(Received for publication, September 10, 1996, and in revised form, February 24, 1997)
From the Laboratory of Molecular Endocrinology, Department of
Medicine, University of Maryland School of Medicine and the Institute
of Human Virology, Medical Biotechnology Center, Baltimore, Maryland
21201, the § Molecular and Cellular Endocrinology Branch,
NIDDK, National Institutes of Health, Bethesda, Maryland 20892, the
¶ Wadsworth Center, New York State Department of Health, Albany,
New York 12201, and the Department of Molecular Biology,
University of Wyoming, Laramie, Wyoming 82071
The region between the 10th and 12th cysteine
(Cys88-Cys105 in human
thyroid-stimulating hormone -subunit (hTSH
)) of the glycoprotein hormone
-subunits corresponds to the disulfide-linked seat-belt region. It wraps around the common
-subunit and has been implicated in regulating specificity between human choriogonadotropin (hCG) and
human follicle-stimulating hormone (hFSH), but determinants of hTSH
specificity are unknown. To characterize the role of this region for
hTSH, we constructed hTSH chimeras in which the entire seat-belt region
Cys88-Cys105 or individual intercysteine
segments Cys88-Cys95 and
Cys95-Cys105 were replaced with the
corresponding sequences of hCG and hFSH or alanine cassettes. Alanine
cassette mutagenesis of hTSH showed that the
Cys95-Cys105 segment of the seat-belt was more
important for TSH receptor binding and signal transduction than the
Cys88-Cys95 determinant loop region. Replacing
the entire seat-belt of hTSH
with the hCG sequence conferred full
hCG receptor binding and activation to the hTSH chimera, whereas TSH
receptor binding and activation were abolished. Conversely,
introduction of the hTSH
seat-belt sequence into hCG
generated an
hCG chimera that bound to and activated the TSH receptor but not the
CG/lutropin (LH) receptor. In contrast, an hTSH chimera bearing hFSH
seat-belt residues did not possess any follitropic activity, and its
thyrotropic activity was only slightly reduced. This may in part be due
to the fact that the net charge of the seat-belt is similar in hTSH and
hFSH but different from hCG. However, exchanging other regions of
charge heterogeneity between hTSH
and hFSH
did not confer follitropic activity to hTSH. Thus, exchanging the seat-belt region between hTSH and hCG switches hormonal specificity in a mutually exclusive fashion. In contrast, the seat-belt appears not to
discriminate between the TSH and the FSH receptors, indicating for the
first time that domains outside the seat-belt region contribute to
glycoprotein hormone specificity.
Thyrotropin (thyroid-stimulating hormone
(TSH))1 choriogonadotropin (CG),
follitropin (follicle-stimulating hormone (FSH)), and lutropin
(luteinizing hormone (LH)) are structurally related heterodimers that
together form the glycoprotein hormone family (1). These hormones
belong to the superfamily of cystine-knot growth factors (2, 3) and
activate specific G-protein-coupled receptors notable for large
extracellular domains containing multiple leucine-rich motifs (4). The
primary structure of the -subunit, which is encoded by a single
gene, is identical in these hormones. The distinct
-subunits,
despite conservation of all 12 cysteine residues and similar overall
folding, are sufficiently different to confer specificity to each
hormone (1-3).
The molecular mechanisms whereby glycoprotein hormones activate their
receptors are largely unknown, but multiple contact points between
ligand and receptor, perhaps in a stepwise fashion, appear necessary to
induce conformational changes favoring receptor G-protein coupling and
subsequent second messenger generation (5-9). Recently, we have
described several -subunit domains important for hTSH activity
(10-13), but there is little information on how the hTSH
-subunit
contributes to receptor activation.
