Certain Activating Mutations within Helix 6 of the Human Luteinizing Hormone Receptor May Be Explained by Alterations That Allow Transmembrane Regions to Activate Gs
Amy N. Abell,
Daniel J. McCormick and
Deborah L. Segaloff
Department of Physiology and Biophysics (A.N.A., D.L.S.) The
University of Iowa College of Medicine Iowa City, Iowa 52242
Department of Biochemistry and Molecular Biology (D.J.M.)
Mayo Clinic/Mayo Foundation Rochester, Minnesota 55905
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ABSTRACT
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Male-limited gonadotropin-independent precocious
puberty (MPP) is frequently associated with mutations of the human
LH/CG receptor (hLHR) that result in constitutively active hLHRs. Many
such activating mutations have been identified in transmembrane 6 of
the hLHR, with the substitution of Asp-578 being the most frequently
observed mutation. Mutagenesis of a transmembrane helix of a G
protein-coupled receptor can cause local alterations in the
conformation near the mutated residue, allosteric changes elsewhere in
the protein, and/or changes in the interhelical packing of the
receptor. Therefore, while it has been hypothesized that activation of
the receptor by mutations of Asp-578 may arise via alterations in the
interactions of helix 6 with other transmembrane helices and/or by
allosterically altering the conformation of the third intracellular
loop, it has not been possible to ascertain the role of the sixth
transmembrane helix per se in activating Gs in the mutated
full-length receptor. Recently, however, we have shown that a peptide
KMAILIFT, corresponding to the juxtacytoplasmic portion of helix 6 of
the hLHR, is capable of activating Gs. These results suggest that helix
6 itself can directly interact with Gs. Importantly, the KMAILIFT
peptide did not include Asp-578, which lies just C-terminal to this
sequence. We show herein that a peptide extended to include Asp-578
(KMAILIFTDFT) is a poor activator of Gs. However, if the peptide is
synthesized with the aspartate replaced with either a glycine or
tyrosine, substitutions that are found in some patients with MPP, these
peptides have Gs-stimulating activity. Additionally, a transmembrane 6
peptide with the substitution of Ile-575 with leucine, another mutation
found in MPP, mimicked the activating effects of this mutation in the
full-length receptor. The ability of peptides in which Asp-578 or
Ile-575 is substituted to mimic the activating effects of these
mutations in the full-length receptor suggests that the sixth
transmembrane helix represents a site for direct interaction with Gs.
In addition to the stimulatory effects of transmembrane 6 peptides,
peptides corresponding to the juxtacytoplasmic portions of the fourth,
fifth, and seventh helices were also able to stimulate Gs. These
results are consistent with the hypothesis that the transmembrane
helices may form a pocket for interaction with Gs and that constitutive
activation of the hLHR may involve the opening of the pocket formed by
these helices, thus exposing Gs-binding sites on these helices.
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INTRODUCTION
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The LH/CG receptor (LHR) is a member of the superfamily of G
protein-coupled receptors having seven membrane-spanning regions
connected by alternating intracellular and extracellular loops, an
extracellular N terminus, and an intracellular cytoplasmic tail (1).
LHR occupation by either LH or hCG results primarily in the activation
of Gs and increased cAMP production (1, 2). LHR signaling is absolutely
required for testosterone production and masculinization of the male.
Recent studies have identified several mutations of the human (h)LHR
that cause constitutive activation of the hLHR, resulting in
male-limited gonadotropin-independent precocious puberty (MPP) (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14).
The substitution of Asp-578 in helix 6 with glycine is the most
prevalent mutation (3, 4). However, substitution of Asp-578 with
tyrosine, a rarer mutation, causes an earlier presentation of MPP (4).
Both D578G and D578Y mutations cause constitutive activation of the
hLHR (15), resulting in testosterone secretion by Leydig cells in the
context of low prepubertal levels of LH and the premature onset of
puberty in boys.
The mechanism by which mutations produce constitutively active hLHRs is
not understood. A mutation may produce constitutive activation
indirectly through changes in receptor conformation that expose distant
Gs contact sites or directly by altering a site that physically
contacts Gs. Although the sites of the hLHR that contact Gs remain
mostly undetermined, studies of other Gs-coupled receptors have led to
the prediction that the carboxyl-terminal region of the third
intracellular loop of the hLHR will be involved in coupling to Gs
(16, 17, 18). Recent studies of the hLHR have suggested that interhelical
interactions of helix 6 with helix 5 (19) and helix 7 (20) are
important in the ligand-independent activation of the hLHR. Thus, based
on the location of Asp-578 in the middle of helix 6 of the hLHR, it has
been postulated that mutations of Asp-578 produce constitutively active
hLHRs by altering interhelical interactions, thus exposing regions of
the third intracellular loop. An alternative possibility,
i.e. that mutations may alter the direct interaction of
helix 6 with Gs, although not excluded, had not until recently been
addressed (21).
