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-Subunit
Department of Molecular Biology University of Wyoming Laramie, Wyoming 82071-3944
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-subunit and a hormone-specific ß-subunit.
Similarly, its receptor is also composed of two domains, an
extracellular N-terminal half (exodomain) and a membrane-associated
C-terminal half (endodomain). hCG initially binds the exodomain of the
receptor after which the resulting hCG/exodomain complex is thought to
interact with the endodomain. This secondary interaction is considered
responsible for signal generation. Despite the importance, it is
unclear which hormone subunit interacts with the exodomain or the
endodomain. As a step to determine the mechanisms of the initial and
secondary interactions and signal generation, we investigated the
interaction of the hormone-specific ß-subunit in hCG with
the receptor’s exodomain. A photoactivable hCG derivative consisting
of the wild-type -subunit and a photoactivable
ß-subunit derivative was prepared and used to label the
exodomain. The analysis and immunoprecipitation of photoaffinity
labeled exodomain demonstrate that the ß-subunit in hCG
makes the direct contact with the exodomain. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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40 amino acids), to exoloops
and N-terminal exodomain (polypeptides 90 amino acids), or
exclusively to the exodomain (glycoprotein hormones 
30 kDa)
(1). The distinction between ligand binding and receptor activation is
supported by the existence of antagonists that competitively inhibit
agonist binding. However, it is experimentally difficult to distinguish
ligand binding from receptor activation. Fortunately, a few receptor
systems such as glycoprotein hormone receptors and protease-activated
receptors are available as good models to distinguish ligand binding
from receptor activation and, thus, investigate the transition between
these two important steps.
hCG is a heterodimeric glycoprotein hormone, consisting of a common
-subunit and a hormone-specific ß-subunit (2). It binds to the LH
receptor (LHR) that comprises two equal halves, an extracellular
N-terminal half (exodomain) and a membrane-associated C-terminal half
(endodomain) (3, 4). The exodomain is approximately 350 amino acids
long, which alone is capable of high-affinity hormone binding (5, 6, 7, 8)
with hormone selectivity (9, 10, 11) but without hormone action (7, 12).
Mutational analysis of the exodomain also implicates its involvement in
hormone binding (13, 14, 15). On the other hand, receptor activation occurs
in the endodomain (16), which is structurally equivalent to the entire
molecule of many other G protein-coupled receptors (1). The existing
evidence suggests that hCG initially binds to the exodomain with high
affinity and hormone specificity, and then the resulting
hormone/exodomain complex is thought to interact with the endodomain
(16). This secondary interaction is considered to generate hormone
signals (1, 15, 16). Sometime between the initial interaction and the
signal generation, hCG undergoes a crucial structural change involving
the intersubunit interaction (17). Although the structural change is an
important part of the hormone-receptor interaction and signal
generation, it is unknown when and where it takes place. This study was
launched to determine whether the ß-subunit in hCG contacts the
exodomain of the receptor. There are numerous mutations of hCGß,
which resulted in the binding-defective hCG. Such results are
consistent with the view that the mutated amino acid residues are
important for hCG binding to the receptor: they cannot
distinguish whether the mutated amino acids were indeed involved in
interacting the receptor or the mutations might have caused a (global)
conformational change in the hormone (which prevented receptor
binding). Our study gets around this ambiguous point and proves the
direct interaction between hCGß and the exodomain.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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on autoradiographs as will be shown later in a
number of figures. There are five amino groups at the N terminus,
Lys2, Lys20, Lys104, and
Lys122, of the hCG ß-subunit (18) that may react with the
reagent. The resulting AB-125I-ß was reconstituted with
untreated to produce /AB-125I-ß. Because both the
photoactivable group and radioactive tracer are attached only to the
ß-subunit of the derivatized hCG, radioactive labeling of receptors
would indicate the labeling by the ß-subunit. However, for the
photoaffinity labeling to be biospecific, /AB-125I-ß
should be bioactive and specific to the LHR and its exodomains.
