(Received for publication, January 17, 1996)
From the
Human choriogonadotropin (hCG) consists of an subunit and
a
subunit. The existing evidence from various studies using
truncation, substitution, synthetic hormone peptides, and hCG crystals
suggests that the C-terminal region of the
subunit contacts the
luteinizing hormone/choriogonoadotropin (LH/CG) receptor and is
involved in receptor activation. Despite a deluge of the speculation
and the important role of the
C-terminal region, direct evidence
for its interaction with the receptor has been elusive. Because of the
significant biological activity, it is imperative to prove the
interaction of the
C-terminal region. For this purpose, decamer
peptides corresponding to the
subunit sequence from His
to Ser
(
) were
derivatized with the N-hydroxysuccinimide ester of
4-azidobenzoylglycine (ABG) and radioiodinated. The resulting
ABG-
I-
was capable of binding
and activating the LH/CG receptor. Furthermore, UV-sensitive
ABG-
I-
exclusively
photoaffinity-labeled an
86-kDa molecule. This labeled molecule
was shown to be the LH/CG receptor by various methods including
immunoprecipitation by anti-LH/CG receptor antiserum. In addition,
evidence is presented that the amino group of
Lys
of
is in such close proximity to a carboxyl
group of the receptor that this pair is cross-linked to form an amide,
a zero length cross-link. This low affinity contact of
and the receptor is sufficient for receptor
activation and is crucial for the full understanding of the
mechanistics of the receptor activation steps.
hCG ()is a placental hormone and is involved in
maintenance of the corpus luteum during pregnancy in human females. It
is a member of the glycoprotein hormone family which includes LH, FSH,
TSH and equine choriogonadotropin. These hormones consist of a common
subunit and a distinct
subunit which are noncovalently
associated(1) .
Photoaffinity labeling studies demonstrated
that both and
subunits affinity-labeled the LH/CG
receptor(2) . The truncation or substitution of hCG
C-terminal amino acid residues reduces the receptor-binding affinity
and abolishes cAMP
induction(3, 4, 5, 6) . This is
consistent with the observation that a dodecamer peptide corresponding
to the hCG
C-terminal region,
,
inhibited
I-hCG binding to the receptor (7) and
that a decamer peptide,
, was capable of
binding to cells possessing the LH/CG receptor and inducing cAMP
synthesis(6) . Also, the crystal structure of HF treated hCG
suggests the
C-terminal region as part of the potential receptor
binding site(8, 9) . Recently, a study using a set of
reciprocal mutants of hCG and the LH/CG receptor,
hCG
As shown in Fig. 1, I-
,
ABG-
I-
and
I-NAc/CONH
-
bound
to the LH/CG receptor on 293
cells with K
values of 126 µM (p <
0.05), 264 µM (p < 0.05) and 253 µM (p < 0.05), respectively. They were capable of
inducing cAMP synthesis with EC
values of 86 µM (p < 0.05), 135 µM (p < 0.05)
and 100 µM, respectively. These results demonstrate the
bioactivity of the peptide and its derivatives. To determine the
identity of their binding molecule, increasing numbers of
293
cells were incubated with
I-ABG-
, irradiated with xenon
flashes, and electrophoresed. The autoradiogram in Fig. 2shows
free
I-ABG-
which was
originally bound to 293
cells but dissociated from
them during solubilization in sodium dodecylsulfate. In addition to the
free
I-ABG-
band, a
86-kDa band was labeled by
I-ABG-
. The intensity of bound
I-ABG-
and the labeled
86-kDa band increased in parallel with increasing numbers of
293
cells. In contrast, the band was absent in the
sample lacking 293
cells. In addition to this
293
cell dependence, the formation of the
86-kDa
band requires photolysis as it is dependent on the number of xenon
flashes and
I-ABG-
bound to
293
cells failed to produce the
86-kDa band when
the sample was not photolyzed. These results suggest that the
radioactive
86-kDa band was generated by the photoactivation of
I-ABG-
and photoaffinity
labeling of a
86-kDa molecule. The band was not produced by
nonspecific association of
I-ABG-
with a
86-kDa molecule. Furthermore, the data indicate that
I-ABG-
exclusively labeled a
86-kDa molecule on 293 cells expressing the LH/CG receptor. As
expected, the size of the
86-kDa band material corresponds to the
mature 85-86-kDa LH/CG receptor(13, 14) .
