The Differential Binding Affinities of the Luteinizing Hormone (LH)/Choriogonadotropin Receptor for LH and Choriogonadotropin Are Dictated by Different Extracellular Domain Residues
Colette Galet and
Mario Ascoli
Department of Pharmacology, Roy J. and Lucille A. Carver College of Medicine, The University of Iowa, Iowa City, Iowa 52242-1109
Address all correspondence and requests for reprints to: Dr. Mario Ascoli, Department of Pharmacology, Roy J. and Lucille A. Carver College of Medicine, The University of Iowa, 2319B Bowen Sciences Building, 51 Newton Road, Iowa City, Iowa 52242-1109. E-mail: mario-ascoli{at}uiowa.edu.
 |
ABSTRACT
|
---|
The high degree of amino acid sequence homology and the divergent ligand binding affinities of the rat (r) and human (h) LH receptors (LHRs) allowed us to identify amino acid residues of their extracellular domain that are responsible for the different binding affinities of bovine (b) and hLH, and human choriogonadotropin (hCG) to the hLHR and rLHR. Because of the proposed importance of the ß-sheets of the leucine-rich repeats (LRRs) of the extracellular domain of the LHR on hormone binding, we examined 10 divergent residues present in these regions by analyzing two complementary sets of mutants in which hLHR residues were substituted with the corresponding rLHR residues and vice versa. These experiments resulted in the identification of a single residue (a Ile or Ser in the C-terminal end of LRR2 of the hLHR or rLHR, respectively) that is important for hLH binding affinity. Surprisingly, however, this residue does not affect hCG or for bLH binding affinity. In fact, the results obtained with bLH and hCG show that several of the divergent residues in the ß-sheets of LRR19 affect bLH binding affinity, but none of them affect hCG binding affinity. Importantly, our results also emphasize the involvement of residues outside of the ß-sheets of the LRRs of the LHR in ligand binding affinity. This finding has to be considered in future models of the interaction of LH/CG with the LHR.
 |
INTRODUCTION
|
---|
THE LH RECEPTOR (LHR) is present in the Leydig cells in the testes and in the granulosa/luteal and theca cells in the ovary, and it plays a critical role in reproductive physiology (reviewed in Refs.1, 2, 3). In men and normal cycling women the LHR binds LH, an anterior pituitary hormone that controls testosterone synthesis in males and is responsible for ovulation in women. The LHR also binds choriogonadotropin (CG), a placental hormone that maintains progesterone synthesis by the corpus luteum during the first trimester of pregnancy and stimulates the fetal Leydig cells to produce testosterone (reviewed in Ref.1). When engaged by these hormones, the LHR couples to a number of G proteins, and it stimulates the cAMP- and inositol phosphate-signaling cascades (reviewed in Ref.2). The functional consequences of the stimulation of the inositol phosphate cascade are not clearly understood, but the cAMP pathway appears to be the principal mediator of ovulation and steroid biosynthesis (reviewed in Refs.1, 2, 3).
The LHR, FSH receptors (FSHRs), and TSH receptors are the initial members of a growing subfamily of G protein-coupled receptors that are characterized by the presence of a large extracellular domain containing several leucine-rich repeats (LRRs) flanked by cysteine-rich regions. These are now known as the LRR-containing G protein-coupled receptor subfamily (4). Among the different members of the LRR-containing G protein-coupled receptor subfamily, the LHR, FSHR, and TSH receptor are highly homologous (3, 4), and they bind highly homologous and complex glycoprotein hormones that are composed of a common
-subunit and a hormone-specific ß-subunit joined by noncovalent interactions (3, 5). Although atomic structures of the extracellular domains of the glycoprotein hormone receptors are not yet available, a number of models of the LRR portions have been constructed using the crystal structure of the ribonuclease inhibitor as the template (2, 3, 6, 7, 8, 9). These models predict that each LRR is composed of 2024 residues that form a ß-strand and a
-helix arranged in parallel to a common axis and organized into a horseshoe-shaped structure with the ß-strands on the concave side and the
-helices on the convex side. These models have also led to the proposal that the glycoprotein hormones may bind to the extracellular domain of the LHR mostly through contact points present in the ß-sheets on the concave side of this horseshoe-shaped structure (2, 3, 6, 8, 9).
A number of studies have already conclusively shown that the extracellular domain of the LHR is sufficient to bind LH or CG with high affinity and with appropriate specificity (reviewed in Refs.2, 3 , and 10), and several studies have shown that conserved residues present in the ß-strands of some of the LRRs are essential for human (h) CG binding and for specificity (8, 9, 11, 12, 13, 14). Nonconserved residues are also likely to modulate ligand binding affinity, however, because LHRs from different species that display limited amino acid sequence divergence often exhibit substantial differences in their binding affinities for LH and CG (reviewed in Refs.2 and 10).
In the studies presented here we took advantage of the high degree of amino acid sequence identity and divergent ligand-binding affinities of the human and the rat (r) LHR to identify LRR residues that contribute to the differential binding affinities of LH and CG, the two hormones that bind to the LHR.
 |
RESULTS
|
---|
A number of studies have demonstrated that the rLHR has a higher binding affinity for hCG, hLH, and other animal LHs than the hLHR (reviewed in Refs.2 and 10). This phenomenon is illustrated by the [125I]hCG competition binding assays shown in Fig. 1
. The concentrations of hLH, bovine (b) LH, or hCG required to inhibit [125I]hCG binding are at least 1 order of magnitude higher in cells transiently expressing the hLHR than with cells transiently expressing the rLHR. This difference is particularly pronounced with bLH, which binds well to the rLHR but binds very poorly (if at all) to the hLHR (see right panel of Fig. 1
and Refs.15 and 16).