Therefore, /AB-125I-ß was tested for binding to the
cells expressing the LHR, LHR295, or LHR336 in
the presence of increasing concentrations of unlabeled hCG. As shown by
the displacement assay and corresponding Scatchard plot in Fig. 1
, intact cells expressing the LHR bound
/AB-125I-ß. However, /AB-125I-ß did
not bind the cells expressing LHR295 or LHR336
because the exodomains were trapped in the cells and not transported to
the cell surface (6, 7). When the cells expressing the LHR,
LHR295 or LHR336 were solubilized in NP-40 and
assayed, /AB-125I-ß bound to solubilized exodomains
and wild-type receptor. The binding affinity of
/AB-125I-ß was comparable to the affinity of
125I-hCG. These results indicate that
/AB-125I-ß is capable of binding the LHR,
LHR295, and LHR336 with high affinity.
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/AB-125I-ß and Cross-Linking
of the - and ß-Subunits of hCG/AB-125I-ß was irradiated with UV for
increasing periods (Fig. 2). The
intensity of the cross-linked ß-dimer band increased, after longer
UV irradiation and reached a plateau after irradiation for more than 1
min. The maximum level of the cross-linked ß-dimer band was
significant: approximately 39% of the total activity (the sum of the
ß-band and ß-band). This result indicates that the majority of
/AB-125I-ß was uniformly derivatized with AB and
effective in reacting with the -subunit. Two minor radioactive bands
had an electrophoretic mobility significantly faster than the gel
mobility of the ß-subunit. They are the proteolytic products of the
ß-subunit that were produced during derivatization, radioiodination,
reconstitution with hCG, fractionation, and storage. To reduce
proteolysis, protease inhibitors were included in the reconstitution
mixture and, without the inhibitors, the proteolysis was more
extensive. The intensity of the two bands increased in parallel to the
storage time and, therefore, experiments were performed soon after
/AB-125I-ß was prepared. Interestingly, the intensity
of the proteolytic product bands diminished after more than 1 min of UV
irradiation and nearly disappeared after UV irradiation for 3 min. We
suspect that the proteolytic products could have adhered to the glass
tubes, as peptides often adsorb to glass upon UV irradiation.
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/AB-125I-ß, irradiated with UV, solubilized in SDS and
dithiothreitol, and electrophoresed. When the exodomains and receptor
are labeled with /AB-125I-ß, the labeled complexes are
expected to appear as bands of higher molecular masses than the
- and ß-bands in autoradiographs. On the autoradiograph (Fig. 3) of the gel, the LHR295
sample showed the 80-kDa and 100-kDa bands in addition to the 30-kDa
hCGß and 50-kDa hCGß bands. Although the 100-kDa band was faint,
it became conspicuous when the autoradiograph was overexposed.
Similarly, the LHR336 sample showed the 82-kDa and faint
102-kDa bands in addition to the 30-kDa hCGß and 50-kDa hCGß
bands. These results are comparable with the result of the LHR sample
that produced the 116-kDa and faint 136-kDa bands. Therefore, the
80-kDa and 100-kDa bands of the LHR295 sample and the
82-kDa and 102-kDa bands of the LHR336 sample are likely to
correspond to the 116-kDa and 136-kDa bands of the LHR sample. The
composition of these bands will be discussed later.
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/AB-125I-ß,
irradiated with UV for increasing times, solubilized in SDS and
dithiothreitol (DTT), and electrophoresed. The autoradiograph of the
gel (Fig. 4A) shows the UV-dependent
appearance of two conspicuous bands (the 50-kDa hCGß dimer band
and the 80-kDa band). Their band intensity was undetectable without UV
irradiation and increased in parallel to increasing UV irradiation.
These results indicate that UV irradiation is necessary for the band
formation.
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. These results indicate that
AB-125I-ß (30 kDa) photoaffinity labeled a 50-kDa
material to produce the 80-kDa band. In addition, when
AB-125I-ß in /AB-125I-ß labeled both the
hCG (20 kDa) and the 50-kDa material, it produced the 100-kDa (20
kDa + 30 kDa + 50 kDa) band. Because each hCGß has five amino groups
that can be derivatized with AB, AB-125I-ß could carry up
to five AB and photoaffinity label both hCG and the 50-kDa material.