Figure 1:
Biological activities of
, ABG-
and
NAc/CONH
-
. For the receptor
binding assay, cells expressing the LH/CG receptor
(293
) were incubated with a constant amount of
I-
,
I-ABG-
or
I-NAc/CONH
-
in the
presence of increasing concentrations of
.
After washing cells, the cell-bound radioactivity was measured. The
data were used in Scatchard plots to determine K
values. For cAMP induction, cells expressing the LH/CG
receptor were incubated with increasing concentrations of
, ABG-
or
NAc/CONH
-
and intracellular cAMP
concentrations were determined. These experiments were repeated three
times, each in triplicate. The data were analyzed using Student's t test.
Figure 2:
Photoaffinity labeling. Increasing
concentrations of 293 cells were incubated with
I-ABG-
and irradiated with
xenon flashes. Cells were solubilized and electrophoresed. The gel was
exposed to x-ray film. The lower band represents
I-ABG-
which was originally
bound to 293
cells but dissociated during
solubilization in sodium dodecyl sulfate. The
86-kDa band
represents the photoaffinity labeled band
material.
Fig. 3demonstrates the specificity of the photoaffinity
labeling. I-ABG-
did not bind
to mock transfected (293
) cells lacking the LH/CG
receptor and did not form the
86-kDa band even after UV
photolysis. This result indicates the requirement of the LH/CG receptor
for
I-ABG-
binding and
labeling the
86-kDa band. Unlabeled hCG or
, but not FSH or TSH, blocked
I-ABG-
binding to
293
cells, another indication of the specificity of
I-ABG-
binding to the LH/CG
receptor. Furthermore,
or
failed to block
I-ABG-
binding to
293
cells. Since
(7) and
(15) inhibit
I-hCG binding to the LH/CG receptor, this result suggests
that there is an exclusive and specific binding site for
I-ABG-
in the LH/CG receptor.
Figure 3:
Specificity of photoaffinity labeling.
Cells expressing (293) or lacking
(293
) the LH/CG receptor were incubated with
I-ABG-
or
I-
in the presence or absence
of 1000
concentrations of competitors. After irradiation with
15 xenon flashes, cells were solubilized and electrophoresed. The gel
was exposed to x-ray film.
Taken together these results clearly indicate I-ABG-
binding to the LH/CG
receptor. Therefore, they suggest that the
86-kDa band represents
photoaffinity labeled LH/CG receptors. To obtain more direct evidence
for this conclusion, the photoaffinity labeled band material was
immunoprecipitated using rabbit anti-LH/CG receptor antiserum (Fig. 4). When 293
cells were incubated with
I-ABG-
, photolyzed,
solubilized in Triton X-100, and immunoprecipitated with anti-LH/CG
receptor antiserum, the
86-kDa-band was precipitated (Fig. 4). However, it was not precipitated when normal rabbit
sera was used, when 293
cells were processed in the
absence of
I-ABG-
, or when
293
cells were used instead of 293
cells. These results, along with the photoaffinity labeling data
in Fig. 1Fig. 2Fig. 3, positively identify the
photoaffinity-labeled
86-kDa band as the LH/CG receptor.
Figure 4:
Immunoprecipitation of affinity-labeled
LH/CG receptor. Cells, 293 or 293
,
were incubated with
I-ABG-
,
washed and irradiated with 15 xenon flashes. Cells were treated with
Triton X-100 solution and the detergent extracts were incubated with
rabbit anti-LH/CG receptor or normal rabbit sera for
immunoprecipitation.
