly, hCG
was preferentially labeled. On the other
hand, denatured hCG was not labeled, and a mutant analog of the peptide
failed to label hCG. Furthermore, the affinity labeling was
UV-dependent and saturable, indicating the specificity of
the photoaffinity labeling. Our results indicate that the region of the
exodomain interacts with hCG and that the contact points are near both
subunits of hCG. Particularly, the alternate residues
(Leu20, Cys22, and Gly24) are
crucial for hCG binding. In addition, the results underscore the fact
that there is a crucial hormone contact site outside of the popularly
believed primary hormone-binding site that is composed of Leu-rich
repeats and is located in the middle of the exodomain. Our observations
are crucial for understanding the molecular mechanism through which the
initial high affinity hormone binding leads to receptor activation in
the endodomain.
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INTRODUCTION |
The LH1/CG receptor
belongs to a subfamily of glycoprotein hormone receptors within the
seven-transmembrane receptor family. Unlike most seven-transmembrane
receptors, it is composed of two equal halves, the 341-amino acid-long
extracellular N-terminal exodomain and the 334-amino acid-long
membrane-associated C-terminal endodomain, which includes seven
transmembrane helices (1, 2). The exodomain binds the hormones with
high affinity (3-7) without hormone action (5, 8). The exodomain-hCG
complex is thought to make a secondary contact with the endodomain,
thus generating a signal (9). Therefore, the high affinity interaction of the exodomain and hCG is the crucial first step leading to signal
generation and hormone action. However, only limited information is
available regarding the precise hormone contact residues and sites in
the exodomain. Three peptide mimics of the exodomain, peptide-(21-38),
peptide-(102-115), and peptide-(253-266), attenuated 125I-hCG binding to membranes expressing the LH/CG receptor
(10). Receptors lacking the 11 amino-terminal residues or one of the leucine-rich motifs 1-6 were trapped in cells and failed to bind hCG
(11).
In this work and the preceding article (12), the exodomain was examined
using several independent methods, including serial truncation from the
C terminus, Ala scanning, peptide mimics of the receptor, photoaffinity
labeling, affinity cross-linking, and immunofluorescence. Our results
show that the Leu20-Pro38 sequence contacts
both the
- and
-subunits of hCG. In addition, three other
sequences near the junctions of exons 3-4, 6-7, and 9-10 are
important for hormone binding.
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EXPERIMENTAL PROCEDURES |
Materials--
The N-hydroxysuccinimide (NHS) esters
of 4-azidobenzoylglycine (ABG) was synthesized as described (13). The
N-hydroxysulfosuccinimide (sulfo-NHS) esters of
4-azidobenzoic acid (AB) and ethylene
glycolbis(sulfosuccinimidylsuccinate) (SES) were purchased from Pierce.
The hCG CR 127 and hCG
- and
-subunits were supplied by the
National Hormone and Pituitary Program (NIDDK, National Institutes of
Health). Denatured hCG was prepared by boiling hCG in 8 M
urea for 30 min. Peptide mimics including wild-type and mutant LH/CG
receptor peptides, LHR18-36 (see Fig. 1), were synthesized
by Biosynthesis (Lewisville, TX) and purified on a Vydac
C18 HPLC 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. The sequence of
LHR18-36 corresponds to
Gly18-Tyr36 of the LH/CG receptor. In
addition, a mutant LHR18-36 was synthesized in which
Leu20, Cys22, and Gly24 were
substituted with Ala.
Derivatization and Radioiodination of Peptides--
NHS-ABG was
freshly dissolved in dimethyl sulfoxide to a concentration of 50 mM, and sulfo-NHS-AB in 0.1 M sodium phosphate (pH 7.5) to a concentration of 20 mM. These reagent
solutions were immediately used to derivatize receptor peptides. In the dark, 10 µl of NHS-ABG or sulfo-NHS-AB was added to 30 µg of
LHR18-36 in 40 µl of 0.1 M sodium phosphate
(pH 7.5). The mixture was incubated for 30 min for NHS-ABG or 60 min
for sulfo-NHS-AB at 25 °C. The following were added to the
derivatization mixture: 1 mCi of Na125I-iodine in 10 µl
of 0.1 M NaOH and 7 µl of chloramine T (1 mg/ml) in 10 mM Na2HPO4 (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
ABG-125I-LHR18-36 or
AB-125I-LHR18-36 solution was mixed with 60 µl of 16% sucrose solution in PBS and fractionated on a Sephadex
Superfine G-10 column (0.6 × 15 cm) using PBS.
