The lutropin/choriogonadotropin receptor is a
seven-transmembrane receptor and consists of two major domains of
similar size, an extracellular exodomain and a membrane-associated
endodomain which includes 3 exoloops. The uniquely large exodomain is
responsible for high affinity hormone binding whereas receptor
activation occurs at the endodomain. However, little is known about the
relationship between the exodomain and endodomain. It was reported that
hormone binding to the exodomain was improved when the endodomain was truncated. This result suggests that hormone binding to the exodomain was influenced by the endodomain. To test this hypothesis, amino acids
of exoloop 2 were examined by Ala substitutions. The binding affinity
was enhanced by some Ala substitutions but attenuated by others. These
results indicate that exoloop 2 influences the hormone binding to the
exodomain. Particularly, the high affinity hormone binding at the
exodomain is constrained by a group of amino acids,
Ser484, Asn485, Lys488,
Ser490, and Ser499. Computer modeling suggests
these residues may be positioned on one side of exoloop 2. It also
influences the affinity for cAMP induction and the maximal cAMP
production in distinct ways, in addition to its influence on the
hormone binding affinity. The distinct ways of influencing these
functions are sometimes in conflict and compromised to attain the
maximal affinity for cAMP induction. As a result, the exodomain attains
the maximal affinity for hormone binding when the endodomain is
truncated and cAMP induction is disengaged.
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INTRODUCTION |
The LH/CG1 receptor
belongs to a subfamily of glycoprotein hormone receptors within
the seven-transmembrane (TM) receptor family. Unlike other
seven-transmembrane receptors, glycoprotein hormone receptors consist
of two equal halves, an extracellular N-terminal half (exodomain) and a
membrane associated C-terminal half (endodomain) (1, 2). The
exodomain alone is capable of high affinity hormone binding (3-5) with
no hormone action (5, 6). On the other hand, the endodomain is the site
for receptor activation (7). However, little is known about the
relationship between the exodomain and endodomain.
In this study, the hormone binding affinity of the truncated exodomain
which lacks the endodomain is shown to be slightly and consistently
higher than that of the wild type receptor. This result suggests that
the endodomain influences the hormone binding at the exodomain. To
identify the responsible amino acid residues the 20 amino acids of
exoloop 2 in the wild type receptor, from Ser484 to
Gln503, were Ala scanned.
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EXPERIMENTAL PROCEDURES |
Mutagenesis and Functional Expression of LH/CG
Receptors--
Mutant LH/CG-R cDNAs were prepared in pSELECT
vector using the Altered Sites Mutagenesis System (Promega), sequenced,
subcloned into pcDNA3 (Invitrogen) as described (8), and sequenced
again to verify mutation sequences. In one of mutants, a stop codon was
introduced immediately after the Gly336 codon to produce
the truncated exodomain, Arg1-Gly336. Mutant
LH/CG receptor constructs were transfected into human embryonic kidney
293 cells by the calcium phosphate method. Stable cell lines were
established in minimum essential medium containing 10% horse serum and
500 µg/ml Geneticin (G-418).
125I-hCG Binding and Intracellular cAMP
Assay--
Stably transfected cells were assayed for
125I-hCG binding in the presence of 150,000 cpm of
125I-hCG (9) and increasing concentrations of cold hCG.
hCG, batch CR 127, was supplied by the National Hormone and Pituitary
Program. Nontransfected cells did not show specific binding of hCG. For intracellular cAMP assay, ~50,000 cells were washed twice with Dulbecco's modified Eagle's media and incubated in the media
containing isobutylmethylxanthine (0.1 µg/ml) for 15 min. Increasing
concentrations of hCG were then added and the incubation was continued
for 45 min at 37 °C. After removing the media, the cells were rinsed once with fresh media without isobutylmethylxanthine, lysed in 70%
ethanol, freeze-thawed in liquid nitrogen, and scraped. After pelleting
cell debris at 16,000 × g for 10 min at 4 °C, the
supernatant was collected, dried under vacuum, and resuspended in 10 µl of the cAMP assay buffer which was provided by the manufacturer
(Amersham). cAMP concentrations were determined with an
125I-cAMP assay kit (Amersham) following the
manufacturer's instructions and validated for use in our laboratory.
