Role of the Third Intracellular Loop for the Activation of Gonadotropin Receptors
Angela Schulz,
Torsten Schöneberg,
Ralf Paschke,
Günter Schultz and
Thomas Gudermann
Institut für Pharmakologie (A.S., T.S., G.S., T.G.) Freie
Universität Berlin 14195 Berlin, Germany
Medizinische Klinik und Poliklinik III (R.P.)
Universität Leipzig 04103 Leipzig, Germany
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ABSTRACT
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Hyperfunctional endocrine thyroid and testicular
disorders can frequently be traced back to gain-of-function mutations
in glycoprotein hormone receptor genes. Deletion mutations in the third
intracellular (i3) loop of the TSH receptor have recently been
identified as a cause of constitutive receptor activity. To examine
whether the underlying mechanism of receptor activation applies to all
glycoprotein hormone receptors, we created deletion mutations in the LH
and FSH receptors. In analogy to the situation with the TSH receptor, a
deletion of nine amino acids resulted in constitutive activity
irrespective of the location of deletions within the i3 loop of the LH
receptor. In contrast, only one (
563566) of four different 4-amino
acid deletion mutants displayed agonist-independent activity.
Systematic examination of the structural requirements for this effect
in the
563566 mutant revealed that only deletions including D564
resulted in constitutive receptor activity. Replacement of D564 by G,
K, and N led to agonist-independent cAMP formation while introduction
of a negatively charged E silenced constitutive receptor activity,
indicating that an anionic amino acid at this position may be required
to maintain an inactive receptor conformation. Insertion of A residues
up- and downstream of D564 did not perturb receptor quiescence, showing
that a certain degree of spatial freedom of the negatively charged
amino acid within the context of the i3 loop is well tolerated. In
contrast to the results obtained with the LH receptor, deletion of the
corresponding D567 from the i3 loop of the FSH receptor did not cause
constitutive receptor activation, highlighting significant differences
in the activation mechanism of gonadotropin receptors.
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INTRODUCTION
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According to the allosteric ternary complex model,
G-protein-coupled receptors (GPCRs) exist in an equilibrium between
inactive and active receptor conformations (1, 2). The model predicts
that, even in the absence of agonist, a certain fraction of receptors
will spontaneously adopt an active conformation, permitting
agonist-independent G-protein activation. In keeping with this current
paradigm, some receptors such as D1B dopamine and TSH
receptors display significantly elevated basal activity even in the
unliganded state when compared with other GPCRs (3, 4). To describe
mutated receptors characterized by a shift of the isomerization
equilibrium toward active conformations, the term constitutive activity
has been coined. By definition, such receptors evoke clearly
discernible second messenger production in the absence of agonist.
Mutational activation of GPCRs has been identified as a
pathophysiological mechanism of human diseases such as retinitis
pigmentosa (5), familial male-limited precocious puberty (6), and
autonomous toxic thyroid adenomas (7). To date, a fair number of
gain-of-function mutations in the receptors for the glycoprotein
hormones TSH and LH have been identified (8, 9), providing valuable
information on structural requirements of receptor quiescence.
Therefore, the subfamily of glycoprotein hormone receptors may serve as
a particularly suitable model system to study conformational changes
accompanying receptor activation.
The structure of glycoprotein hormone receptors is predicted to consist
of a large extracellular hormone-binding domain connected to a
transmembrane (TM) core that shares a common molecular architecture
with other GPCRs. The TM core is composed of seven
-helical TM
domains (TM17) connected by three extracellular and three
intracellular loops (10). Most missense mutations leading to
constitutively active TSH or LH receptors (TSHRs or LHRs) are located
in the C-terminal three TMs. It is hypothesized that interhelical
interactions between TM5/TM6 and TM6/TM7 stabilize the inactive state
of glycoprotein hormone receptors and that gain-of-function mutations
have a destabilizing impact on these contacts (11).
