(Received for publication, October 25, 1996, and in revised form, March 5, 1997)
From the Department of Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, Iowa 52242
The luteinizing hormone/chorionic gonadotropin
receptor (LHR) is a heptahelical receptor that interacts primarily with
Gs. Previous studies by others have shown that some
forms of familial male precocious puberty are associated with mutations
of the human LHR in the sixth transmembrane region that result in
constitutive activation of the receptor. This study demonstrates that a
peptide corresponding to the lower portion of the sixth transmembrane region of the LHR can significantly activate adenylyl cyclase activity.
Experiments with membranes derived from wild-type versus cyc S49 cells demonstrate that the stimulation of
cyclase by this peptide is due to an activation of Gs. As
such, our data demonstrate a direct role for transmembrane region 6 of
the rat LHR in activating Gs and therefore raise the
possibility that mutations in transmembrane region 6 of the LHR may
directly affect the coupling of the receptor to Gs.
Significantly, these data are the first to demonstrate the ability of a
transmembrane portion of a G protein-coupled receptor, in the absence
of any contributions from an intracellular loop region, to activate a G
protein.
The LHR1 is a large cell-surface glycoprotein with the characteristic structure of a member of the superfamily of G protein-coupled receptors (1). This structure includes the presence of seven putative membrane-spanning regions connected by alternating intracellular and extracellular loops, an extracellular N-terminal extension, and an intracellular cytoplasmic tail (1, 2). The rLHR is capable of binding either luteinizing hormone or chorionic gonadotropin with high affinity, resulting in the activation of Gs and adenylyl cyclase and the production of cAMP (1, 3). Although cAMP is responsible for eliciting most of the significant effects of the rLHR, at high receptor densities and in the presence of high concentrations of hormone, the rLHR also activates phospholipase C (4, 5).
Many studies of different members of the G protein-coupled receptor superfamily have focused on the determination of the sites of these receptors that physically interact with G proteins. Utilizing multiple approaches such as chimeric receptors, synthetic peptides, and deletion/substitution mutagenesis, several investigators have determined that the N- and C-terminal portions of the third intracellular loop are essential for the interaction of the adrenergic receptors with their respective G proteins (6-8). Additionally, similar experiments with the muscarinic acetylcholine receptor subfamily of G protein-coupled receptors have revealed that the third intracellular loop determines the subtype-specific coupling of the muscarinic acetylcholine receptors to distinct G proteins, with the N- and C-terminal portions of the third intracellular loop being absolutely required for activation of their respective G proteins (9-11). More important, the third intracellular loop is not the site of G protein interaction for all G protein-coupled receptors. For example, experiments utilizing synthetic peptides and fusion proteins corresponding to regions of the human neutrophil N-formylpeptide receptor demonstrated the importance of the second intracellular loop and the N-terminal portion of the cytoplasmic tail for the coupling of this receptor to Gi (12).
The sites of interaction of the LHR with Gs are not well defined. However, several of the activating mutations found to be associated with familial male precocious puberty have been found to occur in the lower portion of transmembrane 6 and in the C-terminal portion of the third intracellular loop of the hLHR (13-17). These mutations are thought to activate the receptor by altering the packing of the transmembrane helices and/or by exposing intracellular regions, which can then interact with Gs. The identification of these activating mutations has led investigators to hypothesize that the third intracellular loop of the LHR may be important for the coupling of this receptor to Gs (13).
This study was undertaken to identify regions of the LHR that interact with Gs. Because mutation of the LHR frequently results in intracellular retention of mutant receptors (3, 18-25), we chose to utilize an alternate approach. Hence, the following studies were performed by examining the ability of synthetic peptides corresponding to regions of the rLHR to stimulate adenylyl cyclase activity. Based upon the studies of the adrenergic receptors and upon mutations of the LHR causing familial male precocious puberty, we chose to focus initially on the third intracellular loop and sixth transmembrane region of the rLHR. As shown herein, our results clearly demonstrate a role for transmembrane 6 of the LHR in the activation of Gs.
