Hormone Interactions to Leu-rich Repeats in the Gonadotropin
Receptors
III. PHOTOAFFINITY LABELING OF HUMAN CHORIONIC GONADOTROPIN WITH
RECEPTOR LEU-RICH REPEAT 4 PEPTIDE*
MyoungKun
Jeoung
,
Tzulip
Phang
,
Yong Sang
Song
§,
Inhae
Ji
, and
Tae H.
Ji
¶
From the
Department of Chemistry, University of
Kentucky, Lexington, Kentucky 40506-0055 and the
§ Cancer Research Center, Seoul National University College
of Medicine, Seoul 110-744, Korea
Received for publication, May 3, 2000, and in revised form, June 12, 2000
 |
ABSTRACT |
Human chorionic gonadotropin (hCG) binds to the
extracellular N-terminal domain, exodomain, of its receptor, and the
resulting hCG-exodomain complex is thought to modulate the
membrane associated domain, endodomain, of the receptor to generate
hormone signal. The bulk of the exodomain is speculated to assume a
crescent structure consisting of eight to nine Leu-rich repeats (LRRs),
which may provide the hormone contact sites. Unfortunately, little
experimental evidence is available for the precise hormone contact
points in the exodomain and the endodomain. The two preceding articles
(Song, Y., Ji, I., Beauchamp, J., Isaacs, N., and Ji, T. (2001)
J. Biol. Chem. 276, 3426-3435; Song, Y., Ji,
I., Beauchamp, J., Isaacs, N., and Ji, T. (2001) J. Biol.
Chem. 276, 3436-3442) show that putative LRR2 and LRR4
are crucial for hormone binding. In particular, the N-terminal region
of LRR4 assumes the hydrophobic core of the LRR4 loop, whereas the
C-terminal region is crucial for signal generation. However, it is
unclear whether LRR4 interacts hCG and the endodomain and how it might
be involved in signal generation. In this article, our affinity
labeling results present the first evidence that the N-terminal region
of LRR4 interacts with hCG, preferentially the hCG
subunit and that
the hCG/LRR4 complex interacts with exoloop 2 of the endodomain. This
interaction offers a mechanism to generate hormone signal.
 |
INTRODUCTION |
The luteinizing hormone/chorionic gonadotropin receptor
(LHR)1 consists of an
extracellular N-terminal half (exodomain) and a membrane-associated
C-terminal half (endodomain) (1, 2). The ~350-amino acid-long
exodomain has high affinity hormone contact sites (3-5) and shows
eight to nine repeats of 22-29 amino acids with several conserved
Leu/Ile residues (1, 6-10). These Leu/Ile-rich repeats (LRRs)
represent a common structural motif found in a large family of
proteins, which includes glycoprotein hormone receptors (11). In the
crystal structure of ribonuclease inhibitors, the LRRs assume the
horseshoe structure in which individual LRRs form a loop consisting of
a
strand connected to parallel
helices. The
strands in
ribonuclease inhibitors are involved in the interaction with
ribonuclease. However, it is unclear whether the putative LRR sequences
of LHR and other glycoprotein hormone receptors are indeed LRRs and
function as such. In the preceding articles (12, 13), we have shown
that some, but not all, LRRs of LHR and the follicle-stimulating
hormone receptor are crucial for hormone binding. In particular, LRR2
and LRR4 of LHR are most crucial, but it is unclear whether these LRRs
make direct contacts with the hormone. In this article, the evidence is
presented for the interaction of the residues around the Leu-Ser-Ile
motif, the putative
strand, in LRR4 with hCG, in particular with
the hCG
subunit. In addition, our data suggest the interaction of the LRR4-hCG complex with the endodomain, in particular exoloop 2, which is likely to modulate signal generation.
 |
EXPERIMENTAL PROCEDURES |
Materials--
The N-hydroxysuccinimide (NHS) ester
of 4-azidobenzoic acid (AB) was synthesized as described
previously (14). 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. The peptide mimics include the wild type receptor peptide
corresponding to the LHR sequence of Asn96-Asp115 (LHR96-115), a
mutant LHR96-115 with Leu103Ala and
Ile105Ala mutations (LHR96-115(L103A/I105A), a
mutant LHR96-115 with the Lys101
Ala mutation (LHR96-115(K101A)), a mutant
LHR96-115 with the Lys112
Ala mutation
(LHR96-115(K112A)), a wild type peptide encompassing the
sequence upstream of LHR96-115 (LHR85-104),
and a wild type peptide covering the sequence downstream of LHR96-115 (LHR113-132).