Previous studies have shown that the region between the 10th and 12th
cysteine of the -subunit is important not only for subunit
association, receptor binding, as well as activation (14-16), but also
for specific receptor recognition (17-20) of hCG and hFSH. In the
crystal structure of hCG (2, 3), this region corresponds to the
"seat-belt" region (Cys88-Cys105 in
hTSH
), so-called because it wraps around the
-subunit and orients
it in the heterodimer while remaining covalently bonded to the
-subunit through disulfide linkages between
Cys9-Cys90 and
Cys26-Cys110. This seat-belt consists of two
intercysteine segments, a surface-exposed hydrophilic loop between the
10th and 11th cysteine (Cys88-Cys95 in hTSH
)
and a carboxyl-terminal segment between the 11th and 12th cysteine
(Cys95-Cys105 in hTSH
), and is in close
proximity to
-subunit domains important for the structural integrity
and activity of the glycoprotein hormones (2, 3).
In contrast to the work on the gonadotropins, the role of the seat-belt
for hTSH is not known. A single study using a set of overlapping
synthetic peptides spanning the entire hTSH subunit (21) showed that
none of the peptides encompassing the seat-belt region, hTSH 81-85
or hTSH
91-105, inhibited TSH receptor binding, but a peptide
containing the carboxyl terminus (
101-112) possessed the highest
TSH receptor binding activity. However, the role of the seat-belt
region in the context of the intact hTSH heterodimer has not been
investigated. Interestingly, recent studies on a naturally occurring
hTSH
mutation from patients with secondary hypothyroidism have shown
the importance of Cys105 (corresponding to
Cys110 in hCG) for hTSH activity (22).
In the present study, using a chimeric mutagenesis approach, we demonstrate the importance of the seat-belt for hTSH action as well as specificity. Moreover, our findings reveal previously unrecognized differences in the regulation of specificity among the glycoprotein hormones.
The following materials were generous gifts. CHO cells stably transfected with the rhTSH receptor (clone JP09) was from Dr. G. Vassart (Brussels, Belgium) (23); the gonadotropin-responsive murine Leydig cell line MA-10 was from Dr. M. Ascoli, (Iowa City, IA) (24); and cAMP antibody was from Dr. J. L. Vaitukaitis, National Institutes of Health (Bethesda, MD). Embryonic kidney 293 cells (FSH-R/293 cells) and Y-1 cells expressing the human FSH receptor have been described previously (20, 25). Cell culture media and reagents were purchased from Life Technologies, Inc. (Gaithersburg, MD); 125I-cAMP (specific activity, 40-60 µCi/µg) and 125I-hCG (specific activity, 50-70 µCi/µg) were from Hazleton (Vienna, VA), and polymerase chain reaction (PCR) reagents were from Boehringer Mannheim and New England Biolabs (Beverly, MA).
Site-directed MutagenesisThe chimeric hTSH were
constructed with the PCR-based megaprimer method of site-directed
mutagenesis (26), as described (11, 13). Individual intercysteine
segments C10-C11 (hTSH Cys88-Cys95) or
C11-C12 (hTSH
Cys95-Cys105) of the hTSH
-minigene were replaced with nucleotides coding for the respective
sequence of hCG and hFSH or Ala cassettes. To replace the entire
seat-belt (hTSH
Cys88-Cys105), chimeras with
individually mutated intercysteine segments were used as templates for
subsequent PCR reactions. The hTSH
seat-belt was introduced into the
hCG
-subunit in a single PCR reaction using a primer coding for the
entire hTSH
seat-belt. Further hTSH/hFSH chimeras were constructed
in which the carboxyl-terminal residues hTSH
105-112 or amino acids
hTSH
44-52 were replaced with the sequence of hFSH. In addition,
Asp94 of the determinant loop was replaced with Lys
(TSH
Lys94) or Glu (TSH
Glu94). After
subcloning into the expression vectors, the entire PCR products of all
constructs were sequenced to verify the mutations and to rule out any
undesired polymerase errors. Construction of the quadruple
-subunit
mutant bearing Lys residues at positions
13, 14, 16, and 20 (
4K)
was described previously (13).
