The placental hormone hCG
and the pituitary
gonadotropins hLH and hFSH are essential for vertebrate reproduction.
These hormones are heterodimers composed of a conserved
-subunit
and a hormone-specific
-subunit(1) . The crystal structure
of deglycosylated hCG (2) is likely to be representative of
each glycoprotein hormone. Both subunits are composed of three large
loops held together by a cysteine knot. Loops one and three of the
-subunit form a pair held together by a disulfide at one end of
the molecule; loop two is at the opposite end (Fig. 1). The
subunits of hCG are aligned such that paired loops from one subunit are
adjacent to the single loop of the other subunit.
-Subunit
residues 91-110 form an additional loop termed the ``seat
belt'' that wraps around the second loop of the
-subunit to
stabilize the heterodimer. The seat belt loop is primarily responsible
for the ability of the
-subunit to control receptor binding
specificity(3, 4) . While the amino acid sequences of
several gonadotropins(5, 6, 7, 8, 9, 10, 11, 12, 13) and
their receptors (14, 15, 16) have been known
for some time, little is known about the portions of the hormone that
are responsible for their abilities to bind tightly to their receptors.
Figure 1:
Ribbon diagram of hCG
-subunit folding pattern. The loops described in the text are
noted (dark blue). The oligosaccharides were added by modeling (red sticks). They are expected to be highly mobile at least
until the hormone binds to the receptor. A few key residues in three
major epitopes are illustrated here. Those in epitopes for
dimer-specific antibodies are illustrated in red. Antibodies
to these regions bind to hCG at the same time as antibodies to other
residues noted on the diagram. Arg
(orange label)
is located at the tip of the third loop and is a key residue in the
binding site of B105. Asn
(cyan blue) is located
nearby and is a key residue in the binding sites of antibodies B108 and
B112. Residues 108-114 (green label) are found near the
C terminus (C) and form the binding sites for B111. Note that
residues 112-114 are not seen in the crystal structure and are
not shown here. The upper and lower arrows noted for
the seat belt point to its N and C termini.
Many efforts to identify gonadotropin residues that contact the
receptor have employed hormone analogs that have lost all or part of
their receptor binding abilities. Gonadotropin analogs made by
chemical, enzymatic, and genetic
modifications(1, 3, 4, 17, 18, 19, 20, 21, 22, 23) have
shown that modification of several residues reduce receptor binding.
These could be involved in essential receptor contacts. Unfortunately
it is not always easy to interpret the results of this type of study
since mutagenesis can alter the structure of the hormone in an unknown
fashion. Since the free subunits do not bind to receptors(1) ,
any mutation that alters the interaction of the subunits has the
potential to disrupt binding.
Other investigators have made
extensive use of synthetic peptide pieces of the hormone to identify
receptor binding
sites(24, 25, 26, 27, 28, 29, 30) .
A peptide corresponding to the second loop of the hCG
-subunit has
the greatest activity albeit only at concentrations exceeding 0.01
mM(24) . However, this region of hCG can be changed to
the sequence found in the hFSH
-subunit without reducing its
ability to bind to LH receptors or to induce testosterone synthesis (3) . This observation suggests that the second
-subunit
loop does not participate in essential high affinity receptor contacts.
At the high concentrations needed for activity in studies employing
synthetic peptides (i.e. 0.01-10 mM), it is
often difficult to distinguish specific and nonspecific effects.
Interpretation of data obtained using synthetic peptides is usually
based on the assumption that the peptide binds to the receptor to block
its interaction with the labeled hormone. However, the peptide might
function by binding to the hormone and disrupting the interaction
between the
- and
-subunits. In the absence of data
indicating that the peptide has no effect on the hormone, conclusions
as to the binding site of the peptide may be misleading. Efforts to
make more active compounds by combining several peptides that have low
activity have been very disappointing(31, 32) .
The
only methods to directly identify hCG residues that contact LH
receptors involve use of x-ray crystallography and/or NMR spectroscopy.
However, is it not yet clear that crystals of the hormone-receptor
complex can be obtained that include the transmembrane domain. The
complex is likely to be too big for current NMR techniques. To surmount
these difficulties we have used a process of elimination to distinguish
portions of the
-subunit involved in key receptor contacts. This
involves (i) finding mutations that do not alter the ability of the
hormone to bind to receptors or elicit signal transduction and (ii)
identifying the binding sites of monoclonal antibodies that bind
receptor-bound hCG. Because the data obtained do not depend on analogs
that have altered receptor binding or signal transduction activity,
there is no risk that the results will be confounded by the influence
of a mutation on the conformation of the hormone or the interaction
between the subunits. Although this approach will not identify specific
receptor contacts, it can be used to orient the hormone in the receptor
and to delineate portions that are available to make high affinity
receptor contacts.
