(Received for publication, April 17, 1995; and in revised form, May 12, 1995)
From the
The goal of these studies was to devise a model that explains
how human chorionic gonadotropin (hCG) interacts with lutropin (LH)
receptors to elicit a hormone signal. Here we show that -subunit
residues near the N terminus, the exposed surface of the cysteine knot,
and portions of the first and third loops most distant from the
-subunit interface were recognized by antibodies that bound to
hCG-receptor complexes. These observations were combined with similar
data obtained for the
-subunit (Cosowsky, L., Rao, S. N. V.,
Macdonald, G. J., Papkoff, H., Campbell, R. K., and Moyle, W. R.(1995) J. Biol. Chem. 270, 20011-20019), information on
residues of hCG that can be changed without disrupting hormone
function, the crystal structure of deglycosylated hCG, and the crystal
structure of a leucine-repeat protein to devise a model of hCG-receptor
interaction. This model suggests that the extracellular domain of the
LH receptor is ``U-'' or ``J''-shaped and makes
several contacts with the transmembrane domain. High affinity hormone
binding results from interactions between residues in the curved
portion of the extracellular domain of the receptor and the groove in
the hormone formed by the apposition of the second
-subunit loop
and the first and third
-subunit loops. Most of the remainder of
the hormone is found in the large space between the arms of the
extracellular domain and makes few, if any, additional specific
contacts with the receptor needed for high affinity binding. Signal
transduction is caused by steric or other influences of the hormone on
the distance between the arms of the extracellular domain, an effect
augmented by the oligosaccharides. Because the extracellular domain is
coupled at multiple sites to the transmembrane domain, the change in
conformation of the extracellular domain is relayed to the
transmembrane domain and subsequently to the cytoplasmic surface of the
plasma membrane. While the model does not require the hormone to
contact the transmembrane domain to initiate signal transduction, small
portions of both subunits may be near the transmembrane domain and
assist in initiating the hormonal signal. This is the first model that
is consistent with all known information on the activity of the
gonadotropins including the amounts of the hormone that are exposed in
the hormone-receptor complex, the apparent lack of specific contacts
between much of the hormone and the receptor, and the roles of the
oligosaccharides in signal transduction. This model differs from
existing models of hormone action in that the extracellular domain has
a much larger role in hormone action than serving as a high affinity
hormone trap.
The glycoprotein hormones form a family of structurally related
trophic factors that regulate the gonads and the thyroid(1) .
In humans these include the placental hormone hCG ()and the
pituitary hormones hLH, hFSH, and human thyroid-stimulating hormone.
Each is an
heterodimer containing a common
-subunit and
a hormone-specific
-subunit. Hormone function is initiated by
binding of the hormones to a plasma membrane receptor that contains a
large extracellular domain and a plasma membrane domain composed of
seven hydrophobic
-helices(2, 3) . This leads to
activation of G-proteins and subsequent production of second
messengers.
The mechanism by which hormone-receptor interaction leads to signal transduction is not known. In a widely assumed model of hCG-receptor interaction that we term the ``tether'' model, hCG binds with high affinity to the extracellular domain of the LH receptor(4) . Because the extracellular domain is tethered to the transmembrane domain, this brings the hormone close to the transmembrane domain. Signal transduction starts when a second site on the hormone binds to the transmembrane domain. Support for this model is based on the observations that the extracellular domain has high affinity for hCG (4, 5, 6, 7) and the report that hCG can elicit signal transduction from an LH receptor analog containing only the transmembrane domain(7, 8) . The tether model predicts that one large or two smaller regions of hCG on at least two faces of the protein would interact with the receptor. The portions of hCG that interact with the LH receptor are unknown.
One way to test the model
would be to identify portions of the hormone that contact specific
parts of the receptor. Most putative contact sites have been identified
by scanning the hormone to find amino acid substitutions that cause a
loss in hormone function or by measuring the abilities of synthetic
hormone peptides to block hormone activity (9, 10, 11) . As noted in a companion
study(53) , the complexity of these hormones may confound
interpreting the results of these studies. For example, truncation of
the -subunit at residue 87 leads to a hormone analog with very low
affinity for the LH receptor, suggesting that this region may be
involved in receptor contacts(1, 12) . However, as
will be shown here, this analog is recognized better than hCG by
antibodies that have higher affinities for the free
-subunit than
for hCG. This implies that the mutation has altered the interaction
between the subunits and that it will be impossible to distinguish
effects on hormone conformation from those on receptor binding without
using high resolution techniques such as x-ray crystallography or NMR
spectroscopy.