Highly purified hCG (CR-127) was provided by the
National Hormone and Pituitary Agency of NIDDK, National Institutes of
Health. GDPS, alumina WN-3, and reagents for adenylyl cyclase assays were obtained from Sigma. AG-50W-X4 resin was purchased from Bio-Rad, and [
-32P]ATP was from DuPont NEN. Tissue culture
reagents and plasticware were obtained from Life Technologies, Inc. and
Corning Inc. (Corning, NY), respectively.
rLHR peptides were synthesized as C-terminal amides and were purified by reverse-phase high pressure liquid chromatography to >95% purity. Peptides were synthesized either by Multiple Peptide Systems (San Diego, CA) or by the Mayo Protein Core Facility, Mayo Foundation (Rochester, MN). As a control for the different peptide sources, some peptides were synthesized by both vendors and were found to have identical properties.
CellsHuman embryonic kidney 293 cells (ATCC CRL 1573, American Type Culture Collection, Rockville, MD), which do not express
the rLHR, were maintained in a high glucose Dulbecco's modified
Eagle's medium supplemented with 50 µg/ml gentamicin, 10 mM Hepes, and 10% newborn calf serum and incubated at
37 °C in 5% CO2. The clonal stable 293 cell line
rLHR-wt12, expressing ~150,000 rLHR receptors/cell, was generously
donated by Dr. Mario Ascoli (University of Iowa) (5). rLHR-wt12 cells
were maintained as described above and supplemented with 700 µg/ml
Geneticin. S49 wild-type and cyc mouse
lymphoma cell lines were obtained from the Cell Culture Facility,
University of California, San Francisco. S49 wild-type cells were grown
in stationary suspensions in high glucose Dulbecco's modified Eagle's
medium supplemented with 50 µg/ml gentamicin, 10 mM
Hepes, and 10% heat-inactivated horse serum and incubated at 37 °C
in 5% CO2. S49 cyc
cells, which
lack G
s (26), were derived from S49 wild-type cells (27)
and were maintained as described above for S49 wild-type cells.
For the preparation of 293 membranes, confluent 100-mm dishes were set on ice for 15 min and
washed twice with 5 ml of ice-cold buffer A (250 mM
sucrose, 25 mM Tris-Cl, and 1 mM EDTA, pH 7.4). Cells were scraped from the dishes; the dishes were rinsed with cold
buffer A; and the contents were combined. The cell suspension was
centrifuged (2200 × g, 10 min, 4 °C); the
supernatant was aspirated; and the cell pellet was resuspended in 1 mM Tris-Cl and 1 mM EDTA, pH 7.4, to a density
of 2 × 107 cells/ml and vortexed. 8-ml aliquots (1.6 × 108 cells/tube) were vortexed and kept on ice for 15 min to
lyse the cells. Each aliquot was homogenized (Ultra-Turrax T25, Kandel IKA Labortechnick, Staufen, Germany) at 22,000 rpm using 10 bursts of
5 s each with 10 s between each burst. Homogenates were
centrifuged (470 × g, 15 min, 4 °C), and the
post-nuclear supernatant was then centrifuged (100,000 × g, 45 min, 4 °C) to obtain a membrane pellet. The
membrane pellet was resuspended in buffer B (0.8 ml of 125 mM Tris-Cl and 5 mM EDTA, pH 7.4) by hand
homogenization 10 times using a Teflon-glass homogenizer. The membranes
were pelleted (2200 × g, 10 min, 4 °C) and
resuspended in buffer A, and aliquots were stored in liquid nitrogen.
Before use, aliquots were thawed on ice, and membranes were pelleted
(2200 × g, 10 min, 4 °C) and resuspended by
vortexing in buffer B. Crude preparations of S49 wild-type and
cyc cell membranes were prepared as described
by Ross et al. (28).