Derivatization and Radioiodination of Peptides--
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 LHR96-115 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-LHR96-115 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-LHR96-115
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 PBS), and
125I-LHR96-115 (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 of 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, 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 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-LHR96-115 (10 ng/µl) in PBS. 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 (14),
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--
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
LHR96-115.
Inhibition of 125I-hCG binding to LHR--
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
LHR96-115 peptides as described previously (15). After
several times washing the cells, the radioactivity associated with the
cells was counted, and percent bound 125I-hCG was plotted
against the nonradioactive receptor peptides. The results were
converted to Scatchard plot by plotting bound/free peptide
versus bound peptide. The plot was used to calculate the Kd value following the Scatchard equation (16).
 |
RESULTS |
In the preceding articles (12, 13), we showed the crucial roles of
LRRs of LHR in hormone binding, particularly LRR4. This raises the
question as to whether the LRRs directly interact with the hormone or
indirectly influence the hormone/receptor interaction by impacting the
global structure of the receptor exodomain. To examine these
possibilities a peptide mimic corresponding to the receptor sequence
encompassing the
-stranded Leu103-Ile105,
LHR96-115, was synthesized and tested for its ability to
bind and affinity label hCG. For affinity labeling, we employed two
complementary affinity labeling methods. In the first approach,
125I-LHR96-115 incubated with hCG, and the
resulting 125I-LHR96-115-hCG complexes were
cross-linked using SES, a homobifunctional reagent that is capable of
cross-linking two amino groups up to 13 Å apart (17). In the second
approach, 125I-LHR96-115 was derivatized with
AB, an UV-activable reagent, to produce AB-125I-LHR96-115 and incubated with hCG. The
resulting 125I-LHR96-115-hCG complex was
irradiated with UV to photoaffinity label hCG with
AB-125I-LHR96-115. The advantages and
disadvantages of both methods will be discussed later.
To determine whether AB-125I-LHR96-115 and
125I-LHR96-115 would bind and label hCG, they
were incubated with hCG and treated with UV or SES, respectively. The
samples were solubilized in SDS under the reducing condition and
electrophoresed, as described under "Experimental Procedures." The
autoradiographic phosphoimage of the gel shows that both
AB-125I-LHR96-115 and
125I-LHR96-115 labeled both the
and
subunits in hCG (Fig. 1). In addition, the hCG 
dimer was cross-linked and labeled with
125I-LHR96-115 when the
125I-LHR96-115-hCG complex was treated with
SES. 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 (Fig. 1,
lanes 1 and 5).

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Fig. 1.
Autoradiograph of affinity-labeled hCG
subunits. hCG was incubated with
AB-125I-LHR96-115 (lanes 2 and
3) or 125I-LHR96-115 (lanes
4 and 5) and treated with UV for 1 min (lane
3) or 0.3 mM SES (lane 5), respectively, as
described under "Experimental Procedures." After electrophoresis of
the samples, the gel was dried and autoradiographed using
PhosphoImager. Lane 1, 125I-hCG showing
radiolabeled and subunits as standards.