Another way to test the tether model is to identify portions of the hormone that are exposed in the hormone-receptor complex. We have used this approach because it does not depend on loss of function mutants and provides data that are more easily interpreted. The tether model predicts that more than one surface of hCG would be hidden in the hormone-receptor complex. Thus, it can be tested by scanning the surface of the hormone to find regions that are exposed or that are hidden in the hormone-receptor complex. This can be accomplished by identifying hormone epitopes that are recognized by monoclonal antibodies after hCG binds to LH receptors. Since antibodies are much larger molecules than hCG, they will bind to only those surfaces of hCG that do not contact the receptor or other nearby proteins.
In a companion study(53) , we showed that a large
portion of the hormone -subunit is exposed in the hormone-receptor
complex and that the conformation of the region most likely to make
high affinity contacts with the receptor is changed following hormone
binding. Here we report that a similar large portion of the
-subunit can also be detected in the hormone-receptor complex. The
crystal structure of deglycosylated hCG (13) shows that the
-subunit is formed from three large loops held together by a
cysteine knot (Fig. 1). Loops one and three are adjacent, and
loop two is found at the other end of the protein. Loop two is the most
conserved part of the
-subunit and is located near
-subunit
loops one and three. To identify
-subunit residues involved in
-subunit antibody binding sites, we measured the abilities of the
antibodies to bind heterodimers composed of hCG
-subunit and
bovine/human
-subunit chimeras. Because bovine
-subunit had
low affinity for many antibodies made against the human
-subunit,
chimeras having human
-subunit residues at an antibody binding
site would be expected to bind an antibody much better than chimeras
with bovine
-subunit residues at these same sites. By comparing
the sequences of analogs that bound antibodies with those that did not,
we identified key residues likely to be involved in binding of a large
panel of anti-
-subunit antibodies. The relative locations of these
residues determined in epitope maps were consistent with the crystal
structure of deglycosylated hCG(13) . The
-subunit
residues that are exposed in the hormone-receptor complex include
residues in the N terminus and portions of the first and third loops.
In combination with data for the
-subunit in a companion study (53) and knowledge of residues that can be changed without
disrupting hormone function, these observations enabled us to devise a
model that explains how hCG interacts with LH receptors and initiates
signal transduction.
Figure 1:
Ribbon diagram of the -subunit.
This figure illustrates the location of the three
-subunit loops (dark blue) and the two oligosaccharide chains (red
sticks). In the heterodimer, loop two of the
-subunit is
located near the front of loops one and three of the
-subunit(13) . The
-subunit seat belt loop that
surrounds the second
-subunit loop passes over the second loop
midway down its length. The oligosaccharide shown at the bottom
(Asn
), but not the top (Asn
) is essential for
full signal transduction. Residues noted on the figure illustrate the
relative locations of key antibody binding sites summarized in Table 2. Red, green, blue, and orange labels refer to some of the key residues in the
epitopes of type I, II, IV, and V antibodies,
respectively.
Figure 2:
Diagram of the chimeras and analogs used
in these studies. These analogs were produced by a combination of
cassette and polymerase chain reaction mutagenesis as outlined in the
text. The nomenclature refers to the locations of residues derived from
the human -subunit. The amino acid sequence of the bovine and
human
-subunits are
FPDGEFTMQGCPECKLKENKYFSKPDAPIYQCMGCCFSRAYPTPARSKKTMLVPKNITSEATCCVAKAFTKATVMGNVRVENHTECHCSTCYYHKS
and
APDVQDCPECTLQENPFFSQPGAPILQCMGCCFSRAYPTPLRSKKTMLVQKNVTSESTCCVAKSYNRVTVMGGFKVENHTACHCSTCYYHKS,
respectively. Solid bars are residues derived from bovine
-subunit and stippled bars are from human
-subunit.