We have recently shown that the deletion of amino acid residues
from the third intracellular (i3) loop of the TSHR results in
agonist-independent receptor activation (12). This constitutive
activity is independent of the exact position of the deleted amino
acids, yet is influenced by the length of the deleted region. Since
previously identified gain-of-function mutations in intracellular
receptor portions were point mutations, our finding highlights a new
mechanism of TSHR activation. Primary sequence comparison of
glycoprotein hormone receptors reveals an overall amino acid identity
of approximately 50% and of more than 70% among putative TM helices
(13). The remaining structural differences most likely impinge upon
receptor activation and signal transduction properties that display
characteristic differences between members of the glycoprotein hormone
receptor family (4, 13, 14). Our previous observation, that deletions
within the i3 loop constitutively activate the TSHR, prompted us to
study whether such an activation mechanism would be a general
phenomenon and apply to all glycoprotein hormone receptors. Thus, we
systematically studied the importance of the i3 loop for the activation
of the LHR and FSH receptor (FSHR). We demonstrate in the present study
that similar to the TSHR, large deletion mutations in the i3 loop of
the LHR result in constitutive activation. In contrast to findings
obtained with the TSHR, however, deletions of five (
558562) or
four amino acids (
563566) resulted in a similar
agonist-independent receptor activity as noted for the 9-amino acid
(
558566) deletion mutant. To analyze the structural requirements
for constitutive receptor activation, various LHR deletion mutants were
tested. Interestingly, an active conformation was only achieved if a
conserved D residue at position 564 was included in the deleted region.
Further analysis of the functional role of D564 revealed that the
presence of a negatively charged amino acid at this position is
required to stabilize an inactive conformation. Similar mutational
studies with the FSHR did not allow the design of a receptor with
comparable constitutive activity, thus suggesting a more constrained
inactive conformation and an activation mechanism clearly set apart
from the other two glycoprotein hormone receptors.
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RESULTS
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Functional Characterization of LHR Deletion Mutants
Using a site-directed mutagenesis approach we systematically
introduced deletion mutations varying in length and location into the
i3 loop of the LHR. Functional analysis of a 9-amino acid deletion
(
558566) equivalent to a deletion mutation found in the i3 loop of
the TSHR in a toxic thyroid nodule (15) resulted in a 2.6-fold
elevation of basal cAMP levels when compared with the wild-type LHR
[LHR(wt)]. Concomitantly with constitutive receptor activity, we
observed a reduced maximal cAMP response [
40% of the response seen
with the LHR(wt)] to stimulation with 100 nM human CG
(hCG) (Table 1
).
This observation may relate to the significantly reduced membranous
expression of LHR deletion mutants (see Table 1
). Constitutive receptor
activation was also observed after shifting the 9-amino acid deletion
toward the N-terminal loop portion (
554562), demonstrating that a
9-amino acid deletion does not have to be exactly positioned at a
certain location within the i3 loop to cause constitutive receptor
activation (see Table 1
). Since both LHR deletion mutants displayed
functional properties similar to the corresponding TSHR mutants (12),
we set out to study in depth the importance of distinct regions of the
i3 loop for maintaining an inactive state of the LHR.
The
558566 deletion spans a stretch of amino acids that can be
subdivided into a C-terminal portion highly conserved among
glycoprotein hormone receptors and a less conserved N-terminal part
(see Fig. 1
). In the absence of the less
conserved 5 (
558562) or of the 4 conserved amino acids
(
563566), the mutant receptors displayed comparable constitutive
activity. Maximal hCG-induced cAMP levels amounted to 55% of the
LHR(wt) control (see Table 1
). In contrast to previous findings with
the TSHR, the extent of constitutive receptor activation was equivalent
to the one observed with the 9-amino acid deletion mutant
(
558562). Next we asked whether a 4-amino acid deletion per
se or the exact location of this deletion within the i3 loop would
account for constitutive activity. Thus, we generated three additional
4-amino acid deletion mutants located in the very N-terminal
(
550553,
554557) and C-terminal (
567570) portions of the
i3 loop. None of these mutant LHRs displayed elevated basal cAMP
levels. Maximal hCG-induced cAMP levels, however, were markedly
reduced. In accord with the latter findings, all four-amino acid
deletion mutants showed decreased membranous expression levels in
binding studies (see Table 1
). Whereas membrane expression of most
4-amino acid deletion mutants amounted to approximately 16% of wt
receptor levels, expression of the deletion mutant
567570 located
at the i3/TM6 transition was hardly detectable (see Table 1
).