CHO-K1 cells maintained as described
(10) were transiently cotransfected with the various constructs using a
transient transfection protocol based on a liposome formulation
(LipofectAMINE reagent, Life Technologies, Inc.) (10). After culture in
CHO serum-free medium (CHO-SFM, Life Technologies, Inc.) for 48 h,
conditioned media including control medium from mock transfections were
harvested, concentrated with Centriprep 10 concentrators (Amicon,
Beverly, MA), and stored at 70 °C to prevent neuraminidase
digestion.
Wild type and mutant hTSH analogs were quantified with a panel of four different hTSH immunoassays, which were described in detail previously (12). hCG immunoreactivities were measured with two different specific third-generation immunoassays without crossreactivity to other glycoprotein hormones (Nichols Institute, San Juan Capistrano, CA; ICN, Costa Mesa, CA), and hFSH immunoreactivity was measured with an hFSH-specific third-generation immunoassay (Nichols Institute).
Hormone Binding AssaysThe TSH receptor-binding activity of wild type and hTSH mutants was determined by their ability to displace 125I-bTSH from a solubilized porcine thyroid membrane receptor preparation (Kronus, Dana Point, CA), as described previously (10). Binding to the CG/LH receptor was studied in MA-10 cells following a previously employed protocol (13, 24), and FSH receptor binding was analyzed using a rat testis membrane radioreceptor assay as described in detail previously (20).
Hormone Activity AssaysThe ability of the various chimeras to induce cAMP production was studied at the TSH receptor using JP09 cells (23), at the CG/LH receptor using MA-10 cells (24), and at the FSH receptor using FSH-R/293 cells (25). Briefly, confluent cells in 96-well tissue culture plates were incubated in a modified Krebs Ringer buffer for 2 h at 37 °C, 5% CO2 with serial dilutions of wild type and mutant hTSH, as well as control medium from mock transfections. The amount of cAMP released into the medium was assayed by radioimmunoassay (10). Progesterone production at the CG/LH or FSH receptor was determined using a commercially available progesterone radioimmunoassay kit (ICN) after incubation of the chimeric constructs with MA-10 cells or Y-1 cells, respectively, as detailed previously (20, 24).
Replacing individual intercysteine segments C10-C11 of
the hTSH -subunit (the determinant loop, hTSH
Cys88-Cys95) or C11-C12 (the carboxyl-terminal
segment, hTSH
Cys95-Cys105) (Fig.
1) with Ala cassettes or with the corresponding
sequences of hCG and hFSH generated hTSH
constructs designated
89Ala94, 96Ala104,
89CG94, 96CG104,
89FSH94 and 96FSH104,
and exchange of the entire hTSH
seat-belt chimeras
89CG104 and 89FSH104.
The hCG/hTSH chimera 94TSH109 was obtained by
introduction of the hTSH
seat-belt residues into the hCG
-subunit
(Fig. 2). In addition, hTSH
Asp94, which
is conserved in all known
-subunits and essential for hCG activity
(27), was replaced with Lys (hTSH
Lys94) or with Glu
(hTSH
Glu94). Finally, sequences of the hTSH
-subunit
outside the seat-belt were replaced with the corresponding sequence of
hFSH to create 44FSH52 and
105FSH112. These regions were chosen because
they display the greatest charge heterogeneity among these
-subunits, based on the proposed role of variable charges for
glycoprotein hormone specificity (17) (and see below). Receptor binding
and biological properties of these analogs, described in detail below,
are summarized in Table I. A comparison of the receptor
specificity of glycoprotein hormone seat-belt chimeras from this and
other studies (18-20) is given in Table II.