A few anti-hCG
-subunit antibodies to an
overlapping epitope have been reported to bind to hCG-receptor
complexes(33) . Using a series of hCG/hLH
-subunit
chimeras, we identified
-subunit Asn
as a key residue
in the binding sites for two of these antibodies(33) . This
approach was successful for identifying key residues in the binding
sites of antibodies that recognize hCG much better than hLH. However,
it could not be used with antibodies that had high affinities for both
human hormones. Using hCG/bLH and hCG/hFSH
-subunit chimeras we
have now identified additional
-subunit residues that form parts
of epitopes that are exposed in hCG-receptor complexes. When these
observations are combined with information on residues that can be
changed without disrupting hormone function and with the locations of
residues in the crystal structure of deglycosylated hCG(2) ,
they suggest that
-subunit residues in the first and third loops
adjacent to the second loop of the
-subunit are the parts of the
-subunit most likely to form key contacts with the LH receptor.
The conformation of this portion of the hormone is changed when
lutropins bind to LH receptors.
MATERIALS AND METHODS
Hormones
Highly purified hCG (CR119 (11600
IU/mg) and CR121 (13450 IU/mg)) were obtained from Dr. Robert Canfield,
Columbia University, New York, NY. The other species of vertebrate LH
and CG were all prepared (H.P.) essentially by methods previously
described (34, 35, 36) . These included
several mammalian lutropins (ovine, equine, bovine, porcine, canine,
leporine, rodent, and kangaroo), equine CG, three species of
non-mammalian lutropins (ostrich, turtle, and bullfrog), and two
non-mammalian follitropins (ostrich and bullfrog).
Antibodies
B410 was obtained from Dr. Janet Roser,
University of California, Davis, Davis, CA, and corresponds to antibody
518B7 that has been described previously(37) . The sources of
A113, B105, B111, and B112 (33) and the IODO-GEN procedure used
to radioiodinate these proteins have also been described
previously(38) . Radiolabeled antibodies had a specific
activity of approximately 50 µCi/µg (i.e. 3-4
molecules of iodine per molecule of antibody).
Construction of Hormone Analogs
The structures of
the bLH/hCG
-subunit chimeras and the other analogs used in this
study are illustrated in Table 1. Preparation of hCG/hFSH
chimeras has been described elsewhere(3, 4) .
Preparation of the bLH/hCG
-subunit chimeras began with vectors
pSVL-hCG
and pSVL-hCG
` that express hCG
- and
-subunits, respectively, and that have been previously
described(3) . The bLH
-subunit cDNA was obtained from Dr.
Scott Chappel (Ares Advanced Technology, Randolph MA). We prepared
pSVL-bLH
, a vector that expressed bLH
-subunit in COS-7
cells, by removing the cDNA from the source vector by HindIII
digestion, filling in the ends using the Klenow fragment of DNA
polymerase, and ligating the blunt-end fragment into the SmaI
site of pSVL (Pharmacia Biotech Inc.). Vectors which expressed hCG/bLH
-subunit chimeras CbL
36-145, CbL
1-35,
CbL
88-145, CbL
1-87, CbL
103-145, and
CbL
1-102 were constructed using three conserved restriction
sites (i.e. NaeI, PvuII, and PpuMI) present
in the hCG and bLH
-subunit cDNAs (Fig. 2). These enabled
us to scan the
-subunit to identify regions likely to contain the
B105 epitope. Analogs containing fewer hCG or bLH
-subunit
residues were prepared by polymerase chain reaction-directed
mutagenesis using a technique termed ``splicing by overlap
extension'' (37) and the primers shown in Table 2.
In this procedure the N-terminal (i.e. ``upstream'')
and C-terminal (i.e. ``downstream'') coding regions
of the desired analog were made in separate polymerase chain reactions
and then these polymerase chain reaction products were combined to give
the full-length construct.
Figure 2:
Schematic illustration of the
restriction site swapping that was used to prepare bLH and hCG
chimeras. Note the chimeras are named by their content of hCG
-subunit residues. Thus, CbL
1-35 represents a chimera
in which residues 1-35 are derived from hCG
-subunit and the
remainder are from bLH
-subunit.