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Cross-linking of 125I-LHR96-115 to
hCG--
As a first step to determine the specificity of affinity
labeling, 125I-LHR96-115 was incubated with
hCG and treated with increasing concentrations of SES. Electrophoresis
of the treated hCG/125I-LHR96-115 mixture
(Fig. 2A) shows that
125I-LHR96-115 was cross-linked to hCG
,
hCG
, and the hCG
dimer. The extent of cross-linking was
dependent on the SES concentration, reaching the maximum level at
0.3-1 mM SES. Under this condition, ~20% of
125I-LHR96-115 was cross-linked to hCG
and
~10% to hCG
. At higher SES concentrations, for example 10 mM, the extent of cross-linking decreased. This decrease
was due to noncross-linking, monofunctional reactions (only one of the
two NHS groups reacting with a target amino group while the other NHS
group undergoing hydrolysis) of excess SES with
125I-LHR96-115, hCG, and its subunits (18). In
conclusion, our results indicate that
125I-LHR96-115 was covalently cross-linked to
hCG
and hCG
. Furthermore, either or both amino groups of
Lys101 and Lys112 of
125I-LHR96-115, the only amino groups of the
peptide, were cross-linked to an amino group(s) 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.
Affinity labeling is saturable. hCG was
incubated with 125I-LHR96-115 and treated with
SES (A-C). In this series of experiments, the increasing
concentrations of SES (A),
25I-LHR96-115 (B), or hCG (C)
were applied, while the other two conditions were kept constant. In
D-F, hCG was incubated with
AB-125I-LHR96-115 and irradiated with UV. In
this series, the UV irradiation time (D),
AB-125I-LHR96-115 concentration
(E), or hCG concentration (F) was varied, while
the other two factors were kept constant. 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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Saturable Cross-linking of 125I-LHR96-115
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-LHR96-115
while maintaining hCG at a constant concentration (Fig. 2B). Conversely, 125I-LHR96-115 and hCG were
cross-linked at increasing concentrations of hCG and a constant
concentration of 125I-LHR96-115 (Fig.
2C). If cross-links are specific, they should reach
saturation under both conditions. The results 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.
Photoaffinity Labeling of hCG--
Despite the indication for
saturable and specific cross-links between the receptor peptide and
hCG, there were a series of minor cross-linked complexes larger than
the complex of 125I-LHR96-115 and the hCG
dimer. They suggest that a minor population of the 125I-LHR96-115-hCG dimer complex may be
further cross-linked to another hCG subunit or hCG dimer. Although this
is not entirely unexpected, as random collisional cross-links are
possible (18), it raises a concern on the specificity of
homobifunctional cross-links between 125I-LHR96-115 and hCG. A simple way to reduce
or eliminate such random collisional cross-links is photoaffinity
labeling (18). To photoaffinity label hCG with
125I-LHR96-115, the receptor peptide was
derivatized with AB to produce
AB-125I-LHR96-115. When the derivatized
peptide binds to hCG and is irradiated with UV, the cross-link will be
restricted between 125I-LHR96-115 and hCG
or between 125I-LHR96-115 and hCG
. The
reagent, however, will not be able to cross-link an hCG subunit to
another. AB can reach and label target molecules up to 7 Å (19). The
distance is considerably shorter than the maximum cross-linkable 13 Å of SES and therefore, the labeling reaction by AB is more restricted
than the cross-linking reaction by SES. On the other hand,
cross-linking with SES can be useful when AB attached at the contact
point might interfere with the interaction.
As shown in Fig. 2, D-F,
AB-125I-LHR96-115 was capable of photoaffinity
labeling either hCG
or hCG
but not both subunits at the same time. The labeling is generally confined to hCG
with the labeling of
hCG
being faint. This result is consistent with the SES
cross-linking results. One possible explanation is that the peptide is
bound closer to
than
. The labeling required UV irradiation and
was dependent on the irradiation time, reaching the maximum labeling after 30~60-s irradiation. This UV dependence clearly indicates photoaffinity labeling. In addition, the preferential labeling of
hCG
without simultaneously labeling of both subunits suggests a
labeling specificity. To further examine the specificity of photoaffinity labeling, the concentration of either hCG or the peptide
derivatives was changed. When a constant concentration of
AB-125I-LHR96-115 was incubated with
increasing concentrations of hCG, the intensity of labeled hCG
and
bands gradually increased and plateaued (Fig. 2E). A
similar result was obtained in a converse experiment when a constant
amount of hCG was incubated with increasing concentrations of
AB-125I-LHR96-115 (Fig. 2F). These
results indicate that the photoaffinity labeling is dependent on both
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.