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All chimeric hTSH heterodimers
were secreted from the transfected CHO cells, and relative secretion,
compared with hTSH-wt, ranged from 35.3 ± 7.6% in the case of
89Ala94 to 100.2 ± 14.7% in the case of
96CG104. As evidence for accurate quantitation,
hTSH immunoreactivity of each chimera was comparable in four different
hTSH immunoassays, which recognize different epitopes of the hTSH
molecule (10-13) (data not shown). Similarly, secretion of
94TSH109 bearing the hTSH seat-belt sequence
within the hCG -subunit was 94.3 ± 17.7% that of hCG-wt, as
determined by two different hCG immunoassays. Therefore, in accord with
previous studies on hCG and hFSH (18-20), intercysteine loops appear
to be interchangeable between the different
-subunits without major
changes in subunit assembly and heterodimer secretion of hTSH or global
conformational changes of the heterodimer. Secretion of
44FSH52 and 105FSH112
with replacements outside the seat-belt was less than 25% for both
chimeras. The reasons for the reduced secretion of these mutants are
unclear but could be related to decreased stability of messenger RNA,
improper folding of the mutant subunit, or decreased efficiency of
subunit assembly.
TSH receptor binding as well as
thyrotropic activity of both 89Ala94 and
96Ala104 was substantially reduced (Fig. 3,
A and B), showing that the native
seat-belt sequence is important for hTSH activity. Interestingly, maximal cAMP stimulation of the 96Ala104 mutant
was significantly lower than that of the
89Ala94 mutant (28.3 ± 3.2 versus 63.0 ± 4.6% of hTSH-wt, respectively), indicating that the carboxyl-terminal segment of the seat-belt is more
important for hTSH activity than the determinant loop.
Role of Asp94 for hTSH Activity
A single mutation
of the conserved Asp94 to Lys (hTSHLys94)
completely abolished TSH receptor binding and activation, whereas preserving the negative charge at this position by mutating
Asp94 to Glu (hTSH
Glu94) did not have a
significant effect on TSH receptor binding or activation (Table I).
This confirmed the importance of a negative charge in this particular
position for glycoprotein hormone activity (27).
Replacement of the hTSH seat-belt segments
with the respective sequences of hCG either substantially decreased
(89CG94) or abolished measurable TSH receptor
binding and activation (96CG104,
89CG104) of the chimeras (Fig.
4, A and B). hTSH/hCG chimeras
89CG94 and 96CG104
showed only very little CG/LH receptor binding and activation, both
with cAMP stimulation as well as progesterone production. Thus, at the
highest doses possible within the limitations of the transient
transfection system, between 100-200 ng/ml, cAMP or progesterone
production was only 10-18% that of hCG-wt (Fig. 5,
A-C). To more conclusively test whether the
Cys88-Cys95 determinant loop was important for
differential hormonal activity, we constructed a hTSH mutant
4K/89CG94. In
4K/89CG94, residues
13, 14, 16, and 20 were substituted with Lys residues in addition to the replacement of
Cys88-Cys95 with the respective hCG residues.
We had previously shown that introduction of positive charges into this
11-20 domain led to substantial increases of glycoprotein hormone
receptor binding affinity (13). We therefore expected that increasing
the binding affinity of 89CG94 (Fig.
5A) should accentuate its gonadotropic properties. Maximal cAMP and progesterone production with this
4K/89CG94 combination chimera increased to
35% and 50% of hCG-wt levels, respectively (Fig. 5, B and
C). At the same time, the thyrotropic activity of
4K/89CG94 remained unchanged (data not
shown).
Remarkably, replacement of the entire seat-belt of hTSH with the hCG
sequence (89CG104) resulted in a chimera with
hCG receptor binding comparable to hCG-wt, suggesting that the two
individual intercysteine segments confer hCG specificity in a
synergistic fashion (Fig. 5A). Further, the
89CG104 chimera was able to induce biological
responses in MA-10 cells expressing the CG/LH receptor (Fig. 5,
B and C). Whereas potency and efficacy of
progesterone production as well as efficacy of cAMP induction were
similar to hCG-wt, the potency of 89CG104 for
cAMP production was 10-fold less than that of hCG-wt. These differences
may stem in part from differences in the cAMP and progesterone assay
conditions in MA-10 cells (see "Experimental Procedures").