All assays were carried out in duplicate and repeated 4-6 times. Means
and standard deviations were calculated and analyzed by Student's
t test to determine the statistical significance
(p1) of repeats of the same samples. In
addition, values for mutants and the exodomain were compared with the
corresponding values of the wild type receptor using ANOVA with 95%
confidence to determine the statistical significance
(p2) of the differences.
125I-hCG Binding to Solubilized LH/CG
Receptor--
Transfected cells were washed twice with ice-cold 150 mM NaCl, 20 mM HEPES, pH 7.4 (buffer A). Cells
were scraped on ice and collected in buffer A containing protease
inhibitors (1 mM phenylmethylsulfonyl fluoride, 5 mM N-ethylmaleimide, and 10 mM EDTA)
and pelleted by centrifugation at 1,300 × g for 10 min. Cells from a 10-cm plate were resuspended in 0.6 ml of buffer A
containing 1% Nonidet P-40, 20% glycerol, and the above protease
inhibitors (buffer B), incubated on ice for 15 min, and diluted with
5.4 ml of buffer A containing 20% glycerol plus the protease
inhibitors (buffer C). The mixture was centrifuged at 100,000 × g for 60 min. The supernatant (500 µl) was mixed with
150,000 cpm of 125I-hCG and 6.5 µl of 0.9% NaCl and 10 mM Na2HPO4 at pH 7.4 containing increasing concentrations of unlabeled hCG. After incubation at 4 °C
for 12 h, the solution was thoroughly mixed with 250 µl of buffer A containing bovine
-globulin (5 µg/ml) and 750 µl of buffer A containing 20% polyethylene glycol 8,000. After incubation at
4 °C for 10 min, samples were pelleted at 1,300 × g
for 30 min and supernatants removed. Pellets were resuspended in 1.5 ml
of buffer A containing 20% polyethylene glycol 8,000, centrifuged, and
counted for radioactivity.
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RESULTS |
hCG Binds Better to the Truncated Exodomain Than to the Wild Type
Receptor--
The truncated exodomain,
Arg1-Gly336, was expressed in 293 cells. Since
it remains within the cells and is not secreted (4, 5, 10), the cells
expressing it were solubilized in Nonidet P-40 and assayed for
125I-hCG binding (Fig. 1).
The mean Kd value, 571 pM
(p1 < 0.01), of the truncated exodomain in
solution was determined from six repeat experiments, each in duplicate.
This is different from the Kd value of the wild type
receptor solubilized in Nonidet P-40, 943 pM
(p1 < 0.01). To determine whether the
difference in the two Kd values was statistically
significant, they were examined using ANOVA with 95% confidence. The
result indicates that the two Kd values and the
difference between them are statistically significant (p2 < 0.01). Furthermore, the difference could
not be attributed to experimental variations due to varying receptor
concentrations, since the concentrations of the truncated exodomain and
wild type receptor were within the acceptable range (Fig. 1). The
results are also consistent with the previous reports by several
laboratories (4, 5, 10), although the difference was not emphasized and
explored at the time. These results, taken together, demonstrate that
the hormone binding affinity of the truncated exodomain is slightly and
consistently better than that of the wild type receptor.

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Fig. 1.
hCG binding to solubilized LH/CG receptor and
the exodomain of the receptor. The full-length LH/CG receptor and
the exodomain were separately expressed in 293 cells, solubilized in
Nonidet P-40, and used for 125I-hCG binding in the presence
of increasing concentrations of nonradioactive hCG (A) and
Scatchard analysis (B) was plotted against specific binding.
Experiments were repeated 4-6 times in duplicate, and mean and S.D.
were calculated as presented in the table section of the figure. In
addition, the statistical significance of the each mutant data was
analyzed twice for different purposes. First to determine the
statistical significance (p1 values) of repeat
data for each, mutants were analyzed by Student' t test. In
addition, the values for the exodomain were compared with the
corresponding values of the wild type receptor using ANOVA with 95%
confidence to determine the statistical significance of the difference
(p2 values).