Labeling Specificity--
Specific labeling should be displaced by
wild type peptide but not by a peptide that could not bind hCG. We have
shown in the previous reports (12), (13) that the Leu103
Ala or Ile105
Ala substitution in LHR abrogated
hormone binding. Therefore, Leu103 and Ile105
were substituted with Ala in LHR96-115 to produce a
mutant peptide, LHR96-115(L103A/I105A). To test
whether the wild type and mutant LHR peptides could inhibit affinity
labeling, hCG was incubated with
AB-125I-LHR96-115 in the presence of
increasing concentrations of nonderivatized wild type peptide (Fig.
3A) and nonderivatized mutant
peptide (Fig. 3C). Increasing concentrations of
LHR96-115 inhibited photoaffinity labeling in a
dose-dependent manner and eventually, completely blocked
it. These results indicate the specificity of LHR96-115
for the photoaffinity labeling. In contrast, the inhibition by mutant
LHR96-115(L103A/I105A) was significantly less effective
(Fig. 3C). Similar results were obtained with affinity
cross-linking of 125I-LHR96-115 to hCG (Fig.
3, B, D, and F). Although these
results indicate the labeling specificity of
AB-125I-LHR96-115 and
125I-LHR96-115, the futile inhibition could be
interpreted as the mutant peptide binding to a site in hCG
different from the AB-125I-LHR96-115
binding site. To test this hypothesis and test whether the mutant peptide could label hCG, LHR96-115(L103A/I105A) was
radioiodinated or derivatized and then radioiodinated to prepare
125I-LHR96-115(L103A/I105A) or
AB-125I-LHR96-115(L103A/I105A), respectively.
As shown in Fig. 4,
AB-125I-LHR96-115(L103A/I105A) and
125I-LHR96-115(L103A/I105A) labeled the hCG
subunits significantly less. Only trace amounts of labeling were
detected, indicating the labeling affinities were significantly low.
These results are consistent with the observation that the highest
concentrations of nonderivatized LHR96-115(L103A/I105A)
slightly attenuated the labeling by
AB-125I-LHR96-115 and
125I-LHR96-115.

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Fig. 3.
Competitive inhibition of affinity labeling
by unlabeled wild type peptide and mutant peptide.
AB-125I-LHR96-115 was incubated with hCG in
the presence of increasing concentrations of wild type
LHR96-115 (A) or mutant
LHR96-115(L103A/I105A) (B) and irradiated with
UV for 30 s. Samples were processed as described in the legend to
Fig. 2. In addition, 125I-LHR96-115 was
incubated with hCG in the presence of increasing concentrations of wild
type LHR96-115 (C) or mutant
LHR96-115(L103A/I105A) (D) and treated with 0.3 mM SES. The samples were electrophoresed and processed to
determine the percent labeling of the hCG and subunits. The
percent intensities of the labeled and bands were determined
and plotted against increasing concentrations of unlabeled wild type
and mutant peptide (E plotted with the percent labeling data
from A and C, and F plotted with the
percent labeling data from B and D).
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Fig. 4.
Futile affinity labeling of hCG by mutant
LHR96-115. hCG was incubated with mutant
AB-125I-LHR96-115(L103A/I105A) (lanes
3 and 4) or
125I-LHR96-115(L103A/I105A) (lanes
7 and 8) and treated with UV for 1 min (lane
4) or 0.3 mM SES (lane 8), respectively.
These samples were processed as described in the legend to Fig. 1. The
autoradiograph shows no affinity labeling of hCG as compared with
successful labeling of hCG by wild type LHR96-115
(lanes 2 and 6). Lanes 1 and
5 show the control hCG samples that were incubated with the
wild type peptide but without UV or SES treatment.
