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Department of Biochemistry & Molecular Biology University of Georgia Athens, Georgia 30602-7229
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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heterodimer released from
Gi and Gs (9). There is considerable interest in elucidating the mechanism by which binding of the heterodimeric glycoprotein hormones, with molecular masses between 30–37 kDa, to the extracellular domain leads to receptor activation. Ji and co-workers (10) have proposed a model in which high-affinity binding of ligand occurs to the extracellular domain, followed by a conformational change in which the ligand/exodomain complex interacts with the endodomain, leading to a reorientation of helices. Little, however, is known about the nature of the conformational changes of the transmembrane helices (TMHs) required for LHR activation. Lin et al. (11) have proposed a model involving reorientation of TMHs 6 and 7, and Schoneberg et al. (12) proposed a major change in the relative positions of TMHs 5 and 6. The generalized gonado-TSH receptor model proposed by Hoflack et al. (13) has an interior cleft, largely hydrophobic, that could also form a series of hydrogen bonds, contributed by all TMHs, with a portion of the bound glycoprotein hormone penetrating into the cleft. A number of studies have been reported on the functional consequences of replacing, via site-directed mutagenesis, certain amino acid residues in the TMHs (14, 15, 16, 17, 18, 19). Moreover, there are now numerous reports of naturally occurring mutations in the TMHs, some of which are loss-of-function mutations leading to hypogonadism and pseudohermaphroditism, while others lead to constitutive receptor activation, as manifested, for example, in familial and sporadic male-limited precocious puberty (see reviews in Refs. 20, 21, 22, 23). The most common mutation in the human LHR gene resulting in male-limited precocious puberty is a replacement of Asp578 (corresponding to Asp556 in rat LHR in which the 22-amino acid residue signal peptide is not included in the numbering as it is with human LHR) in TMH 6 with Gly.
We suggested earlier that TMH 7 of LHR contains a number of polar and
hydrophobic side chains that can function as hydrogen bond donors
and/or acceptors, e.g. Tyr590,
Asn593, Ser594,
Asn597, and Tyr601, and,
interestingly, these amino acid residues map to a common face of the
helix (17). In a rigorous molecular modeling study of helical packing
in LHR, Lin et al. (11) proposed that interactions involving
TMHs 6 (Thr555 and Asp556)
and 7 (Asn593 and Asn597)
are important in receptor activation. A number of reports have also
indicated the functional importance of TMH 7 in LHR function. For
example, our laboratory reported on two replacements of conserved
residues in TMH 7 of rat LHR, P591L and Y601A, that diminished
ligand-mediated signaling, but not ligand binding (17), as was also
found for Lys583, located at the interface
between exoloop 3 and TMH 7 (24, 25). Another point mutant in TMH 7 was
examined, P598L, which fails to localize properly at the cell surface
(17). Two reports of a naturally occurring mutation in TMH 7 of the
human LHR, S616Y, corresponding to Ser594 of rat
LHR, have been reported in 46,XY individuals presenting with a
micropenis and Leydig cell hypoplasia (16, 26). When examined in
transfected cells, human LHR with the S616Y mutation is nonfunctional,
probably due to the lack of proper membrane localization (16, 26, 27).
Two siblings, one 46,XX and the other 46,XY, presenting with gonadal LH
resistance, were found to have a deletion of two amino acid residues in
LHR, corresponding to Leu608 and
Val609 (Leu586 and
Val587 of rat LHR) (28). As with the Pro
Leu
and SerTyr replacements, the Leu-Val deletion mutant of LHR appears
to be retained intracellularly. Finally, there is a preliminary report
that replacement of Asn619 with Gln in human LHR
(Asn597 in rat LHR) results in constitutive
activation of LHR (29).