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 1. The rLH and hLHR Bind hCG, hLH, and bLH with Different Affinities
HEK293T cells transiently transfected with the rLHR or hLHR were incubated overnight at 4 C with a trace concentration of [125I]hCG alone or together with increasing concentrations of hCG, hLH, or bLH as indicated. The amount of [125I]hCG bound was determined as described in Materials and Methods and is expressed as percent of the amount of [125I]hCG bound to the cells incubated without nonradioactive hormones. Each point shows the average ± SEM of eight to 16 independent transfections. Displacement curves were calculated using the nonlinear regression algorithms included with the Prism Software Package. The dashed lines mark the concentrations of each hormone that inhibit [125I]hCG binding by 50% (i.e. IC50 values).
|
|
Figure 2A
shows an alignment of the amino acid sequences of the extracellular domains of the rLHR and the hLHR. In this figure NCR and CCR refer to the N- and C-terminal cysteine-rich regions, respectively and LRR1LRR9 refer to LRRs 19. Each LRR folds into a ß-strand and a
-helix (Fig. 2B
and Ref.7) and the nine LRRs are thought to be organized into a horseshoe-shaped structure with the ß-strands on the concave side and the
-helices on the convex side (Fig. 2C
and Refs.2, 3, 8 , and 9). The ß-strands of each LRR (highlighted in gray in Fig. 2A
) are composed of a highly conserved X1X2(L)X3(L)X4X5 sequence in which X denotes any amino acid and L denotes a hydrophobic residue, usually leucine or isoleucine (7). In the ribonuclease inhibitor the side chains of the leucine/isoleucine residues face the helical segments and form the hydrophobic core of the LRR, whereas the side chains of the X residues are exposed to the solvent (Fig. 2B
and Ref.7). In the LHR these residues have been proposed to directly interact with the ligands (3, 7, 8, 9).
Figure 2A
shows that the extracellular domains of the rLHR and the hLHR diverge by 50 amino acid residues, but only 10 of these are present at or near the ß-strands of the LRRs. These divergent residues are highlighted in red and are present in LRR1, 2, 3, 7, 8, and 9.
In the experiments described below we took advantage of the differences in binding affinities illustrated in Fig. 1
and of the high degree of amino acid sequence identity between the extracellular domains of the rLHR and the hLHR (illustrated in Fig. 2A
) to identify divergent LHR residues that may be important determinants of ligand binding affinity. We concentrated on the 10 divergent residues present at or near the ß-strands of the LRRs because these have been proposed to be involved in ligand binding (reviewed in Refs.2, 3 , and 10). To test for their potential involvement in ligand binding affinity, we prepared a complementary set of mutants in which each of the divergent residues were sequentially exchanged between the hLHR and the rLHR as shown in Table 1
.1 Each of these mutants was then transiently expressed in human embryonic kidney (HEK) 293T cells, and the cells were used in a simple competition binding assay in which they were incubated with a trace concentration of [125I]hCG alone or together with a single concentration of hLH or bLH. We chose to test hLH and hCG because these are the two physiologically important hormones that bind to the hLHR (1, 2, 3). We also tested bLH because there is a higher degree of amino acid sequence homology between hLH and bLH than between hLH and hCG (5, 17, 18).
Figure 3
summarizes the results obtained with hLH and the rLHR exchange mutants containing the corresponding hLHR residues (panel A) or the hLHR exchange mutants containing the corresponding rLHR residues (panel B). Figure 3A
shows that 0.9 nM hLH inhibits [125I]hCG binding to the wild-type rLHR (rLHR-wt) by approximately 70%, but it has little or no effect on [125I]hCG binding to the hLHR-wt. Thus, if any of the hLHR residues introduced into the rLHR are involved in binding hLH we would expect 0.9 nM hLH to become less effective in inhibiting [125I]hCG binding. Figure 3A
shows that 0.9 nM hLH prevents the binding of [125I]hCG to rLHR M1, M2, and M3 to about the same extent as that detected with the rLHR-wt. In contrast, the inhibitory effect of 0.9 nM hLH on rLHR M4, M5, M6, M8, M9, and M10 is similar to that detected with the hLHR-wt.

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 3. Divergent Residues in LRR2 and LRR3 Contribute to the Differential Binding of Affinity of the rLHR and the hLHR for hLH
HEK293T cells transiently transfected with the rLHR-wt, hLHR-wt, or mutants thereof were incubated overnight at 4 C with a trace concentration of [125I]hCG alone or together with 0.9 or 9 nM hLH as indicated. The amount of [125I]hCG bound was determined as described in Materials and Methods and is expressed as percent of the amount of [125I]hCG bound to the cells incubated without nonradioactive hormones. Each bar shows the average ± SEM of three to eight independent transfections. *, Significantly different (P < 0.05) than the original construct (i.e. rLHR-wt in panel A and hLHR-wt in panel B).
|
|
Figure 3B
summarizes the results obtained for the complementary hLHR exchange mutants containing rLHR residues. For these experiments we chose to perform the [125I]hCG competition binding assays using 9 nM hLH because this concentration of hLH inhibits [125I]hCG binding to the hLHR-wt by approximately 30% but it inhibits the binding of [125I]hCG binding to the rLHR-wt by about 90%. Thus, if any of the rLHR residues introduced into the hLHR are involved in binding hLH we would expect 9 nM hLH to become more effective in inhibiting [125I]hCG binding. In agreement with the data presented in Fig. 3A
, the results presented in Fig. 3B
show that hLHR M1, M2, and M3 behave like the hLHR-wt. Starting with hLHR M4, however, all subsequent exchange mutants show a displacement similar (but not identical) to that seen with the rLHR-wt.