Because mutagenesis of Asp-578 may cause local changes in the
conformation of helix 6 and/or conformational changes elsewhere, it has
not been feasible to address the direct role of helix 6 in Gs
activation using this approach. Therefore, we have turned to an
alternate approach in which peptides corresponding to intracellular or
transmembrane regions of the hLHR have been tested for their ability to
directly activate Gs (21). With synthetic peptides, the direct effect
of regions of a protein can be examined without concerns for
conformational changes induced by mutagenesis of the protein. Several
studies in other systems using synthetic peptides have identified short
stretches of sequence that mimic the function of the corresponding
region of the protein (18, 22, 23, 24, 25, 26). For example, Neubig and co-workers
(25, 27) identified a peptide corresponding to the C-terminal half of
the third intracellular loop of the
2-adrenergic
receptor that stimulates Gi/o. Using peptides corresponding to regions
of G proteins, Hamm and co-workers (28, 29, 30, 31) identified regions of the
visual system G protein transducin (Gt) that interact with rhodopsin,
and regions of Gs that interact with the ß-adrenergic receptor.
Recent studies from our laboratory have demonstrated that the peptide
KMAILIFT, corresponding to the juxtacytoplasmic region of helix 6 (see
Fig. 1
), activates adenylyl cyclase by
stimulating Gs (21). These data clearly point to a role for helix 6 of
the hLHR in activating Gs. Interestingly, this initially described
stimulatory peptide does not contain the aspartate corresponding to
Asp-578, which lies just C-terminal to the KMAILIFT sequence (Fig. 1
).
This observation, coupled with reports of constitutive activation of
the full-length hLHR caused by certain substitutions of Asp-578, led us
to hypothesize that whereas helix 6 can activate Gs, Asp-578 may
normally impede this activation. The following experiments used
synthetic peptides corresponding to wild-type vs. mutated
forms of helix 6 to examine the mechanism by which mutation produces
constitutively active receptors. Additionally, peptides corresponding
to the juxtacytoplasmic portions of the other helices were examined to
identify other hLHR contact sites with Gs.

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Figure 1. Orientation and Proposed Topology of the hLHR
The deduced sequence, orientation, and proposed topology of hLHR (32 )
from which the peptides are derived are illustrated. The shaded
region represents the transmembrane regions
above which are the extracellular regions and
below which are the intracellular regions. The
boundaries for the transmembrane regions are based on the
hydrophobicity plots of the rLHR (2 ). While this manuscript was in
preparation, a molecular model of the hLHR based on the two-dimensional
electron density map of bovine rhodopsin was published (50 ).
Bolded lines indicate the proposed transmembrane
boundaries suggested by this computer model (50 ). The extracellular
domain and the cytoplasmic tail are not shown in this picture.
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RESULTS
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In a previous report, it was demonstrated that a peptide KMAILIFT
corresponding to the juxtacytoplasmic region of transmembrane 6 of the
rat (r)LHR-stimulated basal adenylyl cyclase activity 2-fold in
membranes of untransfected 293 cells (21). This maximal stimulation
elicited by the KMAILIFT peptide is approximately 75% of the maximal
stimulation observed when membranes from 293 cells transfected with the
rLHR are incubated with a maximally stimulatory concentration of hCG
(21). The stimulation of adenylyl cyclase by the KMAILIFT peptide was
further shown to be through the activation of Gs, rather than through a
direct effect on adenylyl cyclase (21). For comparative purposes, the
activity of the KMAILIFT peptide in membranes prepared from
untransfected 293 cells is shown in Fig. 2A
. It can be seen that concentrations of
this peptide up to 100 µM stimulate cyclase activity,
whereas at very high concentrations of peptide the stimulation is
attenuated (Fig. 2A
). Asp-578, the residue most frequently mutated in
the hLHRs of patients with MPP, is located just C-terminal to the
KMAILIFT sequence (3, 4, 8, 15).1 Based on these
observations, we hypothesized that, in addition to the interactions of
Asp-578 with other transmembrane helices (19, 20), Asp-578 may also
directly affect the interaction of helix 6 with Gs.

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Figure 2. Effect of Asp-578 on the Ability of hLHR Helix 6
Peptides to Stimulate Adenylyl Cyclase Activity
Membranes prepared from 293 cells that do not express the hLHR were
incubated for 20 min at 37 C with increasing concentrations of hLHR
peptides and assayed for basal adenylyl cyclase activity as previously
described (21 ). A, Data shown are expressed as a percentage of basal
cyclase activity in the absence of peptide (i.e. 100%
represents no change from basal activity). Basal cyclase activity for
these experiments was 5.54 ± 0.73 pmol/min/mg protein. Data shown
are the mean ± SEM of four experiments. B, Data shown
are expressed as a percentage of basal cyclase activity in the absence
of peptide (i.e. 100% represents no change from basal
activity). Basal cyclase activity for these experiments was 9.86
± 1.00 pmol/min/mg protein. Data shown are the mean ± range of
two experiments.