I-ABG-
has two amino groups
which could have been derivatized with ABG. They are the amino group of
Lys
and the N-terminal amine. Our data cannot specify
whether or not the amino group of Lys
was involved in the
photoaffinity labeling of the receptor. Furthermore, it is difficult to
predict the chemical group(s) of the receptor which was labeled as the
phenyl nitrene reacts nonspecifically with a variety of functionalities (16) . Since this information is necessary to define the
cross-linked point of the peptide and the receptor, we have synthesized
I-NAc/CONH
-
of
which the N-terminal amine of
was
acetylated and the C-terminal carboxylate was amidated. This modified
peptide was used for affinity labeling the receptor. Transiently
transfected 293
cells were incubated with
I-NAc/CONH
-
and
washed to remove unbound peptide. The cells complexed with
I-NAc/CONH
-
were
treated with EDC and solubilized for gel electrophoresis. The sample
treated with EDC showed the
86-kDa band of the receptor which was
labeled with
I-NAc/CONH
-
(Fig. 5). FSH or TSH did not prevent binding and
cross-linking of
I-NAc/CONH
-
. On
the other hand, hCG or unlabeled
blocked
binding of
I-NAc/CONH
-
to the cells. Also
I-NAc/CONH
-
did
not bind to 293
(data not shown). These results
indicate the specificity of
I-NAc/CONH
-
binding to the LH/CG receptor. Carbodiimides including EDC react
with carboxyl groups to produce O-acylisoureas which in turn
react with amino groups to produce amide bonds(16) . Therefore,
the primary reactive group of
I-NAc/CONH
-
with
EDC is the amino group of Lys
. To verify this reaction
specificity the cells complexed with
I-NAc/CONH
-
were
treated with the N-hydroxysuccinimde ester of caproic acid which
monofunctionally reacts with amino group(13) . As expected, it
prevented cross-linking of
I-NAc/CONH
-
to the
receptor as did acetate. These data indicate that the cross-linking
reaction of EDC involves both amino and carboxyl groups, consistent
with the reaction specificity of carbodiimides(16) .
Carbodiimides may also react with phenolic groups of Tyr and
sulfhydryls of Cys. These reactions are, however, monofunctional and
therefore, do not produce cross-linked products(17) . Our data
indicate that the amino group of
I-NAc/CONH
-
is
cross-linked to a carboxyl group to form an amide. They suggest that
the amino group and side chain of hCG
Lys
is in the
proximity of a carboxyl group of the receptor in the hormone receptor
complex. EDC induced a zero length cross-link between these two counter
ions suggests the existence of a salt bridge between them. This
conclusion is consistent with the complementarity of hCG
Lys
and LH/CG receptor's
Asp
(10) .
Figure 5:
Affinity cross-linking. Cells,
293, were incubated with
I-NAc/CONH
-
in the
presence or absence of 1000
concentrations of FSH or TSH. After
washing cells were treated with EDC, solubilized and electrophoresed.
The gel was exposed to x-ray film. As controls, cells complexed with
I-NAc/CONH
-
were
treated with EDC in the presence of sodium acetate, a carboxyl specific
competitor for EDC induced cross-link, or the N-hydroxysuccinimide of caproic acid, a monofunctional reagent
specific to amino groups. The cells did not bind
I-NAc/CONH
-
in the
presence of hCG or unlabeled
. Also
293
cells did not bind
I-NAc/CONH
-
. Since
these sample lanes were blank, the data are not
shown.
The binding and labeling site of I-ABG-
appears to be specific
since it was blocked by
, but not by two
other peptides,
and
which are known to inhibit
I-hCG binding to the
receptor. This is consistent with the x-ray crystal structure of HF
treated hCG(8, 9) . In the crystal, the
C
terminus shows a structure up to
Tyr
. The last three
residues of the
C terminus,
His
-Lys
-Ser
does not show a
structure, despite their presence in an open
5 nm solvent channel.
This result suggests that
His
-Lys
-Ser
are flexible.
It is interesting to speculate the
C-terminal positions up to
Tyr
, in relationship with
and
.
is
part of a loop in the hCG crystal and
Met
, the
residue closest to
Tyr
and the
C-terminal
peptide backbone, is 16.5 Å away from
Tyr
.
Therefore, it is not expected for
to
compete with
I-ABG-
for
receptor binding, consistent with the data in Fig. 3. On the
other hand,
is closer to
Tyr
,
Gln
being 8.6 Å away
from
Tyr
. Therefore, it may be possible for
to weakly interfere with, although may not
completely block, binding of
I-ABG-
to the receptor. In fact, the intensity of the photoaffinity
labeled
86-kDa-band is somewhat reduced in the presence of
in Fig. 3.
In this study we
demonstrate that the specifically interacts
with the LH/CG receptor. In this complex of
and the receptor, the amino group of hCG
Lys
is
near to a carboxyl group of the receptor to form an ion pair. This
interaction between the peptide and the receptor is sufficient to
activate the receptor to induce cAMP synthesis as does the hormone
itself.