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Figure 1. Localization of LHR Mutations within the i3 Loop
Putative arrangement of the LHR within the lipid bilayer highlighting
amino acid sequences of the third intracellular loop of the wt human
LHR and the mutants constructed. Deleted amino acids are indicated by
dashes; amino acid substitutions are highlighted in
italics, and amino acid residues that are conserved within
the group of glycoprotein hormone receptors are shown in
boldface. Acronyms for each mutation are indicated on
the left.
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The results obtained so far indicated that the amino acid cluster
563566 contains determinants responsible for maintaining the inactive
receptor conformation. Subsequently, we attempted to identify relevant
structural elements within this portion by further dissecting the
563566 portion of the i3 loop, deleting only 2 and then single amino
acids. Membrane insertion of the 2-amino acid deletion mutants
563564 and
565566 was 45.9 and 27.6% of wt levels,
respectively, and hCG stimulation yielded similar maximal cAMP levels
(see Table 1
). However, only the mutant LHR-
563564 displayed
constitutive activity (see Table 1
). Functional analysis of the
single-amino acid deletion mutants LHR-
563 and -
564 proved that
the absence of a D at position 564 is responsible for the constitutive
activity (see Table 1
).
To test whether the agonist hCG elicited cAMP formation in cells
expressing the LHR(wt) or various constitutively active mutants
with different potencies, concentration-response studies were
performed. As shown in Fig. 2
, EC50 values for the LHRs that were investigated differed at
most by a factor of 2.6 [LHR(wt): 0.31 nM;
558566:
0.44 nM;
563566: 0.41 nM;
563564:
0.87 nM;
564: 0.79 nM].

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Figure 2. Concentration-Dependent hCG-Stimulated cAMP
Accumulation in COS-7 Cells Expressing the wt and Constitutively Active
Mutant LHRs
cAMP accumulation assays were performed in COS-7 cells transiently
transfected with wt and mutant LHR plasmids. Cells were incubated with
increasing concentrations of hCG, and increases in cAMP levels were
determined as described in Materials and Methods. All
data are presented as means (-fold of basal cAMP levels of the wt LHR)
of two independent experiments each performed in duplicate.
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Mutational Characterization of Amino Acid Position 564
To further characterize the importance of D564 for receptor
quiescence, we replaced D564 by an E residue. Expression of LHR-D564E
did not reveal signs of constitutive receptor activity, but rather led
to a reduced basal cAMP formation when compared with the LHR(wt) (see
Table 1
). Next, we replaced the free carboxyl moiety of D564 by the
corresponding carboxyl amide by changing D to N (D564N). In contrast to
the results obtained with the D564E mutant, LHR-D564N showed
ligand-independent cAMP accumulation. Introduction of a noncharged G
and a positively charged K at position 564 conferred constitutive
activity upon the mutated receptor (see Table 1
). Maximal
hCG-stimulatable cAMP formation, as well as membranous receptor
expression of the latter three missense mutants, closely resembled the
situation encountered in the case of wt receptor expression (see Table 1
).
Next, we speculated that the immediate conformational environment
within the i3 loop of position D564 may have a bearing on the activity
state of the receptor. To address this issue, we inserted three
additional A residues upstream (insA562) or downstream (insA564) of
D564. None of these mutations entailed significant changes in basal or
in agonist-induced intracellular cAMP levels upon functional expression
of the respective receptors (see Table 1
).
Screening for a Basic Amino Acid in the i2 Loop That Interacts with
D564
In a first attempt to identify a positively charged amino
acid potentially involved in a salt bridge with D564, all conserved
basic amino acids in the second intracellular (i2) loop were replaced
by A residues, and the resulting mutants were tested for constitutive
activity (Fig. 3A
). None of the tested
LHR mutants imparted constitutively elevated basal cAMP levels on the
transfected cells. All i2 mutants were functionally expressed at the
cell surface as illustrated by maximal agonist-dependent cAMP formation
that resembled that observed with the LHR(wt) (Fig. 3B
).