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To examine potential direct effects of Asp-578 on the coupling of the
hLHR to Gs, a longer peptide C-terminally extended to include the next
three residues of helix 6 was examined (see Fig. 1
and Table 1
). As shown in Fig. 2
and in Table 2
, the KMAILIFTDFT peptide was a poor
stimulator of 293 membrane adenylyl cyclase activity as compared with
the shorter KMAILIFT peptide. These data suggest that the additional
residues, DFT, interfere with the ability of the sequence KMAILIFT to
activate adenylyl cyclase. The KMAILIFTDFT peptide was then used as a
template to prepare peptides in which the aspartate residue was
substituted with either glycine or tyrosine (Table 1
). These peptides
were designed to reflect the substitutions of Asp-578 of the
full-length hLHR associated with constitutive activation of the
receptor as seen in MPP (3, 4, 8). As shown in Fig. 2A
, both
KMAILIFTGFT and
KMAILIFTYFT peptides stimulated adenylyl cyclase
activity to a greater degree than the
KMAILIFTDFT peptide. At high concentrations, the
KMAILIFTGFT and
KMAILIFTYFT peptides were even more efficacious
than the shorter KMAILIFT peptide because, unlike the KMAILIFT peptide,
they did not inhibit cyclase activity at very high concentrations. As
shown in Fig. 2B
, the reduced activity of the
KMAILIFTDFT peptide is not due to the negative
charge of Asp-578. Thus, the KMAILIFTEFT
peptide, in which the aspartate was substituted with glutamate, was
also stimulatory. Interestingly, a peptide in which the aspartate was
substituted with an asparagine (KMAILIFTNFT) was
also active (Fig. 2B
). Together, these data suggest that it is the
addition of the aspartate, not the phenylalanine or threonine, to the
KMAILIFT peptide that reduces the ability of the
KMAILIFTDFT peptide to stimulate adenylyl
cyclase activity. Additionally, these data suggest that substitution of
the aspartate in the KMAILIFTDFT peptide alters
the peptide, allowing it to better stimulate adenylyl cyclase activity.
The increased activity of the helix 6 peptides corresponding to D578G,
D578Y, and D578E mutations correlates well with the reported
constitutive activity induced by these mutations in the full-length
hLHR (Table 1
). However, whereas the helix six peptide corresponding to
the D578N mutation exhibited increased activity, the comparable
substitution in the full-length receptor is without effect (Table 1
).
To confirm that the hLHR helix 6 peptides that stimulate basal adenylyl
cyclase activity in 293 cell membranes are doing so via an activation
of Gs, the ability of the LHR peptides to stimulate adenylyl cyclase
activity was examined in S49 wild-type (S49-wt) cell membranes as
compared with S49 cyc- cell membranes that lack
Gs
(33). Previous studies from our laboratory (21) confirmed that
the S49-wt and S49 cyc- cell membranes behaved
as predicted from initial characterizations of these cells (34, 35).
Thus, S49-wt membranes exhibited basal, NaF-, and forskolin-stimulated
cyclase activity. However, cyc- membranes
exhibited low levels of basal cyclase activity and did not respond to
NaF, but displayed forskolin-stimulated cyclase activity at comparable
levels to that observed in wild-type membranes (21, 33, 35). In each
experiment presented herein utilizing S49-wt and
cyc- cell membranes, basal, NaF-, and
forskolin-stimulated cyclase activities were routinely included as
internal controls. As shown in Fig. 3
, in
S49-wt cell membranes the KMAILIFTDFT peptide
was much less potent than the KMAILIFTGFT and
KMAILIFTYFT peptides. Importantly, even at very
high (300 µM) peptide concentrations,
KMAILIFTDFT, KMAILIFTGFT,
and KMAILIFTYFT peptides caused no detectable
increases in adenylyl cyclase activity in S49
cyc- cell membranes (Fig. 3
), although
comparable levels of forskolin-stimulated adenylyl cyclase activity
were observed in the S49 cyc- cell membranes as
compared with the S49-wt cell membranes, confirming that the levels of
adenylyl cyclase in the two membrane preparations were equivalent (Fig. 3
).

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Figure 3. Effect of hLHR Helix 6 Peptides on S49-wt and S49
cyc- Adenylyl Cyclase Activity
Crude membranes were prepared from S49-wt or S49
cyc- mouse lymphoma cells as previously
described (21 ). S49-wt membranes (open symbols) were
incubated for 20 min at 30 C with increasing concentrations of hLHR
peptides and assayed for basal adenylyl cyclase activity as previously
described (21 ). S49 cyc- membranes
(closed symbols) were incubated for 20 min at 30 C with
300 µM of hLHR peptides and assayed for basal adenylyl
cyclase activity as previously described (21 ). Data shown are a
representative experiment of three such experiments. All points
represent the mean ± range of duplicate determinations. The
control data for this experiment, expressed as the mean ± range
for duplicate determinations of adenylyl cyclase activity in picomoles
per min/mg of protein, were the following: for S49-wt membranes basal,
2.06 ± 0.13; NaF, 36.27 ± 1.21; forskolin, 55.80 ±
1.14; for S49 cyc- membranes basal, 0; NaF,
0; forskolin, 46.87 ± 1.23.
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Although the general effects of LHR peptides on membranes from 293
cells and from S49 cells were similar, there were differences in the
absolute stimulatory activities of the peptides in these two cell
types.2 Table 2
compares the
effects of incubation of 293 and S49-wt membranes with 300
µM LHR peptides on basal cyclase activity. In all cases,
LHR peptides produced a greater percent increase in basal cyclase
activity in S49-wt cell membranes as compared with 293 membranes. For
example, defining basal cyclase activity as 100%, incubation of 293
membranes with 300 µM KMAILIFTYFT
peptide resulted in a cyclase activity of 284.5 ± 10.1%, whereas
incubation of S49-wt membranes with this peptide resulted in a cyclase
activity of 428.8 ± 30.3% (Table 2
). However, the fold
stimulation of cyclase activity by either the
KMAILIFTYFT or
KMAILIFTGFT peptides as compared with the
KMAILIFTDFT peptide was the same for both cell
types (Table 2
), indicating that the relative effects of each peptide
were similar in the two cell types.