Adenylyl cyclase assays
were performed as described by Salomon (29). For experiments with 293 cell membranes, 10 µg of membranes in 10 µl of buffer B were
preincubated with increasing concentrations of the individual rLHR
peptides (dissolved in 10 µl of 10% Me2SO) for 15 min at
37 °C, followed by 15 min at 4 °C. After preincubation, the
following components were added: 10 µl of 75 µg/ml hCG in 150 mM NaCl, 20 mM Hepes, and 1% bovine serum
albumin, pH 7.4, or 10 µl of buffer only; 10 µl of 100 µM GTP; and 10 µl of a reaction mixture containing 0.5 mM ATP, 20 mM MgCl2, 5 mM cAMP, 100 mM phosphocreatine, 200 units/ml
creatine kinase, 200 units/ml myokinase, 3 µCi/ml
[3H]cAMP, and 140 µCi/ml [-32P]ATP.
The reaction mixtures were incubated for 20 min at 37 °C. Reactions
were terminated by the addition of 0.5 ml of stop solution (40 mM ATP, 10 mM cAMP, and 1% SDS) and by boiling
for 3 min. [32P]cAMP formed was separated from other
32P-nucleotides by sequential chromatography through Dowex
anion-exchange columns and alumina columns as described previously
(29). Scintillation fluid was added to the eluant, and samples were
counted for 5 min. Cyclase assays with S49 cell membranes were
performed essentially as described above for 293 membranes with the
following modifications. 10 µg of S49 membranes in 10 µl of 20 mM Hepes, 2 mM MgCl2, and 1 mM EDTA, pH 8.0, were preincubated with rLHR peptides for
15 min at 30 °C with shaking, followed by 15 min at 4 °C. After
preincubation, the following components were added: 10 µl of 230 mM Hepes and 4 mM EDTA, pH 8.0; 10 µl of 250 µM GTP; and 10 µl of the reaction mixture described
above, except that MgCl2 was included at 50 mM
instead of 20 mM. The reaction mixture was incubated for 20 min at 30 °C with shaking. Reactions were terminated; radiolabeled nucleotides were separated; and samples were counted as described above
for 293 membrane cyclase assays.
For assays measuring the
GDPS-induced inhibition of rLHR peptide-stimulated adenylyl cyclase
activity, GTP (final concentration of 20 µM) was
substituted with GDP
S (final concentration of 6 µM).
Membranes were incubated with the indicated rLHR peptide (final
concentration of 30 µM), with a saturating concentration of hCG (final concentration of 15 µg/ml), or with 100 µM forskolin. Adenylyl cyclase activity was measured
essentially as described above.
To determine the role of the third intracellular loop and
transmembrane 6 regions of the rLHR in the activation of
Gs, peptide I3/TM6, corresponding to the C-terminal portion
of the third intracellular loop and the lower portion of transmembrane
region 6, was synthesized (Fig. 1 and Table
I). As a control for I3/TM6, peptide E1/TM2 was also
synthesized. As shown in Fig. 1, peptide E1/TM2 represents the
N-terminal portion of the first extracellular loop and the upper
portion of transmembrane 2, a region that should not be directly
involved in the coupling of the rLHR to Gs. These peptides were then tested for their ability to either activate Gs or
competitively inhibit the interaction of the rLHR with Gs.
Membranes prepared from 293 cells expressing the wild-type rLHR were
incubated with increasing concentrations of either peptide E1/TM2 or
I3/TM6 in the absence or presence of a maximally stimulatory
concentration of hCG, and adenylyl cyclase activity was measured. As
shown in Fig. 2, E1/TM2 had no effect on either basal or
hormone-stimulated cyclase activity, whereas I3/TM6 markedly altered
adenylyl cyclase activity. Low concentrations of I3/TM6 (30 µM) stimulated basal cyclase activity ~3-fold (Fig.