Cross-linking of 125I-LHR18-36 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-LHR18-36 (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 Me2SO 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% SDS, 100 mM
dithiothreitol, and 8 M urea. The solubilized samples were
electrophoresed on 8-12% polyacrylamide gradient gels. Gels were
dried and exposed to Eastman Kodak 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
ABG-125I-LHR18-36 (10 ng/µl) in PBS. For
labeling with AB-125I-LHR18-36, 20 µl of
PBS, 10 µl of hCG (20 ng/µl) in PBS, and 10 µl of
AB-125I-LHR18-36 (15 ng/µl) in PBS were
mixed. The mixtures were incubated at 37 °C for 90 min in the dark;
irradiated with a Mineralight R-52 UV lamp for 3 min as described
previously (13); 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, which was 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
profile 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 .
Competitive Inhibition of Photoaffinity Labeling of
hCG--
Competitive inhibition experiments were carried out as
described for the 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 LHR18-36.
Inhibition of 125I-hCG Binding to the LH/CG
Receptor--
A human embryonic kidney 293 cell line stably expressing
the rat LH/CG receptor was incubated with 100,000 cpm of
125I-hCG in the presence of increasing concentrations of
nonradioactive wild-type or mutant LHR18-36 as described
previously (14). After washing the cells several times, the
radioactivity associated with the cells was counted to determine the
Kd value.
Trichloroacetic Acid Precipitation of
125I-LHR18-36 Complexed with hCG--
20 µl
of PBS, 10 µl of 150,000 cpm of
125I-LHR18-36 (10 ng/µl) in PBS, 10 µl of
hCG (10 ng/µl) in PBS, and 10 µl of increasing concentrations of
unlabeled LHR18-36 in PBS were sequentially introduced to
siliconized glass tubes. After incubation at 37 °C for 90 min, 5 µl of 50% trichloroacetic acid was introduced to the tubes, and the
mixture was incubated at 4 °C for 10 min. The mixture was
centrifuged at 2500 × g for 20 min at 4 °C, and the
radioactivity of the pellet was counted.
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RESULTS |
In the preceding article (12), we showed that the amino acid
sequence near the N terminus of the LH/CG receptor, particularly Leu20-Arg21-Cys22-Pro23-Gly24,
is crucial for hCG binding. This raises the question as to whether the
peptide sequence directly interacts with the hormone or indirectly influences the hormone/receptor interaction by impacting on the global
structure of the receptor. To examine these possibilities, a peptide
mimic corresponding to the receptor sequence from Gly18 to
Tyr36, LHR18-36 (Fig.
1), was synthesized and tested for its
ability to bind and to affinity label hCG. The sequence of
LHR18-36 corresponds to
Gly18-Tyr36 of the LH/CG receptor. In
addition, a mutant LHR18-36 was synthesized in which
Leu20, Cys22, and Gly24 were
substituted with Ala.

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Fig. 1.
Sequences of LHR peptide mimics.
LHR18-36 was synthesized, with its sequence corresponding
to Gly18-Tyr36 of the LH/CG receptor. In
addition, a mutant LHR18-36 was synthesized in which
Leu20, Cys22, and Gly24 were
substituted with Ala.
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Cross-linking of 125I-LHR18-36 to
hCG--
125I-LHR18-36 was incubated with hCG
and treated with a homobifunctional reagent, SES, which specifically
reacts with amino groups to covalently cross-link two amino groups
(15). Electrophoresis of the treated
125I-LHR18-36/hCG mixture showed that
125I-LHR18-36 was primarily cross-linked to
hCG
, hCG
, and the hCG
dimer (Fig.
2A, lane 6). The
positions of hCG
, hCG
, and the hCG
dimer were determined by
comparing with the respective positions of 125I-hCG
,
125I-hCG
, and the cross-linked
125I-hCG
dimer on the autoradiograph (Fig.