As described above, the KMAILIFTDFT peptide
exhibited a reduced ability to activate Gs as compared with the
KMAILIFT, KMAILIFTGFT, and
KMAILIFTYFT peptides. Clearly, the
KMAILIFTDFT peptide binds and activates Gs, as
measurable activity is present in cells that have Gs, but absent in S49
cyc- cells which lack Gs
. The reduced
activity of the KMAILIFTDFT peptide may be due
to the decreased affinity of this peptide for Gs or to a decreased
ability of the peptide to activate Gs. As shown above (see Fig. 2
), the
KMAILIFTDFT peptide stimulated cyclase activity
only at high (300 µM) concentrations of peptide, whereas
low (10 µM) concentrations of the
KMAILIFTYFT peptide were stimulatory. To
determine whether the KMAILIFTDFT peptide binds
to Gs at lower concentrations than are necessary for it to activate Gs
and to further study the association of the
KMAILIFTDFT peptide with Gs, we examined whether
the KMAILIFTDFT peptide could prevent the
ability of the KMAILIFTYFT peptide to activate
adenylyl cyclase. Membranes from 293 cells were incubated with a
minimal stimulatory concentration of the active
KMAILIFTYFT peptide (10 µM) and
increasing concentrations of the KMAILIFTDFT
peptide. As shown in Fig. 4
, stimulation
of adenylyl cyclase activity by the KMAILIFTYFT
peptide was attenuated by coincubation with increasing concentrations
of the KMAILIFTDFT peptide. Although the
inhibition by the KMAILIFTDFT peptide was not
complete at 100 µM KMAILIFTDFT
peptide (Fig. 4
), it was difficult to examine higher concentrations of
the KMAILIFTDFT peptide in this competition
assay. Importantly, inhibition occurred at concentrations of the
KMAILIFTDFT peptide as low as 30
µM. These data suggest that the
KMAILIFTFT peptide may bind to Gs at lower
peptide concentrations, but the KMAILIFTDFT
peptide is a poor activator of Gs, and thus, requires higher
concentrations to activate Gs.

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Figure 4. The hLHR Helix 6 KMAILIFTDFT
Peptide Attenuates the Stimulation of Gs by the Active
KMAILIFTYFT Peptide
Membranes prepared from 293 cells that do not express the LHR were
incubated for 20 min at 37 C with 10 µM
KMAILIFTYFT peptide and increasing
concentrations of KMAILIFTDFT peptide and
assayed for basal adenylyl cyclase activity as previously described
(21 ). Data shown are expressed as a percentage of basal cyclase
activity in the absence of peptide (i.e. 100% represents no
change from basal activity). Data shown are the mean ± range of
two experiments. Basal adenylyl cyclase activity was 8.25 ± 0.03
pmol/min/mg protein. In these experiments the
KMAILIFTYFT peptide alone stimulated adenylyl
cyclase activity 149 ± 20%.
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Since substitution of Asp-578 in the KMAILIFTDFT
peptide to glycine or tyrosine, substitutions that produce
constitutively active full-length LHRs, resulted in peptides with
enhanced abilities to activate Gs, it was important to determine
whether other constitutively activating mutations found in the lower
portion of helix 6 could similarly alter the activity of the
KMAILIFTDFT peptide. To address this question,
three additional peptides were synthesized where a residue in the
KMAILIFTDFT peptide was substituted with one corresponding to that
found in patients with constitutively activated LHRs (Table 1
) (6, 7, 36). As shown in Fig. 5A
, peptides with
either the M571I or T577I substitutions had almost no effect on 293
membrane cyclase activity. However, the peptide with the I575L
substitution stimulated 293 membrane cyclase activity to a greater
extent than the wild-type peptide (Fig. 5A
). Similar activities were
observed in S49-wt membranes (Fig. 5B
), and no activity was seen in S49
cyc- membranes. Therefore, of the three other
activating helix 6 mutations examined, peptides corresponding to the
M571I and T577I substitutions did not show increased activity. However,
the peptide corresponding to the I575L substitution activated Gs to a
greater extent that the helix 6 peptide corresponsing to the wild-type
sequence. The ability of certain helix 6 peptides to exhibit greater Gs
stimulatory activity does not readily correlate with the isoelectric
point (pI) of the peptide or the relative contributions of
polar, nonpolar, acidic, or basic residues to the peptide composition
(Table 1
). For example, whereas the I575L peptide is active and the
M571I peptide is relatively inactive, the general physical properties
of the two peptides are the same.