DNA Isolation, Cell Culture, and
Transfections
Plasmid DNA was isolated by alkaline lysis and
purified by polyethylene glycol precipitation(39) . COS-7 cells
maintained in Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum and 2 mM glutamine
were co-transfected with pSVL-hCG
and the appropriate
-subunit coding vector using a calcium phosphate precipitation
procedure(40) . After a 24-h incubation at 37 °C the cell
monolayers were washed, and the media were replaced with fresh
serum-free Dulbecco's modified Eagle's medium. Three days
later, the media were collected, centrifuged to remove cellular debris,
and concentrated approximately 50-fold. This was accomplished by
placing the media into a dialysis bag and laying the bag in Aquacide II
(Calbiochem). Residual salts were removed by dialysis against 0.02 M phosphate buffer (pH 7.4).
Antibody Assays
To learn which vertebrate
gonadotropins bind B105, we employed a solid phase assay that measured
the ability of radiolabeled antibodies to bind hormone adsorbed to the
wells of a polystyrene microtiter plate. Wells were incubated with 1
µg of hormone diluted in 50 µl of 0.02 M sodium
phosphate, 0.9% NaCl solution (pH 7.2). This permitted the hormone to
adsorb to the surface. After 1 h at 37 °C, the solution was
replaced with 200 µl of the buffer containing 200 µg of bovine
serum albumin for an additional hour at 37 °C to block the
adsorption sites remaining on the plastic surface. Next, the albumin
solution was replaced with 50 µl of albumin solution containing
2-3 ng of radiolabeled B105 (approximately 50,000-150,000
cpm) to permit the B105 to bind to the adsorbed hormone. After a final
hour at 37 °C, the radiolabeled solution was aspirated, the wells
were rinsed with albumin containing buffer, the plates were cut apart
with scissors, and each well was analyzed in a gamma counter. For
controls we measured the binding of labeled antibody to wells that had
not been exposed to hormone and, in some cases, to wells that contained
an excess (1 µg) free hormone or unlabeled antibody added together
with the radiolabeled antibody. All three controls had the same level
of background.To quantify the hCG/bLH
-subunit chimeras made
in cell culture we used a monoclonal sandwich immunoassay procedure
that has been described previously (41) except that A113, an
antibody to the
-subunit of hCG, was used to capture the analogs,
and either radiolabeled B105 or B410 were used as detection reagents.
B410 is an antibody that binds hCG and bLH with high affinity (42) and was used to estimate the amount of dimer in the media.
The amounts of culture media that gave similar activity for the analogs
in the B410 sandwich assay were used to assess B105 binding.
Receptor Binding Assays
We employed a
bio-immunoradiometric assay (Bio-IRMA) to determine the position of the
antibody binding site relative to that of the receptor(43) .
This assay depends on the ability of a radiolabeled monoclonal antibody
to bind to hormone-receptor complexes and has been described in detail
elsewhere (43) . To detect differences in the affinity of
antibodies for free and receptor bound hormones we performed the assay
in a simultaneous fashion (i.e. the membrane bound receptors,
hormone, and radiolabeled B105 were mixed together at the start of the
experiment). After 1 h at 37° the reaction mixture was diluted to 2
ml with a 0.9% NaCl solution containing 1 mg of bovine serum
albumin/ml, and the membranes were pelleted by centrifugation at 1000
g. The supernatant was aspirated, and the radiolabel
remaining in the pellet was analyzed in a gamma counter. Bio-IRMA
designed to characterize the abilities of B105, B108, B111, and B112 to
bind to hCG-receptor complexes were also performed in a sequential
fashion. Briefly, we incubated homogenates of rat ovaries with amounts
of hCG that permit them to bind to LH receptors in the membranes. The
membranes were collected by sedimentation at 1000
g and resuspended in hormone-free buffer. Radiolabeled antibody was
added and permitted to bind to hormone-receptor complexes in the
membranes. Bound and free labels were separated by sedimenting the
membranes at 1000
g and the label bound to membrane
hormone-receptor complexes was analyzed in a gamma counter. Procedures for measuring the abilities of hormone analogs to bind to
LH receptors by competition with
I-hCG have been
described elsewhere(41) . Procedures for measuring the
abilities of the antibodies to inhibit binding of radioiodinated hCG to
cells expressing LH receptors or membranes from rat corpora lutea have
also been described earlier(41, 43, 44) .
Briefly, the antibody was incubated with hCG for 30-60 min prior
to addition of cells. After further incubation at 37 °C for 60 min,
the cells or membranes were collected by centrifugation and
radiolabeled hCG that had been bound was measured in a gamma counter.