Modeling of
hCG-receptor complexes will be described in detail elsewhere. Briefly,
we modeled the LH receptor on the crystal structure of of porcine
ribonuclease inhibitor (30) obtained from the Protein Data Bank (i.e. 1bnh.ent). We aligned the sequences of the leucine
repeats in the LH receptor with those of the crystal structure of
ribonuclease inhibitor. In this alignment residues encoded by the
intron-exon junctions of the receptor become located in solvent exposed
loops furthest from the portion of the extracellular domain that
contacts the transmembrane domain. Five of the six glycosylation
signals found in the LH receptor are also located in these loops. After
adding oligosaccharides, we subjected the structure to energy
minimization and molecular dynamics at 300 K using the modeling package
Sybyl (Tripos, St. Louis, MO). The crystal structure of hCG was
``docked'' to this model of the extracellular domain of the
receptor using biological information on the parts of the hormone that
are exposed in the hormone-receptor complex reported in this and in a
companion study(53) . The groove of the hormone between the
- and
-subunits thought to make the high affinity contacts
identified in the companion study (53) was placed over receptor
residues 93-170 which have been shown to control LH receptor
binding specificity(31) . The hormone was rotated slightly
around this putative contact until the exposed and hidden surfaces of
the hormone were in positions that agreed sterically with the
observations on antibody binding made in this and a companion
study(53) . The fully glycosylated complex was then subjected
to energy minimization and molecular dynamics until it reached a stable
minimum energy. Finally, to illustrate the transmembrane domain, we
visually docked the structure of rhodopsin (i.e. 1bac.ent from
the Protein Data Bank) to the plasma membrane side of the
hormone-receptor complex. During docking, the N-terminal end of the
first
-helix was placed beneath the C-terminal end of the
extracellular domain. The structure of rhodopsin was then rotated until
the helices were perpendicular to the central cavity of the
extracellular domain. In this configuration helices 1-5 of
rhodopsin made a direct contact with the extracellular domain of the
receptor while helices 6 and 7 were under the open space created by the
``U'' shape of the extracellular domain.
Figure 3:
Ability of antibodies to inhibit binding
of I-hCG to LH receptors. Increasing amounts of
antibodies as shown on the abscissa were incubated with radiolabeled
hCG for 30 min at 37 °C. After the antibody had bound to the
hormone, the mixture was added to homogenates of ovarian corpora lutea
and the binding of
I-hCG to LH receptors was measured as
described in the text. Radiolabel that became bound to the membrane
receptors is illustrated here. Values are means of closely agreeing
duplicates (all antibodies except A101) or triplicates
(A101).
Previous studies including those using antisera to hCG
-subunit have failed to detect exposed residues of the
-subunit and led to models of hormone binding in which the entire
-subunit is close to the receptor(32) . The observations
that two
-subunit antibodies bound to hormone-receptor complexes
show that these models are incorrect. However, we have also found that
hCG can bind to membranes and other surfaces
``nonspecifically''(23) . Binding of A407 was readily
detected with virtually every preparation of radiolabeled antibody;
binding of A105 to receptor complexes was seen only with freshly
iodinated preparations. To be certain that we were not observing the
binding of A105 to hCG that became bound ``nonspecifically''
to the membranes, we repeated the studies using analogs of hCG that had
related structures but different abilities to bind to LH receptors.
A105 bound to receptor complexes prepared by incubating membrane
receptors with hCG analogs having lutropin activity. It did not bind
receptor preparations that had been incubated with similar amounts of
closely related analogs that have low LH activity (Table 1). This
showed that A105 recognized an exposed portion of the
-subunit of
hCG after the hormone was specifically bound to LH receptors.
Figure 4: Epitope map describing the binding sites of the antibodies used in these studies. These maps illustrate the relative properties of the antibodies to bind to hCG as discussed in the text. They were determined by measuring the abilities of the antibodies to bind to hCG at the same time.
Heterodimers lacking the C terminus of the -subunit have low
affinities for their receptors and this has been interpreted as support
for the idea that C terminus of the
-subunit has a role in hormone
binding(1) . To learn if the C terminus of the
-subunit
was involved in the binding sites of any of the
-subunit
antibodies, we tested the abilities of each antibody to bind an analog
lacking
-subunit residues 88-92. All of these antibodies
bound to this analog indicating that residues 88-92 were not part
of their binding sites (Table 5). Unexpectedly, antibodies that
had higher affinity for the heterodimer recognized the analog lacking
the C terminus better than hCG (Table 5). This suggested that
this mutation distorted the interaction between the subunits without
causing them to dissociate. We also found that deletion of the C
terminus led to an increased ability of hCG to be recognized by A105,
an antibody that bound to hormone-receptor complexes (Table 4).
The binding site for A105 appears to be near the C terminus, and we
anticipate that the mobility of the C terminus that prevents it from
being seen in the crystal structure (13) also interferes with
binding of A105. These observations indicate that it may be premature
to conclude that the C terminus of the
-subunit contacts the LH
receptor based on the reduced abilities of analogs with mutations in
this region to bind to LH receptors. In addition, these observations
suggested that the ability of mutant subunits to combine into a
heterodimer is not sufficient to detect the influences of mutations on
hormone conformation.