Moreover, such generation of full hormonal responses at submaximal cAMP
levels, termed "the cAMP superfluity concept," has been well
recognized in studies on structure-function relationships of
glycoprotein hormones (8, 28). Analogous findings for recombinant
analogs with substantially higher progesterone-inducing than
cAMP-inducing ability have been described by others (29). Interestingly, hTSH/hCG specificity appeared mutually exclusive since
the 89CG104 chimera did not possess significant
thyrotropic activity (Fig. 4, A and B).
Conversely, the reciprocal chimera
94TSH109, which bears the hTSH seat-belt in
the context of the hCG-
subunit, bound to the TSH receptor and was
able to activate cAMP production in JP09 cells, with an
EC50 that was 26.7 ± 4.7-fold higher than that of
hTSH-wt (Fig. 6, A and B). At the
same time, 94TSH109 did not bind to the CG/LH
receptor, nor did it stimulate cAMP or progesterone production in MA-10
cells at concentrations up to 1000 ng/ml (Table I).
hTSH/hFSH Chimeras
Analogous replacement of individual
intercysteine segments of hTSH with the corresponding hFSH residues
only slightly reduced TSH receptor binding or activation of these
hTSH/hFSH chimeras (Fig. 7, A and
B). Further, in contrast to 89CG104,
the 89FSH104 construct bearing the entire hFSH
seat-belt sequence was able to significantly bind to the TSH receptor
and induce 50% of maximal hTSH-wt cAMP production in JP09 cells.
Interestingly, none of the three hTSH/hFSH chimeras showed significant
follitropic activity. Unlike hFSH-wt, the chimeras did not stimulate
cAMP production at the hFSH receptor expressed in 293 cells (Fig.
8). Further, they did not show significant binding in a
rat testis FSH radioreceptor assay and did not stimulate progesterone
production in Y-1 cells expressing the hFSH receptor (Table I).
Since charge heterogeneity could play a role for hTSH/hFSH specificity
(17), we replaced candidate regions hTSH 44-52 and the
carboxyl-terminal residues 105-112 with hFSH sequences. Thus, hTSH
44KYALSQDVC52 has a net charge of 0, and the
corresponding hFSH
sequence ARPKIQKTC has a charge of +3. hTSH
105CTKPQKSY112 has a net charge of +2, and the
corresponding hFSH
sequence CSFGEMKE has a charge of
1. The
hTSH
44-52 region corresponds to the carboxyl-terminal part of the
long
2 loop identified by Keutmann et al. (30), which
forms a wedge-shaped and partly surface-exposed extrusion in proximity
to the determinant loop (2). However, none of the resulting hTSH/hFSH
chimeras showed any follitropic activity in the three different assay
systems (Table I). Interestingly, the thyrotropic activity of
105FSH112, but not that of
44FSH52 was significantly reduced (Fig.
9), in accord with previous studies on hTSH
structure-function using a synthetic peptide approach (21).
Previous studies on glycoprotein hormone specificity had focused
on analogs that bound either to the CG/LH or the FSH receptor. These
studies had suggested that domains within the seat-belt region of the
-subunit are involved in directing gonadotropin specificity
(18-20). It is unknown, however, how hTSH specificity is achieved and
whether the seat-belt is critical for interaction with the TSH
receptor. We directly compared the effects of systematically replacing
the hTSH
seat-belt and its individual intercysteine segments with
the corresponding regions of two different hormones, hCG and hFSH in
parallel. Conversely, the hTSH
seat-belt residues were introduced
into hCG. This strategy allowed us to characterize the role of the
seat-belt for hTSH activity as well as specificity and to reveal
divergent principles of specificity regulation among the members of the
glycoprotein hormone family (see Table II).