As a control, we monitored the ability of unlabeled hCG to block the
binding of the radiolabeled hCG.
Model of hCG
A crystal structure of deglycosylated
hCG has recently been published (2) and the coordinates were
kindly given to us by Dr. Neil Isaacs. We added oligosaccharides to
this structure using the Sybyl modeling package (Tripos Associates,
Inc., St. Louis, MO). Following energy minimization and molecular
dynamics calculations in which the positions of the C
coordinates
were held constant, we arrived at a structure that had no bad contacts
and that should be reflective of glycosylated hCG.
RESULTS
The Most Conserved
-Subunit Epitope Does Not
Contact the Receptor
When these studies were started, we
expected the most conserved part of the lutropin
-subunit to be
nearest the LH receptor interface. This is because a large number of
lutropins are able to bind to rat LH receptors and stimulate
steroidogenesis. Surprisingly, antibodies that bound to several
different mammalian lutropins (i.e. B105, B110, and B410) also
bound to these hormones when they had bound to rat LH receptors,
indicating that a conserved epitope was exposed in the hormone-receptor
complex (see Fig. 3for data on B105, remainder of data not
shown). Studies were then designed to identify amino acid residues that
were part of this epitope. To accomplish this we focused on identifying
the binding site for antibody B105, an antibody that had high affinity
for hLH and hCG and very low affinity for most other mammalian
lutropins including those from cattle, rabbits, pigs, dogs, horses,
rats, and kangaroos (data not shown). Except for sea turtle LH, B105
did not bind to LH from most non-mammalian species including those from
ostriches or bullfrogs. B105 did not bind to hFSH, equine FSH, ovine
FSH, ostrich FSH, or bullfrog FSH. This indicated that the binding we
observed was specific to a conserved mammalian lutropin epitope that
remains exposed when these hormones bind to rat LH-receptor complexes.
Figure 3:
Simultaneous Bio-IRMA of hCG and several
mammalian LH. hCG and various mammalian LH were incubated with rat
luteal LH receptors and radiolabeled B105 in a simultaneous Bio-IRMA as
previously described(43) . Values shown are the amount
(count/min ± S.E.) of radiolabeled B105 that was bound to the
rat receptors in the presence of different amounts of hormone. The bars
above the arrows indicate values obtained in the presence of 0.01 ng of
hormone. The bars illustrated sequentially to the right of that marked
by the arrow refer to 0.1 ng (&cjs2090;), 1 ng (&cjs2089;), 10
ng (
), 100 ng (
), and 1000 ng (
) of hormone,
respectively. Neither FSH nor any of the non-mammalian hormones tested
gave measurable activity in this assay (not
shown).
To identify residues likely to be in the B105 epitope, we used a
homology scanning mutagenesis procedure that depended on measuring
antibody binding to chimeras of hCG and bLH
-subunits. The
-subunits of hCG and bLH were used to make the chimeras because
these hormones had high and low affinities for B105, respectively.
Thus, chimeras that bound B105 well would contain hCG residues in the
B105 epitope, respectively; whereas those that bound B105 poorly would
have bLH residues in the B105 epitope. As a control to determine the
amount of chimera made by the transfected COS-7 cells, we measured the
binding of the chimeras to B410, an antibody that had high affinity for
both hCG and bLH. B105 bound well only to those LH analogs that
contained Arg
(Table 3). This included hCG/bLH
-subunit chimeras that had hCG residues 36-145, 1-87,
and 1-102 (i.e. CbL
36-145,
CbL
1-87, and CbL
1-102). B105 did not bind to
hCG/bLH chimeras that contained bLH residues in these regions (i.e. CbL
1-35, CbL
88-145, and
CbL
103-145). An analog of hCG (hCG
:R74P) containing a
single substitution (i.e. Arg
was replaced by
Pro, the corresponding residue from bLH
-subunit) did not bind
B105 any better than bLH. Conversely, an analog of bLH (bLH
:P74R)
in which Pro
was converted to Arg bound B105 with the same
high affinity as hCG. This indicated that the conserved epitope was
formed by a region of hCG near Arg
, namely the third
-subunit loop(2) .
Other Surfaces of the
-Subunit Are also Exposed in
the hCG-Receptor Complex
We observed that several antibodies
bound to hCG-receptor complexes (see Table 4for a partial list).