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Why Does the Truncated Exodomain Bind Better?--
There are two
notable differences in the truncated exodomain and the extracellular
exodomain of the wild type receptor: the location and structure. The
former is present within the cell and lacks the endodomain, whereas the
latter is present on the cell surface and covalently linked to the
endodomain. One might raise an unlikely possibility that the location
of the exodomain prior to the solubilization in Nonidet P-40 solution
affected the binding affinity. To test this hypothesis, the
Kd values of several LH/CG receptors with various
Pro to Phe substitutions (11) were examined. These mutants are
expressed both at the cell surface (extracellular) and in cells
(intracellular) but the ratios of the extracellular/intracellular
concentrations vary depending on which Pro is mutated (Table
I). The Kd ratio of
receptors on intact cells and those in Nonidet P-40 solution increases
as does the intracellular receptor concentration. This result indicates
that the Kd values of intracellular receptors are
higher than those of extracellular receptors. Therefore, the binding
affinity of intracellular receptors is worse than that of the
corresponding extracellular receptors. Furthermore, it indicates that
the extracellular or intracellular location of the exodomain alone is
not responsible for the different Kd values.
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Table I
Comparison of receptors present on the plasma membrane and those
trapped in the cell
Several LH/CG receptors with Pro to Phe substitution. P463F, P562F, and
P591F, show varying levels of surface and intracellular expression
(11). The extracellular concentration of receptors present on the
plasma membrane was determined from the receptors which
125I-hCG bound to intact cells. The total receptor
concentration was determined with 125I-hCG binding to receptors
present in Nonidet P-40 solution of whole cells. The intracellular
concentration of receptors present in cells was estimated by
substracting the extracellular receptor concentration from the total
receptor concentration. R stands for receptor.
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Alternatively, the different Kd values of the
truncated intracellular exodomain and the extracellular exodomain of the wild type receptor may come from the interaction with the endodomain. For example, the exodomain of the wild type receptor may
interact with the endodomain which in turn influences the hormone
binding affinity whereas the truncated intracellular exodomain does not
have any exodomain to interact with. Such an influence by the
endodomain on the exodomain is likely to occur at the extracellular portion of the endodomain, including the three exoloops. To determine such a region(s) and amino acid residues, the 20 amino acids of exoloop
2 were individually substituted with Ala to produce 20 substitution
mutants. They are Ser484, Asn485,
Tyr486, Met487, Lys488,
Val489, Ser490, Ile491,
Cys492, Leu493, Pro494,
Met495, Asp496, Val497,
Glu498, Ser499, Thr500,
Leu501, Ser502, and Gln503. The
resulting mutants are LH/CG-RS484A,
LH/CG-RN485A, LH/CG-RY486A, LH/CG-RM487A, LH/CG-RK488A,
LH/CG-RV489A, LH/CG-RS490A,
LH/CG-RI491A, LH/CG-RC492A,
LH/CG-RL493A, LH/CG-RP494A,
LH/CG-RM495A, LH/CG-RD496A, LH/CG-RV497A, LH/CG-RE498A,
LH/CG-RS499A, LH/CG-RT500A,
LH/CG-RL501A, and LH/CG-RS502A,
LH/CG-RQ503A (Figs. 2 and
3). For the convenience of data
presentation and analysis, exoloop 2 amino acids are divided into two
groups, 11 upstream (Fig. 2) and 9 downstream amino acids (Fig. 3).

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Fig. 2.
Ala scan of upstream residues of exoloop
2. The upstream 11 amino acids of exoloop 2, from
Ser484 to Pro494, were individually substituted
with Ala and the resulting mutant receptors were stably expressed on
human 293 cells. The cells were assayed for hormone binding and
hCG-dependent cAMP induction. After experiments were
repeated 4-6 times in duplicate, the statistical significance of the
each mutant data was analyzed. The p1 values
(the statistical significance of repeats) are presented as:
a for p1 < 0.001; b for
p1 < 0.01; and c for
p1 < 0.05. ND indicates not
detectable.
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Fig. 3.
Ala scan of downstream residues of exoloop
2. The downstream 9 amino acids of exoloop 2, from
Met495 to Gln503, were individually substituted
with Ala and the resulting mutant receptors were stably expressed on
human 293 cells. They were assayed and the data analyzed as described
in the legend to Fig. 2. In addition, a double substitution mutant was
generated in which both Asp496 and Glu498 were
replaced with Ala.
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Effects of Ala Substitution for Upstream Amino Acids of Exoloop
2--
Among 11 mutant receptors with Ala substitution for the
upstream amino acids, 6 were surface-expressed in reasonable receptor concentrations, 12,100-64,800 receptors/cell (Fig. 2).