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Figure 3. Functional Analysis of Conserved Basic Amino Acids
within the LHR i2 Loop by Alanine Scanning Mutagenesis
To identify a cationic contact site for D564, all conserved basic amino
acids within the i2 loop were replaced by A using a site-directed
mutagenesis approach (A). The various LHR mutants were expressed in
COS-7 cells, and cAMP accumulation assays were performed as described
in Materials and Methods (B). Basal (open
bars) and agonist-induced cAMP levels (100 nM hCG;
filled bars) are presented as means ±
SEM of two independent experiments, each carried out in
triplicate.
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hCG-Induced Phospholipase C Activation
To study the influence of constitutively activating mutations on
the phospholipase C effector system (13, 16), we examined all LHR
mutants for their ability to generate inositol phosphates (IPs).
Expression of the LHR(wt) consistently led to a 3- to 4-fold increase
in IP levels after stimulation with 100 nM hCG (see Table 1
). Most of the LHR mutants (
558566,
563566,
550553,
554557, and
567570) that were expressed at considerably lower
receptor densities than the wt receptor did not respond to agonist
challenge with significant IP formation. It should be noted, however,
that none of the mutant LHRs showed constitutive activity toward the
IP-signaling pathway. Surprisingly, expression of several receptor
mutants that constitutively activated the Gs/adenylyl
cyclase pathway (
564, D564G, D564K, and D564N) was accompanied by a
profound enhancement of maximal hCG-induced IP accumulation. However,
we had to abandon our initial suspicion of a causal relationship
between ligand-independent cAMP production and a more efficient
coupling to the phospholipase C system, because expression of
additional mutant receptors (insA562 and insA564) not constitutively
activating Gs also entailed a marked increase of maximal
agonist-induced IP levels when compared with the wt receptor (Fig. 4
and Table 1
). These observations
reflect an increased efficacy of the agonist hCG acting at the mutant
receptors. On the contrary, the EC50 values of the wt and
mutant LHRs [LHR(wt): 7.0 nM;
564: 11.2 nM;
D564E: 7.2 nM; insA562: 7.4 nM; insA564: 7.2
nM] showed that there was no significant change in the
potency of hCG to stimulate phospholipase C via various LHR mutants
(see Fig. 4
).

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Figure 4. hCG-Stimulated IP Accumulation in COS-7 Cells
Expressing the wt and Selected Mutant LHRs
Determination of IP levels was performed in COS-7 cells, transiently
transfected with wt and mutant LHR plasmids. Cells were incubated with
increasing hCG concentrations, and increases in IP levels were
determined as described in Materials and Methods. All
data are presented as means (-fold of basal) of two independent
experiments each performed in duplicate.
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Expression and Characterization of FSHR i3 Loop Deletion
Mutants
Since deletions within the i3 loop of two glycoprotein hormone
receptors, i.e. TSHR and LHR, can result in constitutive
activity, we sought to investigate whether such a receptor activation
mechanism also applies to the third member of this receptor subfamily,
the FSHR (Fig. 5A
). Expression of
FSHR-
561569 did not confer elevated basal cAMP levels to
transfected COS-7 cells (Table 2
and Fig. 5B
). The mutant receptor
showed a poor membranous expression of [125I]FSH binding
sites and responded only weakly to porcine (p) FSH (800
nM). Subsequently, we omitted only the C-terminal 3 amino
acids (to preserve the four-S stretch in the FSHR i3 loop) from the
conserved portion of the original 9-amino acid deletion to generate
FSHR-
567569 (see Fig. 5A
). In addition, we deleted D567
corresponding to D564 in the LHR (see Fig. 5A
). Whereas
FSHR-
567569 showed a drastically reduced membranous expression of
binding-competent receptors and poorly responded to FSH challenge (see
Table 2
and Fig. 5B
), deletion of D567
resulted in a mutant receptor that did not constitutively couple the
mutant FSHR to the Gs/adenylyl cyclase system.
Subsequently, we replaced D567 in the i3 loop by a G residue to
generate FSHR-D567G, which has been reported to epitomize the only
constitutively active FSHR known so far (17). If anything, expression
of FSHR-D567G showed the tendency to slightly increase basal cAMP
production, although this effect was not significant (see Table 2
).