Since antibodies are large proteins, this suggested that a substantial
percentage of the
-subunit surface was not obscured by the LH
receptor or other nearby proteins. To test this idea and to identify
those antibodies that bound to a different region of the
-subunit,
we mapped the binding sites of all the hCG antibodies relative to one
another (see Fig. 4for a partial map). These maps showed that
the exposed surface was large enough to be recognized by antibodies
that could bind to hCG at the same time. For example, antibodies B105,
B111, and B112 were all found to bind to hCG-receptor complexes (Table 4). B112 and B111 bound to hCG at the same time, yet each
competed for binding to hCG with B105. The B112 binding site has been
shown to involve Asn
(33) and we focused on
identifying residues that were involved in the B111 site.
Figure 4:
Epitope map of selected
-subunit
antibodies. Sandwich assays were performed in which hCG was captured
using each of the antibodies listed and detected using radiolabeled
B105, B108, B111, or B112. Epitopes for antibodies that bind to hCG at
the same time are shown in different or non-overlapping circles.
Epitopes for antibodies that do not bind hCG at the same time are shown
in the same or overlapping circle.
To
identify key residues in the B111 binding site, we used a series of
hCG/hFSH chimeras that contained hCG
-subunit residues 1-93
and varying numbers of hFSH residues between amino acids 94 and 145.
While all of these were capable of binding B105, only a few were able
to bind to B111 (Table 5). Comparisons of the sequences of the
analogs (Table 1) with their abilities to bind B111 (Table 5) showed that the B111 binding site involved residues
108-114. For example, B111 did not bind CFC94-114 and had
only minimal ability to bind to CFC101-114, suggesting that
substitution of FSH residues in place of hCG
-subunit residues
94-114 or 101-114 destroyed the B111 binding site. B111
bound CFC101-106 like hCG and much better than
CF101-109
114 and hCG
G103V
114. Because residue 107
is conserved in hCG and the hCG/hFSH
-subunit chimeras, these
observations suggest that residues 108-114 account for most of
the B111 binding site. Residues C-terminal of 114 also appear to have a
small influence on B111 binding since B111 had reduced affinity for
analogs that were truncated at residue 114 (i.e. CF101-109
114 and hCG
G102V
114). Because B111
binds to hCG-receptor complexes and recognizes residues 108-114,
we conclude that the region of hCG that includes the C-terminal portion
of the seat-belt loop does not contact the LH receptor.
Antibodies That Bind to hCG-Receptor Complexes Inhibit
hCG Binding to Receptors
We observed that most antibodies that
bind to hCG-receptor complexes can also inhibit binding of hCG to rat
LH receptors (Fig. 5). This suggests that antibodies can inhibit
receptor binding even though they recognize epitopes that are not part
of the high affinity hormone binding site. Most of these block binding
only partially, yet those that appear to bind closest to the part of
the hormone that interacts with the receptor (e.g. B108 and
B112) block receptor binding completely. Not all antibodies bind to
hCG-receptor complexes. Key residues in the binding sites of many
antibodies that do not bind to hCG-receptor complexes are known (33) , and we had hoped that these data would provide
additional information on portions of the
-subunit likely to
contact LH receptors. Most antibodies that do not bind to hCG-receptor
complexes recognize part of the second loop and residues 91-94 of
the seat belt. They bind to hCG at the same time as B105 (Fig. 4) and are good inhibitors of hCG receptor binding. While
these observations do not prove that these portions of the
-subunit contact the receptor, they are consistent with the idea
that the second loop and the N-terminal part of the seat belt are near
the hCG receptor interface.
Figure 5:
Comparison of the abilities of antibodies
B105, B108, B111, and B112 to inhibit binding of hCG to rat LH
receptors. The antibodies were added to
I-hCG prior to
adding the radiolabel to CHO cells expressing rat LH receptors or to
rat luteal membranes. Values are means of triplicates and the bars extend to the limit of the S.E.
The Conformation of the
-Subunit Is Altered when hCG
Binds to LH Receptors
Using a Bio-IRMA(43) , we found
that the affinity of B105 for most non-human mammalian lutropins was
increased when they were complexed with rat LH receptors (Fig. 3). While we did not measure this increase in affinity
directly, it must have been substantial because the assay was performed
by incubating the hormone, membrane-bound rat LH receptors, and
radiolabeled antibody B105 in the same tube. In this format the
antibody can bind to free hormone as well as to hormone-receptor
complex; the receptor can bind to free hormone or antibody-hormone
complex. When less than 4 ng of hCG (i.e. 0.1 pmol) were used
in the assay, the amount of radiolabeled B105 bound to the
hormone-receptor complex was proportional to the amount of hCG bound to
the receptor. When a higher amount of hCG was used in the assay, the
amount of hCG bound to both the antibody and the receptor was also
increased, but binding of labeled B105 to hormone-receptor complexes
was reduced. This is because B105-hCG-complexes are unable to bind to
LH receptors that are already occupied by hCG. We estimated the
magnitude of the increase in affinity of B105 for the receptor-bound
hormone by comparing the binding of B105 to hCG- and lutropin-receptor
complexes when large amounts of the hormones were used in the assays.