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Biological Specificity of Affinity Labeling--
Although the
affinity labeling is specific, our data do not show the biological
significance of the affinity labeling. To test this concern, two
different experiments were performed. In the first test, denatured hCG
was tested for affinity labeling, and in the second the peptides were
examined whether they could inhibit 125I-hCG binding to the
receptor on intact cells. For the first test, denatured hCG was
incubated with increasing concentrations of AB-125I-LHR96-115or
125I-LHR96-115 and treated with UV or SES,
respectively (Fig. 5). Denatured hCG was
not labeled at all by either of the LHR peptide derivatives, despite
high concentrations of the peptide probes. The results suggest the
specificity of the affinity labeling for biologically active hCG. Since
SES failed to cross-link 125I-LHR96-115 to
denatured hCG, 125I-LHR96-115
appears to have a difficulty to recognize denatured hCG. To test this
possibility, 125I-hCG was incubated with intact cells
expressing LHR in the presence of increasing concentrations of the wild
type or mutant peptide, LHR96-115 or
LHR96-115(L103A/I105A) (Fig.
6). The wild type LHR96-115
inhibited 125I-hCG binding to the receptor with a
Kd value of 43.4 µM, suggesting its
binding to the receptor with a reasonable affinity for a peptide (20,
21). In contrast, the Kd value of mutant
LHR96-115(L103A/I105A) was 5 mM, which is
insignificant. This result, taken together with the futile labeling of
denatured hCG (Fig. 5), shows the biological specificity of the binding
and labeling of LHR96-115 to hCG. Furthermore, the results
show that the interaction between hCG and LHR96-115
simulates the interaction between hCG and the receptor.

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Fig. 5.
Denatured hCG is not affinity-labeled.
Denatured hCG (200 ng) was incubated with increasing concentrations of
AB-125I-LHR96-115 (A) or
125I-LHR96-115 and treated with UV for 60 s or 0.3 mM SES (B). The samples were processed
as described in the legend to Fig. 1.
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Fig. 6.
Inhibition of 125I-hCG binding to
the receptor by LHR peptides. 125I-hCG was incubated
with intact 293 cells expressing LHR in the presence of increasing
concentrations of unlabeled wild type and mutant LHR96-115
peptides. After washing cells several times to remove unbound
125I-hCG, cells were counted for the bound
125I-hCG as described under "Experimental Procedures."
The results were plotted against the concentrations of unlabeled
peptides (left panel) and converted to Scatchard plots
(right panel). The Kd values of
individual peptides in the table were determined with standard
deviations based on the bound/free and bound peptide values as
described previously (16).
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Affinity Labeling Site--
LHR96-115 has two Lys
residues, Lys101 and Lys112, which are
derivatized with AB or reacted with SES. Since these two Lys are 11 amino acids apart and located in the opposite side of the LRR4 (Fig. 7), it is important to know whether both
or only one of them is involved in the affinity labeling. The
information will be crucial for defining the orientation and hormone
interacting phase of LRR4. To determine the labeling activity of the
two Lys residues, one of them was substituted with Ala in
LHR96-115 to produce LHR96-115(K101A) and
LHR96-115(K112A). These two peptides were capable of
inhibiting 125I-hCG binding to the receptor on intact
cells, but their Kd values were 12-15-fold higher
than the corresponding Kd value of the wild type
LHR96-115 (Fig. 6). The result is consistent with the
effect of Ala substitution for Lys101 or Lys112
in intact receptor on hCG binding. The Kd value for hCG binding of LHR increased by 2.6-3.4-fold when Lys101
or Lys112 was substituted with Ala (13). Since they were
capable of binding hCG, we examined whether the two mutant peptides
were also capable of inhibiting photoaffinity labeling of hCG by
AB-125I-LHR96-115 and affinity cross-linking
of 125I-LHR96-115 to hCG (Fig.
8). The results show their ability to
inhibit the labeling, but the potency was noticeably less than the
inhibition potency of the wild type peptide. Again, this result is
consistent with the lower affinity of the two mutant peptides to hCG as
compared with the affinity of the wild type peptide to hCG. All of
these results show their specific interaction with hCG. Finally, we attempted to photoaffinity label hCG with
AB-125I-LHR96-115(K101A) and
AB-125I-LHR96-115(K112A) (Fig.