2B). The magnitude of this response is similar to that
elicited by a maximal concentration of hCG (Fig. 2A). When
membranes were incubated in the presence of both a maximal
concentration of hCG and 30 µM I3/TM6, this peptide produced a further 3-fold increase in cyclase activity above the 3-fold
stimulation by hCG alone (Fig. 2C). In addition to the stimulatory effects of low concentrations of I3/TM6, high
concentrations (300 µM) of I3/TM6 produced a significant
inhibition of both basal and hormone-stimulated cyclase activity (Fig.
2, B and C). Both the activating and inhibitory
actions of the I3/TM6 peptide on basal adenylyl cyclase activity were
independent of the presence of the full-length rLHR as similar
activities were observed in membranes prepared from untransfected 293 cells that do not express the rLHR (Fig. 3).
|
Experiments with other G protein-coupled receptors such as the
2-adrenergic receptor would suggest that it is the I3
portion of I3/TM6 that is responsible for the activity of this peptide (6, 7). To address this possibility, peptide I3 (Table I), corresponding to the third intracellular loop of the rLHR, was tested
for activity. Over a range of 1-300 µM, however, I3 was found to have no effect on either basal or hCG-stimulated cyclase activity (data not shown). Since it is possible that the lack of
activity of the I3 peptide was due to the absence of a hydrophobic sequence that would enhance its association with membranes, an I3/TM2
peptide (Table I) was also examined. This peptide, in which the
C-terminal portion of the I3 sequence is now followed by a hydrophobic
sequence corresponding to the upper portion of the second transmembrane
helix, however, was also devoid of any stimulatory or inhibitory
activity (data not shown).
The lack of activity of the I3 and I3/TM2 peptides and the identification of several activating mutations in the lower portion of transmembrane 6 of the hLHR (13, 15) raised the possibility that the TM6 portion itself may be responsible for at least some of the activity of the I3/TM6 peptide (13, 15). To address this question, peptide TM6, corresponding to the lower portion of transmembrane 6, was synthesized (Fig. 1 and Table I). When incubated with membranes expressing the rLHR, 10-56 µM TM6 peptide stimulated both basal and hormone-stimulated cyclase activity ~2-fold (Fig. 2, B and C). Much higher concentrations of peptide TM6 inhibited both basal and hormone-stimulated cyclase activity, but to a lesser extent than the I3/TM6 peptide (Fig. 2, B and C). Similar to the I3/TM6 peptide, the activity of the TM6 peptide was independent of the presence of the full-length rLHR as activity was present in 293 membranes (Fig. 3). These data show that the residues corresponding to the lower portion of transmembrane 6 are responsible for a significant part of the activity of the I3/TM6 peptide.
Control peptides were synthesized to demonstrate the specificity of the
TM6 amino acid sequence for the activation of Gs. For these
purposes, peptide TM6(L552P), corresponding to peptide TM6 but with the
substitution of a leucine with a disruptive proline, was synthesized
(Table I). In addition, peptide TM2up, corresponding to amino acids
391-398 (LLIASVDS), representing the upper portion of transmembrane 2, was synthesized (Table I). More important, unlike the TM6 peptide, the
TM6(L552P) and TM2up peptides had no stimulatory or inhibitory effects
on either basal (Fig. 4A) or
hormone-stimulated (data not shown) cyclase activity, suggesting a
structural basis for the activity of TM6. In addition to these two
control peptides, randomly scrambled versions of the TM6 peptide, designated TM6-scr1 and TM6-scr2 (Table I), were prepared.
Interestingly, both scrambled peptides were capable of stimulating
basal (Fig. 4B) and hormone-stimulated (data not shown)
cyclase activity, identical to the parent TM6 peptide; however, both
peptides inhibited cyclase activity at much lower concentrations of
peptide.