Kd values of these surface-expressed receptors were
diverse in the range of 250 to 840 pM.
LH/CG-RN485A, LH/CG-RK488A, and
LH/CG-RS490A, displayed lower Kd values,
indicating that their hormone binding affinities are better than that
of the wild type receptor. On the other hand, the Kd
value of LH/CG-RV489A was 2-fold higher, an indication for
a low binding affinity. In contrast to these surface-expressed
mutants, hCG binding to the cells transfected with the other 5 mutants
was marginal or not detected. For example, hCG bound to
LH/CG-RI491A on intact cells with a normal affinity but the
receptor concentration was <10% of the concentration of the
wild type receptor. 125I-hCG binding to intact cells was
hardly detectable for LH/CG-RS484A, LH/CG-RY486A, LH/CG-RC492A, and
LH/CG-RP494A.
To determine whether any of these mutant receptors were trapped inside
the cells or defective in hCG binding, stably transfected cells were
solubilized in Nonidet P-40 and assayed for hormone binding. As shown
in Fig. 2, C and D, all five mutants in the detergent solution bound hCG (p1 < 0.05) and
the receptor concentrations were not significantly different from the
concentrations of the wild type receptor (p1 < 0.05). These results indicate that the five mutant receptors were
expressed but not efficiently transported to the cell surface.
The Kd value of LH/CG-RS484A was 2-fold
lower than that of the wild type receptor, an indication of a 2-fold
improved affinity for hCG binding. In contrast to the improved binding affinity, LH/CG-RP494A showed a 2-fold lower affinity as
the Kd value was 2-fold higher. The other three
mutants in solution showed slightly higher Kd
values. These results indicate that the Ala substitutions somewhat
impacted the hormone binding affinity of the mutant receptors, in
addition to impairing their expression on the cell surface.
In contrast to the diverse Kd values,
EC50 values for cAMP induction by most of surface-expressed
mutant receptors except LH/CG-RV489A are 3-5-fold higher
than the EC50 value of the wild type receptor (Fig.
2E). This is a significant reduction in the affinity for
cAMP induction and indicates that these Ala substitutions impaired, but
did not improve, the affinity for cAMP induction. In contrast to this
normal binding with poor cAMP induction, LH/CG-RV489A bound
hCG with a 2-fold lower affinity yet induced cAMP normally
(p1 < 0.001).
Ala Substitution for Downstream Amino Acids of Exoloop 2--
All
9 mutant receptors with Ala substitution for the downstream amino acids
were surface-expressed and the receptor concentrations were reasonable,
being in the range of 12,200-37,000/cell (Fig. 3). Their affinities
for hormone binding were generally similar to that of the wild type
receptor. However, the Kd value of
LH/CG-RS499A was lower (280 pM) than that of
the wild type receptor. This result indicates that the S499A
substitution improved the binding affinity as did the S484A, K488A, and
S490A substitutions. In contrast to the improved binding affinities,
the Kd values of LH/CG-RM495A and
LH/CG-RS502A were higher, 840 and 630 pM,
respectively. All of the mutants were capable of inducing cAMP and the
EC50 values for cAMP induction were either similar to or
slightly higher, up to 3-fold, than that of the wild type receptor. The
maximum cAMP induction levels were similar to or lower than that
produced by the wild type receptor.
Effect of Surface Receptor Concentrations--
Surface
concentrations of mutant receptors shown in Figs. 2 and 3 were diverse.
To determine whether levels of surface receptor concentrations had any
effect on hormone binding, cells were transiently transfected with
varying concentrations of receptor plasmids and selected for those
expressing ~40,000 receptors/cell (Table
II). Cells were assayed for hormone
binding to intact cells. The Kd values (Table II)
for the wild type receptor LH/CG-RN485A and LH/CG-RM487A, LH/CG-RK489A, and
LH/CG-RD496A were similar to the Kd
values of the same receptors which were expressed at diverse levels
(Figs. 2 and 3). These results show that the surface receptor
concentration did not impact the hormone binding affinity. To test
whether receptor transport to the surface membrane might have had any
effect on the hormone binding affinity, cells expressing the receptors
were solubilized and assayed for hormone binding to solubilized
receptors (Table II). The results show that the % Kd values of receptors were similar to those of
receptors expressed on the cell surface, an indication for no
significant effect of receptor transport on hormone binding.