9).
AB-125I-LHR96-115(K112A) photoaffinity labeled
hCG similar to the photoaffinity labeling of hCG by
AB-125I-LHR96-115, whereas the labeling of hCG
with AB-125I-LHR96-115(K101A) was
less (Fig. 9A). This result indicates that the photoaffinity labeling is significantly more effective when AB is attached to Lys101 than to Lys112.

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Fig. 8.
Inhibition of affinity labeling by Lys to Ala
mutant peptides. hCG was affinity-labeled with
AB-125I-LHR96-115 and
125I-LHR96-115 in the presence of increasing
concentrations of LHR96-115(K101A) or
LHR96-115(K112A) as described in the legend to Fig. 3.
After processing the samples, the phosphoimage of the gel was analyzed
to determine the percent labeling the hCG subunits. The results were
plotted against the concentration of peptides for the inhibition of
photoaffinity labeling hCG (A) and hCG (B)
and affinity cross-linking to hCG (C) and hCG
(D). In addition, the inhibition of unlabeled wild type
LHR96-115 was presented for comparison.
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Fig. 9.
Affinity labeling of hCG with Lys to
Ala mutant peptides. A, hCG was photoaffinity-labeled
with AB-125I-LHR96-115,
AB-125I-LHR96-115(K101A), or
AB-125I-LHR96-115(K101A) as described in the
legend to Fig. 3. B, likewise, hCG was
affinity-cross-linked to 125I-LHR96-115,
125I-LHR96-115(K101A), or
215I-LHR96-115(K101A) as described in the
legend to Fig. 3. "+" and " " indicate the treatment with or
without UV or SES.
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If this is true, one would expect the same trend with affinity
cross-linking using the two peptides. Indeed,
125ILHR96-115(K112A) was cross-linked to
hCG with SES significantly better than
125I-LHR96-115(K101A) (Fig. 9B).
However, neither of the derivatized peptides labeled denatured hCG,
indicating a specificity of affinity labeling of hCG by
AB-125I-LHR96-115(K112A) and
125I-LHR96-115(K112A) (data not shown). Taken
together, these results indicate that Lys101 is more
suitable for affinity labeling hCG than Lys112 is. They
also suggest that Lys101 is at or near the hCG contact
point as suggested by the computer model that the short
strand is a
ligand contact site, and the Lys101 is projected toward
ligand (Fig. 7). In contrast, Lys112 is located near the
helix as part of the outer lining of the donut structure, at the
opposite side from the ligand binding site. Since Lys101 is
in the N-terminal area of LHR96-115, whereas
Lys112 is in the C-terminal region, one way to verify the
conclusion is to use peptide mimics covering the sequences upstream and
downstream of LHR96-115. To this end, we synthesized two
peptide mimics, LHR84-104 and LHR113-132, and
tested them for their ability to inhibit photoaffinity labeling of hCG
by AB-125I-LHR96-115 and affinity
cross-linking of 125I-LHR96-115 to hCG (Fig.
10). LHR84-104 and
LHR113-132 inhibited the affinity labeling of hCG, but
their potency was less than that of LHR96-115.
LHR84-104 was more effective in inhibiting hCG
than
LHR113-132 was. On the other hand, LHR85-104 was similar
to LHR113-132 in inhibiting the labeling of
hCG
.

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|
Fig. 10.