The locations of all the antibody
binding sites are consistent with the crystal structure of
deglycosylated hCG(13) . This is most convincingly illustrated
by predictions about the locations of residues in the twin loops of the
-subunit made on the basis of the A109 and A407 binding sites. For
example, as predicted from the ability of A109 to bind to
chimeras(33) , residues 14-17 and 73-75 were found
to be adjacent in the crystal structure. Also, the proximity of
residues near the N terminus of the protein and residue 81 had been
predicted from the A407 binding site prior to the crystal
structure(33) . This suggested that the locations of the
antibody binding sites have been correctly identified.
Figure 5:
Composite figure illustrating the
relative binding sites of several anti--subunit antibodies. The
-subunit is illustrated in white and the
-subunit is shown in yellow. The oligosaccharides are illustrated in red.
The relative locations of the antibody binding sites are shown by the
positions of the space filled coordinates of the crystal structure of
anti-lysozyme Fab fragments that have been placed visually. This was
done by docking a high resolution structure for a
lysozyme-anti-lysozyme Fab fragment complex perpendicular to hCG over
residues that were identified as being in the antibody binding sites (Table 2). The view shown in the upper panel explains the
relative abilities of the antibodies to bind to hCG at the same time (cf. epitope map, Fig. 4). Top panel, view
from above the
-subunit looking down on the tips of the first and
third loops. Lower panel, view of molecule turned 90° and
illustrating the second loop of the
-subunit and the
-subunit. Color code:
-subunit, white;
-subunit, yellow; oligosaccharides, red; type IV
site, orange; type V site, blue; type II site, green; and type I site, purple.
Aside from
monoclonal antibodies made against synthetic peptides, the binding
sites for most anti--subunit antibodies have not been determined.
The identification of key residues in the binding sites of antibodies
we report here should have several practical applications. Many of the
antibodies we have used in these studies are widely used for research
and clinical diagnoses. Knowledge of the portions of hCG recognized by
these antibodies will facilitate identifying unknown analytes present
in serum or that are produced by tissues. Epitope maps prepared with
these antibodies as reference compounds can also be used to predict the
binding sites of other antibodies. Finally, since their binding sites
are now known, these antibodies can be used to study the influence of
mutations on specific regions of the protein and to detect changes in
hormone conformation that may influence receptor binding (cf. Table 2and Table 5).
Figure 6:
Model of hCG binding to the extracellular
domain of the LH receptor that illustrates a mechanism of signal
transduction. Note, panels in the left column illustrate a
side view of the receptor and those in the right column illustrate the corresponding top view obtained by rotating the
side view forward 90°. The free LH receptor (top left and right panels) ribbon model is based on the structure of RNase
inhibitor (2) and was prepared by replacing residues in the
leucine-rich repeats of RNase inhibitor with those of the corresponding
repeats found in the LH receptor. Following extensive energy
minimization and molecular dynamics, the receptor model was
``docked'' onto the structure of bacteriorhodopsin (52) to obtain a view illustrating the approximate sizes and
orientations of the extracellular and transmembrane domains. The
extracellular and cytoplasmic loops of the transmembrane domain are not
shown. During docking, the C-terminal residue of the extracellular
domain (Arg) was placed adjacent to the N-terminal end of
the first
-helix of rhodopsin (white). The remaining six
helices of rhodopsin (shown in the order green, red, yellow, purple, orange, and magenta) were rotated to make maximal contact with the
extracellular domain. The extracellular domain of the receptor is
illustrated in blue (residues 1-93 and 170-341)
and orange (residues 94-169). The orange-colored section
of the extracellular domain illustrates a portion of the LH receptor
that appears to control its ligand binding specificity. When this
section of the LH receptor was substituted for the homologous region of
the FSH receptor, the resulting chimera bound both hCG and hFSH with
high affinity(31) . Five of the six receptor oligosaccharides
are illustrated in yellow. In the hormone-receptor complex (middle left and right panels) note that, to improve
the clarity of these panels, we have omitted the oligosaccharides and
the transmembrane domain of the receptor. To dock the hormone to the
receptor we moved the groove in hCG formed by
-subunit loop two,
-subunit loop three, and, to a lesser extent,
-subunit loop
one (13) over the orange-colored portion of the receptor
(
-subunit, red ribbon;
-subunit, green
ribbon; hormone oligosaccharides, red sticks). We then
rotated the hormone about this intersection with the receptor to expose
hormone residues found to be exposed and hidden based on the antibody
binding data described in this and a companion study(53) .