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Table II
Effect of constant receptor concentrations
Cells were transiently transfected with varying amounts of receptor
plasmids and selected for those expressing ~40,000
receptors/cell (Table III). Cells were assayed for hormone binding to
intact cells and to solubilized receptors.
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Double Ala Substitutions for Nearby Asp and Glu--
Ionic amino
acids of the LH/CG receptor have been implicated for important roles
(8, 12-14). However, the D496A substitution and E498A substitution
individually had marginal effects on surface expression, hCG binding,
and cAMP induction. Therefore, both Asp496 and
Glu498 were substituted with Ala to produce a double
substitution mutant, LH/CG-RD496A/E498A. These double
substitutions substantially attenuated the surface expression of the
mutant and reduced the affinity for cAMP induction. Remarkably, the
hormone binding affinity improved by more than 2-fold, underscoring the
importance of their potential role to attenuate the high affinity
hormone binding of the natural receptor. It is not clear whether these
two nearby anionic residues play an important and mutually
substitutable role in the hormone binding affinity.
Verification of Mutagenesis--
Our site-directed mutagenesis
requires a synthetic oligonucleotides with a mutant sequence for each
mutation and furthermore, does not involve polymerase chain reaction.
After mutagenesis, the mutant and flanking sequences are verified by
sequencing. In addition, the same sequence is confirmed once more after
a mutant cDNA is subcloned into the expression vector. Therefore, it is highly unlikely that a mutant cDNA might have undergone an
unintended mutation(s) during the mutagenesis and subcloning. To
confirm this, mutant cDNAs were reverted to the wild type cDNA which was in turn used to transfect cells. All of the revertants behaved the same as the wild type receptor in surface expression, hormone binding, and cAMP induction (data not included), indicating that there were no mutations other than the intended Ala
substitutions.
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DISCUSSION |
Influence of Exoloop 2 on Hormone Binding--
Some Ala
substitutions for exoloop 2 amino acids impacted the hormone binding
affinity, noticeably enhancing or reducing the binding affinity whereas
others did not have a significant effect (Fig.
4). To facilitate the comparison of these
diverse effects, percent Kd values for mutants were
calculated by dividing their Kd values with that of
the wild type receptor (Table III and
Fig. 4). Most noticeable were the S484A, K488A, S490A, S499A, and
D496A/E498A substitutions. They improved the binding affinity up to
2-fold and these improvements were statistically significant
(p2 < 0.05 to p2 < 0.001) according to ANOVA analysis. This is specific since the same Ala
substitutions mostly attenuated, but never improved, the affinity for
cAMP induction and other substitutions did not improve the binding
affinity. It is interesting and could be significant that the extent of
the improvement in the binding affinity of some of the mutant receptors
is in the range of the enhanced binding affinity of the truncated
exodomain as compared with the binding affinity of the extracellular
exodomain of the wild type receptor. This correlation of the
improvement and the extent of the improvement in the binding affinity
after truncation of the endodomain and some Ala substitutions suggest
that the high affinity hormone binding to the exodomain is influenced
and furthermore, attenuated by the endodomain, including exoloop 2, of
the wild type receptor. Exoloop 2 may interact with the exodomain or
the exodomain-hormone complex to modulate the structure. If so, the
exodomain may assume a structure more favorable for hormone binding in
some mutants, including LH/CG-RS484A and
LH/CG-RD496A/E498A. This is consistent with the recent
suggestion that exoloop 2 contacts the hormone (15).

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Fig. 4.
Percent Kd and
EC50 values for exoloop 2 mutants. Percent
Kd and percent EC50 values for
individual mutants were calculated by dividing the values for the wild
type receptor with the corresponding values for each mutant. The
statistical significance of p2 (the differences
between the mutant values and the wild type values) was determined by
ANOVA as described in the legend to Fig. 2. The resulting p
values are presented as: A for p2 < 0.001; B for p2 < 0.01; and
C for p2 < 0.05. Significantly
higher values are shown in black bars and significantly
lower values shown in gray bars. Open bars represent those
values which are not significantly different from the wild type
value.