This interpretation is also consistent with the sequential appearance
of the photoaffinity-labeled 82-kDa and 102-kDa complexes. For example,
the 80-kDa ß-band preceded the appearance of the 100-kDa band.
Therefore, the 50-kDa component corresponds to LHR296,
which has an apparent molecular mass of approximately 50 kDa including
six chains of N-oligosaccharides (19) with terminal sialic
acids (20). The percent radioactivities of the hormone/exodomain
complexes are low in this and later figures. This is because the total
radioactivities in these gel lanes include unbound
[125I]hCG present in the samples of solubilized
exodomains. Therefore, we suspect that the actual percentage of
labeled hormone/exodomain complexes among total
hormone/exodomain complexes is significantly higher, being in the range
of 20%.
Dependence of Photoaffinity Labeling on the Concentration of
/AB-125I-ß and
LHR295
When a constant concentration of LHR295 was incubated
with increasing concentrations of /AB-125I-ß and
irradiated with UV, the intensity of the 80-kDa band increased (Fig. 4B
). In addition, the faint 100-kDa band also appeared at high
/AB-125I-ß concentrations. The result indicates that
the labeling is dependent on the concentration of
AB-125I-ß. The intensity of the 80-kDa band also
increased when increasing concentrations of LHR295 were
photoaffinity labeled with a constant concentration of
/AB-125I-ß (Fig. 4C), indicating the dependence of the
photoaffinity labeling on the LHR295 concentration.
Hormone Specificity of Photoaffinity Labeling
LHR295
Glycoprotein hormones consist of a common -subunit and a
hormone-specific ß-subunit. Despite this structural similarity, they
have cognate receptors and only cross-activity. To examine the hormone
specificity of the photoaffinity labeling, LHR295 was
photoaffinity labeled with /AB-125I-ß in the presence
of an excess amount of unlabeled hCG, denatured hCG, FSH, and TSH (Fig. 5). Untreated hCG completely blocked the
photoaffinity labeling of LHR295. However, denatured hCG,
FSH, and TSH did not prevent the photoaffinity labeling although FSH
and TSH slightly reduced the affinity labeling. It is clear that the
photoaffinity labeling is specific to active hCG. The results
are also consistent with the low-affinity cross-activity of FSH and TSH
with hCG.
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/AB-125I-ß, immunoprecipitated with anti-Flag
antibody and Protein-G Sepharose, solubilized in SDS and DTT, and
electrophoresed on polyacrylamide gel. The autoradiograph of the gel
(Fig. 6A, gel lane 7) shows the 80-kDa
ß/LHR295 and 100-kDa ß/LHR295 bands,
indicating that the band materials contain immunospecific
Flag-LHR295 and radioactive AB-125I-ß. The
bands were not visible if any of /AB-125I-ß,
Flag-LHR295, UV irradiation, anti-Flag antibody, or
protein-G Sepharose was omitted in photoaffinity labeling and
immunoprecipitation. More specifically, the sample that lacked only the
UV irradiation showed the AB-125I-ß band but not the
80-kDa ß/LHR295 and 100-kDa ß/LHR295
bands. These results indicate that /AB-125I-ß was
associated and immunoprecipitated with Flag-LHR295 but was
not covalently linked to it. Normal mouse sera could not substitute
anti-Flag antibody for immunoprecipitating the 80-kDa
ß/LHR295 and 100-kDa ß/LHR295 bands.
Furthermore, LHR295 lacking the Flag epitope could not be
immunoprecipitated even if all of the necessary factors, except
Flag-LHR295, were included in photoaffinity labeling and
immunoprecipitation (data not shown). These results indicate that the
80-kDa ß/LHR295 and 100-kDa ß/LHR295
bands were properly and specifically immunoprecipitated with
AB-125I-ß and that Flag-LHR295 was present in
the bands.
