Functional Role of Transmembrane Helix 7 in the Activation of the Heptahelical Lutropin Receptor
Krassimira Angelova,
Prema Narayan,
J. Paul Simon and
David Puett
Department of Biochemistry & Molecular Biology University of
Georgia Athens, Georgia 30602-7229
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ABSTRACT
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A member of the G protein-coupled receptor
superfamily, the LH receptor (LHR), and the two other glycoprotein
hormone receptors are distinguished from the other members by the
presence of a relatively large N-terminal extracellular domain that is
responsible for high-affinity ligand binding. Transmembrane helix (TMH)
7 of LHR is amphipathic, with an extended face containing only
hydrophobic side chains and another containing both hydrophobic and
polar side chains with potential hydrogen bond donor and acceptor
functions. Since several reports have shown the importance of
this helix in ligand-mediated signaling, we have used Ala scanning
mutagenesis to study eight amino acid residues of rat LHR that are
invariant in the three glycoprotein hormone receptors,
Leu586, Val587,
Asn593, Ser594,
Cys595, Asn597,
Phe604, and Thr605. The
wild type (WT) and mutant cDNAs were transiently transfected into COS-7
cells for characterization by human CG (hCG) binding and cAMP
production. No differences were detected in dissociation
constants (Kds) or basal cAMP production
relative to WT LHR, but three categories of LHR mutants were
distinguished from WT LHR based upon their expression levels and
responsiveness to hCG: 1) comparable or higher expression but reduced
ligand responsiveness (N593A and C595A), 2) reduced expression and
ligand responsiveness (N597A and T605A), and 3) comparable expression
and responsiveness (L586A, V587A, S594A, and F604A). Three other
mutants, C595M, F604Y, and T605Y, were comparable to WT LHR in ligand
responsiveness. To provide more information on
Asn593 and Asn597, a
total of 12 replacements were investigated. Of considerable interest
and potential significance was the finding that many of the
replacements in LHR resulted in either loss of function (N593A, Q, S;
N597R) or gain of function (N593R and N597Q), this being the first
evidence of a position in LHR that, depending upon the nature of the
amino acid residue, can result in constitutive activation and/or
diminished responsiveness to ligand. The results of molecular modeling
and energy minimization of TMHs 6 and 7, based on a postulated
interaction between Asp556 (TMH 6) and
Asn593/Asn597 (TMH 7),
indicated that, while there is not a correlation between function and
predicted energies of WT LHR and the mutants, reorientation of one or
both helices is responsible for the functional changes observed.
Possible interactions of TMHs 3 and 4 and of 5 and 6 were suggested by
molecular modeling. Ten mutants were prepared of two amino acid
residues that are invariant in the glycoprotein hormone receptors and
have side chain hydrogen bond donor and acceptor function,
Glu429 in TMH 3 and
Asn513 in TMH 5. Expression levels and
hCG-mediated signaling were reduced in most of the LHR mutants, but
none of these exhibited constitutive receptor activation. We conclude
that Glu429 is not critical for receptor
function, while Asn513 appears to be
particularly important in receptor folding and/or trafficking. The
results reported herein indicate an important role for TMH 7, and
particularly Asn593 and
Asn597, in the process of receptor activation.
Moreover, these two asparagines, although in close proximity to each
other in TMH 7, are quite distinct in function as evidenced by certain
replacements that can lead to loss of function in one and gain of
function in the other.
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INTRODUCTION
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The LH receptor (LHR) (1), along with the receptors for FSH and
TSH, form a subfamily of the superfamily of G protein-coupled receptors
(2). These particular heptahelical receptors are distinguished by their
large extracellular N-terminal domain that accounts for roughly half
the size of the receptor and is responsible for high-affinity ligand
binding (3, 4, 5, 6, 7). LHR is expressed on gonadal cells and is required for
normal reproductive function in males and females and for male sexual
differentiation (8). In addition to the well established signaling
pathway involving Gs and activation of adenylyl
cyclase (1, 2), it has been reported that phospholipase Cß is also
activated, perhaps by the ß
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 3037 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 Ser
Tyr 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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Molecular modeling was performed on TMH 7 of rat LHR, and
schematic representations of the helix are shown in Fig. 1
. 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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Figure 1. Schematic Model of TMH 7 (Residues 584605)
of LHR
A side view of TMH 7 (ILLVLFYPVNSCANPFLYAIFT) showing only the amino
acid residues replaced in this study (A). A perfect -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 -carbons from each
residue connected to generate the helical wheel diagram viewed from the
extracellular side of the cell membrane looking toward the cytoplasmic
side, and the amino acid residues investigated in this study are shown
in boldface (B).