Ala cassette mutations showed that the primary sequence of the
seat-belt is essential for hTSH receptor binding and activation. Of
central importance was the negatively charged Asp94 of the
determinant loop since a single mutation of this residue to Lys, but
not to Glu, abolished hTSH receptor binding and activity. The critical
role of the negative charge of Asp94, which is conserved in
all known glycoprotein hormone -subunits and forms a non-bonded
interaction with
Thr54 (2, 3), was first identified in
hCG by Chen et al. (27), suggesting that this residue is
universally important for the members of the glycoprotein hormone
family.
Our chimeric studies demonstrated that the seat-belt region of the hTSH
-subunit, if placed into the context of the hCG
-subunit, confers
thyrotropic activity although the seat-belt alone was not sufficient
for full thyrotropic activity. This suggests that additional hTSH
domains beside the seat-belt may contribute to hTSH specificity or that
the hCG
-subunit contains segments that restrict interaction with
the TSH receptor. In this respect, it had been shown that removal of
the C-terminal extension peptide of hCG (31) as well as of the
N-linked carbohydrate side chain at
Asn52
increased the weak inherent thyrotropic activity of hCG (11). This
thyrotropic activity of native hCG however, unlike the chimera described here, requires 1000-fold higher concentrations than TSH
itself in most systems (11, 31). In contrast to the results with the
hCG/hTSH chimera, the hCG
seat-belt, in the context of the hTSH
-subunit, was sufficient for full CG/LH receptor binding and
secretory response. This reciprocal exchange of hCG/hTSH receptor
specificity was mutually exclusive as both chimeras possessed no
significant residual activity at their native receptor.
Remarkably, introduction of the hFSH seat-belt into the hTSH
-subunit did not result in FSH receptor binding or follitropic activity, and the hTSH/hFSH chimera retained most of its thyrotropic activity. In contrast to this finding, hCG could be converted to hFSH
by placing the hFSH
seat-belt into the hCG
-subunit (18, 19), and
hFSH adopted partial CG/LH receptor binding after exchange of its
determinant loop with the corresponding hCG sequence (20) (see Table
II). Hence, the role of the seat-belt in conferring specificity appears
to depend on the particular subunit into which it is introduced. These
findings are best reconciled by considering the concept of "negative
specificity," which proposes that specificity of glycoprotein
hormones evolved independently from signal transduction by the
introduction of domains that block inappropriate ligand-receptor
interactions (9, 19). In this respect, it is interesting to note that
the net charge of the determinant loop, the N-terminal part of the
seat-belt region, is similar in hTSH (
2) and hFSH (
3) but different
from that in hCG (+1). Thus, it is conceivable that a net positive
charge of the determinant loop, in conjunction with the
carboxyl-terminal segment of the seat-belt, interferes with hormone
binding to the TSH as well as FSH receptor; whereas a net negative
charge, again in conjunction with carboxyl-terminal residues reduces
interaction with the CG/LH receptor. From an evolutionary standpoint,
it is justifiable to assume that diversification and ligand selectivity did not evolve by development of new mechanisms of receptor activation but rather by the emergence of inhibitory domains that impose steric
hindrances, thus allowing only the intended ligand to interact with the
common activation domain. This concept of negative specificity is not
without precedent among cystine-knot growth factors. Thus, binding
specificity among members of the neurotrophin family is achieved by the
cooperation of distinct active and inhibitory binding determinants that
restrict ligand-receptor interactions, enabling the creation of analogs
with multiple specificities (32).
Our findings extend the original observation of Moore et al.