Taking into account, however, that membrane expression of
binding-competent receptors after transfection of COS-7 cells with
FSHR-D567G cDNA is reduced by 60% when compared with FSHR(wt) (see
Table 2
), we cannot exclude with certainty that functional
characteristics of the latter mutant may indeed mirror some degree of
constitutive activity. Upon FSHR(wt) expression in COS-7 cells, we did
not observe FSH-stimulatable phosphoinositide breakdown. Therefore,
this signaling pathway was not analyzed further for the various FSHR
mutants.

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Figure 5. Localization of FSHR Mutations and Functional
Characterization of Mutant Receptors
To characterize mutant FSHRs (A), COS-7 cells were transfected with the
different constructs, and cAMP accumulation assays were performed as
outlined in Materials and Methods. (B) Data obtained
from three independent experiments, each performed in triplicate, are
presented as -fold of basal cAMP levels of the FSHR(wt) (means ±
SEM).
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DISCUSSION
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We recently identified deletion mutations within the i3 loop of
the TSHR resulting in marked constitutive receptor activation (12). As
the number of deleted amino acids, rather than their exact position
within the i3 loop, is the major determinant of constitutive TSHR
activation and because 75% of the i3 loop could be deleted without
rendering the receptor unresponsive to agonist, we speculated that the
deletion per se led to constitutive receptor activity by
allowing critical amino acid residues within TM6 to productively
interact with Gs (12, 18). To investigate whether this
activation mechanism applied to all glycoprotein hormone receptors, we
functionally characterized i3 loop deletion mutants of the human
LHR and FSHR.
Functional analysis of an LHR 9-amino acid deletion (
558566)
corresponding to the one originally described in the TSHR (12), as well
as of an N-terminally shifted deletion (
554562), revealed
pronounced agonist-independent cAMP formation, thereby supporting a
mechanism of receptor activation initially worked out for the TSHR
(12). Sequence comparison within the family of glycoprotein hormone
receptors reveals that the 9-amino acid deletion (
558566) can be
subdivided into a highly conserved C-terminal and a less conserved
N-terminal portion. In contrast to previous findings with the TSHR,
deletion of the nonconserved (
558562) or conserved (
563566)
stretch of amino acids from the LHR i3 loop did not result in a
reduction of constitutive activity when compared with the original
558566 mutant. These initial findings prompted us to hypothesize
that, in the LHR, deletion of 4 amino acid residues may already be
sufficient for maximal constitutive receptor activity. However,
expression of only one 4-amino acid deletion mutant (LHR-
563566)
of 4 (
550553,
554557,
567570) was accompanied by
constitutive activation of the Gs/adenylyl cyclase system.
This indicates that a loss of distinct amino acids is responsible for
constitutive activity. Subdividing the
563566 mutation into 2- and
single-amino acid deletions finally revealed that the loss of D564
resulted in agonist-independent cAMP formation comparable to that
observed with the original
558566 mutant. A point mutation at
amino acid position 564 (D564G) was first identified in a patient with
familial male-limited precocious puberty (19). The corresponding
mutation (D619G) in the TSHR causing a toxic thyroid nodule (20) in
conjunction with the marked constitutive activity elicited by deleting
D619 (12) underline the importance of this conserved D within the i3
loop for maintaining an inactive receptor conformation. Replacement of
D564 by E (D564E) extended the carboxylate side chain of D present in
the LHR(wt) by one methylene group while preserving the negative
charge. This conservative substitution did not lead to constitutive
receptor activity. However, introduction of a positively charged amino
acid (D564K) and conversion of D564 to N, a similarly sized but
uncharged residue that retains the ability to serve as a hydrogen bond
acceptor, resulted in constitutive activity. These data indicate that a
negative charge at position 564 in the LHRs i3 loop may be necessary
to keep the receptor in the inactive conformation. Thus, one may
hypothesize that the negatively charged D564 stabilizes a positively
charged amino acid residue within the receptor via a salt bridge
constraint. While this manuscript was being reviewed, Kosugi and
colleagues (21) also stressed the importance of D564 for the
stabilization of an inactive state of the LHR.