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/AB-125I-ß and processed for immunoprecipitation. The
82-kDa ß/LHR336 and 102 kDa ß/LHR336
bands were precipitated (Fig. 6B). They were not immunoprecipitated
when any of /AB-125I-ß, Flag-LHR295, UV
irradiation, anti-Flag antibody, or protein-G Sepharose was omitted. In
addition, normal mouse sera could not replace anti-Flag antibody in the
immunoprecipitation, an indication of the specificity for anti-Flag
epitope. Another positive control (Flag-LHRwt) was
successfully and specifically immunoprecipitated, whereas the
corresponding negative control lacking the Flag sequence
(LHRwt) was not immunoprecipitated (Fig. 6C). Taken
together, our results unequivocally demonstrate that the
immunoprecipitation was specific for the Flag epitope and that the
material (
50 kDa) photoaffinity labeled with
/AB-125I-ß was LHR295. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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/AB-125I-ß
specifically interacted to LHR295 with a high affinity and
photoaffinity labeled it. In a similar manner,
/AB-125I-ß also photoaffinity labeled
LHR336 and LHRwt. These results indicate the
direct contact of the ß-subunit in hCG with LHR295 and
therefore, the exodomain of the LHR. This result is consistent with our
earlier observation that the peptide mimic corresponding to an
exodomain sequence affinity labeled the ß-subunit of hCG (22).
It has been shown that hCG undergoes a structural change involving the
interaction between the two subunits after it binds the LHR on intact
cells (17). However, it was unclear when the change occurs,
e.g. when hCG initially interacts with the exodomain of the
receptor or when the hCG/exodomain complex makes the secondary contact
with the endodomain and generates a signal (1). Another question
concerns where the structural changes in hCG occur. These are questions
pertinent to understanding the mechanisms of the two-step hCG-receptor
interactions and signal generation. Our observations in the present
study shed some light on these issues. However, before discussing them,
it is helpful to review the structure of /AB-125I-ß.
NHS-AB can react to the five amino groups at the N terminus,
Lys2, Lys20, Lys104, and
Lys122 of the hCG ß-subunit (18). Based on the crystal
structure of hCG (23), it is possible to predict the
potential cross-linking activity of AB attached to each of the
amino groups. The reagents attached to the N-terminus and
Lys2 could cross-link the ß-subunit to the N-terminal
region of the -subunit since these two groups are adjacent (Fig. 7). The two N-terminal regions are,
however, located at the rear side of the putative receptor-binding
phase of the hormone. ßLys20 is in the ß-loop 1 that is
part of the putative receptor binding site and therefore, the AB
attached to ßLys20 could label the receptor. However,
ßLys20 is beyond the maximum labeling distance (7 Å)
from the -subunit, and its side chain projects away from the
-subunit. ßLys104 is in the seat belt that is near
-loop 2 and part of the putative receptor-binding site. This is
consistent with the results of the mutational analysis of
ßLys104 (24) and chemical modification (25). The AB
coupled to ßLys104 could label the -subunit or the
receptor. ßLys122 is in the C-terminal region, which is
unnecessary for the interaction with the -subunit and receptor.
Therefore, our data narrow the potential photoaffinity labels for the
receptor to those attached to ßLys20 and
ßLys104. On the other hand, the potential intersubunit
labels are the reagents attached to the ßN-terminus,
ßLys2, and ßLys104. Overall, our results
are consistent with previously reports on amino group labeling,
accessibility, and cross-linking as summarized by Gordon and
Ward (26).