2A, lanes 1-3). Without SES, ~2% of
125I-LHR18-36 remained associated with hCG
under reducing electrophoretic conditions. The band became conspicuous
on the autoradiographs when large amounts of radioactive samples were
applied and x-ray film was overexposed. This result suggests the
inherent affinity of the peptide for hCG. However, cross-linking of
125I-LHR18-36 to hCG required SES, hCG, and
125I-LHR18-36 since it did not occur in the
absence of any of the three. The extent of cross-linking was dependent
on the SES concentration (Fig. 2, B and C),
reaching the maximum level at 0.3-1 mM SES. Under this
condition, ~20% of 125I-LHR18-36 was
cross-linked to each hCG
and hCG
. At a higher SES concentration, e.g. 3 mM, the extent of cross-linking
decreased. This decrease was due to a non-cross-linking, monofunctional
reaction (only one of the two NHS groups reacting with a target amino
group while leaving the other NHS group unused) of excess SES with
125I-LHR18-36, hCG, and its subunits (16). In
conclusion, our results indicate that
125I-LHR18-36 was covalently cross-linked to
hCG
and hCG
. Furthermore, the N-terminal amino group of
125I-LHR18-36, the only amino group of the
peptide, was cross-linked to an amino group of either hCG
or hCG
.
The distance between the pair of two cross-linked amino groups is
expected to be <13 Å.

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Fig. 2.
Autoradiography of cross-linking of
125I-LHR18-36 to hCG. After incubating
125I-LHR18-36 with hCG, the mixture was
treated with increasing concentrations of SES, solubilized under
reducing conditions, and electrophoresed (C). Gels were
dried and autoradiographed (B) as described under
"Experimental Procedures." In addition, 125I-hCG ,
125I-hCG , and cross-linked 125I-hCG
were electrophoresed as standards (A).
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Specificity of Cross-linking of LHR18-36 to
hCG--
To determine whether the cross-links are specific between the
receptor peptide and hCG, cross-linking was performed under increasing
concentrations of 125I-LHR18-36 while
maintaining hCG at a constant concentration (Fig.
3A). Conversely,
125I-LHR18-36 and hCG were cross-linked at
increasing concentrations of hCG and a constant concentration of
125I-LHR18-36 (Fig. 3B). If
cross-links are specific, they should reach saturation under both
conditions. The results (Fig. 3, D and E) indeed
show plateaus under both conditions, an indication of saturable and specific cross-linking. This specific cross-linking is not expected to
occur with peptides that do not recognize hCG. In the preceding article
(12), Ala substitution for Leu20, Cys22, or
Gly24 of the receptor resulted in the loss of hCG binding
by the receptor. Therefore, a mutant LHR18-36 was
synthesized in which Leu20, Cys22, and
Gly24 were substituted with Ala (Fig. 1). As expected,
mutant LHR18-36 was not cross-linked to either subunit of
hCG (Fig. 3, C and F). It is not clear whether
the lack of observed cross-linking of mutant LHR18-36 was
caused by a lack of mutant LHR18-36 binding to hCG or by a
lack of a cross-linking reaction by SES due to the putative steric
hindrance even though mutant LHR18-36 successfully bound
to hCG. To distinguish these possibilities, 125I-LHR18-36 was cross-linked to hCG in the
presence of increasing concentrations of unlabeled wild-type
LHR18-36 (Fig.
4A) and mutant
LHR18-36 (Fig. 4B). Wild-type
LHR18-36 attenuated the cross-linking, but mutant
LHR18-36 was significantly less effective (Fig.
4C), an indication of the less efficient binding of mutant
LHR18-36 to hCG.

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Fig. 3.
125I-LHR18-36 and
hCG concentration-dependent cross-linking. A constant
concentration of hCG was incubated with increasing concentrations of
125I-LHR18-36 and treated with SES
(A and D). Conversely, a constant concentration
of 125I-LHR18-36 was incubated with increasing
concentrations of hCG and treated with SES (B and
E). Wild-type (WT) and mutant (Mut)
LHR18-36 were incubated with hCG and treated with SES
(C and F).
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Fig. 4.
Inhibition of cross-linking of
125I-LHR18-36 to hCG by unlabeled wild-type
and mutant LHR18-36.
125I-LHR18-36 was incubated with hCG in
the presence of increasing concentrations of wild-type (WT)
LHR18-36 (A) or mutant LHR18-36
(B) and treated with SES. Samples were processed as
described in the legend to Fig. 2. The intensities of the and bands were plotted against increasing concentrations of unlabeled wild
type (WT) and mutant peptides (C).
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Affinity of LHR18-36 Binding to hCG--
To determine
the binding affinity, 125I-LHR18-36 was
incubated with hCG in the presence of increasing concentrations of
unlabeled LHR18-36 (Fig.