To assess the importance of the conformational environment of D564
within the i3 loop for maintaining the inactive receptor conformation,
we inserted three additional A residues up- and downstream of position
564, generating LHR-insA562 and -insA564. The relative shift of the
conserved D within the i3 loop did not result in constitutive receptor
activity. Our results show that while a negatively charged amino acid
residue in the C-terminal part of the i3 loop is required for receptor
quiescence, there is some degree of spatial freedom.
As the deletion of D619 from the TSHR i3 loop, as well as a D619G
replacement, is sufficient to render the receptor constitutively
active (12), a common activation mechanism appears to apply to both
glycoprotein hormone receptors. Therefore, we initiated a search for a
positively charged amino acid residue in the cytoplasmic portions of
the LHR that could potentially provide for a counter ion for D564 to
form a salt bridge. Because the i2 loop of the TSHR and LHR has been
suggested to participate in Gs activation (22, 23), we
initially focused on basic amino acids within this loop that are
conserved among glycoprotein hormone receptors and replaced these
residues by A. However, none of the five A mutants showed signs of
ligand-independent activation and, therefore, most probably does not
represent the site of a positively charged contact partner for D564.
Systematic mutagenesis studies are underway to probe other locations at
the cytoplasmic receptor aspect to identify a basic amino acid residue
involved in maintaining a salt bridge constraint. Identification of
such an amino acid would greatly enhance our knowledge on the spatial
arrangement of intracellular receptor loops.
Experimental evidence combined with molecular modeling suggested that
the central R residue in the conserved DRY motif, a structural hallmark
of all GPCRs belonging to the rhodopsin family, is stabilized in a
polar pocket formed by several highly conserved polar amino acids
located at the cytoplasmic aspect of different TMs (24). Thus, we
considered R464 in the i2 loop of the LHR representing the center piece
of the DRY motif (permutated to ERW in the glycoprotein hormone
receptors) to be a potential contact partner for D564. Our A scanning
approach revealed the noteworthy fact that R464 at the TM3/i2
transition can be replaced by A without major disturbance of LHR
signaling upon transient expression of the mutant in COS-7 cells. An
exchange of the conserved R for H, however, led to a drastic decrease
of maximal hCG-induced cAMP formation in stably transfected HEK 293
cells (25). Functional studies with mutated m1 muscarinic receptors
have shown that a charge-conserving R-for-K exchange results only in
modest impairment of receptor function (26), suggesting that the nature
of the replacing amino acid residue, and not only the loss of the
conserved R, significantly contributes to functional properties of the
resulting mutant receptors. Mutational studies with rhodopsin (27), the
V2 vasopressin (28), and
1B-adrenergic receptors (24)
indicated that replacement of the conserved R in the DRY motif by
various different amino acids virtually abolishes G-protein coupling,
and the conserved R has been implicated as a central trigger of GDP
release from the G protein
-subunit (29). Our results clearly show
that although the conserved R enhances LHR coupling to Gs,
it cannot be regarded as the general and indispensable molecular switch
for G-protein activation via all GPCRs.
In addition to the Gs/adenylyl cyclase system, the
activated LHR is also known to stimulate phospholipase C (13, 16). We
noticed that several LHR mutants (D564G, D564N, D564K,
564) modified
at position 564 caused constitutive coupling to Gs and
additionally profoundly enhanced agonist-induced IP accumulation,
suggesting a connection between constitutive cAMP formation and
agonist-dependent phospholipase C activation. Interestingly, two A
insertion mutants, LHR-insA562 and -insA564, which do not cause
constitutive cAMP formation, also responded to hCG challenge with a
markedly increased phosphoinositide breakdown. These results show that
ligand-independent adenylyl cyclase stimulation does not represent a
prerequisite for an efficient coupling to the phospholipase C-signaling
pathway, yet both signaling events appear to be independent.