In the assays illustrated in Fig. 3there was a massive excess
of hormone relative to receptor when 1 µg of hormone (i.e. 25 pmol of hCG or 30 pmol of lutropin) was mixed with 0.1 pmol of
receptors and 0.01-0.02 pmol of radioiodinated B105. When 1
µg of hCG was used in this assay, the B105 and the receptors were
almost fully occupied by hCG, and less than 0.3% of the radiolabeled
B105 became bound to the hormone-receptor complex. In contrast, when 1
µg of any non-human mammalian LH was mixed with receptors and B105,
we observed that 7-10% of the B105 became bound to
hormone-receptor complexes. By dividing the percentage of B105 bound
when 1 µg (30 pmol) of non-human mammalian LH was used in the assay
by that percentage observed when 1 µg (25 pmol) of hCG was used in
the assay, it can be seen that the affinity of B105 for
receptor-complexes containing any non-human lutropin was at least
20-fold greater than that for the free hormone, and probably much more.
This strongly suggests that binding of bLH and other non-human
lutropins to receptor complexes is accompanied by a change in the
molecule that reduces the inhibitory influence of Pro
on
B105 binding to mammalian lutropins.Unexpectedly, eCG was much less
active than eLH in the Bio-IRMA (Fig. 6). This was surprising
since (i) both eLH and eCG have the same primary amino acid
sequences(45) , (ii) both can be recognized by B105 (not
shown), and (iii) both bind to LH receptors (Fig. 5). The only
difference between eLH and eCG is the extent and type of their
glycosylation. This observation suggests that the oligosaccharides of
eCG block binding of B105 to eCG, but only when the hormone has bound
to LH receptors.
Figure 6:
Panel A, inhibition by hCG, eLH and eCG of
radiolabeled hCG binding to the rat luteal receptor. All the other
mammalian LH (not illustrated) used in these studies had activities
equal to or lower than that of eLH. Panel B, activities of
hCG, eLH, and eCG in the sequential Bio-IRMA. See the text and Moyle et al.(43) for the details of this assay. Values are
means of duplicates and the bars extend to the individual
measurements.
DISCUSSION
The Binding Sites Reported Here Are Consistent with the
Crystal Structure of Deglycosylated hCG and with the Epitope Map
A summary of the antibody binding site data described here is
shown in Table 6. Use of this approach to identify portions of
hCG that are exposed in hormone-receptor complexes depends on the
correct identification of antibody binding sites. The consistency of
the data with the crystal structure of hCG provides strong support for
the conclusions that we have drawn about the identities of
-subunit regions that are recognized by antibodies and the
proposed orientation of the antibody binding sites. The idea that
Arg
is an important component of the B105 epitope agrees
with the observations that all bLH/hCG chimeras containing Arg at
residue 74 bound B105 well and that all chimeras containing Pro at this
site bound B105 poorly. However, given that antibody combining sites
are usually much larger(46) , other residues including those
common to both bLH and hCG
-subunits are likely to be involved in
the B105 epitope. While we did not attempt to identify other residues
in the B105 site, we observed that B105 competed for binding to hCG
with B111 and B112, two antibodies that can bind to hCG at the same
time. Thus, the surface recognized by B105 would be expected to lie
between those recognized by B111 and B112. This is consistent with the
crystal structure of chemically deglycosylated hCG and the location of
the key residues in the B105, B111, and B112 binding sites(2) .