5A). Unlabeled
LHR18-36 inhibited 125I-LHR18-36
binding to hCG, with a Kd value of 29 µM. It is not clear whether this inhibition was caused by
competitive binding of LHR18-36 and the receptor to hCG or
by a putative allosteric effect of LHR18-36 binding to
hCG. To examine these possibilities and the relevance of the inhibition
of hCG binding to the LH/CG receptor, 125I-hCG was
incubated with a 293 cell line stably expressing the receptor (14) in
the presence of increasing concentrations of unlabeled
LHR18-36 (Fig. 5B). Unlabeled wild-type
LHR18-36, but not mutant LHR18-36,
competitively attenuated binding of 125I-hCG to the
receptor, with a Kd value of 25 µM.
This result indicates that the interaction of LHR18-36 and
hCG is specific and simulates the interaction between hCG and the
receptor. Furthermore, the two independent Kd values
(29 and 25 µM) are not only close, but are also similar to the value of 15 µM for the inhibition of
125I-hCG binding to the receptor by LHR21-41
(10).

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Fig. 5.
Inhibition of
125I-LHR18-36 binding to hCG and of
125I-hCG binding to the LH/CG receptor. The affinity
of LHR18-36 binding to hCG was determined using two
independent methods. 125I-LHR18-36 was
incubated with hCG in the presence of increasing concentrations of
unlabeled LHR18-36 (A) as described under
"Experimental Procedures." Alternatively, 125I-hCG was
incubated with 293 cells stably expressing the LH/CG receptor in the
presence of increasing concentrations of unlabeled
LHR18-36 (B). WT, wild-type;
NS, not significant.
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Photoaffinity Labeling of hCG--
Despite the indication for
specific cross-links between the receptor peptide and hCG, there were a
series of minor cross-linked complexes larger than the complex of
125I-LHR18-36 and the hCG dimer. This suggests
that a minor population of the 125I-LHR18-36·hCG dimer complex was further
cross-linked to other hCG subunits or the hCG dimer. Although this is
not entirely unexpected, as random collisional cross-links are possible
(16), it raises concern regarding the specificity of homobifunctional
cross-links between 125I-LHR18-36 and hCG. A
simple way to reduce or eliminate such random collisional cross-links
is photoaffinity labeling (16).
To photoaffinity label hCG with 125I-LHR18-36,
the receptor peptide was derivatized with either AB or ABG (13) to
produce AB-125I-LHR18-36 or
ABG-125I-LHR18-36, respectively. When these
peptides bind to hCG and are irradiated with UV, cross-linking will be
restricted between 125I-LHR18-36 and hCG
or
between 125I-LHR18-36 and hCG
. The reagent,
however, will not be able to cross-link one hCG subunit to another. AB
and ABG can reach and label target molecules up to 7 and 10 Å,
respectively (17). These distances are considerably shorter than the
maximum cross-linkable 13 Å of SES, and therefore, the labeling
reaction by AB and ABG is more restricted than the cross-linking
reaction by SES. As shown in Fig. 6,
AB-125I-LHR18-36 and
ABG-125I-LHR18-36 were capable of
photoaffinity labeling either hCG
or hCG
, but not both subunits
at the same time to produce the labeled hCG
complex.
Interestingly, AB-125I-LHR18-36 labeled them
more efficiently than ABG-125I-LHR18-36 (Fig.
6, C and D). In addition, hCG
was more
preferentially labeled than hCG
. This result is consistent with the
SES cross-linking results. One possible explanation is that the N
terminus of the LHR18-36 derivatives is <7 Å from the
hCG
- and
-subunits and that the peptide derivatives are bound
closer to
than
. The labeling required UV irradiation and was
dependent on the irradiation time, reaching the maximum labeling after
~1 and 0.5 min of irradiation of the
ABG-125I-LHR18-36·hCG and
AB-125I-LHR18-36·hCG complexes,
respectively. Unlike SES cross-links, the maximum levels were sustained
after longer UV exposure. This UV dependence clearly indicates
photoaffinity labeling. In addition, the sustained maximum levels and
the preferential labeling of hCG
without simultaneous labeling of
both subunits suggest a labeling specificity. To further examine the
specificity of photoaffinity labeling, the concentration of either hCG
or the peptide derivatives was changed.

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Fig. 6.