In view of the important structural and functional role of TMH 7 of LHR indicated in these studies, we have investigated several polar amino acid residues that can serve as hydrogen bond donors and acceptors, Asn593 and Asn597, and as hydrogen bond donors, Ser594 and Thr605. In addition, we have studied the role of several hydrophobic side chains, Leu586, Val587, Cys595, and Phe604. These amino acid residues were chosen because all are invariant in the three glycoprotein hormone receptors and are conserved in all G protein-coupled receptors; moreover, all but Val587 and Cys595 map to a common face of the helix. Our results demonstrate that replacements of Asn593 and Asn597 can result in loss of function, i.e. reduced responsiveness to ligand, or gain of function, i.e. constitutive activation, depending upon the chemical structure of the side chain. These experimental observations were complemented with molecular modeling and energy minimization of TMHs 6 and 7.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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. The two prolines at positions 591 and
598 introduce kinks, and panel A presents a side view of the helix,
with only the side chains displayed for those amino acid residues
replaced in this study. Asn593,
Ser594, and Asn597 form a
patch of polar amino acid residues on one extended face of the helix
near its center. Although not shown for purposes of clarity, the side
chains of the two tyrosines at positions 590 and 601 fall on an
extended common face of the helix, as does
Thr605. Thus, all six of the side chains with
hydrogen bond donor function on TMH 7, Tyr590,
Asn593, Ser594,
Asn597, Tyr601, and
Thr605, the two asparagines also having hydrogen
bond acceptor function, are interspersed with hydrophobic side chains
on one side of the helix, while the opposite side contains all
hydrophobic side chains. The partial amphipathic nature of TMH 7 was
noted in an earlier study (17). A top view of an energy-minimized
structure of TMH 7 is shown in Fig. 1B. This representation emphasizes
the kinking introduced by the two prolines and also shows the partial
amphipathic nature of TMH 7.
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, and representative data are
shown in Fig. 2 for competitive binding
and ligand-mediated signaling of the N593A, C595A, and N597A LHR
mutants. In all cases, the Kds and basal cAMP
levels are equivalent to those of WT LHR. The results can be considered
in three categories based upon expression levels and ligand
responsiveness. 1) For the N593A and C595A mutants, receptor expression
was comparable to or exceeded that of WT LHR, but responsiveness to hCG
was diminished; 2) for the N597A and T605A mutants, both expression
levels and hCG responsiveness were reduced relative to WT LHR; and 3)
for the mutants L586A, V587A, S594A, and F604A, receptor expression and
responsiveness were like those of WT LHR.
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, there is only a
very slight dependence of Rmax on receptor
density for WT LHR. The receptor mutants L586A, V587A, and S594A, but
not N593A, C595A, N597A, F604A, or T605A (data not shown for the
latter), also showed a slight dependence of Rmax
on receptor expression levels, and there was no significant change in
basal cAMP values with receptor density for WT and the mutant LHRs
(data not shown).
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, the
receptor expression levels, basal cAMP values, and hCG-mediated cAMP
increases were comparable to WT LHR, with the exception of the C595M
mutant that expressed at a lower level. Thus, it appears that the more
hydrophobic Met, compared with Ala, restores signaling at position 595
(Cys); position 604 (Phe) can tolerate Ala or Tyr replacements equally
well; and the more hydrophobic side chain, Tyr, is tolerated better at
position 605 (Thr) than Ala.
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In these studies, as with the C595M, F604Y, and T605Y LHR mutants,
signaling was based on measurements of basal cAMP and maximally
hCG-stimulated cAMP. As shown in Table 3,
the Asn593Ala, Gln, and Ser replacements
yielded LHR mutants that expressed at levels comparable to WT LHR, yet
responsiveness to hCG was blunted; the
Asn593Lys replacement exhibited properties
like those of WT LHR. The Asn593Asp and
Glu replacements resulted in reduced expression and responsiveness, the
latter perhaps attributable to the low receptor number and/or inherent
loss of function. Of considerable interest is the
Asn593Arg substitution. This LHR mutant, which
expresses at a level comparable to WT LHR, is constitutively active and
responsive to hCG. Figure 4 shows the
basal and stimulated cAMP levels for two mutant forms of LHR relative
to WT LHR and represents a loss-of-function mutant, N593A, and a
gain-of-function mutant, N593R. Comparable studies were performed with
Ala, Gln, Asp, Lys, and Arg replacements of
Asn597 (Table 3). Compared with WT LHR, the
N597A, N597D, and N597K mutants expressed at lower levels and exhibited
reduced responsiveness to hCG; basal cAMP values were similar to that
of WT LHR. The N597R mutant exhibited a basal cAMP level like that of
WT LHR, but responded poorly to hCG. Thus, the
Asn597Arg replacement represents a
loss-of-function mutant. The N597Q mutant expressed at a level
comparable to that of WT LHR and, like the N593R mutant, exhibited
constitutive receptor activation as judged by the increase in basal
cAMP; moreover, responsiveness to hCG was like that of WT LHR. The
basal and cAMP levels of the N597R (loss-of-function mutant) and the
constitutively active mutant, N597Q, are shown in Fig. 5.