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Ala scanning mutagenesis was initially conducted on eight amino acid
residues in TMH 7 of rat LHR. After transient transfection into COS-7
cells, each of the mutant LHRs was characterized by competitive ligand
binding with 125I-human (h) CG and hCG to
determine the IC50, from which the
Kd could be obtained. Since amino acid residue
replacements in TMH 7 were found to have no effect on
Kd values, the specific binding at a fixed
concentration of 125I-hCG,
Bo, is proportional to receptor density. We also
measured basal cAMP production and changes thereof in response to hCG
to obtain the ED50 and maximal cAMP produced at a
saturating concentration of hCG after subtraction of the basal level
(Rmax). In each case, wild-type (WT) LHR was
included as control, and the results are reported for
Bo and Rmax as a percentage
of WT LHR. The results for each of the Ala replacements are summarized
in Table 1
, 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.
To address the question of basal cAMP levels and
Rmax dependence on receptor density over the
range investigated, we determined maximal ligand-mediated signaling at
various levels of expression for the Ala-containing receptor mutants.
From the data presented in Fig. 3
, 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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Figure 3. Relationship between Rmax and
Receptor Density (Proportional to Bo) for Transfected COS-7
Cells Expressing WT and Mutant LHRs
Plots (log10-log10) of Rmax
vs. Bo were constructed for WT and mutant
LHRs. Each point represents a separate transfection, and
the means ± range of duplicate measurements are shown. The data
were fit by linear regression, and the solid line shows
the best fit with 95% confidence limits (P <
0.05) given by the dashed lines. There is a slight
dependence of maximal cAMP response to hCG and apparent receptor
density for WT LHR.
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Other point mutants of LHR were then characterized, C595M, F604Y, and
T605Y, with the replacements chosen for the following reasons. In view
of the finding that the C595A mutant exhibited loss of responsiveness,
we chose a Met replacement since it is more hydrophobic than Ala and
has a sulfur-containing side chain, but is devoid of thiol function.
The F604Y replacement was made to ask if an aromatic side chain with
hydroxyl function would alter expression and signaling since the Ala
replacement did not. Finally, since the T605A mutant exhibited
reductions in both receptor expression and signaling, we replaced the
side chain with an aromatic side chain, while retaining the hydroxyl
function of Thr. As shown in Table 2
, 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.
Additional studies focused on Asn593 and
Asn597 for several reasons. The N593A mutant
exhibited interesting properties in that expression was greater than
that of WT LHR, yet signaling was diminished. Also, we showed earlier
that the two Asns in TMH 7 mapped to a face of the helix that contained
the other side chains with hydrogen bond donor or acceptor
function, e.g. Tyr590,
Ser594, Tyr601, and
Thr605, and suggested that this region may be
involved in ligand-mediated signaling (17). Finally, using molecular
modeling Lin et al. (11) suggested that
Asp556 in TMH 6 may interact with the two Asns in
TMH 7 as part of receptor activation.
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 Asn593
Ala, Gln, and Ser replacements
yielded LHR mutants that expressed at levels comparable to WT LHR, yet
responsiveness to hCG was blunted; the
Asn593
Lys replacement exhibited properties
like those of WT LHR. The Asn593
Asp 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
Asn593
Arg 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
Asn597
Arg 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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Table 3. Expression and Signaling Parameters of
Asn593 and Asn597 Replacements in TMH 7 of
LHR1
and Relative Energies from Energy
Minimization of TMHs 6 and 7
Interactions
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Figure 4. Basal and Maximal cAMP Levels for LHR Mutants in
TMH 7 with Replacements at Asn593
The two-mutant forms of LHR have comparable receptor densities (cf.
Table 3 ): one is a loss-of-function mutant (N593A) (A), and the other
is a gain-of-function mutant (N593R) (B). The cAMP levels measured in
the presence of hCG have not been corrected for basal levels.
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Figure 5. Basal and Maximal cAMP Levels for LHR Mutants in
TMH 7 with Replacements at Asn597
The two mutant forms of LHR have similar receptor levels (cf. Table 3 ).
The N597R mutant represents a loss-of-function mutant (A), and N597Q is
a gain-of-function mutant (B).
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Energy minimization of the interaction between TMHs 6 and 7 gave the
results presented in Table 3
. 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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Figure 6. Schematic Model of TMHs 6 and 7 in WT LHR
Emphasizing the Predicted Interhelical Interactions
A side view of the energy-minimized structure of TMHs 6 and 7 with the
putative interaction (11 ) between Asp556 (TMH 6) and
Asn593/Asn597 (TMH 7) highlighted.