To determine whether the I3/TM6 and TM6 peptides were interacting
directly with Gs to stimulate cyclase activity, as opposed to nonspecifically affecting adenylyl cyclase, the GTP dependence of
the stimulation by these peptides was examined. Membranes prepared from
293 cells expressing the rLHR were incubated either in the presence of
GTP or in the absence of GTP and the presence of the G protein
inhibitor GDPS. The ability of GDP
S to inhibit the activation of
cyclase activity by a stimulatory dose (30 µM) of peptide
was then examined. As controls, the effects of GDP
S on forskolin-
and hCG-stimulated cyclase activity were also examined. As would be
expected, GDP
S significantly decreased hCG-stimulated cyclase
activity, whereas it had little effect on forskolin-stimulated cyclase
activity (Fig. 5). The results in Fig. 5 show that
GDP
S markedly decreased the stimulatory activities of both the
I3/TM6 and TM6 peptides, suggesting that low concentrations of these peptides act directly on Gs to stimulate cyclase
activity.
To more definitively demonstrate the interaction of rLHR peptides with
Gs, the effects of the I3/TM6, TM6, and TM6(L552P) peptides
were examined in membranes prepared from wild-type versus cyc S49 cells. As shown in Fig.
6A, S49 wild-type membranes demonstrated both
sodium fluoride- and forskolin-stimulated adenylyl cyclase activity. As
expected, S49 cyc
membranes, which lack
G
s (26), showed no detectable basal cyclase activity or
stimulation of cyclase by NaF, but did exhibit forskolin-stimulated
cyclase activity similar to that measured in S49 wild-type membranes
(Fig. 6A). Examination of I3/TM6 peptide activity showed
that low concentrations of I3/TM6 (10-56 µM) produced a
3-fold stimulation of adenylyl cyclase activity in S49 wild-type membranes similar to that observed in 293 membranes (Fig.
6B). Interestingly, this 3-fold stimulation was maintained
in S49 wild-type membranes even at very high concentrations of peptide
I3/TM6, concentrations that inhibited 293 membrane cyclase activity
(cf. Figs. 3 and 6B). Similar to 293 membranes,
low concentrations of peptide TM6 (10-56 µM) produced an
~2-fold increase in cyclase activity in S49 wild-type membranes (Fig.
6B). However, unlike the moderate inhibition of cyclase
activity in 293 membranes by higher concentrations of peptide TM6,
higher concentrations of peptide TM6 produced a further increase in S49
wild-type cyclase activity to ~4-5-fold of basal cyclase activity
(cf. Figs. 3 and 6B). The control peptide
TM6(L552P) had no effect on S49 wild-type cyclase activity (Fig.
6B). The ability of the TM6 and I3/TM6 peptides to stimulate
adenylyl cyclase activity was then examined in S49
cyc
cell membranes. As shown in Fig.
6B, the cyclase activity observed over all concentrations of
both peptides tested was undetectable. The ability of the TM6 and
I3/TM6 peptides to stimulate adenylyl cyclase activity in S49 wild-type
cell membranes, but not in S49 cyc
cell
membranes, clearly demonstrates the requirement for Gs in the stimulatory activity of the peptides upon adenylyl cyclase. As
such, we conclude that the TM6 and I3/TM6 peptides interact directly
with Gs, thereby causing its activation.