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Figure 5. Effect of Other hLHR
KMAILIFTDFT Peptide Substitutions on 293 and
S49-wt and cyc- Membrane Adenylyl Cyclase
Activity
A, Membranes prepared from 293 cells that do not express the LHR were
incubated for 20 min at 37 C with increasing concentrations of hLHR
peptides containing substitutions corresponding to other activating
mutations of helix 6 in the full-length hLHR. The sequences of the
peptides are shown in Table 1 . Basal adenylyl cyclase activity was
assayed as previously described (21 ). Data shown are expressed as a
percentage of basal cyclase activity in the absence of peptide
(i.e. 100% represents no change from basal activity). Basal
cyclase activity was 7.06 ± 1.77 pmol/min/mg protein. Data shown
are the mean ± SEM of three experiments. B, Crude
membranes were prepared from wt or cyc- S49
mouse lymphoma cells as previously described (21 ). S49-wt or
cyc- membranes were incubated for 20 min at 30
C with a final concentration of 300 µM of the indicated
hLHR peptides and assayed for basal adenylyl cyclase activity as
previously described (21 ). Data shown are the mean ± range of
duplicate determinations of a representative experiment of two such
experiments. The control data for this experiment, expressed as the
mean ± range for duplicate determinations of adenylyl cyclase
activity in picomoles per min/mg of protein, were the following: for
S49-wt membranes basal, 2.57 ± 0.02; NaF, 32.14 ± 2.18;
forskolin, 54.08 ± 1.23; for S49 cyc-
membranes basal, 0.28 ± 0.08; NaF, 0.22 ± 0.01; forskolin,
50.43 ± 6.47.
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It was important to further determine whether the stimulatory effects
of a hLHR juxtacytoplasmic region on Gs activation were limited to
helix 6, or whether peptides corresponding to the juxtacytoplasmic
portions of the other helices can also stimulate Gs. To address this
question, peptides corresponding to the juxtacytoplasmic regions of all
seven transmembrane helices (TM1, TM2, TM3, TM4, TM5, TM6, and TM7)
were synthesized (Fig. 1
and Table 3
). Of
these peptides, the peptide corresponding to helix 1 was insoluble and
was not examined further. The effects of the other peptides on 293
membrane adenylyl cyclase activity were then examined. TM2 and TM3
peptides had no effect on 293 membrane cyclase activity (Fig. 6
). As shown in Fig. 6
, the TM4, TM5,
TM6, and TM7 peptides had varied effects on 293 membrane adenylyl
cyclase activity. The TM4 peptide stimulated 293 cyclase activity, but
required much higher concentrations as compared with the TM6 peptide
(Fig. 6
). The TM5 and the TM7 peptides both stimulated cyclase activity
at low concentrations but were strongly inhibitory at higher
concentrations (Fig. 6
). The effects of these transmembrane peptides
were also examined in S49-wt membranes. TM2 and TM3 peptides did not
stimulate S49 membrane cyclase activity at any concentration of peptide
tested (Fig. 7A
), consistent with their
lack of cyclase stimulation in 293 membranes. However, at very high
concentrations of TM2, there was a slight inhibition of S49-wt cyclase
activity (Fig. 7A
). As seen in 293 membranes, the TM4 peptide
stimulated S49-wt cyclase activity, but again required much higher
concentrations of peptide (Fig. 7A
). Again, as observed in 293
membranes, low concentrations of the TM5 and TM7 peptides stimulated
S49-wt cyclase activity, whereas high concentrations inhibited cyclase
activity (Fig. 7A
). Figure 7B
further shows that whereas maximally
stimulatory concentrations of each peptide stimulated cyclase activity
in S49-wt membranes, they were without effect in S49
cyc- membranes. These data suggest that other
hLHR transmembrane helices may be involved in direct interactions with
Gs.

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Figure 6. Effect of Peptides Corresponding to the
Juxtacytoplasmic Regions of the Transmembrane Helices of the hLHR on
293 Membrane Adenylyl Cyclase Activity
Membranes prepared from 293 cells that do not express the LHR were
incubated for 20 min at 37 C with increasing concentrations of hLHR
peptides shown in Table 3 and assayed for basal adenylyl cyclase
activity as previously described (21 ). Data shown are expressed as a
percentage of basal cyclase activity in the absence of peptide
(i.e. 100% represents no change from basal activity). A
representative experiment of two such experiments is shown and all
points represent the mean of duplicate determinations. Basal cyclase
activity for these experiments was 4.24 ± 0.21 pmol/min/mg
protein.
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Figure 7. Effect of Peptides Corresponding to the
Juxtacytoplasmic Regions of the Transmembrane Helices of the hLHR
on S49-wt and S49 cyc- Membrane
Adenylyl Cyclase Activity
Crude membranes were prepared from S49-wt or S49
cyc- mouse lymphoma cells as previously
described (21 ). A, S49-wt membranes were incubated for 20 min at 30 C
with increasing concentrations of the indicated hLHR peptides and
assayed for basal adenylyl cyclase activity as previously described
(21 ). A representative experiment of two such experiments is shown, and
all points represent the mean of duplicate determinations. Basal
adenylyl cyclase activity in this experiment was 2.76 ± 0.08
pmol/min/mg protein. B, S49-wt or cyc-
membranes were incubated for 20 min at 30 C with the indicated hLHR
peptides at a concentration that was found to be maximally activating
in S49-wt membranes (TM4, 300 µM; TM5, 30
µM; TM6, 100 µM; TM7, 56 µM)
and assayed for basal adenylyl cyclase activity as previously described
(21 ). Data points represent the mean ± range of duplicate
determinations of a representative experiment of either two or three
experiments. The control data for this experiment, expressed as the
mean ± range for duplicate determinations of adenylyl cyclase
activity in picomoles per min/mg of protein, were the following: for
S49-wt membranes basal, 2.76 ± 0.08; NaF, 34.55 ± 0.13;
forskolin, 52.93 ± 3.27; for S49 cyc-
membranes basal, 0.26 ± 0.04; NaF, 0.24 ± 0.01; forskolin,
51.49 ± 7.55.