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Table III
Relative affinities and maximum cAMP levels
Values for percent Kd and percent EC50 were
calculated by dividing Kd and EC50 values
(Figs. 2 and 3) of the wild type receptor with those of the mutant and
wild type receptors. For percent maximum cAMP, maximum cAMP production
levels of mutants were divided by that of wild type receptor. To
compare the relationship between the affinities for hormone binding and cAMP induction, the percent EC50 value of a mutant was divided by the percent Kd value of the same mutant.
Likewise, the percent maximum cAMP was divided by the percent
EC50 value of the same mutant. The percent
Kd values for I491A on cells and in solution were 82 and 84, respectively. DA/EA stands for the double substitution,
D496A/E498A.
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Structure of Exoloop 2--
It is interesting to see that all of
the three Ser to Ala substitutions in exoloop 2, S484A, S490A, and
S499A, improved the binding affinity. It is not clear whether there is
a correlation between the three Ser to Ala substitutions and the
improved binding affinities and whether there is a structural
relationship of the three residues. If there are, the three Ser
residues and possibly some other residues including Lys488
(Fig. 4) might be arranged in the exterior of a structure to influence
hormone binding and the affinity.
To help envision such a structure and to test whether the structure is
possible, exoloop 2 was modeled based on the observation that the
Kd values of the mutant receptors vary according to
their positions (Fig. 4) in putative secondary structures. Exoloop 2 was anchored to the 4th and 5th TMs of bacteriorhodopsin (16). We
focused on Ser484, Asn485, Lys488,
Ser490, and Ser499 for which Ala substitution
improved the binding affinity. One model was developed using the
program "O" (17) with ideal 310 helix and
-sheet
bond angles and lengths, with the most common side chain rotamers
chosen for each residue (the upper models in Fig.
5). No energy minimization calculations
were performed, as ideal geometry was given to the model from the
outset and interactions between secondary structure elements are
minimal. Here, Ser484, Lys488,
Ser490, and Ser499 are present in the exterior
of exoloop 2 and their side chains facing toward the same side.
Asn485, however, is anomalous and orients toward the
interior of the loop. Particular attention was paid to the fact that
Leu493, Asp496, and Ser499 have a
periodicity of 3 and the Ala substitution for them retains or slightly
decreases the wild type Kd. These residues may mark
one side of an
or 310 helix with periodicities of 3.4 and 3.0, respectively. Similarly, S484A, K488A, and S490A substitutions resulted in lower than wild-type Kd values and the
residues might mark one side of a
-pleated sheet. However, in this
case, there is an anomalous increased Kd by the
Y486A substitution and a decrease by the N485A substitution. With these
observations, the structure of exoloop 2 has been modeled to give a
-strand over the first seven residues of the loop followed by a
short coil region and two turns of 310 helix. The loop has
been constructed so that residues Ser484,
Lys488, Ser490, Leu493,
Asp496, and Ser499 have side chains close to
each other. Therefore, all may have similar effects on the interactions
of the loop with the local protein environment, but the exact nature of
these interactions cannot be predicted with the available data. This
simple model ignores the anomalies at Asn485 and
Tyr486.

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Fig. 5.
Computer models of exoloop 2. Two models
were constructed, both in loop (left) and spacefill
(right) structures. Side chains of Ser484,
Asn485, Lys488, Ser490, and
Ser499 are colored in magenta,
Val489, Pro494, and Met495 in
yellow, and the rest in gray. The upper
model was constructed using the program "O" (17) with ideal
310 helix and -sheet bond angles and lengths, with the
most common side chain rotamers chosen for each residue. The
lower model was built using a method similar to that
developed for modeling nucleic acid loop structures (18).
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Another method was developed for modeling nucleic acid loop structures
(18) and incorporated the AMBER energy minimization (19). Crystal
structure of the seven-helix bundle from bacteriorhodopsin (16) was
used for anchoring the loop. Using a reduced-coordinates approach,
random loop structures with the targeted pair of fixed ends were
generated. Those loop structures with energies below a threshold were
selected and subjected to a short run of energy equilibration using
Metropolis Monte Carlo simulation (18) and then, 1,000 cycles of energy
minimization using AMBER (19). It produced a structure with
Ser484, Asn485, Lys488,
Ser490, and Ser499 on one side of the loop
surface (the lower models in Fig. 5). Therefore, it is possible that
Ser484, Asn485, Lys488,
Ser490, and Ser499 are coordinated to position
themselves in a specific loop structure and to influence the hormone
binding affinity. In contrast to the improved binding affinities of
some of the substitutions, the V489A, P494A, and M495A substitutions
attenuated the binding affinity by ~2-fold. Interestingly, these
residues are clustered together in the middle of exoloop 2 in both
models, suggesting their intriguing role in the binding affinity.