To examine whether i3 loop deletions lead to constitutive activity of
all glycoprotein hormone receptors, we also created mutant FSHRs. The
human receptor, however, did not tolerate the deletion of 9 or 3 amino
acid residues from its third intracellular loop. These results were not
unexpected, as extensive mutational studies with the rat FSHR had
already shown that this glycoprotein hormone receptor is particularly
prone to improper folding and intracellular retention when only
slightly modified (30, 31). Deletion from the FSHR i3 loop of D567,
which corresponds to D564 in the LHR and D619 in the TSHR, did not
interfere with membrane expression. However, in contrast to our results
with the TSHR and LHR, the corresponding FSHR mutant was functionally
equivalent to FSHR(wt) and did not present any evidence for
constitutive activity. Therefore, one cannot escape the conclusion
that, in the case of the FSHR, the molecular events underlying receptor
activation differ from those operative in the other two glycoprotein
hormone receptors. Our results are in accord with a recent study on
hybrid LHRs/FSHRs emphasizing the importance of TM5/TM6 interactions
for keeping gonadotropin receptors in an inactive state (14). In the
FSHR, interhelical contacts between TM5 and TM6 appear to be more
constrained to secure an inactive receptor state that cannot be
destabilized by activating mutations or deletions in the i3 loop.
We previously suggested a model of TSHR activation in which deletion
mutations within the i3 loop are assumed to lose the contact of TM5 to
TM6 (12). Considering the present results obtained with LHR, agonist-
or mutation-induced disruption of a salt bridge involving the i3 loop
may also contribute to the activation of LHRs and TSHRs. In the FSHR
the impact of the i3 loop on receptor activation clearly differs from
the scenario that prevails in the TSHR and LHR. In the future, it will
be enlightening to correlate these in vitro findings with
distinct biological functions of the members of the glycoprotein
hormone receptor family.
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MATERIALS AND METHODS
|
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Generation of Mutant LHR and FSHR Genes
The cDNA of the human LHR in the pSG5 vector (Stratagene, La
Jolla, CA) was inserted into the eukaryotic expression vector pcD-PS
(32) via an EcoRI site in the polylinker and is further
referred to as LHR-pcD-PS. A 1-kb fragment of 3'-untranslated region
was removed by site-directed mutagenesis (33). The cDNA of the human
FSHR was amplified by PCR from poly(A+) RNA isolated from
human granulosa lutein cells. Briefly, two overlapping PCR fragments
were amplified using the primer pairs PR1
5'-GCGGAATTCATGGCCCTGCTCCTGGTCTCTTTG-3'/PR2
5'-GCTGGCTTCCATGAGG-GCGACAAG-3' and PR3
5'-CTGCCTACTCTGGAAAAGCTTGTC-3'/PR4
5'-CGCGGATCCTGAGTTTTGGGCTAAATGA-CTTAG-3'. The PCR reactions were
performed with the Expand High Fidelity system (Boehringer, Mannheim,
Germany) under the following conditions (35 cycles): 1 min at 94 C, 1
min at 62 C, and 2 min at 68 C. To obtain full-length cDNA, the two
overlapping PCR fragments were used as template, and the FSHR was
amplified by applying the primer pair PR1/PR4. After a restriction
enzyme digest with EcoRI/BamHI, the PCR product
was initially cloned into the pSG5 vector (Stratagene). The cDNA
sequence was confirmed by direct sequencing. Since all constructs used
in this study were inserted into the pcD-PS vector, the FSHR cDNA was
subsequently subcloned into this expression vector using the
BglII sites within the coding sequence of the FSHR and the
polylinker. The remaining cDNA coding for the N-terminal portion of the
human (h)FSHR was introduced by employing standard PCR mutagenesis
techniques using the SmaI site of the polylinker and the
Bsu36I site in the coding sequence.
To characterize functional properties of different mutations within the
i3 loop of the LHR and FSHR (Figs. 1
and 5A
), mutations were created by
PCR mutagenesis techniques using the LHR-pcD-PS and FSHR-pcD-PS
expression plasmids as a template. PCR fragments containing the
mutations were digested and used to replace the corresponding
BstBI/SpeI and PflMI fragments in the LHR-pcD-PS
and FSHR-pcD-PS vectors, respectively. Point mutations in the i2 loop
were introduced into LHR-pcD-PS using BstBI/XbaI
sites. The identity of the various constructs and the correctness of
all PCR-derived sequences were confirmed by restriction analysis and
dideoxy sequencing with thermosequenase and dye-labeled terminator
chemistry (Amersham, Arlington Heights, IL).