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DISCUSSION
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G protein-coupled receptors comprise an extremely large (>1,000)
superfamily of proteins involved in numerous, diverse biological
systems (see Refs. 37, 38 for recent reviews). Much research has
focused on understanding the mechanism of activation of these receptors
with the goal of using this information to target them for potential
drug development. Recently, numerous constitutively active receptors
characterized by agonist-independent activity have been identified (3, 39, 40, 41, 42, 43, 44, 45, 46). Some of these mutations have been identified through
site-directed mutagenesis studies, whereas many others represent
naturally occurring mutations discovered in patients with several
endocrine disorders (see Refs. 3, 31, 41, 46 for examples). The
mechanism by which mutations produce constitutively active receptors
has been difficult to determine. Some mutations are predicted to alter
a direct interaction site of the receptor with the G protein, whereas
other mutations are thought to alter the general conformation of the
receptor through changes in interhelical interactions or allosteric
changes in the conformation of the receptor that expose interaction
sites for the G protein (15, 20, 44).
The overall goal of this study was to examine the mechanisms by which
mutations in the hLHR cause the constitutive activity of the hLHR. In a
previous report, we identified a peptide KMAILIFT corresponding to the
juxtacytoplasmic region of helix 6 of the LHR that stimulates Gs (21).
Located just C-terminal to the KMAILIFT sequence is Asp-578, the
residue most frequently mutated in patients with MPP (3, 4, 8). Based
on the location of Asp-578 and the effect of mutations of Asp-578, we
hypothesized that some mutations of this residue may alter the direct
activation of Gs by helix 6 of the hLHR. To test this hypothesis, a
peptide KMAILIFTDFT with three additional residues including Asp-578
was examined. The Gs-stimulatory activity of the KMAILIFTDFT peptide
was reduced compared with the original KMAILIFT peptide. Substitution
of the aspartate in the wild-type KMAILIFTDFT
peptide with either glycine or tyrosine, corresponding to substitutions
of the full-length hLHR that cause constitutive activation (15),
resulted in peptides with even greater activity than the original
shorter KMAILIFT peptide. These data suggest that it must be the
aspartate in the DFT sequence, not the phenylalanine or threonine, that
is responsible for the decreased activity of the
KMAILIFTDFT peptide as compared with the
original KMAILIFT peptide.
Other investigators have identified peptides that directly stimulate Gs
(17, 47). For example, Munch et al. (47) identified a
peptide corresponding to eight residues from the C-terminal portion of
the third intracellular loop and four residues from helix 6 of the
avian ß-adrenergic receptor that stimulated cyclase activity.
However, this stimulation required 100 µM concentrations
of peptide and produced only a 30% increase in cyclase activity. The
studies presented herein reveal a strong stimulation of cyclase
activity (
75% of that produced by LHR-expressing 293 membranes in
response to a maximally stimulatory concentration of hCG) by helix 6
peptides such as KMAILIFT, KMAILIFTGFT, and
KMAILIFTYFT at peptide concentrations as low as
10 µM.
Further experiments were performed to address whether there was any
specificity for amino acids with a given property to confer
Gs-stimulatory activity in D578-substituted hLHR helix 6 peptides. The
data presented suggest that the decreased activity of the
KMAILIFTDFT peptide is not simply due to the
negative charge of the aspartate, as substitution of the aspartate with
a negatively charged glutamate resulted in a peptide with strong
stimulatory activity. These results are consistent with a recent report
showing that substitution of Asp-578 with glutamate in the full-length
hLHR resulted in a receptor displaying constitutive activation (15).
Therefore, the increased activity of the helix 6 peptides with D578G,
D578Y, or D578E substitutions correlates well with the reported
constitutive activity of the full-length hLHR harboring these
substitutions (15). However, there is a discrepancy between the helix 6
D578N peptide, which displays increased activity, and the D578N
substitution of the full-length hLHR, which does not cause constitutive
activity (15).
One possible explanation for the effects of the aspartate on the
KMAILIFTDFT peptide is that this peptide is able
to bind to Gs, but is a poor activator of Gs. This explanation is
supported by experiments in which increasing concentrations of the
KMAILIFTDFT peptide inhibited the stimulation of
cyclase activity by the KMAILIFTYFT peptide.
These experiments suggest that the KMAILIFTDFT
peptide may compete with the KMAILIFTYFT peptide
for the interaction with Gs. In a previous study, Wade et
al. (24) demonstrated the ability of some
2-adrenergic peptides to dimerize, with dimerized
peptides possessing even greater potency than the monomeric peptides.
The possibility that the KMAILIFTDFT peptide may
form heterodimers with the KMAILIFTYFT peptide,
and in doing so inhibit the association of the
KMAILIFTYFT peptide with Gs, cannot be excluded
by our experiments. We performed additional experiments to examine
whether the KMAILIFTDFT peptide could inhibit
hCG-stimulated adenylyl cyclase activity in membranes from 293 cells
expressing the LHR. Inhibition of hCG-stimulated cyclase was observed
but only at relatively high concentrations of peptide (600
µM), and under these conditions forskolin-stimulated
cyclase was inhibited as well. Therefore, we were precluded from making
any meaningful conclusions from these experiments.