Roles of peptides flanking
LHR96-115 on affinity labeling. hCG was
photoaffinity-labeled with AB-125I-LHR96-115
and affinity-cross-linked to 125I-LHR96-115
with SES in the presence of increasing concentrations of
LHR96-115, LHR85-104, or
LHR113-132 as described in the legend to Fig. 3. After
processing the samples, the percent labeling of hCG and hCG was
determined. The results are presented for the inhibition of
photoaffinity labeling of hCG (A) and hCG
(B) and the inhibition of affinity cross-linking of hCG
(C) and hCG (D).
|
|
Interaction of LHR96-115-hCG Complex with
Exoloops--
In the preceding article (13), we pointed out the
absolute homology in the 8 residues (boldface) in the
Leu98-Pro-Gly-Leu-Lys-Tyr-Leu-Ser-Ile-Cys-Asn-Thr-Gly109
sequence among cloned LHR, follicle-stimulating hormone
receptor, and thyroid-stimulating hormone receptor of various
species. Furthermore, we showed that the tandem three conserved
residues,
Asn107-Thr-Gly109,
were more important for cAMP induction than hormone binding. This is
unique because the exodomain is responsible of high affinity hormone
binding and mutations in the exodomain impact hormone binding, which in
turn affected cAMP induction, not the other way around. Therefore, we
have raised the possibility that this region may be involved in the
interaction with the endodomain and, thus, in signal generation. This
is a crucial issue, because the exodomain and endodomain are known to
interact (22-25), and this interaction regulates the generation of
hormone signals (22), (23). However, the exact contact points in the
exodomain and endodomain are unknown. Since the three exoloops in the
endodomain are a logical candidate for the exodomain/endodomain
interaction, we have synthesized peptide mimics for the exoloops 1, 2, and 3 of LHR (LHRexo1, LHRexo2, and
LHRexo3) and tested whether they could inhibit the
photoaffinity labeling of hCG by
AB-125I-LHR96-115 (Fig.
11). LHRexo2 effectively
inhibited the photoaffinity labeling, whereas the inhibition by
LHRexo1 was less. In contrast, LHRexo3 did not
inhibit the labeling. These differential effects suggest the
specificity of the inhibition.

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Fig. 11.
Roles of exoloop peptides on affinity
labeling. hCG was photoaffinity-labeled with
AB-125I-LHR96-115 in the presence of
increasing concentrations of LHR peptide mimics corresponding to the
sequences of exoloops 1, 2, and 3 (LHRexo1,
LHRexo2, and LHRexo3) as described in the
legend to Fig. 3. Exoloop 1 connects transmembrane domains 2 and 3, exoloop 2 connects TMs 4 and 5, and exoloop 3 connects transmembrane
domains 6 and 7. The LHRexo1 sequence is
Asp-Ser-Gln-Thr-Lys-Gly-Gln-Tyr-Tyr-Asn-His-Ala-Ile-Asp-Trp-Gln-Thr-Gly-Ser-Gly-Cys-Ser-Thr.
The LHRexo2 sequence is
Ser-Asn-Tyr-Met-Lys-Val-Ser-Ile-Cys-Phe-Pro-Met-Asp-Val-Glu-Thr-Thr-Leu-Ser-Gln.
LHRexo3 comprises Lys-Val-Pro-Leu-Ile-Thr-Val-Thr-Asn-Ser-Lys. Labeled
hCG was processed, and the percent labeling of hCG and hCG
was determined as described in the legend to Fig. 3.
|
|
 |
DISCUSSION |
Our results show that AB-125I-LHR96-115
photoaffinity labels hCG. Ample evidence is presented to support the
specificity of the photoaffinity labeling under rigorous conditions.
The labeling is saturable and dependent on the hCG concentration,
derivatized 125I-LHR96-115 concentration, and
UV activation. AB-125I-LHR96-115 photoaffinity
labels bioactive hCG but not denatured hCG. This labeling is blocked by
nonderivatized wild type LHR96-115 but not by
nonderivatized mutant LHR96-115(L103A/I105A).
The same Ala mutations in LHR abolish the hCG binding activity of LHR.
Furthermore, AB-125I-LHR96-115(L103A/I105A)
does not photoaffinity label bioactive hCG and denatured
hCG. LHR96-115 inhibits 125IhCG
binding to the receptor expressed on intact cells but
LHR96-115(L103A/I105A) is not capable of inhibiting
125I-hCG binding to the receptor. To avoid the potential
interference of the photoactivable group on binding of
AB-125I-LHR96-115 to the receptor and the
subsequent labeling, 125I-LHR96-115 was
affinity-cross-linked to hCG with SES. This affinity labeling is
equally successful with similar specificity.