(17), who proposed that the variable charge of this loop may act as a
determinant of hormone specificity. However, our data show that the
carboxyl-terminal segment of the seat-belt is of similar importance for
specificity, and charge differences of the determinant loop per
se are, therefore, not sufficient for the switch of hormonal
activity. Indeed, conversion of hTSH to a full hCG analog required
concomitant replacement of the determinant loop as well as the
carboxyl-terminal segment of the seat-belt, which displays a high
degree of sequence but not charge heterogeneity among the glycoprotein
hormones. Interestingly, the luteotropic activity of the hTSH/hCG
determinant loop chimera could be increased by concomitant introduction
of a cluster of Lys residues into the spatially unrelated 11-20
domain, previously shown to increase receptor binding of hTSH as well
as hCG (13).
In an attempt to identify domains determining hTSH/hFSH specificity
outside the seat-belt, we focused on regions hTSH 44-52, which
correspond to the carboxyl-terminal part of the long
2 loop
identified by Keutmann et al. (30), and the
carboxyl terminus 105-112. These domains were chosen because they display the
greatest degree of charge heterogeneity between their
-subunits. The
decrease of thyrotropic activity of the
105FSH112 chimera confirmed the importance of
the hTSH
carboxyl terminus, which was identified with a synthetic
peptide approach (21), for hTSH activity. However, introduction of hFSH
residues into these regions of the hTSH
-subunit did not confer FSH
receptor binding or follitropic activity to any of these chimeras. This was in agreement with the findings of Campbell et al., who
showed that the long
2 loop was not important for hCG
versus hFSH specificity (18). Thus, charged residues appear
to play a lesser role in determining hTSH/hFSH specificity. It is
possible that hTSH/hFSH specificity is not located within distinct
segments of the
-subunit but is mediated by a combination of
topically related domains although the present study was not designed
to systematically test this possibility.
Our study cannot define the molecular mechanisms whereby the seat-belt
determines glycoprotein hormone specificity as this will require
complete elucidation of the structure of hormone-receptor complexes. In
this respect, the seat-belt could either directly contact the receptor
or influence the conformation of functionally important but unrelated
portions of the hormone. An indirect effect of the seat-belt on the
conformation of the -subunit would be consistent with antibody
binding studies showing that the conformation of the
-subunit could
change depending upon with which
-subunit it associated (33), as
well as with a recent model of glycoprotein hormone-receptor
interaction predicting that the seat-belt does not directly contact the
receptor (6). It could also explain the lack of TSH receptor binding of
hTSH
peptides spanning the seat-belt region (21), as well as
observations that mutations of identical
-subunit residues have
hormone-dependent effects on activity (10-13, 25, 34). In
this respect, we have shown that identical mutations in the
33-44
domain, which includes a positively charged
-helix at
40-46,
truncation of the
-carboxyl terminus, and deletion of the
carbohydrate consensus sequence at
Asn52, all affected
hTSH subunit association or activity differently than in the analogous
hCG and hFSH mutants (10-12). Intriguingly, these
-subunit domains
are in close proximity to the seat-belt in the crystal structure of hCG
and an hTSH homology model (5, 13). It has thus been proposed that they
may form a composite receptor-binding domain (2, 3). In contrast, a
peripheral
-subunit receptor binding domain located at the tip of
the
1 loop,
11-20, appears to be important for all glycoprotein
hormones (13). It is tempting to speculate that the lack of specificity of the
11-20 domain is related to its distance from the seat-belt region. On the other hand, the
-subunit domains in close proximity to the
-subunit seat-belt may be spatially oriented by the seat-belt to contact the appropriate receptor in a hormone-dependent
fashion. In this respect, a direct cooperation between the
complementary charged residues Lys91 of the
-subunit and
Asp397 of the CG/LH receptor has recently been demonstrated
(35).
Thus our findings suggest that, during the evolutionary divergence of the glycoprotein hormones from a common ancestor gene (36), determinants of ligand specificity have evolved independently and in different ways. The seat-belt region appears to be critical to direct glycoprotein hormone binding to either the CG/LH receptor or to the TSH and FSH receptor. Determinants mediating discrimination between the TSH and FSH receptor remain to be elucidated.