Taken altogether, the data showing the greater activity of the shorter
KMAILIFT helix 6 peptide, as compared with the longer KMAILIFTDFT
peptide containing Asp-578, and the data on the D578-substituted
peptides suggest that the juxtacytoplasmic region of helix 6 has the
potential to directly interact with and activate Gs, but that Asp-578
normally constrains this activity. The data further suggest that one
mechanism by which mutations of Asp-578 cause constitutive activation
of the full-length hLHR may be by relieving the constraint imposed by
Asp-578 in the Gs-activating properties of helix 6. This hypothesis
does not preclude other additional mechanisms, such as disruption of
interhelical interactions, that may also be involved in the
constitutive activation of the hLHR by mutation of Asp-578. However,
our data do suggest a hitherto unreported potential role of helix 6 in
the activation of Gs per se. It should be pointed out that a
recent mutagenesis study on the related TSH receptor also
suggests a role for helix 6 of the TSH receptor in activating Gs (48, 49).
Mutations of several residues within helix 6 of the hLHR have been
reported to cause constitutive activation. Therefore, we also examined
the properties of hLHR helix 6 peptides containing either M571I, I575L,
or T577I substitutions. Of these three peptides, only one (I575L)
showed increased activity. Thus, in addition to the correlation of the
activating properties of D578G, D578Y, and D578E peptides with
the corresponding activating mutations of the full-length hLHR, there
is a correlation between peptides and the full-length receptor with an
additional mutation located elsewhere in helix 6 (I575L). However,
there is a lack of correlation between the helix 6 M571I and T577I
peptides and the corresponding mutations in the full-length hLHR. One
possible hypothesis to explain this apparent discrepancy is that in the
context of the full-length receptor, M571 and T577 may not be
constraining the Gs-stimulatory activity of helix 6. Rather, they may
be inhibiting the Gs-stimulatory activity of the receptor via holding
it in a particular conformation through interhelical interactions. This
is, however, speculative and awaits further investigation.
Furthermore, there does not appear to be a correlation between pI or
percent composition of polar, nonpolar, acidic, and basic residues
among the active or inactive helix 6 peptides (Table 1
). Clearly, the
active peptides do not share similar physical properties (as defined by
these broad categories). It is possible, however, that the secondary
structure adapted by the active peptides is distinct from that of the
inactive peptides, and studies are underway to address this
question.
To examine the potential role of other hLHR transmembrane regions
in interactions with Gs, the effects of the juxtacytoplasmic portions
of the other helices were examined. The precise location of the
transmembrane boundaries of the hLHR are unknown. Because of the strong
homology between the rat and human LHRs, transmembrane peptides were
synthesized based on hydropathy plots of the rLHR (2). While this
manuscript was in preparation, a computer model of the hLHR based on
the two-dimensional electron density map of bovine rhodopsin was
published (50). The transmembrane boundaries suggested by this model
differ somewhat from the boundaries used for our transmembrane peptide
studies (Fig. 1
) (50). Importantly, in both models, the LHR peptides we
used were located within the proposed transmembrane regions (Fig. 1
and
Table 3
). Peptides corresponding to the juxtacytoplasmic portions of
helices 4, 5, 6, and 7 possessed the ability to stimulate Gs to various
extents. As summarized in Table 3
, there does not appear to be a
correlation between the gross physical properties of activating
transmembrane peptides vs. inactive transmembrane peptides.
The stimulation of Gs by peptides corresponding to helices 4, 5, 6, and
7 is consistent with the hypothesis that multiple transmembrane regions
of the hLHR may form a binding pocket for interaction with Gs.
Studies with other G protein-coupled receptors support the general
model that activation of the receptors is associated with increased
movement of the helices (reviewed in Ref. 38). This, in turn, is
thought to open up the cytoplasmic cleft exposing specific sites for G
protein interaction and activation (51, 52, 53). Data presented herein is
consistent with the cleft of the hLHR forming a binding pocket for Gs.
Our data further suggest that the pocket formed by the helices of the
hLHR for the binding and activation of Gs may be deeper than that
previously suggested for other receptors.
 |
MATERIALS AND METHODS
|
---|
Supplies
Highly purified hCG (CR-127) was provided by the National
Hormone and Pituitary Agency of the NIDDK/NIH. Alumina WN3 and reagents
for adenylyl cyclase assays were obtained from Sigma (St. Louis, MO).
AG50W-X4 resin was obtained from Bio-Rad (Richmond, CA), and
[
32P]ATP was purchased from New England Nuclear
(Boston, MA). Tissue culture reagents and plasticware were purchased
from Life Technologies, Inc. (Grand Island, NY and Corning, NY,
respectively).
Peptides
LHR peptides were synthesized as C-terminal amides and were
purified by reverse-phase HPLC to >95% purity by the Mayo Protein
Core Facility, Mayo Foundation (Rochester, MN). Peptides were initially
dissolved in dimethylsulfoxide and diluted further with water to
yield a 10% dimethylsulfoxide stock solution. The physical properties
of each of the peptides were calculated using the computer program
MacVector.
Cells
Human embryonic kidney 293 cells were obtained from American
Type Culture Collection (ATCC CRL 1573; Rockville, MD). The clonal cell
line rLHR-wt-16 expressing approximately 9,000 rLHRs per cell was
kindly donated by Mario Ascoli (University of Iowa). S49-wt and S49
cyc- mouse lymphoma cell lines were obtained
from Dr. Henry Bourne (University of California, San Francisco). S49
cyc- cells, which lack G
s (33),
are a clonal derivative of S49-wt cells (35). All cell lines were
maintained as described previously (21).