Thus, this screening of the rLHR and hLHR complementary exchange mutants hLHR shows that the divergent residues in LRR1 and the first divergent residue in LRR2 (i.e. those mutated in the M1, M2, and M3 series of mutants, see Table 1
) are not involved in hLH binding whereas the second divergent residues in LRR2 and/or the single divergent residues in LRR3 (i.e. those mutated in the M4 and M5 series of mutants, see Table 1
) are involved in hLH binding. We can also conclude that the divergent residues in LRR7, LRR8, and LRR9 are not involved in hLH binding because the M6, M8, M9, and M10 series of mutants behave similarly to the M4 and M5 series.
The same mutants were also used for competition binding assays with bLH instead of hLH. For the same reasons outlined above, bLH was used at a concentration of 3 nM with the rLHR mutants containing hLHR residues (Fig. 4A
) and at 6 µM with the hLHR mutants containing rLHR residues (Fig. 4B
). Similarly to the data obtained when hLH was used as a competitor, a switch between the efficiency of bLH to inhibit [125I]hCG binding to the two sets of mutants is detected starting with the M4 series. There are two differences between the two sets of mutants, however. First, bLH is a better competitor for [125I]hCG binding to the rLHR M3 mutant when compared with the rLHR-wt (Fig. 4A
) without a complementary change (i.e. a decrease in competition) when the hLHR M3 mutant is compared with the hLHR-wt (Fig. 4B
). Second, bLH seems to be a worse competitor for [125I]hCG binding to the hLHR M1 mutant when compared with the hLHR-wt without a complementary change when the rLHR M1 mutant is compared with the rLHR-wt. These differences were not further investigated because of the lack of complementarity of the results obtained with the two sets of mutants.

View larger version (29K):
[in this window]
[in a new window]
|
Fig. 4. Divergent Residues in LRR2 and Beyond Contribute to the Differential Binding Affinity of the rLHR and hLHR for bLH
HEK293T cells transiently transfected with the rLHR-wt, hLHR-wt, or mutants thereof were incubated overnight at 4 C with a trace concentration of [125I]hCG alone or together with 3 nM or 6 µM bLH as indicated. The amount of [125I]hCG bound was determined as described in Materials and Methods and is expressed as percent of the amount of [125I]hCG bound to the cells incubated without nonradioactive hormones. Each bar shows the average ± SEM of three to 10 independent transfections. *, Significantly different (P < 0.05) than the original construct (i.e. rLHR-wt in panel A and hLHR-wt in panel B).
|
|
Based on the results presented in Figs. 3
and 4
, we prepared and analyzed an additional series of mutants in which the two divergent residues in LRR2 and the single divergent residue in LRR3 (i.e. those mutated simultaneously in the M4 and M5 series of mutants) were individually exchanged between the rLHR and the hLHR. These were again used for competition binding assays as described above, and results are shown in Figs. 5
for hLH and Fig. 6
for bLH.

View larger version (29K):
[in this window]
[in a new window]
|
Fig. 5. A Single Divergent Residue in LRR2 Contributes to the Differential Binding Affinity of the rLHR and hLHR for hLH
HEK293T cells transiently transfected with the rLHR-wt, hLHR-wt, or mutants thereof were incubated overnight at 4 C with a trace concentration of [125I]hCG alone or together with 0.9 or 9 nM hLH as indicated. The amount of [125I]hCG bound was determined as described in Materials and Methods and is expressed as percent of the amount of [125I]hCG bound to the cells incubated without nonradioactive hormones. Each bar shows the average ± SEM of three to 10 independent transfections. *, Significantly different (P < 0.05) than the original construct (i.e. rLHR-wt in panel A and hLHR-wt in panel B).
|
|

View larger version (31K):
[in this window]
[in a new window]
|
Fig. 6. Multiple Divergent Residues in LRR2 and Beyond Contribute to the Differential Binding Affinity of the rLHR and the hLHR for bLH
HEK293T cells transiently transfected with the rLHR-wt, hLHR-wt, or mutants thereof were incubated overnight at 4 C with a trace concentration of [125I]hCG alone or together with 3 nM or 6 µM bLH as indicated. The amount of [125I]hCG bound was determined as described in Materials and Methods and is expressed as percent of the amount of [125I]hCG bound to the cells incubated without nonradioactive hormones. Each bar shows the average ± SEM of three to 10 independent transfections. *, Significantly different (P < 0.05) than the original construct (i.e. rLHR-wt in panel A and hLHR-wt in panel B).
|
|
Figure 5A
shows that the individual exchange of the first divergent residue in LRR2 of the rLHR for the corresponding hLHR residue (rLHRV54I) has no effect on the ability of hLH to prevent [125I]hCG binding (i.e. this mutant behaves like the rLHR-wt) whereas the individual exchange of the second divergent residue in LRR2 (rLHRS61I) shifts the magnitude of the inhibitory effect of hLH toward that detected with the rLHR M4, M5, or M6 mutants. The individual exchange of the single divergent residue in LRR3 (rLHRL81I) has no effect on the ability of hLH to prevent [125I]hCG binding. The results obtained with the complementary mutants revealed the same trend (Fig. 5B
). The individual exchange of the first divergent residue in LRR2 of the hLHR for the corresponding rLHR residue (hLHRI76V) has no effect on the ability of hLH to prevent [125I]hCG binding, whereas the individual exchange of the second divergent residue in LRR2 (hLHRI83S) shifts the magnitude of the inhibitory effect of hLH toward that detected with the hLHR M4, M5, or M6 mutants. Lastly, the individual exchange of the single divergent residue in LRR3 (hLHRI103L) has no effect on the inhibitory efficiency of hLH.