UV-dependent photoaffinity
labeling of hCG by AB-125I-LHR18-36 or
ABG-125I-LHR18-36. A constant
concentration of hCG was incubated with a constant concentration of
AB-125I-LHR18-36 (B and
D) or ABG-125I-LHR18-36
(A and C). The mixture was irradiated with UV for
increasing time periods, solubilized, and electrophoresed as described
in the legend to Fig. 2. Dried gels were analyzed on a phosphoimager,
and radioactive band intensities were determined as described under
"Experimental Procedures." In addition, gels were
autoradiographed.
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Concentration Effect of hCG and Peptide Derivatives--
When a
constant amount of hCG was incubated with increasing concentrations of
AB-125I-LHR18-36 or
ABG-125I-LHR18-36, the intensity of labeled
hCG
- and
-bands gradually increased and plateaued (Fig.
7). A similar result was obtained in a
converse experiment when a constant concentration of
AB-125I-LHR18-36 or
ABG-125I-LHR18-36 was incubated with
increasing concentrations of hCG (Fig.
8). These results indicate that the
photoaffinity labeling is dependent on both of the derivatized peptides
and hCG as they are limiting factors. In both cases, the derivatized
peptides labeled hCG
more than hCG
, an indication of a labeling
specificity.

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Fig. 7.
Photoaffinity labeling of hCG with increasing
concentrations of AB-125I-LHR18-36 or
ABG-125I-LHR18-36. A constant
concentration of hCG was incubated with increasing concentrations of
AB-125I-LHR18-36 (B and
D) or ABG-125I-LHR18-36
(A and C). The mixture was irradiated with UV,
solubilized, electrophoresed, and analyzed as described in the legend
to Fig. 6.
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Fig. 8.
hCG concentration-dependent
photoaffinity labeling. Increasing concentrations of hCG were
incubated with a constant concentration of
AB-125I-LHR18-36 (B and
D)or ABG-125I-LHR18-36
(A and C). The mixture was irradiated with UV,
solubilized, and electrophoresed as described in the legend to Fig.
6.
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Competitive Inhibition of Photoaffinity Labeling by Nonderivatized
Peptides--
A nonderivatized peptide is expected to displace
specific labeling. Therefore, hCG was incubated with
AB-125I-LHR18-36 or
ABG-125I-LHR18-36 in the presence of
increasing concentrations of nonderivatized peptide (Fig.
9, A and D).
Increasing concentrations of LHR18-36 inhibited
photoaffinity labeling in a dose-dependent manner and eventually completely blocked it. These results indicate the
specificity of LHR18-36 for the photoaffinity labeling.
However, this specific labeling should not be blocked by a peptide that
does not recognize hCG. Increasing concentrations of mutant
LHR18-36 failed to significantly block the photoaffinity
labeling of hCG by AB-125I-LHR18-36 or
ABG-125I-LHR18-36 (Fig. 9, B and
E). Only at the highest concentrations of the mutant peptide
was labeling by AB-125I-LHR18-36 slightly
reduced. Although these results indicate the labeling specificity of
AB-125I-LHR18-36 and
ABG-125I-LHR18-36, the futile inhibition could
be interpreted as the mutant peptide binding to a site in hCG different
from the AB-125I-LHR18-36- or
ABG-125I-LHR18-36-binding site. To test this
hypothesis, mutant LHR18-36 was derivatized and
radioiodinated to prepare AB-125I-mutant
LHR18-36 and ABG-125I-mutant
LHR18-36.

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Fig. 9.
Inhibition of photoaffinity labeling by
nonderivatized wild-type or mutant LHR18-36. hCG was
photoaffinity-labeled with AB-125I-LHR18-36
(D-F) or ABG-125I-LHR18-36
(A-C) in the presence of increasing concentrations of
nonderivatized wild-type (WT) or mutant
LHR18-36. Samples were processed as described in the
legend to Fig. 6.
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Photoaffinity Labeling by Mutant LHR18-36--
As
shown in Fig. 10,
AB-125I-mutant LHR18-36 and
ABG-125I-mutant LHR18-36 failed to
conspicuously label the hCG subunits. Only trace amounts of labeling
were detected, indicating that the labeling affinities were
significantly low. These results are consistent with the observation
that the highest concentrations of nonderivatized mutant
LHR18-36 slightly attenuated the labeling by
AB-125I-LHR18-36. In addition,
AB-125I-LHR18-36 and
ABG-125I-LHR18-36 did not photoaffinity label
denatured hCG that was boiled in 8 M urea and did not bind
to the receptor (data not shown).