Transient Expression of Mutant LHRs and FSHRs and Functional
Assays
COS-7 cells were grown in DMEM supplemented with 10% FBS, 100
U/ml penicillin, and 100 µg/ml streptomycin at 37 C in a humidified
7% CO2 incubator. For transient transfections of COS-7
cells, a calcium phosphate coprecipitation method (34) was applied. In
general, 20 µg of plasmid DNA per 10-cm dish or 5 µg/well (12-well
dish) were transfected. For cAMP measurements, cells were split into
12-well plates (2 x 105 cells per well), transfected,
prelabeled with [3H]adenine (2550 Ci/mmol, Dupont-NEN,
Brussels, Belgium), and assays were performed 3 days after
transfection. For cAMP assays, cells were washed once in serum-free
DMEM, followed by a preincubation with the same medium containing 1
mM 3-isobutyl-1-methylxanthine (Sigma Chemical Co., St.
Louis, MO) for 20 min at 37 C in a humidified 7% CO2
incubator. Subsequently, cells were stimulated with appropriate
concentrations of hCG (from pregnancy urine, 3,000 U/mg, Sigma) and
porcine FSH (pFSH, isolated from porcine pituitary, 50 U/vial, Sigma)
for 1 h. Reactions were terminated by aspiration of the medium and
addition of 1 ml 5% trichloric acid. cAMP content of the cell extracts
was determined as described previously (35).
To measure IP formation, transfected COS-7 cells were incubated with 2
µCi/ml of [myo-3H]inositol (18.6 Ci/mmol, Amersham) for
18 h. Thereafter, cells were washed once with serum-free DMEM
without antibiotics containing 10 mM LiCl. hCG-induced
increases in intracellular IP levels were determined by anion exchange
chromatography as described (36).
To account for experimental variability inherent to transient
transfection procedures, wt receptor cDNAs were included as internal
controls in each transfection assay, and functional data were expressed
as -fold increase of wt basal unless stated otherwise in the respective
figure legends.
[125I]hCG and
[125I]hFSH Binding
For radioligand binding studies, cells were harvested 72 h
after transfection, and saturation binding assays were performed using
membrane homogenates. Incubations were carried out for 1 h at 22 C
in a 0.25 ml volume in the presence of 1.2 x 106 cpm
of [125I]hCG (1800 Ci/mmol; Dupont-NEN, Brussels,
Belgium) or 4 x 105 cpm of [125I]hFSH (1287
Ci/mmol; Dupont-NEN). Nonspecific binding was defined as binding in the
presence of 1 µM hCG and 1.6 µM pFSH,
respectively. Purchased [125I]hCG was characterized by
RRA using the murine LHR stably expressed in L cells (13). Saturation
binding experiments yielded a Kd value of 0.25
nM for [125I]hCG. [125I]hFSH
was initially tested in binding experiments using hFSHRs expressed in
COS-7 cells. Kd values of 0.6 nM were obtained
for [125I]hFSH. Protein concentrations were determined by
the bicinchoninc acid protein assay system (Pierce, Rockford,
IL). Binding data were analyzed by a nonlinear least squares
curve-fitting procedure using the program Ligand (37).
 |
ACKNOWLEDGMENTS
|
---|
We would like to thank Robert Grosse for his help to clone the
cDNA encoding the hFSHR. The cDNA of the human LHR was a generous gift
of Dr. A. Shenker, Northwestern University Medical School, Chicago.
 |
FOOTNOTES
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---|
Address requests for reprints to: Torsten Schöneberg, Institut für Pharmakologie, Freie Universität Berlin, Thielallee 6973, D-14195 Berlin, Germany. E-mail: schoberg{at}zedat.fu-berlin.de
This work was supported by the Deutsche Forschungsgemeinschaft and
Fonds der Chemischen Industrie.
Received for publication May 6, 1998.
Revision received October 8, 1998.
Accepted for publication October 23, 1998.
 |
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