Finally, all the atoms in the hormone and extracellular domain of the
receptor including those in their oligosaccharides were subjected to
energy minimization and molecular dynamics at 300 K to eliminate bad
contacts and optimize side chain interactions. This occurred without
major structural changes in either the hormone or the receptor and led
to the stable complex shown here. hCG
-subunit loops one and three
are immediately above the portion of the receptor known to convey
lutropin binding specificity (i.e. colored orange).
The second
-subunit loop contacts the concave portion of this
region of the extracellular domain on its inner surface. hCG
-subunit loops one and three and
-subunit loop two project
into the large cavity formed by the U shape of the receptor
extracellular domain and are nearest its C-terminal arm. The
N-terminal ends of both hormone subunits (free ends of the
- and
-subunit ribbons shown in middle left panel) and the
oligosaccharides of the
-subunit (red oligosaccharide chains that
point upward in middle left panel) are on the surface of the
hormone furthest from the plasma membrane. Only those oligosaccharides
on the
-subunit are shown in the top view (middle right
panel). Note the proximity of the oligosaccharide needed for
signal transduction (i.e. that at Asn
) and the N
terminus of the extracellular domain of the receptor (middle right
panel). Signal transduction results from the steric effect of the
oligosacharide at Asn
on the N-terminal end of the
extracellular domain to widen the distance between its N- and
C-terminal arms. This may be accentuated by steric interaction of
-subunit loops one and three with the C-terminal end of the
extracellular domain. The model does not require a direct interaction
between the hormone and the transmembrane domain for signal
transduction. Given the proximity of the hormone and the transmembrane
domain in this model, these cannot be excluded. In antibody-hCG
receptor complexes (lower left and right panels) the
crystal structure of an Fab fragment-lysozyme complex was
``docked'' to residues of epitopes known to be exposed in
hCG-receptor complexes. To minimize complexity, we illustrate only four
of the five known antibody binding sites and none of the
oligosaccharides on the hormone or the receptor. The orange and yellow
ribbons correspond to Fab fragments docked over exposed residues of the
-subunit (i.e. A105, yellow; A407, orange; cf. Table 2). The red and blue ribbons correspond to Fab fragments docked over exposed
residues of the
-subunit (i.e. B111, red; B112, blue). Key residues in the binding sites of these antibodies
include 108-114 and 77, respectively. Not shown is the binding
site of B105, an antibody that recognizes a portion of hCG containing
residue 74 (cf. (53) ). The binding site for this
antibody is most easily visualized as lying between that of the red and blue ribbons on the lower left
panel.
There are few other configurations of the receptor that would account for the portions of the hormone in the receptor complex that can be detected by antibodies and that would also permit the hormone to be near the transmembrane domain. As discussed later, the conformation of another leucine-rich repeat protein, RNase inhibitor is horseshoe-shaped(30) . This provides considerable precedent for the model we have proposed. In addition, the model is consistent with the known interactions between hCG and the LH receptor discussed as follows.
First, the model of hCG-receptor interaction accounts for
portions of the hormone that can be recognized by antibodies that bind
to hormone-receptor complexes described here and in a companion study (53) (Fig. 6). In the -subunit this includes
residues near the N terminus and most of the portions of the first and
third
-subunit loops furthest from the
-subunit interface. In
the
-subunit this includes residues on the surfaces of the first
and third loops that are furthest from the
-subunit interface.
Surfaces recognized by antibodies face away from all parts of the
receptor and are not likely to be near other proteins in the cell
membrane.
Second, the model explains why many residues in both
- and
-subunits usually proposed to be near the receptor
interface can be replaced without altering receptor binding or hormone
activity(24, 31) . In the
-subunit these include
most of the residues found in loops one and three near the
-subunit interface, a few residues in the second loop including
Arg
-Lys
(38) , and a residue that can
be cross-linked to the
-subunit (i.e. that corresponding
to human
-subunit Lys
in pig and bovine
LH)(39, 40) . In the
-subunit these residues
include nearly all the amino acids found in the second loop (24) and in the seat belt, the region that has the major
influence on receptor binding specificity (24, 31) .