The results obtained when bLH was used as a competitor are shown in Fig. 6
. These results are different than those obtained when hLH is used as a competitor and not always complementary for the two sets of mutants. The only pair of mutants that behaved in a complementary fashion were the S/I exchange mutants in LRR2 (i.e. rLHR-S61I or hLHR-I83S). The V/I exchange mutants of LRR2 and the L/I exchange mutants in LRR3 affected the ability of bLH to compete for [125I]hCG binding to the hLHR (Fig. 6B
) but not to the rLHR (Fig. 6A
).
A more complete analysis of the two exchange mutants containing a single-point mutation highlighted above (i.e. rLHR-S61I and hLHR-I83S) as well as the multiple M5 and M10 mutants was next accomplished by performing full competition binding assays using hLH and bLH as shown in Figs. 7
and 8
. This allowed us to calculate the inhibition constants (Ki) for each hormone and thus perform a more quantitative comparison as summarized in Table 2
.

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 7. A Single Divergent Residue in LRR2 Contributes to the Differential Binding of Affinity of the rLHR (A) and the hLHR (B) for hLH
HEK293T cells transiently transfected with the rLHR-wt, hLHR-wt, or mutants thereof were incubated overnight at 4 C with a trace concentration of [125I]hCG alone or together with increasing concentrations of hLH as indicated. The amount of [125I]hCG bound was determined as described in Materials and Methods and is expressed as percent of the amount of [125I]hCG bound to the cells incubated without nonradioactive hormones. Each point shows the average ± SEM of three to 12 independent transfections. Displacement curves were calculated using the nonlinear regression algorithms included with the Prism Software Package.
|
|

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 8. Several Divergent Residues in the Extracellular Domain of the LHR Contribute to the Differential Binding of Affinity of the rLHR and the hLHR for bLH
A, HEK293T cells transiently transfected with the rLHR or mutants thereof were incubated overnight at 4 C with a trace concentration of [125I]hCG alone or together with increasing concentrations of bLH as indicated. The amount of [125I]hCG bound was determined as described in Materials and Methods and is expressed as percent of the amount of [125I]hCG bound to the cells incubated without nonradioactive hormones. Each point shows the average ± SEM of three to 16 independent transfections. Displacement curves were calculated using the nonlinear regression algorithms included with the Prism Software Package. B, HEK293T cells transiently transfected with the hLHR-wt or mutants thereof were incubated overnight at 4 C with a trace concentration of [125I]hCG alone or together with 6 µM bLH as indicated. The amount of [125I]hCG bound was determined as described in Materials and Methods and is expressed as percent of the amount of [125I]hCG bound to the cells incubated without nonradioactive hormones. Each bar shows the average ± SEM of four to 16 independent transfections.
|
|
View this table:
[in this window]
[in a new window]
|
Table 2. Distinct Amino Acids Contribute to the Differential Binding Affinity of the rLHR and the hLHR for hLH, bLH, and hCG
|
|
The Ki values for hLH for the rLHR-wt and hLHR-wt are 0.09 and 8.9 nM, respectively (Table 2
). Exchanging all divergent residues of the extracellular domain between the rLHR and the hLHR (i.e. the M10 mutants) or only those in LRR1, LRR2, and LRR3 (i.e. the M5 mutants) increases the Ki of hLH for the rLHR to 0.40.6 nM and decreases the Ki of hLH for the hLHR to 13 nM (Table 2
). Remarkably, the exchange of a single divergent residue, the serine/isoleucine pair present in LRR2, causes a complementary change in the affinity of hLH for the rLHR and hLHR that is of the same magnitude as the change observed when all divergent residues in the extracellular domain were exchanged (compare rLHRS61I with rLHRM10 and hLHRI83S with hLHRM10 in Table 2
and Fig. 7
). It is important to note, however, that although this change in affinity is complementary in nature and similar in magnitude (i.e. a
4-fold change), it does not completely reverse the affinity of the two receptors for hLH (Fig. 7
and Table 2
). These results clearly show that residues outside of the ß-sheets of the LRRs can also modulate the binding affinity of hLH.
The results obtained with bLH as a competitor are presented in Fig. 8
and Table 2
. The Ki values for bLH with the rLHR-wt and hLHR-wt are 1.5 and more than 10,000 nM, respectively. For the rLHR mutants the individual exchange of the second divergent residue in LRR2 (i.e. rLHR S61I) causes no significant change in the Ki whereas the simultaneous exchange of more divergent residues as exemplified by the rLHR M5 and rLHR M10 mutants induces a progressive shift in Ki values toward that observed with the hLHR-wt (Fig. 8A
and Table 2
). The Ki values for bLH for the complementary hLHR mutants could not be determined because the concentrations of bLH required to prevent [125I]hCG binding to the hLHR-wt are very high (see Fig. 8A
). When a single, high concentration of bLH (6 µM) was used, however, a progressive increase in its ability to displace [125I]hCG was detected as expected (Fig. 8B
). Thus, in contrast to the binding affinity for hLH, which is dictated, in large part, by the divergent S/I pair in LRR2, the binding affinity for bLH appears to be influenced mostly by divergent residues present in LRR3 as well as LRR7 through 9. Because the simultaneous mutation of the 10 divergent residues present in the ß-sheets of LRR19 of the rLHR do not fully change the bLH binding affinity to that of the hLHR (Table 2
), these results also implicate residues outside of the ß-sheets of the LRRs as modulators of the binding affinity of bLH.