This study was undertaken to better define the role of the third
intracellular loop and transmembrane region 6 of the LHR in the
activation of G proteins. Toward this end, peptides corresponding to
various portions of these regions of the rLHR were tested for their
ability to activate adenylyl cyclase activity in membranes prepared
from 293 cells. Since the peptides were also assayed for potential
stimulatory or inhibitory activity in membranes derived from 293 cells
expressing the recombinant rLHR, we chose to use the rLHR sequence as
the basis for the design of these peptides. It should be noted,
however, that there is a very high degree of amino acid sequence
identity between the rat and human LHRs within the third intracellular
loop and transmembrane 6 regions. As such, peptide I3/TM6
(corresponding to the C-terminal portion of the third intracellular
loop and the lower portion of transmembrane 6) is identical to the hLHR
except for two residues within the C-terminal portion of the third
intracellular loop. Consequently, peptide TM6, which corresponds only
to the lower portion of transmembrane 6, is the same for the rLHR and
hLHR. The data presented provide evidence of a direct interaction of
transmembrane region 6 of the LHR with Gs. When present at
concentrations of 10-100 µM, the TM6 peptide
(corresponding to the lower portion of TM6) was able to stimulate both
basal and hCG-stimulated adenylyl cyclase activity. This stimulatory
activity is mediated by Gs, as peptide activity is present
in S49 wild-type membranes, but is absent in S49
cyc membranes, which lack G
s
(26). More important, experiments with control peptides for the TM6
peptide suggest that the activity of the TM6 peptide requires some
specific, disruptable sequence. For example, the equally hydrophobic
peptide TM2up failed to alter cyclase activity, demonstrating that a
string of predominantly hydrophobic amino acids is not sufficient to
activate Gs. In addition, a leucine to proline substitution
in the TM6 peptide resulted in a peptide with no activity, indicating
that the TM6 peptide has a specific structure that is disruptable.
However, clearly the amino acid sequence requirements of the TM6
peptide are not absolute as scrambled TM6 peptides had similar
activating properties as the parent TM6 peptide. Results from
experiments with the scrambled TM6 peptides were not entirely
unexpected since the scrambled TM6 peptides consist almost entirely of
homologous hydrophobic substitutions, and many researchers have shown
that homologous substitutions are often well tolerated (30-32). Taken
all together, the results presented suggest that the TM6 peptide
possesses some specific amino acid sequence and structure necessary for
its activation of Gs. More important, these results are the
first to demonstrate an interaction of a transmembrane region of a G
protein-coupled receptor with a G protein.
In addition to the stimulation of adenylyl cyclase activity by TM6, a
longer peptide, I3/TM6 (corresponding to the C-terminal portion of the
third intracellular loop and the lower portion of transmembrane 6),
stimulated cyclase activity. The effects of the I3/TM6 peptide appear
to be specific since a control peptide, E1/TM2 (corresponding to a
portion of the first extracellular loop and the upper portion of
transmembrane 2), had no effect on cyclase activity. Also, the effects
of the I3/TM6 peptide were independent of the presence of the
full-length rLHR. In addition, the I3/TM6 peptide acted directly on
Gs as activation was present in S49 wild-type membranes,
but was absent in S49 cyc membranes. However,
the I3/TM6 peptide stimulated cyclase activity more effectively than
the TM6 peptide, suggesting that the I3 portion of the peptide may be
responsible for some of the activity of the I3/TM6 peptide and that the
I3 portion may interact with Gs. Surprisingly, examination
of other peptides revealed that neither the I3 peptide (corresponding
to the full third intracellular loop of the rLHR) nor the I3/TM2
peptide (corresponding to the C-terminal portion of the third
intracellular loop followed by a hydrophobic anchor derived from the
upper portion of transmembrane region 2) had any effects on basal or
hormone-stimulated cyclase activity. Based upon the
2-adrenergic receptor paradigm, it is generally assumed
that the C-terminal portion of the third intracellular loop of the LHR
is involved in the activation of Gs. However, the only data
thus far on the LHR to support this hypothesis come from the
observations that some constitutively activating mutations of the hLHR
result from single amino acid substitutions within the C-terminal
portion of the third intracellular loop (14, 17). On the other hand, it
has been shown that substitution of the three lysine residues in the
C-terminal portion of the third intracellular loop of the rLHR (present
also in the hLHR) to alanines had no adverse effects on either basal or
hormone-stimulated cAMP accumulation in cells expressing the mutant
receptor (20). While these results do not necessarily rule out a role
for this region of the LHR in activating Gs, they do
demonstrate that an amphiphilic helical structure of the C-terminal
region of the third intracellular loop is certainly not required for
activation of Gs by the rLHR. Clearly, more studies need to
be performed to address the role of the third intracellular loop of the
LHR in Gs activation. There are therefore several possible
reasons why the I3 and I3/TM2 peptides did not exhibit any stimulatory activity in this study. These include the possibility that the C-terminal portion of the third intracellular loop of the LHR does not
directly interact with Gs (and the constitutively
activating mutations may be causing allosteric changes in transmembrane
6). Alternatively, although the C-terminal portion of the third
intracellular loop of the LHR may interact with Gs,
stimulation of Gs may occur only by the coordinated
interaction of this region with one or more other regions of the
receptor. It may also be that these peptides are interacting with
Gs, but in a nonproductive manner distinct from the
interaction of the LHR with Gs. Finally, one cannot exclude
the possibility that these peptides are simply not assuming the same
conformation as the C-terminal portion of the third intracellular loop
of the full-length LHR.