Effects of Ala Substitutions on cAMP Induction--
The
significant attenuation by Ala substitution of the affinity for but not
maximal level of cAMP induction indicate that once the mutant receptors
were activated, even if poorly, they effectively produced cAMP.
Overall, exoloop 2 is more important for the affinity for cAMP
induction than for the maximal cAMP induction. This further suggests
distinct mechanisms for the affinity and maximal level of cAMP
production. These distinct mechanisms are more obvious when percent
values for Kd, EC50, and maximal cAMP
production as well as their ratios are compared (Table III and Fig. 4).
The ratios of percent EC50/percent Kd for most of the mutants were <1, indicating more severe reductions in
the affinities for cAMP induction than the hormone binding affinities.
Integral Roles of Exoloop 2 on the Hormone Binding Affinity, cAMP
Induction Affinity, and Maximal cAMP Production--
Our data indicate
the importance of exoloop 2 for the hormone binding affinity, the
affinity for cAMP induction, and the maximal cAMP production. This is
probably accomplished by interaction of the exodomain and endodomain,
particularly exoloop 2. The roles of exoloop 2 on each of these
functions are distinct as are the mechanisms of exoloop 2 to influence
them. Some of them appear to be in conflict. As a result, the hormone
binding affinity, the affinity for cAMP induction, and the maximal cAMP
production seem to be compromised. Ala substitutions both positively
and negatively impact the hormone binding affinity and the maximal level of cAMP. However, the affinity for cAMP induction is never augmented by the substitutions. Therefore, the hormone binding affinity
and the maximal level of cAMP were compromised to reach the best
affinity for cAMP induction. For example, the hormone binding affinity
for the exodomain reaches the maximum only when the endodomain is
removed and cAMP induction is completely disengaged from the hormone
binding.
Disulfide Bridge between Exoloops 1 and 2--
The C492A
substitution resulted in the loss of surface expression but not the
high affinity hormone binding as found in other substitutions for Cys
(20). It indicates the importance of Cys492 in surface
expression but not in hormone binding. Cys residues corresponding to
Cys492 of exoloop 2 and Cys416 of exoloop 1 of
the LH/CG-R have been implicated to form a disulfide bridge in the
other seven TM receptors (21). If this is true for the LH/CG receptor,
the disulfide may not be essential for hormone binding. Another
possibility is that Cys492 of exoloop 2 and
Cys416 of exoloop 1 may not form a disulfide or may form
disulfides with other Cys residues of the exodomain. It will be of
interest to see whether the putative disulfide plays a role in surface expression of the LH/CG receptor, as do disulfides of hCG in hCG processing (22).
Effects of Ala Substitutions on the Surface Expression--
Some
of the substitutions attenuated or blocked surface expression of the
resulting mutants. As a result, most of LH/CG-RS484A, LH/CG-RY486A, LH/CG-RI491A,
LH/CG-RC492A, LH/CG-RP494A, and
LH/CG-RD496A/LH/CG-RE498A were trapped in the
cells. Therefore, these amino acids are crucial for surface expression
of the receptor.
Conclusion--
Our data demonstrate that exoloop 2 of the LH/CG-R
distinctly influences the hormone binding affinity, the affinity for
cAMP induction, and the maximal cAMP production, probably by the
interaction of exoloop 2 with the exodomain and other parts of the
endodomain such as TM 6 and TM 7. These distinct ways of influencing
the functions are sometimes in conflict and compromised to attain the
maximal affinity for cAMP induction. The high affinity hormone binding
at the exodomain is constrained by some of the exoloop 2 residues,
particularly, Ser484, Asn485,
Lys488, Ser490, and Ser499. As a
result, the exodomain attains the maximal affinity for hormone binding
when the endodomain is truncated and cAMP induction is disengaged.
We thank Roger L. Gilchrist and Charles
Murrieta for reading this manuscript and helpful
suggestions.