Lastly, because the LHR also binds hCG we performed displacement experiments with this hormone (Fig. 9
) and calculated the Ki values for each of the mutants discussed above. The Ki values for hCG with rLHR-wt and hLHR-wt are 0.16 and 4.2 nM, respectively (Table 2
). The exchange of the single S/I pair in LRR2 and the simultaneous exchange of all divergent residues in LRR13 (i.e. the M5 mutants) or of all divergent residues in LRR19 (i.e. the M10 mutants) have no effect on the Ki for hCG and the rLHR (Fig. 9A
and Table 2
) or the Ki for hCG and the hLHR (Fig. 9B
and Table 2
).

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 9. None of the Divergent Residues in the LRRs of the LHR Contribute to the Differential Binding of Affinity of the rLHR (A) and the hLHR (B) for hCG
HEK293T cells transiently transfected with the rLHR-wt, hLHR-wt, or mutants thereof were incubated overnight at 4 C with a trace concentration of [125I]hCG alone or together with increasing concentrations of hCG as indicated. The amount of [125I]hCG bound was determined as described in Materials and Methods and is expressed as percent of the amount of [125I]hCG bound to the cells incubated without nonradioactive hormones. Each point shows the average ± SEM of three to 15 independent transfections. Displacement curves were calculated using the nonlinear regression algorithms included with the Prism Software Package.
|
|
 |
DISCUSSION
|
---|
In the present study we used a complementary mutagenesis approach to identify residues that contribute to the differential binding affinities of the rLHR and hLHR for LH and CG. Because this mutagenesis approach used is based on exchanging two divergent residues that are known to support LH and CG binding to the LHR of humans or rodents, the results obtained cannot be confounded by gross changes in overall structure of the receptor. For the same reasons the results obtained could not be due to a potentially wrong choice of the amino acid chosen for mutagenesis or the nature of the amino acid mutation introduced. Moreover, because we used two complementary sets of mutants (i.e. rat residues into the hLHR and human residues into the rLHR), the results obtained with these two sets of mutants are also expected to be complementary. Thus, if any of the divergent residues contribute to the binding affinity of LH or CG we would expect an increase in binding affinity when the hLHR residues are replaced with the corresponding rLHR residues and a complementary decrease in binding affinity when the same rLHR residues are replaced with the corresponding hLHR residues.
Although there are 50 divergent residues in the extracellular domains of the rLHR and hLHR, we chose to pursue the exchange mutagenesis studies only on the 10 divergent residues present in the ß-strands of LRR19 (Fig. 2
) because residues located in these regions have been proposed to directly contact the ligands (2, 3, 6, 8, 9). In fact, mutagenesis studies of conserved residues present in some of these LRRs clearly support this proposal (8, 9). Our studies are novel, however, because they were aimed solely at the nonconserved residues present in these regions. As such, this approach should not identify residues that are essential for the binding of LH and CG. Instead, it should (and it did) identify residues that modulate binding affinity.
There are three unexpected conclusions that can be drawn from the data presented here.
Conclusion 1. The Residues Involved in Determining the Differential Binding Affinity of the rLHR and the hLHR for hCG Are Not Located in the LRRs
Our data show that the simultaneous exchange of the 10 divergent residues of the ß-sheets of LRR19 between the hLHR and the rLHR does not affect the binding affinity of either one of these two receptors for hCG. This does not mean that ß-sheet residues of LRR19 are not involved in hCG binding. In fact, previous studies have already shown that mutation of charged residues in the ß-strands of LRR58 prevent hCG binding (8, 9). Because these previously identified residues are conserved between the rLH and the hLHR, they were not examined here (Fig. 2A
and Refs.8 and 9).
In this respect it is also interesting to note that a recent study employing exchange mutagenesis between the hFSHR and the hLHR resulted in the identification of two residues present in position X5 (i.e. at the edge of the ß-strands) of LRR3 and LRR6 of the hFSHR that, when substituted with the corresponding hLHR residues (Asp107 or Gly183, respectively), greatly increases the binding affinity of the hFSHR for hCG without compromising its affinity for hFSH (12, 13, 14). Thus, Asp107 in position X5 of LRR3 and Gly183 in position X5 of LRR6 of the hLHR must also represent contact points between the hLHR and hCG. These two residues are also conserved between the rLHR and the hLHR (Figs. 2
and 10
), and therefore they do not contribute to the differential binding affinities of these two receptors for hCG.

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 10. Structural Models Depicting the ß-Sheets of LRR1-LRR3 of the hLHR (A) and the rLHR (B)
The predicted structures (6 7 ) of the ß-sheets of LRR1-LRR3 are shown in blue, and the side chains of the amino acids of the hLHR (A) or rLHR (B) are colored according to their physical properties: green for hydrophobic, yellow for polar, and red for acidic. The ß-sheet of LRR1 is on the right side and the ß-sheet of LRR3 is on the left side of each panel, respectively. Ile83 (in panel A, the hLHR) or Ser61 (in panel B, the rLHR) are marked with a white arrow. This is the residue pair located in position X5+1 of LRR2 that was identified here as being important for hLH binding affinity. All amino acids within a 4 Å radius of this residue are enclosed in the orange oval. Asn107 (in panel A, the hLHR) or Asn85 (in panel B, the rLHR) are marked with a double white arrow. This is a conserved residue in position X5 of LRR3 of the LHR that has been shown to participate in hCG binding selectivity (12 13 14 ).
|
|
When considered together, these three sets of studies show that several conserved residues present in ß-sheets of the LRRs are the main binding determinants for hCG but that the differential binding affinity of hCG for the rLHR and the hLHR is dictated by one or more of the 40 divergent residues that are not located in or near these ß-strands.