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Fig. 10.
Photoaffinity labeling by wild-type and
mutant AB-125I-LHR18-36 or
ABG-125I-LHR18-36. hCG was incubated with
wild-type (WT) and mutant (Mut)
AB-125I-LHR18-36 (B and
D) or ABG-125I-LHR18-36
(A and C) and photoaffinity-labeled as described
in the legend to Fig. 6.
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DISCUSSION |
Our results show that AB-125I-LHR18-36
and ABG-125I-LHR18-36 photoaffinity label hCG.
The
-subunit is preferentially labeled. Ample evidence was presented
to support the specificity of the photoaffinity labeling of hCG. The
labeling is saturable and dependent on the hCG concentration,
derivatized 125I-LHR18-36 concentration, and
UV exposure. AB-125I-LHR18-36 and
ABG-125I-LHR18-36 photoaffinity label
bioactive hCG, but not denatured hCG. This labeling is blocked by
nonderivatized wild-type LHR18-36, but not by
nonderivatized mutant LHR18-36. Furthermore,
AB-125I-mutant LHR18-36 and
ABG-125I-mutant LHR18-36 do not photoaffinity
label bioactive hCG and denatured hCG.
Both subunits of hCG are labeled, indicating that the UV-activable
group coupled to LHR18-36 can reach them. This is
consistent with other studies (18-20) and not surprising since the two
subunits are closely intertwined in the crystal structure (21, 22).
Interestingly, hCG
was preferentially labeled. Since only one
photosensitive group is attached to the N terminus of each derivatized
peptide, AB-125I-LHR18-36 and
ABG-125I-LHR18-36 bound to hCG can
photoaffinity label only one, but not both, of the subunits. Obviously,
the reagent more readily reaches and labels the
-subunit than the
-subunit. Since the maximum labeling distances of AB and ABG are 7 and 10 Å, respectively (17), and AB-125I-LHR18-36 labels hCG more efficiently
than ABG-125I-LHR18-36, the N terminus of the
LHR18-36 derivatives is <7 Å from both subunits.
Therefore, both subunits of hCG are likely to contact the
LHR18-36 derivatives. Our results are not consistent with
the unlikely possibility that the peptide associates with hCG at sites
other than the receptor contact site, impacts on the global structure of hCG, and interferes with the hormone/receptor interaction. Clearly,
LHR18-36 interacts with hCG at or near a contact site of
hCG and the LH/CG receptor.
The recent crystallization of Leu-rich repeats (23, 24) and their
presence in the middle of the exodomain of all glycoprotein hormone
receptors (1) generated a deluge of the popular and probable thoughts
(11, 25-27) that eight to nine Leu-rich repeats compose the primary
contact site for the ligand. They compose the bulk of the exodomain at
its center and are computer-modeled to show a crescent structure (Fig.
11). The inner surface of the crescent
consists of
-sheets of the repeats and is thought to be the ligand
contact site (24-26), perhaps interacting with the putative
receptor-binding C-terminal and seat belt side of hCG (21). Our results
in this work and the preceding article (12) indicate that there is a
crucial hormone contact site outside of this Leu-rich crescent. It will
be interesting to see if the Gly18-Tyr36
sequence of the receptor reaches the opposite face of the seat belt
side (Fig. 11). In the Gly18-Tyr36 sequence,
the alternate Leu20, Cys22, and
Gly24 residues are crucial for hormone binding (12). These
three residues appear to be at one side of a
-like structure and
could face the hormone and provide a direct contact site.

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Fig. 11.
Schematic model for the interaction of the
LH/CG receptor exodomain with hCG. To facilitate the visualization
of the interaction of the exodomain with hCG, a schematic model was
prepared. hCG interacts with the inner face of the crescent structure
of the receptor as suggested by computer modeling (25, 28, 29).
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Our results are consistent with the observations of others indicating
multiple contact sites for the hCG/receptor interaction (10, 11, 28,
29). Each contact site is likely to contribute to the overall
interaction and affinity. For complete understanding of the
interaction, it is necessary to know whether the multiple contact sites
are independent or related and whether they interact with hCG
simultaneously or sequentially. This information will be useful for
designing agonists and antagonists of hCG.