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Figure 7. Schematic Models of TMHs 6 and 7 in WT and Four
Mutant LHRs
A top view, i.e. from the cell exterior toward the
cytoplasmic side, between positions 556 and 593/597 for WT LHR (A) and
the four point mutants N593Q(B), N593R(C), N597Q(D), and N597R(E). All
putative hydrogen bonds between D556 and N593/N597, Q593/N597,
R593/N597, N593/Q597, and N593/R597 were between 2.62.7 Å; moreover,
the N593R mutant is predicted to have an intramolecular hydrogen bond,
between R593 and N597 of 2.8 Å.
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In summary, the Asn593
Ala, Gln, and Ser and
Asn597
Arg replacements represent
loss-of-function mutants; the Asn593
Asp, Glu
and the Asn597
Ala, Asp, and Lys replacements
may as well, but the reduced level of receptor expression compromises
such a conclusion. The Asn593
Arg and
Asn597
Gln 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
Asn513
Gly, 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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This study has identified three amino acid residues of TMH 7 of
LHR that appear to be involved in ligand-mediated signaling,
Asn593, Cys595, and
Asn597. In earlier work we identified similar
roles for Pro591 and
Tyr601, but not Tyr590
(16). Thus, of the six side chains in TMH 7 that map to a common face
of the helix and exhibit potential hydrogen bond donor and/or acceptor
function, Tyr590, Asn593,
Ser594, Asn597,
Tyr601, and Thr605,
three seem to have a functional role in signaling.
Interestingly, the three functional side chains,
Asn593, Asn597, and
Tyr601, are in close proximity to each other.
Pro591 and Cys595 map to a
common segment on the extended hydrophobic face of TMH 7 opposite that
of the
Asn593/Asn597/Tyr601
patch. The role(s) for Pro591 and
Cys595 may be structural only; alternatively, the
peptide carbonyl group of proline and the side chain of cysteine may
serve as hydrogen bond acceptor and donor, respectively. It is quite
possible that these two opposite faces of TMH 7 may be required for
helix reorientation in ligand-mediated signaling. The naturally
occurring loss-of-function human LHR mutant, S616Y
(Ser594 in rat LHR), has been reported to result
in poor cell surface localization (16, 26, 27). Our results on the
S594A mutant in rat LHR show that WT-like expression and function
occur; thus, the bulky side chain of tyrosine probably interferes with
proper helix packing and perhaps trafficking to the cell surface. There
is no evidence that the hydroxyl group of this serine is essential for
signaling.
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,
Asn670
Ser (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. 24 kcal/mol higher energy. The
other replacements such as Ala at positions 593 and 597 and Ser at 593
result in unfavorable energies of 1830 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.
Asn593
Arg 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
Glu429
Gln 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.
 |
MATERIALS AND METHODS
|
---|
Cell Culture and Transient Transfection
COS-7 cells, obtained from American Type Culture Collection (Manassas, VA), were grown in a monolayer culture in
DMEM supplemented with 10% (vol/vol) FBS, 50 U/ml penicillin, 50
µg/ml streptomycin, 50 µg/ml gentamycin, and 0.125 µg/ml
Amphotericin (Life Technologies, Inc., Gaithersburg,
MD). Cells were maintained at 37 C in humidified air
containing 5% CO2 and transiently transfected
with 10 µg of the WT or mutant cDNA using Lipofectamine as
recommended by Life Technologies (Gaithersburg, MD).
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
Asn597
Gln 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 1618 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 1618 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.
 |
ACKNOWLEDGMENTS
|
---|
We thank Drs. Neil Bhowmick and Adrian Lapthorn for their
interest and assistance in the initial modeling studies of TMH
7.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. David Puett, Department of Biochemistry & Molecular Biology, Life Sciences Building, Green Street, University of Georgia, Athens, Georgia 30602-7229.
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.