Conclusion 2. The ß-Sheet LRR Residues Involved in Determining the Differential Binding Affinities of the rLHR and hLHR for bLH, hLH, and hCG Are Different
Our data show that there is little (if any) overlap in the identity of the divergent amino acids of the ß-sheets of the LRRs that determine the binding affinities of the LHR for bLH, hLH, and hCG. We identified one LRR residue (Ser61 or Ile83 in the connecting loop of LRR2 of the hLHR or rLHR, respectively) that contributes to the divergent binding affinity of hLH, but this residue does not contribute to the divergent binding affinity of bLH or hCG (Table 2
). In fact, our studies show that the binding affinity of the LHR for bLH is not influenced by the divergent residues in the ß-sheets of LRR1 or 2. A change in the binding affinity for bLH is first observed only when the divergent residues in LRR13 are simultaneously exchanged. The magnitude of this change in affinity becomes much more pronounced when all divergent residues in LRR19 are exchanged. These data suggest that only those divergent residues located in the ß-sheets of LRR3, LRR7, LRR8, and/or LRR9 play an important contribution to bLH binding affinity. These results are in agreement with those of Moyle and co-workers (16), who previously concluded that the C-terminal portion of the extracellular domain of the hLHR inhibits bLH binding. When considered together, these results clearly support the hypothesis that there are multiple contacts between bLH and some of the LHR residues present in the ß-sheets of the LRRs (2, 3, 6, 8, 9). It should be clear, however, that other divergent residues that are not located in or near the ß-strands of the extracellular domain of the LHR must also be involved in bLH and hLH binding because exchanging all divergent residues in the ß-strands of LRR19 between the human and rat LHR does not completely reverse their binding affinity for these two hormones (Table 2
).
Conclusion 3. Only One of the 10 Divergent Residues Present in the ß-Sheets of LRR19 of the LHR Contributes to the Differential Binding Affinity of the rLHR and the hLHR for hLH
Of the 10 divergent residues present in or near the ß-strands of LRR19, only one residue (Ser61 in the rLHR or Ile83 in the hLHR) located immediately after position X5 of LRR2 (Figs. 2
and 10
) contributes to the differential binding affinity of the hLHR and the rLHR for hLH. A Ser present in this position of the rLHR or an Ile at this position of the hLHR supports a high or a low binding affinity for hLH, respectively. This important residue is just beyond the boundary of LRR2, and thus it is believed to be in one of the loops that connect the LRR ß-strand to the parallel
-helix rather than on the ß-strand itself (Figs. 2
and 10
). We speculate that an isoleucine in this position of the hLHR would favor an interaction with the hydrophobic core formed by the two conserved leucines of LRR2 (Fig. 10A
), whereas a serine in this position of the rLHR would discourage this interaction and perhaps allow more flexibility of this LRR and/or establish a direct contact with hLH (Fig. 10B
). It is also worth noting that although this residue is an important determinant of hLH binding affinity, it is certainly not the only residue involved in hLH binding. The binding affinities of hLH for the rLHR and the hLHR differ by 2 orders of magnitude, but the exchange of this residue collapses that difference by only 1 order of magnitude (Table 2
). Clearly, then one or more of the other 40 divergent residues (cf. Fig. 2A
) that are not located in or near the ß-strands of the extracellular domain of the LHR must also modulate the binding affinities of the rLHR and the hLHR for hLH.
It is interesting to note that the LRR residues that have been identified so far as being involved in ligand specificity (12, 13, 14) or binding affinity (this paper) are located in the C-terminal ends of the LRR2, -3, or -6. Ligand specificity is affected by a conserved Asp in position X5 of LRR3 of the hLHR (12, 13, 14), and the binding affinity of the LHR for hLH is influenced by a divergent residue in position X5+1 of LRR2 (this paper). We speculate that the proximity of the conserved Asp in position X5 of LRR3 of the hLHR to the divergent Ile in position X5+1 of LRR2 of the hLHR stabilizes an interaction between the ß-strands of LRR1 and -2 in the hLHR (Fig. 10A
), whereas the proximity of the conserved Asp in position X5 of LRR3 of the rLHR to the divergent Ser in position X5+1 of LRR2 of the rLHR could contribute to the polar environment surrounding the conserved Asp and would not favor an interaction between LRR2 and LRR3 of the rLHR (Fig. 10B
).