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. Since the calculations are based on
charged side chains of the amino acid residues, Asp, Glu, Lys, and Arg,
it is not surprising that Asp-Lys and Asp-Arg interactions appear to be
more favorable, e.g. -18 to -30 kcal/mol relative to the
putative Asp-Asn interactions in WT LHR (11). Figure 6 shows a side-view schematic of the
energy-minimized structures of native TMHs 6 and 7. This structure was
used as a starting point to evaluate the effect of replacement of
Asn593 and Asn597 with Gln
and Arg to give the four point mutants, N593Q, N593R, N597Q, and N597R.
These were chosen since N593R and N597Q are constitutively activating
mutants and N593Q and N597R are loss-of-function mutants. The
appropriate amino acid residues were substituted in TMH 7, and the new
helix 6-helix 7 interaction was minimized. The results of these
calculations, along with WT LHR as control, are presented in Fig. 7 as viewed from the cell exterior toward
the interior. This vantage point was chosen since there is little shift
in the vertical arrangement of the helices in the energy-minimized
structures of these four mutants, but noticeable reorientation,
e.g. twisting or turning, does occur. The Gln replacements,
not surprisingly, are accommodated more readily than the Arg
replacements. The two activating mutations, N593R and N597Q, lead to
dramatic shifts in TMHs 6 and 7, respectively. The two loss-of-function
mutants, N593Q and N597R, also lead to helix reorientation, but of a
somewhat different nature. The Arg597 side chain,
in particular, cannot be fully extended as can the
Arg593 side chain, and thus projects into the
interior of the helical array presenting a hydrogen bond donor to this
microenvironment. In all cases, reorientation of TMHs 6 and 7 can alter
the conformation of the third intracellular and extracellular loops, as
well as the cytoplastic tail of LHR.
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Ala, Gln, and Ser and
Asn597Arg replacements represent
loss-of-function mutants; the Asn593Asp, Glu
and the Asn597Ala, Asp, and Lys replacements
may as well, but the reduced level of receptor expression compromises
such a conclusion. The Asn593Arg and
Asn597Gln replacements yield constitutively
activating LHR mutants that can also respond to hCG.
As indicated by molecular modeling (11), there are large interhelical
contact surfaces between TMHs 3 and 4 and potential interactions
between Glu429 in TMH 3 and
Ser472 in TMH 4; moreover,
Asn513, one of the few polar amino acid residues
in TMH 5, is unique in the three glycoprotein hormone receptors and may
interact with TMH 6. Thus, we investigated the functional roles of
Glu429 and Asn513 in TMHs 3
and 5, respectively, by site-directed mutagenesis. These two amino acid
residues are invariant in the three glyco-protein hormone receptors
(2). As shown in Table 4, the E429D LHR
mutant expressed poorly and, not surprisingly, the responsiveness to
hCG was minimal. Other replacements with Ala and Gln yielded mutants
that gave expression levels and Rmax values
comparable to those of WT LHR. The E429S mutant exhibited a reduction
in both expression and signaling. The
Asn513Gly, Ala, Gln, Leu, Asp, and His
replacements resulted in receptor mutants that expressed poorly and
failed to respond well to hCG as monitored by
Rmax values. There was no evidence of
constitutive action in any of the Glu429 or
Asn513 LHR mutants, and the reduced expression
levels for many of the replacements preclude any conclusion regarding
loss of responsiveness.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The most surprising and significant observations from the current study are that replacement of either Asn593 or Asn597 can lead to loss-of-function mutants (Ala593, Gln593, Ser593, and Arg597) or to constitutively activating mutants (Arg593 and Gln597). The observation that replacement of a given side chain can result in either loss of function or gain of function, depending upon the nature of the side chain, is indeed intriguing. The importance of Asp556 (Asp578 in human LHR) in TMH 6 has been well documented in LHR function (18); our results argue for pivotal roles, albeit less dramatic, of Asn593 and Asn597 as well. The most common naturally occurring mutation leading to familial or sporadic male-limited precocious puberty is replacement of Asp in TMH 6 with Gly, which results in a 4.7-fold increase in basal cAMP over that of WT LHR (18). However, not all naturally occurring gain-of-function mutations increase basal cAMP to this extent. The A572V mutation in TMH 6 of human LHR also leads to male-limited precocious puberty, but gives only a 3-fold increase in basal cAMP levels (30). The fold-increase in basal cAMP levels noted with the N593R mutant is comparable to this value.