 |
REFERENCES
|
---|
-
Dufau ML 1998 The luteinizing hormone receptor. Annu Rev
Physiol 60:461496[CrossRef][Medline]
-
Segaloff DL, Ascoli M 1993 The lutropin/choriogonadotropin
receptor. 4 years later. Endocr Rev 14:324347[Abstract]
-
Tsai-Morris CH, Buczko E, Wang W, Dufau ML 1990 Intronic
nature of the rat luteinizing hormone receptor gene defines a soluble
receptor subspecies with hormone binding activity. J Biol Chem 265:1938519388[Abstract/Free Full Text]
-
Xie Y-B, Wang H, Segaloff D 1990 Extracellular domain of
lutropin/choriogonadotropin receptor expressed in transfected cells
binds choriogonadotropin with high affinity. J Biol Chem 265:2141121414[Abstract/Free Full Text]
-
Braun T, Schofield PR, Sprengel R 1991 Amino-terminal
leucine-rich repeats in gonadotropin receptors determine hormone
selectivity. EMBO J 10:18851890[Abstract]
-
Ji I, Ji TH 1991 Exons 110 of the rat LH receptor encode a
high affinity hormone binding site and exon 11 encodes G-protein
modulation and a potential second hormone binding site. Endocrinology 128:26482650[Abstract]
-
Moyle WR, Bernard M, Myers RV, Marko OM, Strader CD 1991 Lutropin/beta-adrenergic receptor chimeras bind choriogonadotropin and
adrenergic ligands but are not expressed at the cell surface. J
Biol Chem 266:1080710812[Abstract/Free Full Text]
-
Jaffe RB 1999 Disorders of Sexual Differentiation. In: Yen
SCC, Jaffe RB, Barbieri RL (eds) Reproductive Endocrinology, ed. 4. WB
Saunders Co, Philadelphia, pp 363387
-
Herrlich A, Kuhn B, Grosse R, Schmid A, Schultz G, Gudermann
T 1996 Involvement of Gs and
Gi proteins in dual coupling of the luteinizing
hormone receptor to adenylyl cyclase and phospholipase C. J Biol
Chem 271:1676416772[Abstract/Free Full Text]
-
Ji TH, Murdoch WJ, Ji I 1995 Activation of membrane receptors.
Endocrine 3:187194
-
Lin Z, Shenker A, Pearlstein R 1997 A model of the
lutropin/choriogonadotropin receptor: insights into the structural and
functional effects of constitutively activating mutations. Protein Eng 10:501510[Abstract]
-
Schoneberg T, Schultz G, Gudermann T 1999 Structural basis of
G protein-coupled receptor function. Mol Cell Endocrinol 151:181193[CrossRef][Medline]
-
Hoflack J, Hibert MF, Trumpp-Kallmeyer S, Bidart J-M 1993 Three-dimensional models of gonado-thyrotropin hormone receptor
transmembrane domain. Drug Des Discov 10:157171[Medline]
-
Ji I, Ji TH 1991 Asp383 in the second
transmembrane domain of the lutropin receptor is important for high
affinity hormone binding and cAMP production. J Biol Chem 266:1495314957[Abstract/Free Full Text]
-
Quintana J, Wang H, Ascoli M 1993 The regulation of the
binding affinity of the luteinizing hormone/choriogonadotropin receptor
by sodium ions is mediated by a highly conserved aspartate located in
the second transmembrane domain of G protein-coupled receptors. Mol
Endocrinol 7:767775[Abstract]
-
Laue LL, Wu S-M, Judo M, Bourdony CJ, Cutler Jr GB, Hsueh AJW,
Chan W-Y 1996 Compound heterozygous mutations of the luteinizing
hormone receptor gene in Leydig cell hypoplasia. Mol Endocrinol 10:987997[Abstract]
-
Fernandez LM, Puett D 1996 Identification of amino acid
residues in transmembrane helices VI and VII of the
lutropin/choriogonadotropin receptor involved in signaling.
Biochemistry 35:39863993[CrossRef][Medline]
-
Kosugi S, Mori T, Shenker A 1996 The role of
Asp578 in maintaining the inactive conformation
of the human lutropin/choriogonadotropin receptor. J Biol Chem
271: 3181331817
-
Hong S, Ryu K-S, Oh M-S, Ji I, Ji TH 1997 Roles of
transmembrane prolines and proline-induced kinks of the
lutropin/choriogonadotropin receptor. J Biol Chem 272: 4166- 4171
-
Themmen APN, Martens JWM, Brunner HG 1998 Activating and
inactivating mutations in LH receptors. Mol Cell Endocrinol 145:137142[CrossRef][Medline]
-
Shenker A 1998 Disorders caused by mutations of the
lutropin/choriogonadotropin receptor gene. In: AM Spiegel (ed)
Contemporary Endocrinology: G Proteins, Receptors and Disease. Humana
Press, Inc., Totowa, NJ, pp 139152
-
Chan WY 1998 Molecular genetic, biomedical, and clinical