In summary, our experiments have unexpectedly revealed that the divergent ß-sheet LRR residues that serve as binding affinity determinants of the hLHR and the rLHR for hLH, bLH, and hCG are different. Although the amino acid sequences of these three glycoproteins are quite similar, they have different carbohydrate moieties (3, 5). These differences may lead to subtle changes in the three-dimensional structures of the hormones resulting in the different binding modalities reported here. Lastly, the data presented here also highlight the importance of residues that are not located in or near the ß-sheets of the LRRs of the LHR in ligand binding affinity. This finding is unexpected, and it has important implications in future modeling of the interaction of LH/CG with the LHR because the most widely accepted model assumes that these two hormones bind mostly through contact points present in the ß-sheets on the putative concave side of the extracellular domain of the LHR (2, 3, 6, 8, 9).
 |
MATERIALS AND METHODS
|
---|
Hormones and Supplies
HEK293T cells are a derivative of HEK293 cells that express the simian virus 40 T antigen (19) and were provided to us by Dr. Marlene Hosey (Northwestern University, Chicago, IL). Purified hCG (AFP8456A) was kindly provided by Dr. A. Parlow and the National Hormone and Pituitary Agency (National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD). Recombinant hLH and recombinant hCG were kindly provided by the Serono Reproductive Biology Institute (Boston, MA). Purified bLH was kindly provided by Dr. John Pierce (previously of the University of California at Los Angeles). [125I]hCG was prepared as described elsewhere (20). Other cell culture supplies and reagents were obtained from Corning, Inc. (Corning, NY) and Invitrogen (San Diego, CA), respectively. All other chemicals were obtained from commonly used suppliers.
Plasmids and Cells
The preparation and characterization of expression vectors for the myc-hLHR-wt (in pcDNA3.1) and the myc-rLHR-wt (in pcDNA1Neo) have been described elsewhere (21, 22). The different exchange mutants of the myc-hLHR and myc-rLHR used here were constructed by site-directed mutagenesis using the entire coding sequence of the myc-rLHR wt or the N-terminal domain (residues 25358) of the hLHR in pBluescript. The presence of the introduced mutations was verified by DNA sequencing and subcloned in the expression vector pcDNA 1 neo and pcDNA 3.1, respectively. The sequence of the mutants was again determined after subcloning into the appropriate expression vectors.
HEK293T cells were maintained in DMEM containing 10 mM HEPES, 10% newborn calf-serum, and 50 µg/ml gentamicin, pH 7.4. Cells were plated in gelatin-coated 35-mm wells and transiently transfected with 0.5 µg plasmid DNA, using the calcium phosphate methods of Chen and Okayama (23), when 7080% confluent. After an overnight incubation with the transfection mixture, the cells were washed, and they were used 24 h later for binding assays.
Competition Binding Assay
[125I]hCG competition binding assays were performed in assay medium (Waymouths MB752/1 without sodium bicarbonate but containing 20 mM HEPES and 1 mg/ml BSA, pH 7.4). The transiently transfected cells were incubated with a trace concentration of [125I]hCG (0.05 nM) alone or together with an increasing concentration of hCG, hLH, or bLH as indicated in the figures. After an overnight incubation at 4 C, the cells were scraped, collected in polypropylene tubes, and collected by centrifugation at 4 C. The supernatant was aspirated and the pellets were washed with 2 ml cold medium and centrifuged again. The supernatant was aspirated, and the radioactivity remaining with the pellet was measured using a
-counter.
The results of the competition binding assays were plotted as percent of the maximal amount of [125I]hCG bound (i.e. the [125I]hCG bound in the cells incubated without any competing hormone). This value varied between 1,000 and 20,000 cpm/well, depending on the construct used and the specific activity of the different batches of [125I]hCG. Saturation binding analysis done with [125I]hCG and several of the constructs used here showed that HEK293T transiently transfected with any of the rLHR mutants (using 0.5 µg DNA/35-mm well; see above) express 7,00014,000 receptors per cell, whereas those transfected with any of the hLHR mutants (using 0.5 µg DNA/35-mm well; see above) express 50,00080,000 receptors per cell. This expected difference in receptor density (reviewed in Ref.2) should not have an effect on binding affinities because these two parameters are independent (24).
For full competition binding assays (see Figs. 79

) the IC50 and Ki values were calculated using the Prism Software Package from GraphPad Software, Inc. (San Diego, CA). For the calculation of Ki values (Table 2
), we used the concentration of [125I]hCG included as tracer in the competition binding assays (0.05 nM; see above) and the dissociation constants (Kd values) for hCG measured from saturation binding assays done in HEK293T cells transiently transfected with the rLHR-wt or the hLHR-wt. In agreement with previous results (reviewed in Ref.2), these were calculated to be 0.07 and 1.12 nM, respectively. Because the EC50 values calculated from the competition binding curves using hCG as the competitor did not show any statistically significant differences among the rLHR-wt and the three mutants (Fig. 9A
) or among the hLHR-wt and the three mutants (Fig. 9B
), we used the measured Kd value for [125I]hCG binding to the rLHR-wt for the calculations involving the three rLHR mutants and the Kd value for hCG binding to the hLHR-wt for the calculations involving the three hLHR mutants.
Statistical analysis was done using the Mann and Whitney test with the aid of the Instat Software Package from GraphPad Software.
 |
ACKNOWLEDGMENTS
|
---|
We thank Dr. Dario Mizrachi for help with the preparation of Fig. 2
, B and C, and Fig. 10
and for useful discussions on possible structural interpretations of the data presented here. We thank Dr. Deborah L Segaloff for helpful suggestions on the preparation of this manuscript. We also thank the Serono Reproductive Biology Institute for their generous gift of recombinant hLH and hCG and for the full-length hLHR cDNA.
 |
FOOTNOTES
|
---|
This work was supported by Grant CA-40629 from the National Cancer Institute.
First Published Online January 27, 2005
Abbreviations: CG, Choriogonadotropin; FSHR, FSH receptor; HEK, human embryonic kidney; LHR, LH receptor; LRR, leucine-rich repeat; rLHR-wt, wild-type rLHR.
1 Note that by convention residue 1 of the rLHR is the amino terminus of the mature receptor identified by amino acid sequencing. Residue 1 of the hLHR is, however, the first residue of the signal peptide because the amino terminus of the mature hLHR has not been experimentally identified (2 20 ) 
Received for publication October 13, 2004.