In addition to the stimulation of cyclase by low concentrations of peptides I3/TM6 and TM6, very high concentrations of both peptides I3/TM6 and TM6 were able to inhibit 293 membrane adenylyl cyclase activity. However, this inhibition was severely reduced or completely absent in S49 wild-type membranes. Whether the inhibitory activity of these peptides in 293 cells is due to interactions of the peptides with Gi or due to nonspecific inhibition of G protein activity remains to be determined. In either case, the results would not be relevant to the overall conclusion that lower (i.e. more physiologically relevant) concentrations of these peptides exert stimulatory effects on Gs that are independent of cell type.
Many investigators have identified regions of G protein-coupled
receptors that possess the ability to activate G proteins. For example,
peptides corresponding to the C-terminal portion of the third
intracellular loop of both the 1- and
2-adrenergic receptors were each capable of stimulating
Gs (7, 8). In addition, a peptide corresponding to the
C-terminal portion of the third intracellular loop of the
m4-acetylcholine receptor was capable of directly
stimulating both Gi and Go (11). Also, a
peptide corresponding to a cytoplasmic region and a portion of the
transmembrane segment of the single transmembrane insulin-like growth
factor/mannose 6-phosphate receptor was capable of activating Gi, but a peptide corresponding to the transmembrane
portion alone had no effect on Gi activity (33). More
important, this report is the first to our knowledge to identify a
peptide corresponding to a transmembrane region of a G protein-coupled
receptor that has the ability to directly activate a G protein. Recent
studies have suggested that transmembrane 6 of the hLHR is involved in interhelical interactions (34). If this suggestion turns out to be
correct, our results suggest that in addition to contributing to
interhelical interactions, the lower portion of transmembrane 6 of the
LHR is also capable of interacting with and activating Gs.
Interestingly, many mutations associated with familial male precocious
puberty occur in this lower portion of transmembrane 6 of the hLHR
(13-17). Our data would further suggest that constitutive activation
of hLHRs observed with transmembrane 6 mutations that cause familial
male precocious puberty may be due to changes in the interaction of the
lower portion of transmembrane 6 with Gs, as opposed to or
in addition to allosteric changes in the conformation of the third
intracellular loop. Recently, Liu et al. (10) demonstrated that the insertion of one to four alanines into the lower portion of
transmembrane 6 of the m2-acetylcholine receptor resulted
in constitutively activated receptors. They proposed that the
activation of this receptor involved the movement of transmembrane 6 toward the cytoplasm (10). This model is consistent with our
identification of the lower portion of transmembrane 6 of the LHR
directly activating Gs. Whether this region of the rLHR
activates Gs while in the membrane or whether it is exposed
to the cytoplasm when hormone binds remains to be determined.
We thank Drs. Mario Ascoli, Daniel McCormick, Nikolai Artemeyev, and Paul Sternweis for helpful discussions and Mario Ascoli for critically reading the manuscript.