A naturally occurring mutation in TMH 7 of the TSH receptor,
Asn670Ser (corresponding to position 593 in
rat LHR), was found to lead to constitutive activation (31).
Interestingly, in rat LHR we found that the same replacement led to a
loss-of-function mutant. Replacement of Asn593
with Arg yields a gain-of-function LHR mutant and responsiveness to
hCG; hence, the Arg side chain, in addition to its role in constitutive
activation, is able to mimic Asn in LHR responsiveness to hCG.
Likewise, Gln behaves similarly when replacing
Asn597.
It has been suggested that the inactive conformation of the LHR is
stabilized by interactions involving Thr555 and
Asp556 in TMH 6 with Asn593
and Asn597 in TMH 7 and that weakening the
hydrogen bonds between them can cause constitutive receptor activation
(11). Our results on Ala replacements of Asn593
and of Asn597 are not totally consistent with
this model; otherwise, one of the individual Ala replacements should
lead to constitutive receptor activation, and this was not observed. It
is of interest that the formation of an intrahelical hydrogen bond
between Asn593 and Asn597
in TMH 7 may be required for complete ligand-mediated activation of
LHR, and, if so, it is not surprising that Ala replacements of either
of the two asparagines results in a loss-of-function mutant. (We
prepared a double mutant of LHR, N593A/N597A, but it failed to express
at sufficiently high levels for evaluation.) The close proximity of
Asn593 and Asn597 is
demonstrated in Fig. 1A. Movement of TMH 6 has been proposed to be
involved in the activation process (11), and our results strongly argue
that TMH 7 is reoriented as well.
The results of molecular modeling and energy minimization are based on ionizable side chains of Asp, Glu, Lys, and Arg residues. In bacteriorhodopsin, water is present in the interhelical channel, and many of the ionizable side chains are charged (32, 33, 34). Unfortunately, no comparable data exist for LHR. The energy minimizations were also performed considering only interhelical interactions between 6 and 7. The important contributions of other TMHs and bound phospholipids, as well as the constraints imposed by the third intracellular and extracellular loops, were not included in the calculations. Thus, the energies reflect a localized interaction of TMHs 6 and 7. Not surprisingly, Lys and Arg replacements of Asn at positions 593 and 597 in TMH 7 yielded the lowest energies resulting from the formation of ion-ion interactions with Asp556 in TMH 6, while Asp and Glu replacements gave higher energies due to charge repulsion. These results may of course be misleading if the side chains are not ionized. Gln substitutions of Asn at positions 593 and 597 are only slightly less favored than Asn, e.g. 2–4 kcal/mol higher energy. The other replacements such as Ala at positions 593 and 597 and Ser at 593 result in unfavorable energies of 18–30 kcal/mol.
Overall, the predicted energies do not correlate with the functional data. For example, N593R, but not N593K, results in constitutive activation of the LHR mutant, and N597R, but not N597K, leads to a loss-of-function mutant; yet, the energies are comparable. Likewise, N593Q produces a loss-of-function mutant, while N597Q gives constitutive activation. The results of energy minimization, however, argue strongly for helix reorientation and perhaps a minimal displacement concomitant with certain amino acid residue replacements at positions 593 and 597 in TMH 7. Such changes in relative conformation of one or both helices presumably account for the different functional data found such as loss of function or gain of function; moreover, structural changes may adversely affect expression levels of the receptor.