implications of gonadotropin receptor mutations. Mol Genet Metab 63:7584[CrossRef][Medline]
-
Simoni M, Gromoll J, Nieschlag E 1998 Molecular
pathophysiology and clinical manifestations of gonadotropin receptor
defects. Steroids 63:288293[CrossRef][Medline]
-
Fernandez LM, Puett D 1996 Lys583 in the
third extracellular loop of the lutropin/choriogonadotropin receptor is
critical for signaling. J Biol Chem 271:925930[Abstract/Free Full Text]
-
Ryu K-S, Gilchrist RL, Ji I, Kim S-J, Ji, TH 1996 Exoloop 3 of
the luteinizing hormone/choriogonadotropin receptor. J Biol Chem 271:73017304[Abstract/Free Full Text]
-
Latronico A, Anasti J, Arnhold IJP, Rapaport R, Mendonca BB,
Bloise W, Castro M, Tsigos C, Chrousos GP 1996 Testicular and ovarian
resistance to luteinizing hormone caused by inactivating mutations of
the luteinizing hormone-receptor gene. N Engl J Med 334:507512[Free Full Text]
-
Martens JWM, Verhoef-Post M, Abelin N, Ezabella M, Toledo SPA,
Brunner HG, Themmen APN 1998 A homozygous mutation in the luteinizing
hormone receptor causes partial Leydig cell hypoplasia: correlation
between receptor activity and phenotype. Mol Endocrinol 12:775784[Abstract/Free Full Text]
-
Latronico AC, Chai Y, Arnhold IJ, Liu X, Mendonca BB, Segaloff
DL 1998 A homozygous microdeletion in helix 7 of the luteinizing
hormone receptor associated with familial testicular and ovarian
resistance is due to both decreased cell surface expression and
impaired effector activation by the cell surface receptor. Mol
Endocrinol 12:442450[Abstract/Free Full Text]
-
Kosugi S, Lin Z, Pearlstein R, Mori T, Shenker A, Evidence
that interhelical hydrogen bonding between Asp 578, Asn 619 helps
maintain the inactive conformation of the human lutropin receptor
(LHR). Program of the 79th Annual Meeting of The Endocrine Society,
Minneapolis, MN, 1997, p 171 (Abstract)
-
Yano K, Saji M, Hidaka A, Moriya N, Okuno A, Kohn LD, Cutler
Jr GB 1995 A new constitutively activating point mutation in the
luteinizing hormone/choriogonadotropin receptor gene in cases of
male-limited precocious puberty. J Clin Endocrinol Metab 80:11621168[Abstract]
-
Van Sande J, Parma J, Tonacchera M, Swillene S, Dumont J,
Vassart G 1995 Somatic and germline mutations of the TSH receptor gene
in thyroid diseases. J Clin Endocrinol Metab 80:25772585[Medline]
-
Pebay-Peyroula E, Rummel G, Rosenbusch JP, Landau EM 1997 X-ray structure of bacteriorhodopsin at 2.5 angstroms from
microcrystals grown in lipidic cubic phases. Science 277:16761680[Abstract/Free Full Text]
-
Kimura Y, Vassylyev DG, Miyazawa A, Kidera A, Matsushima M,
Mitsuoka K, Murata K, Hirai T, Fujiyoshi Y 1997 Surface of
bacteriorhodopsin revealed by high-resolution electron crystallography.
Nature 389:206211[CrossRef][Medline]
-
Luecke H, Schobert B, Richter H-T, Cartailler J-P, Lanyi JK 1999 Structural changes in bacteriorhodopsin during ion transport at 2
angstrom resolution. Science 286: 255260
-
Gudermann T, Kalkbrenner F, Schultz G 1996 Diversity and
selectivity of receptor-G protein interaction. Annu Rev Pharmacol
Toxicol 36:429459[CrossRef][Medline]
-
Ben-Menahem D, Kudo M, Pixley MR, Sato A, Suganuma N, Perlass
E, Hsueh AJW, Boime I 1997 The biologic action of single-chain
gonadotropin is not dependent on the individual disulfide bonds of
the ß subunit. J Biol Chem 272:68276830[Abstract/Free Full Text]
-
Min K-S, Liu X, Fabritz JE, Jaquette J, Abell AN, Ascoli M 1998 Mutations that induce constitutive activation and mutation that
impair signal transduction modulate the basal and/or agonist-stimulated
internalization of the lutropin/choriogonadotropin receptor. J
Biol Chem 273:3491134919[Abstract/Free Full Text]
-
Bradbury FA, Menon KMJ 1999 Evidence that constitutively
active luteinizing hormone/human chorionic gonadotropin receptors are
rapidly internalized. Biochemistry 38:87038712[CrossRef][Medline]
-
Ji I, Ji TH 1991 Asp383 in the second
transmembrane domain of the lutropin receptor is important for high
affinity hormone binding and cAMP production. J Biol Chem 266:1495314957[Abstract/Free Full Text]