The model suggests that the side chains of these residues do not make
specific contacts with the receptor even though many of these residues
are in the large groove of the extracellular domain. Their functions in
signal transduction, if any, occurs primarily by steric effects.
Mutations of residues that are found in this groove have relatively
little influence on hormone function unless they disrupt the structure
of the hormone.
Portions of the - and
-subunits that
become located between the arms of the receptor extracellular domain
lose their abilities to be recognized by antibodies after they bind to
LH receptors. These epitopes appear to be obscured by the receptor.
Antibodies that bind to these regions are very effective in blocking
hormone binding, a phenomenon that appears due in part to their
abilities to prevent this region of the hormone from entering the space
between the arms of the receptor. As noted earlier, Pantel et al.(41) have found that these antibodies can recognize hCG
that is bound to truncated LH receptors, an observation that is
consistent with this view.
Third, the structure of a U- or J-shaped
extracellular domain could readily create a narrow projection that fits
into the hormone groove created by the apposition of the second
-subunit loop and portions of the first and third
-subunit
loops. Entry of a portion of the receptor into this groove could also
account for the change in conformation of the
-subunit that occurs
on binding to receptors (28, 53) .
Fourth, the
model of hCG-receptor interaction explains why the oligosaccharides are
essential for signal transduction. In the model, binding of hormone to
the receptor is distinct from signal transduction. Signal transduction
occurs by an influence of the hormone on the distance between the arms
of the extracellular domain. While it is possible that the hormone
reduces this distance by binding to both arms of the extracellular
domain, we think this is very unlikely since the residues in the
portion of the hormone near the arms of the extracellular domain (24) and the leucine-rich receptor repeats in these arms (31) can be changed without disrupting signal transduction.
More likely, we anticipate that the hormone will increase the distance
between the two arms and we propose that this is the role played by the
oligosaccharides in signal transduction. The sugars appear to function
primarily by their bulk rather than by a specific interaction with the
receptor, an idea that is consistent with the following observations. (a) It explains why both hLH and hCG are potent lutropins even
though their sugars are quite different(42) . It also explains
the full efficacy of hormones having high mannose sugars that have been
expressed in Baculovirus(43) . (b) The bulk
of the sugars resolves the differences in the influence of the
oligosaccharides on efficacy(44) . That on the -subunit at
Asn
has the least effect on hormone efficacy because it
extends beyond the arms of the receptor. The
-subunit
oligosaccharide at Asn
has the greatest influence on
efficacy because it is closest to the receptor. The oligosaccharides on
the
-subunit have an intermediate influence on efficacy. These
project away from the hormone-receptor complex but are sufficiently
near the arms of the extracellular domain that they could interact with
them. (c) The influence of bulk accounts for the observations
that sequential removal of the oligosaccharides from hCG using
exoglycosidases is accompanied by a graded loss in
efficacy(45) . (d) Finally, the observation that
antibodies to hCG can restore efficacy to deglycosylated hCG (46, 47) is also consistent with the idea that the
oligosaccharides function primarily by steric effects. Binding of
antibodies to the exposed region of the
-subunit would increase
the bulk of the portion of the hormone that is found between the arms
of the receptor extracellular domain.
Fifth, the model explains the observation that hCG will bind and activate FSH receptor analogs that have relatively few residues derived from the LH receptor(31) . Because the arms of the extracellular domain make few specific high affinity contacts with the side chains of the hormone, hCG analogs bind with high affinity to LH/FSH receptor chimeras that are derived mostly from the FSH receptor(31) . Further, because the ``contacts'' needed for signal transduction are ``steric'' in nature, both hCG and hFSH can elicit signal transduction from LH/FSH receptor chimeras(31) . Only a small part of the extracellular domain of the receptor appears essential for hormone binding. Thus, the model also explains why LH receptors that have been truncated at amino acid 206 are able to bind hCG with high affinity (4) .
Sixth, the model does not require the hormone
to interact with the transmembrane domain of the receptor. However,
since much of the hormone is present in the space between the arms of
the extracellular domain, these interactions are not precluded. This
would account for the reports that hCG can bind and activate the
transmembrane domain of the LH receptor (7, 8) and
that a synthetic peptide corresponding to the C terminus of the
-subunit can stimulate cyclic AMP accumulation (48) . The
interaction of a portion of the hormone with the transmembrane domain
might also serve to attract the hormone into the groove in the
extracellular domain and potentiate its effect on the conformation of
the receptor. This would explain the observation that binding of hCG to
a membrane bound receptor occurs with higher affinity than to soluble
receptors.