The simplest scheme that has been proposed to account for G
protein-coupled receptor function involves two conformations, one
inactive, Ro, and one active,
R*, that can undergo interconversion,
Ro
R*. In this model,
ligand is assumed to have a low affinity for the
Ro conformation and a high affinity for that of
R*. Thus, in the presence of ligand, the
equilibrium is shifted toward the right, resulting in increased
signaling. In the absence of ligand, most of the receptors are in the
Ro conformation; the occasional conformational
conversion to R* results in a non-zero level of
basal cAMP. (In the case of LHR and COS-7 cells, however, we do not
find a strong correlation between basal cAMP levels and receptor
density at the levels at which we normally express.) A somewhat more
complex scenario would involve, rather than a cooperative transition
between Ro and R*, a series
of intermediate steps (35) as depicted in the following equation, where
each intermediate conformation is somewhat more active than the
preceding one and j denotes a general intermediate form of which there
may be many: R0 
R1
R2 Rj
R*.
In this model, one could argue that a given constitutively active mutation would convert Ro to a more active form, e.g. R1, R2, or Rj, and that addition of ligand would yield the L·R* state characterized by maximal cAMP production, as obtained, for example, with WT receptor.
We have identified LHR mutants that exhibit WT-like basal cAMP production but cannot respond fully to ligand, i.e. loss-of-function mutants. This may represent a case of the mutant receptor in state Ro that can adopt either state R1, R2, or Rj, but not R*, in the presence of ligand. We have characterized other mutants that are constitutively active and highly responsive to ligand. These mutants may adopt state R1 or R2 in the absence of ligand and thus exhibit an elevated level of cAMP, and furthermore they may be able to complete all of the intermediate steps to form a WT-like L·R* complex.
Realizing that replacement of transmembrane amino acid residues with
quite different structures, e.g.
Asn593Arg in TMH 7, may lead to a conformation
that cannot be adopted by WT LHR, a more general representation
involving separate schema for loss-of-function and constitutively
activating mutations may be more appropriate. Also, it has been
proposed, based upon strong supporting experimental evidence, that the
native conformation of hCG is not required for activity (36); it seems
reasonable that a number of conformations of LHR involving the region
encoded by exon 11, i.e. the transmembrane helices and
intra/extracellular loops, are possible. Another consideration is that
the ligand may induce one or more receptor conformations. Finally, the
different rates of internalization reported for gain-of-function LHR
mutants (37, 38), compared with WT LHR, may also be a factor.
In conclusion, considering our results on Pro591
and Tyr601 from earlier work (17), we have
identified three amino acid residues in close proximity on one face of
TMH 7 of LHR, Asn593,
Asn597, and Tyr601, and two
residues in close proximity on the opposite face of the helix,
Pro591 and Cys595, that are
involved, either directly or indirectly, in ligand-mediated signaling.
In addition, a novel and unusual finding was that replacement of
Asn593 or Asn597 can result
in either a loss-of-function or a gain-of-function mutant, depending
upon the nature of the side chain. Molecular modeling and energy
minimization indicated a reorientation and/or displacement of one or
both TMHs 6 and 7 concomitant with certain functional replacements of
D556, N593, or N597. It seems likely that a reorientation of TMHs 6 and
7 of LHR is required for proper signaling in WT LHR. Finally, we found
no evidence for a role of Glu429 in TMH 3,
consistent with the earlier finding based on a
Glu429Gln replacement (39).
Asn513 appears to be important in receptor
folding and/or membrane trafficking, but there is no evidence that it
is involved in ligand-mediated signaling.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Mutagenesis of LHR
Mutagenesis of rat LHR, cloned in the expression vector pSVL,
was performed by the in vitro site-directed mutagenesis and
Quick Change site-directed mutagenesis kits as recommended by
CLONTECH Laboratories, Inc. (Palo Alto, CA) and
Stratagene (La Jolla, CA), respectively. To overcome the
low expression levels associated with the
Asn597Gln replacement, we replaced the LHR
signal sequence with that corresponding to hCGß followed by the
carboxy-terminal peptide of hCGß. Mutant clones were identified by
sequencing using the Sequenase Version 2.0 DNA sequencing kit
(Amersham Pharmacia Biotech, Arlington Heights, IL).