Both subunits of hCG are labeled, indicating that the UV-activable
group coupled to AB-125I-LHR96-115 can reach
them. This is consistent with other studies (26-28) and not
surprising, since the two subunits are closely intertwined in the
crystal structure (29, 30). Interestingly, hCG
was preferentially
labeled. Obviously, the reagent more readily reaches and labels the
subunit than the
subunit. Since the maximum labeling distances of
AB is 7 Å (19), hCG
is likely to contact AB-125I-LHR96-115. Our results are
inconsistent with the unlikely possibility that the peptide associates
with hCG at sites other than the receptor contact site, impacts the
global structure of hCG, and interferes with the hormone/receptor
interaction. Since LHR96-115 inhibits hCG binding to the
receptor, AB-125I-LHR96-115 interacts with hCG
at or near a contact site of hCG and the LH/CG receptor.
It is significant that only one of the hCG
subunits, but not
both, is labeled, although two AB could be attached to the two Lys
residues of LHR96-115. This suggests that only one of the
Lys residues is close to hCG. Indeed, photoaffinity labeling using
mutant peptides lacking one of the Lys residues shows that the AB
coupled to Lys101 is capable of labeling hCG, whereas the
AB attached to Lys112 is less effective. This is strong
evidence to support the orientation of Lys101 and
Lys112 in the LRR4 loop model (Fig. 7) and implicates the
N-terminal region of LHR96-113, including the putative
strand of LRR4, in the interaction with hCG.
The crystallization of Leu-rich repeats (11, 31) and their presence in
the middle of the exodomain of all glycoprotein hormone receptors (1)
generated much speculation (8, 32-34) that the eight to nine LRRs
provide the primary contact site for the cognate ligands, LH/CG, FSH,
and TSH. They comprise the bulk of the exodomain at its center and are
computer-modeled to show a crescent structure. The inner surface of the
crescent consists of
sheets of the repeats and is thought to be the
ligand contact site (8, 31, 32), perhaps interacting with the putative receptor binding
C terminus and seat belt side of hCG (29). However,
little experimental evidence has been available to support these
popular views. Our results of this and the preceding articles (12, 13)
are the first experimental evidence supporting the LRR structure of LHR
and the direct interaction of the LRR4
strand with hCG. Our studies
have laid the ground work to determine the contact residues of the
receptor and the hormone.
It has been known that LHR interacts hCG initially at the exodomain,
and the exodomain-hCG complex impacts the endodomain. This secondary
contact is thought to generate the hormone signals (22, 23). There is
evidence that the exodomain and endodomain are intimately associated
before and after hormone binding (24, 25). This association is crucial
because it affects the hormone binding affinity and provides a
mechanism for the signal generation (24, 25). Unfortunately, there are
few clues to the site of the interaction between the exodomain and
endodomain except the recent reports implicating exoloops 2 and 3 (24,
25). The observations described in this and preceding articles (12, 13) show the involvement of LRR4 in the signal generation, implicating exoloop 2 and, perhaps, exoloop 1 as contact points of the
exodomain/hCG complex. In fact, our computer modeling shows that the
exoloop 2 projects straight up from the connecting the transmembranes 4 and 5, like a hairpin, toward the exodomain. It will be interesting to
see whether the hairpin structure of exoloop 2 interacts with the
crescent LRR structure of the exodomain, in particular LRR4. Such an
exodomain/endodomain interaction could provide a mechanism for the
mutual modulation of the two distinct domains (24, 25) and signal generation.
 |
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.M003774200
 |
ABBREVIATIONS |
The abbreviations used are:
LH, luteinizing
hormone;
LHR, LH receptor;
CG, choriogonadotropin;
h, human;
LRR, Leu-rich repeat;
AB, 4-azidobenzoyl;
NHS, N-hydroxysuccinimide;
SES, ethylene glycolbis(sulfosuccinimidylsuccinate);
PBS, phosphate-buffered
saline.
 |
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