Substantial Portions of the hCG
-Subunit Face Away
from the Receptor Interface after hCG Binds to LH Receptors
Epitope maps suggest that most anti-
-subunit antibodies
bind to one of three regions in hCG. The first includes the second loop
and the N-terminal part of the seat belt(33) . Most antibodies
to this region (i.e. sites for B101, B107, and B109) bind to
hCG much better than to the free
-subunit. The second epitope
region includes the binding sites for antibodies that recognize
epitopes in loops one and three (i.e. the binding sites for
antibodies B105, B108, B110, B111, B112, and B410). The third region
includes the binding site for antibodies that recognize the C-terminal
part of the seat belt loop (i.e. antibody B111). Surprisingly,
two of these three epitope regions can be detected in hCG-receptor
complexes. Antibodies are large proteins and the observation that at
least two of the three major
-subunit epitope regions can be
detected after hCG binds to LH receptors implies that only a small
fraction of the
-subunit surface is available to participate in
essential high affinity contacts with the LH receptor.All of the
-subunit antibodies that bound to hCG-receptor complexes inhibited
hCG binding, albeit to different degrees. For example, B108 and B112
inhibited hCG binding considerably better than antibodies B105 and
B111. This was related to the surface of the
-subunit to which
they bind. The surface recognized by B108 and B112 is closer to the
-subunit interface than that recognized by either B105 or
B111(2) . Thus, binding of B108 and B112 may be near the
hormone-receptor interface or in the pathway taken by the hormone on
its way to contacting the receptor.
Parts of the
-Subunit That Contact LH Receptors
Our goal was to distinguish portions of hCG
-subunit
that do not contact the LH receptor and to combine this information
with the crystal structure to identify regions that would be available
to interact with the receptor. Portions of the
-subunit noted
earlier that can be recognized by antibodies that bind to hCG-receptor
complexes clearly do not contact LH receptors. However, this strategy
can detect only those portions of the hormone for which we have
antibodies and which are likely to be oriented away from the receptor.
Other parts of the
-subunit near the receptor interface that do
not form contacts needed for high affinity binding can be deduced by
comparing the abilities of mammalian lutropins or mutant analogs to
bind rat LH receptors.
Second
-Subunit Loop (Residues
38-57)
Initially, we thought that the second loop would
contact the receptor and control high affinity binding. The idea that
residues 38-57 contact the receptor was supported by the
observations that peptides from this area can inhibit hCG binding to LH
receptors (24) and by our observation that antibodies
B101(41) , B107, or B109 (
)do not bind to
hCG-receptor complexes. These antibodies recognize the second
-subunit loop and/or residues 91-94 in the
seat-belt(33, 47) . Thus, we were astonished to find
that analogs of hCG in which
-subunit residues were replaced with
the corresponding residues from hFSH
-subunit bound to LH
receptors and elicited signal transduction indistinguishably from
hCG(3) . Only Thr
, Val
, and
Gln
are common to this region of hCG and hFSH. While it is
conceivable that these
-subunit sequences are responsible for high
affinity binding of both hCG and FSH, this seems unlikely. The
abilities of the synthetic peptides to bind LH receptors may be
explained by the observation that they were active only at
superpharmacological concentrations (i.e. >0.01
mM). Our failure to detect B101, B107, or B109 binding to
receptor bound hCG is consistent with the idea that portions of the
second loop and the seat belt are obscured by the LH receptor even
though this region does not appear to make key receptor contacts.
Seat Belt
Because it is responsible for
gonadotropin receptor binding specificity(4) , the seat belt (i.e. residues 91-110) would be expected to contact the
LH receptor. Residues 91 and 92 are unlikely to participate in LH
receptor binding since they differ in hCG, hLH, and in many naturally
occurring analogs that have high affinity for the LH receptor (Table 1). Residues between Cys
and Cys
are also unlikely to contact LH receptors since they can be
changed to the sequence found in FSH
-subunit without disrupting
LH receptor binding or efficacy(4) . The remaining seat belt
region between Cys
and Cys
, termed the
``determinant'' loop, has been proposed to have a role in LH
receptor binding(48) . However, with the exception of residue
Cys
, Asp
, and Cys
, this region
is not well conserved among most lutropins that bind to the rat LH
receptor. Indeed, we have found that most hCG analogs that have at
least one positive charge between residue 94-96, either Ser or
Thr for residues 97 and 98, and Asp
have high affinity for
LH receptors(4) .
The role of Asp
in
receptor contact cannot be determined since mutations of this residue
reduced hCG function(21) . Loss of function mutations have
proven valuable for identifying receptor contacts in growth
hormone(49, 50) . However, these are of limited use in
interpreting the interaction of gonadotropins with their receptors.
Unlike growth hormone, gonadotropins are heterodimers whose activity
depends on the correct interaction of both subunits with one another
and/or with the receptor. Thus, loss of function caused by a mutation
could be due to an effect on a receptor contact or to an influence on
the interaction between the subunits needed for hormone activity.