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- and ß-subunits of a UV activable
derivative of hCG is 3-fold higher when the hCG derivative is free in
solution than bound to the receptor. In that study, hCG was
derivatized and reassociated with untreated hCGß. This suggests that
the interaction between the hCG subunits changes after binding to
the receptor. With regard to the structural change of hCG upon binding
to the receptor, the ß-intersubunit cross-link was 3-fold higher
when hCG was free than it was bound to the receptor (17). This
indicates that the interaction between the two subunits of hCG changed
after hCG bound to the receptor. However, it was unclear when the
change takes place, whether during the initial hCG contact with the
exodomain or during the secondary contact of the hCG/exodomain complex
with the endodomain. The affinity labeling data in this study (Table 1 and Figs. 2 and 4) show that the change
occurred during the initial interaction of hCG with the exodomain. For
example, up to 39% of /AB-125I-ß that was in solution
and not bound to receptor was cross-linked to produce the ß-dimer
(Table 1 and Fig. 2). Therefore, the ratio of cross-linked ß and
uncross-linked ß is 0.64. In contrast to these significant
intersubunit cross-links of free hCG, the intersubunit cross-link of
/AB-125I-ß that was bound to LHR295 was
significantly low. For example, the ratios of cross-linked ß and
uncross-linked ß of /AB-125I-ß bound to
LHR295 were 0.07–0.1 (Table 1). These results indicate a
significant difference in the interaction between the two subunits
before and after binding to LHR295. Therefore, there was a
change in the intersubunit interaction upon /AB-125I-ß
binding to LHR295. Furthermore, this change took place when
hCG bound the exodomain, i.e. before the secondary
interaction of the hCG/exodomain complex with the endodomain and
generation of a hormone signal.
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ß-subunits. |
MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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, hCGß, FSH, and
TSH were supplied by the National Hormone and Pituitary Program
(Baltimore, MD). Denatured hCG was prepared by boiling in 8
M urea, 2.7 M guanidine hydrochloride, and 100
mM DTT for 20 min.
Derivatization and Radioiodination of hCGß and Reconstitution
of Derivatized hCG (/AB-125I-ß
4055)
hCGß was derivatized with 4-azidobenzoic acid (AB) using the
N-hydroxysuccinimide ester of AB, radioiodinated, purified,
and reconstituted with untreated hCG as described previously (28).
Briefly, 33 µg of hCGß were dissolved in 40 µl of 10
mM Na2HPO4 (pH 7.4) and incubated
with 5.3 µl of 16.5 mM NHS-AB at 25 C for 60 min. The
resulting AB-ß was radioiodinated and fractionated on Sephadex G-50
superfine after which the AB-125I-ß fraction (1
x 108 cpm) was incubated with 30 µg of hCG and one
tablet of protease inhibitor cocktail (Roche Molecular Biochemicals, Nutley, NJ) with gentle mixing. The
resulting /AB-125I-ß was fractionated on Sephadex
G-100 superfine column (0.7 x 50 cm). The resulting hCG
derivative (/AB-125I-ß) was capable of binding the LHR
as untreated hCG did (28) and was used for photoaffinity labeling of
the LHR and LHR exodomains. Hormone binding to intact cells expressing
the wild-type LHR as well as solubilized LHR and exodomains was carried
out, and the data were analyzed as described above. Based on the
binding data and Scatchard analyses, receptor concentrations were
determined.
Photoaffinity Labeling of LHR Exodomains with
/AB-125I-ß
Solubilized LHR295, LHR336, and
wild-type LHR (120 µl) were separately placed in siliconized glass
tubes, incubated with 150,000 cpm of /AB-125I-ß at 4 C
for 16 h, irradiated with a MineralightR-52 UV lamp, solubilized
in 2% SDS and 100 mM DTT, and electrophoresed. Gels were
dried on filter paper, autoradiographed, and, in addition,
phosphoimaged on a model GS-525 Molecular Imager System Scanner
(Bio-Rad Laboratories, Inc., Richmond, CA) as described
previously (22).
Immunoprecipitation of Photoaffinity-Labeled Flag Exodomains
For immunoprecipitation of photoaffinity-labeled
LHR295 and LHR336, the Flag epitope was
inserted at the N terminus of mature exodomains (21). The resulting
Flag-LHR295 and Flag-LHR336 were incubated with
/AB-125I-ß and immunoprecipitated using monoclonal
anti-Flag antibody as described previously (21).
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FOOTNOTES |
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This work was supported by NIH Grants HD-18702 and DK-51469.
Received for publication November 25, 1998. Revision received May 6, 1999. Accepted for publication May 13, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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subunit and ß subunit. J
Biol Chem 250:5247–5258 and ß subunits of human
choriogonadotropin photoaffinity label the hormone receptor. Proc Natl
Acad Sci USA 78:5465–5469This article has been cited by other articles:
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