Mutant cDNAs were amplified, and the QIAGEN (Chatsworth,
CA) plasmid maxi kit was used to obtain purified DNA.
Hormone Binding to Transfected Cells
About 16–18 h after transfection, the COS-7 cells were replated
(5 x 105 cells per well) into six-well
tissue culture plates and assayed for binding 24 h later.
125I-hCG (50 pM, DuPont NEN, Boston,
MA) and increasing concentrations of hCG were added to each well for
competitive binding assays; nonspecific binding was determined by
addition of 1000-fold excess of unlabeled hormone. All determinations
were performed in duplicate, and, unless stated otherwise, the data are
given as mean ± SEM of two to eight independent
transfections.
cAMP Assay
About 16–18 h after transfection, cells were replated (1
x 105 cells per well) into 12-well tissue
culture plates. After 24 h, the cells were incubated with
increasing or maximal (100 ng/ml) concentrations of hCG for 30 min at
37 C in the presence of 0.8 mM isobutylmethylxanthine
(Sigma, St. Louis, MO). Incubation medium was then removed
and the cells lysed in 100% ethanol at -20 C overnight. The extract
was collected, dried under vacuum, and resuspended in the buffer of the
125I-cAMP assay kit. cAMP concentrations were
determined by RIA as recommended by DuPont NEN. Duplicate
determinations were made for each experiment, and the results are
presented as mean ± SEM of two to eight independent
transfections unless stated otherwise.
Data Analysis
Both binding and cAMP data were analyzed by the Prism software
(Graph Pad Software, San Diego, CA). To compare the expression levels
of WT and mutant LHRs, the specific binding of the WT receptor was
normalized to 100%, and the specific binding of each of the mutants
was given relative to that value for each transfection. For purposes of
comparison of the signal transduction potency of the WT and mutant
receptors, the maximal hCG-mediated cAMP production over the basal
levels was normalized to 100% for the WT receptor in a given
transfection, and the value obtained with the different receptor
mutants, corrected for basal level, was expressed as a percentage of
that of WT receptor. Basal cAMP levels are given as picomoles/ml for WT
LHR and all mutants. Significance was determined by an unpaired
two-tailed Students t test, with 95% confidence limits
(P < 0.05).
Molecular Modeling
All molecular modeling was performed with SYBYL 6.5 Release
(Tripos Associates, Inc., St. Louis, MO). TMHs 6 and 7 were constructed
as individual helical segments with the following backbone torsion
angles for all residues except proline: ø =
-58o, 
= -47o,
and 
= 180o. Proline residues were
incorporated with a ø angle of -75o with
and identical to the other amino acid residues in the helical
segments. Each TMH was then minimized using the Kollman All-Atom force
field using Kollman point charges. The nonbonded cutoff was set a 8.0
Å, and the default distance dielectric function was used with a
dielectric constant of 1.0. Minimization was automatically terminated
when the gradient fell below 0.05 kcal/mol.
The energy-minimized helices were positioned adjacent to each other such that Asn593 and Asn597 were located within hydrogen bonding distance (<3 Å) to Asp556, and the helical axes were parallel. The interacting helices were then minimized as above with the assumption that the ionizable side chains are charged. This construct is similar to that proposed by Lin et al. (11) and was used as the native, one-state model for comparison with residue replacements at positions 593 and 597.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was supported by NIH Research Grant DK-33973.
Received for publication September 27, 1999. Revision received December 19, 1999. Accepted for publication January 5, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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C. Govaerts, A. Lefort, S. Costagliola, S. J. Wodak, J. A. Ballesteros, J. Van Sande, L. Pardo, and G. Vassart A Conserved Asn in Transmembrane Helix 7 Is an On/Off Switch in the Activation of the Thyrotropin Receptor J. Biol. Chem., June 15, 2001; 276(25): 22991 - 22999. [Abstract] [Full Text] [PDF]
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-helix was
constructed with SYBYL (Tripos Associates Inc.) and then minimized
using the Kolman all-atom force field (electrostatics included) until
default convergence criteria were met. The 
). The cAMP changes in response to hCG
of WT LHR (
) and mutant LHR (
) are shown in the bottom
three panels. The data represent the mean ± M
of three independent transfections.