Therefore, in the absence of data showing that a mutation of Asp
does not alter the interaction between the subunits, conclusions
about its role in receptor contacts are premature.
N Terminus of the
-Subunit
Deletion of the N
terminus of the
-subunit strongly inhibits its ability to combine
with the
-subunit(51) . However, the small amount of dimer
that does form is biologically active, suggesting that this region does
not contact the receptor. This observation also suggests that the
ability of a
-subunit mutant to combine with the
-subunit
conveys little about the conformation of the subunits in the
heterodimer and, by itself, is not sufficient to prove that the
conformation of the heterodimer has not been altered by a specific
mutation.
Portions of the Twin Loops Containing the N-Linked
Oligosaccharides
Another portion of the
-subunit unlikely
to form specific contacts with the LH receptor is the region that
contains N-linked oligosaccharides at residues Asn
and Asn
. These can be removed without reducing the
affinity of the resulting analog for the LH receptor(52) .
Indeed, removal of the oligosaccharides is usually associated with an
increase in affinity. The influence of the oligosaccharides is limited
to hormone efficacy, an observation that is explained by the model of
hCG-receptor interaction in a companion study(53) .
The Remaining Region of hCG
-Subunit Is the Portion
of the Protein in Loops One and Three That Face
-Subunit Loop
Two
Models describing the interaction of the gonadotropin
-subunit and the receptor need to account for the observations
that a substantial portion of the
-subunit of receptor-bound hCG
can be recognized by monoclonal antibodies and that most residues in
the remainder of the molecule can be changed without disrupting high
affinity LH receptor binding or signal transduction. The regions that
remain include residues of loops one and three that face
-subunit
loop two,
-subunit residues that are moderately conserved in most
mammalian glycoprotein hormones.
The Conformation of the Third
-Subunit Loop Is
Changed after hCG Binds to LH Receptors
The observation that B105 binds better to most mammalian
lutropins when they are bound to LH receptors suggests that the
conformations of the hormones are altered after they bind to receptors.
This finding is consistent with the idea that portions of the
-subunit loops one and three nearest the
-subunit interface
participate in receptor binding. To accommodate the receptor, loops one
and three may need to be moved slightly. Given the internal hydrogen
bonds and the disulfide between Cys
and Cys
,
a slight movement of residues in the surface of these loops nearest the
-subunit could be transmitted to the antibody binding sites found
on the ``backside'' of these same loops.Other features of
the lutropins also appear to be changed when they bind to LH receptors.
Unlike the binding of B105 to most mammalian lutropins, the affinity of
B105 for hCG-receptor complexes was lower than that for
hCG(44) . Since hCG contains a long C-terminal extension, the
reduction in affinity might be caused by a change in the position of
the C-terminal extension of hCG after receptor binding. However, other
observations argue against this idea. As shown here (Fig. 3)
B105 bound to eLH-receptor complexes better than to eLH, yet B105 bound
to eCG much better than to eCG-receptor complexes (Fig. 6). This
implies that the N-linked or O-linked
oligosaccharides are more important than the C-terminal extension. Both
eLH and eCG have the same amino acid sequence and contain a C-terminal
extension like hCG. The hormones differ primarily in their
oligosaccharides. The N-linked oligosaccharides of eCG are di-
and tri-branched and contain large amounts of sialic acid whereas those
on eLH are mono- or di-branched and more often sulfated than
sialylated(54) . Thus, the N-linked sugars on eCG are
considerably bulkier than those on eLH and might interfere with the
binding of B105 to eCG LH-receptor complexes. We have proposed a model
of hCG receptor interaction in which the oligosaccharides influence
signal transduction by their bulk (53) . In this model the
positions of the N-linked oligosaccharides would be expected
to be different in the free hormone and in the receptor bound hormone.
If the position of oligosaccharides were constrained such that they
were directed more toward the B105 binding site when the hormone bound
to the receptor, the increased sizes of the oligosaccharides in eCG
might interfere with B105 binding. This would account for the ability
of B105 to bind to receptor complexes that contain eLH but not the
bulkier eCG.
In conclusion, these observations suggest that the
portions of the
-subunit most likely to contact the LH receptor
are nearest its interface with the second
-subunit loop. This is
in a region of the
-subunit that appears to have a different
conformation in receptor-bound hormone. Most other regions of the
-subunit do not appear to make specific high affinity hormone
contacts, an important consideration in the model of hormone-induced
signal transduction proposed in a companion study(53) .