Preparation of Membranes
293 membranes were prepared as described previously (21). S49-wt
and cyc- membranes were prepared as described
by Ross et al. (54).
Adenylyl Cyclase Activity Assay
Adenylyl cyclase activity assays were performed as described
previously (21). Briefly, for 293 membranes, 10 µg of membrane in 10
µl of membrane buffer (125 mM Tris-Cl, 5 mM
EDTA, pH 7.4) were preincubated with increasing concentrations of
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% BSA, pH 7.4, or buffer only; 10
µl of 100 µM GTP; and 10 µl of reaction mixture
containing 0.5 mM ATP, 20 mM MgCl2,
5 mM cAMP, 100 mM phosphocreatine, 200 U/ml
creatine kinase, 200 U/ml myokinase, 3 µCi/ml [3H]cAMP,
and 140 µCi/ml [
-32P]ATP. These components were
incubated 20 min at 37 C, after which reactions were terminated by the
addition of stop solution (40 mM ATP, 10 mM
cAMP, 1% SDS) and boiling for 3 min. [32P]cAMP was
isolated and counted as previously described (21). For S49 membranes,
cyclase assays were peformed essentially as described above for 293
membranes with the following modifications: 10 µg of S49-wt or
cyc- membranes in HME buffer (20 mM
HEPES, 2 mM MgCl2, 1 mM EDTA, pH
8.0) were preincubated with increasing concentrations of LHR 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 the reaction mixture contained 50 mM
MgCl2 instead of 20 mM MgCl2. These
components were incubated for 20 min at 30 C with shaking, after which
reactions were terminated; [32P]cAMP was isolated and
counted as described above. For each experiment in which
peptide-stimulated cyclase activity in S49-wt and
cyc- membranes was compared, basal, NaF-, and
forskolin-stimulated cyclase activities were determined to verify the
absence or presence of Gs
and the presence of comparable levels of
functional adenylyl cyclase. For each adenylyl cyclase assay,
background [32P]cAMP formation (0.95 ± 0.10) was
measured and subtracted from each sample.
It should also be pointed out that the conditions for assaying
cyclase activity differ in incubation temperature (37 C vs.
30 C), in the concentrations of GTP (20 µM vs.
50 µM), and in the concentrations of MgCl2 (4
mM vs. 10 mM) for 293 and S49
membranes, respectively. Because the isoforms of adenylyl cyclase in
S49 membranes are rapidly inactivated by heating (55), it is
technically not possible to examine S49 membrane cyclase activity at
the same incubation temperature used to examine 293 membrane cyclase
activity. Additionally, measurement of cyclase activity in S49
membranes requires higher concentrations of GTP and MgCl2
as compared with those for 293 membranes (55). Our measurements of
cyclase activity in 293 membranes under conditions used for S49
membranes resulted in a 5 to 10-fold elevation of basal cyclase
activity. This increase in basal cyclase activity of 293 membranes
incubated with higher concentrations of MgCl2 and GTP is
consistent with previous reports of activation of Gs by higher
concentrations of MgCl2 and/or GTP in 293 membranes (56)
and other cell types (57). Membranes from 293 cells assayed under S49
adenylyl cyclase assay conditions were stimulated by LHR peptides.
However, the peptide-stimulated activity was reduced by 4050% as
compared with that observed using 293 assay conditions, demonstrating
the decreased sensitivity of the 293 membranes under the conditions
used for S49 membranes.
 |
ACKNOWLEDGMENTS
|
---|
We thank Drs. Mario Ascoli and Nikolai Artemyev for helpful
discussions and Dr. Ascoli for critically reading the manuscript. We
also gratefully acknowledge Harinder Kaur for her technical support.
All peptides were synthesized and purified by the Mayo Protein Core
Facility, Mayo Clinic/Mayo Foundation.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Deborah L. Segaloff, Ph.D., Department of Physiology and Biophysics, The University of Iowa College of Medicine, Iowa City, Iowa 52242. E-mail:
deborah-segaloff{at}uiowa.edu
These studies were supported by NIH Grant HD-22196 (to D.L.S.). During
the course of these studies D.L.S. was a recipient of NIH Research
Career Development Award HD-00968. The services and facilities provided
by the University of Iowa Diabetes and Endocrinology Research Center
Grant DK-25295 are gratefully acknowledged.
1 Although our previous study examined the
coupling of the rLHR to Gs, the amino acid sequence of helix 6 is
identical between the rat and the human LHRs (2 32 ). This complete
identity allows us to extend our conclusions from the previous study of
the rLHR on the role of helix 6 in the direct activation of Gs to the
hLHR. 
2 There are both biological and technical reasons
that could account for the differences in cyclase activity between the
293 and S49-wt cell membranes. Thus, the two cell types may have
differential expression of long and short forms of
s, different
concentrations and/or isoforms of adenylyl cyclase, or may vary in the
degree of compartmentalization of signaling molecules. Furthermore, as
explained in Materials and Methods, the two cell types
require different conditions for the assaying of adenylyl cyclase
activity. 
Received for publication March 11, 1998.
Revision received August 4, 1998.
Accepted for publication August 28, 1998.
 |
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