Accepted for publication January 19, 2005.
 |
REFERENCES
|
---|
- Themmen APN, Huhtaniemi IT 2000 Mutations of gonadotropins and gonadotropin receptors: elucidating the physiology and pathophysiology of pituitary-gonadal function. Endocr Rev 21:551583[Abstract/Free Full Text]
- Ascoli M, Fanelli F, Segaloff DL 2002 The lutropin/choriogonadotropin receptor. A 2002 perspective. Endocr Rev 23:141174[Abstract/Free Full Text]
- Vassart G, Pardo L, Costagliola S 2004 A molecular dissection of the glycoprotein hormone receptors. Trends Biochem Sci 29:119126[CrossRef][Medline]
- Hsu SY, Hsueh AJ 2000 Discovering new hormones, receptors, and signaling mediators in the genomic era. Mol Endocrinol 14:594604[Free Full Text]
- Pierce JG, Parsons TF 1981 Glycoprotein hormones: structure and function. Annu Rev Biochem 50:465495[CrossRef][Medline]
- Jiang X, Dreano M, Buckler DR, Cheng S, Ythier A, Wu H, Hendrickson WA, El Tayar N 1995 Structural predictions for the ligand-binding region of glycoprotein hormone receptors and the nature of hormone-receptor interactions. Structure 15:13411353[CrossRef]
- Kobe B, Kajava AV 2001 The leucine-rich repeat as a protein recognition motif. Curr Opin Struct Biol 11:725732[CrossRef][Medline]
- Bhowmick N, Huang J, Puett D, Isaacs NW, Lapthorn AJ 1996 Determination of residues important in hormone binding to the extracellular domain of the luteinizing hormone/ chorionic gonadotropin receptor by site-directed mutagenesis and modeling. Mol Endocrinol 10:11471159[Abstract]
- Bhowmick N, Narayan P, Puett D 1999 Identification of ionizable amino acid residues on the extracellular domain of the lutropin receptor involved in ligand binding. Endocrinology 140:45584563[Abstract/Free Full Text]
- Segaloff DL, Ascoli M 1993 The lutropin/choriogonadotropin (LH/CG) receptor. 4 years later. Endocr Rev 14:324347[Abstract]
- Smits G, Govaerts C, Nubourgh I, Pardo L, Vassart G, Costagliola S 2002 Lysine 183 and glutamic acid 157 of the TSH receptor: two interacting residues with a key role in determining specificity toward TSH and human CG. Mol Endocrinol 16:722735[Abstract/Free Full Text]
- Vischer HF, Granneman JCM, Noordam MJ, Mosselman S, Bogerd J 2003 Ligand selectivity of gonadotropin receptors. Role of the ß-strands of extracellular leucine-rich repeats 3 and 6 of the human luteinizing hormone receptor. J Biol Chem 278:1550515513[Abstract/Free Full Text]
- Vischer HF, Granneman JCM, Bogerd J 2003 Opposite contribution of two ligand-selective determinants in the N-terminal hormone-binding exodomain of human gonadotropin receptors. Mol Endocrinol 17:19721981[Abstract/Free Full Text]
- Smits G, Campillo M, Govaerts C, Janssens V, Richter C, Vassart G, Pardo L, Costagliola S 2003 Glycoprotein hormone receptors: determinants in leucine-rich repeats responsible for ligand specificity. EMBO J 22:26922703[Abstract/Free Full Text]
- Jia X-C, Oikawa M, Bo M, Tanaka T, Ny T, Boime I, Hsueh AJW 1991 Expression of human luteinizing hormone (LH) receptor: interaction with LH and chorionic gonadotropin from human but not equine, rat, and ovine species. Mol Endocrinol 5:759768[Abstract]
- Bernard MP, Myers RV, Moyle WR 1998 Lutropins appear to contact two independent sites in the extracellular domain of their receptors. Biochem J 335:611617[Medline]
- Strickland TW, Parsons TF, Pierce JG 1985 Structure of LH and hCG. In: Ascoli M, ed. Luteinizing hormone action and receptors. Boca Raton, FL: CRC Press; 116
- Pierce JG 1988 Gonadotropins: chemistry and biosynthesis. In: Knobil E, Neill JD, Ewing LL, Greenwald GS, Markert CL, Pfaff DW, eds. The physiology of reproduction. New York: Raven Press; 13351348
- Margolskee R, McHenry-Rinde B, Horn R 1993 Panning transfected cells for electrophysiological studies. Biotechniques 15:906911[Medline]
- Ascoli M, Puett D 1978 Gonadotropin binding and stimulation of steroidogenesis in Leydig tumor cells. Proc Natl Acad Sci USA 75:99102[Abstract]
- Fabritz J, Ryan S, Ascoli M 1998 Transfected cells express mostly the intracellular precursor of the lutropin/choriogonadotropin receptor but this precursor binds choriogonadotropin with high affinity. Biochemistry 37:664672[CrossRef][Medline]
- Min L, Ascoli M 2000 Effect of activating and inactivating mutations on the phosphorylation and trafficking of the human lutropin/choriogonadotropin receptor. Mol Endocrinol 14:17971810[Abstract/Free Full Text]
- Chen C, Okayama H 1987 High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol 7:27452752[Medline]
- Jenkinson DH 2003 Classical approaches to the study of drug-receptor interactions. In: Foreman JC, Johansen T, eds. Textbook of receptor pharmacology. 2nd ed., Boca Raton, FL: CRC Press; 378