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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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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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563–566) of four different 4-amino
acid deletion mutants displayed agonist-independent activity.
Systematic examination of the structural requirements for this effect
in the 563–566 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. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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 (TM1–7) 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 (558–562) or
four amino acids (563–566) resulted in a similar
agonist-independent receptor activity as noted for the 9-amino acid
(558–566) 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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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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558–566) 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
).
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). Constitutive receptor
activation was also observed after shifting the 9-amino acid deletion
toward the N-terminal loop portion (554–562), 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 558–566 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 (558–562) or of the 4 conserved amino acids
(563–566), 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
(558–562). 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
(550–553, 554–557) and C-terminal (567–570) 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 567–570 located
at the i3/TM6 transition was hardly detectable (see Table 1).
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563–564 and 565–566 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-563–564 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; 558–566:
0.44 nM; 563–566: 0.41 nM; 563–564:
0.87 nM; 564: 0.79 nM].
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). 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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). Most of the LHR mutants (558–566, 563–566, 550–553,
554–557, and 567–570) 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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). Expression of
FSHR-561–569 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-567–569 (see Fig. 5A). In addition, we deleted D567
corresponding to D564 in the LHR (see Fig. 5A). Whereas
FSHR-567–569 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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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Functional analysis of an LHR 9-amino acid deletion (558–566)
corresponding to the one originally described in the TSHR (12), as well
as of an N-terminally shifted deletion (554–562), 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 (558–566) 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 (558–562) or conserved (563–566)
stretch of amino acids from the LHR i3 loop did not result in a
reduction of constitutive activity when compared with the original
558–566 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-563–566)
of 4 (550–553, 554–557, 567–570) 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 563–566 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 558–566 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 LHR’s 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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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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 (25–50 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).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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1B-adrenergic
receptor by all amino acid substitutions at a single site. Evidence for
a region which constrains receptor activation. J Biol Chem 267:1430–14331B-adrenergic
receptor: role of highly conserved polar amino acids in receptor
activation. EMBO J 15:3566–3578[Medline]
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S. Mukherjee, V. V. Gurevich, A. Preninger, H. E. Hamm, M.-F. Bader, A. T. Fazleabas, L. Birnbaumer, and M. Hunzicker-Dunn Aspartic Acid 564 in the Third Cytoplasmic Loop of the Luteinizing Hormone/Choriogonadotropin Receptor Is Crucial for Phosphorylation-independent Interaction with Arrestin2 J. Biol. Chem., May 10, 2002; 277(20): 17916 - 17927. [Abstract] [Full Text] [PDF]
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 M. Ascoli, F. Fanelli, and D. L. Segaloff The Lutropin/Choriogonadotropin Receptor, A 2002 Perspective Endocr. Rev., April 1, 2002; 23(2): 141 - 174. [Abstract] [Full Text] [PDF]
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 S. Nishi, S. Y. Hsu, K. Zell, and A. J. W. Hsueh Characterization of Two Fly LGR (Leucine-Rich Repeat-Containing, G Protein-Coupled Receptor) Proteins Homologous to Vertebrate Glycoprotein Hormone Receptors: Constitutive Activation of Wild-Type Fly LGR1 But Not LGR2 in Transfected Mammalian Cells Endocrinology, November 1, 2000; 141(11): 4081 - 4090. [Abstract] [Full Text] [PDF]
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Y.-Y. Zhou, D. Yang, W.-Z. Zhu, S.-J. Zhang, D.-J. Wang, D. K. Rohrer, E. Devic, B. K. Kobilka, E. G. Lakatta, H. Cheng, et al. Spontaneous Activation of beta 2- but Not beta 1-Adrenoceptors Expressed in Cardiac Myocytes from beta 1beta 2 Double Knockout Mice Mol. Pharmacol., November 1, 2000; 58(5): 887 - 894. [Abstract] [Full Text]
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A. P. N. Themmen and I. T. Huhtaniemi Mutations of Gonadotropins and Gonadotropin Receptors: Elucidating the Physiology and Pathophysiology of Pituitary-Gonadal Function Endocr. Rev., October 1, 2000; 21(5): 551 - 583. [Abstract] [Full Text]
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Y.-X. Tao, A. N. Abell, X. Liu, K. Nakamura, and D. L. Segaloff Constitutive Activation of G Protein-Coupled Receptors as a Result of Selective Substitution of a Conserved Leucine Residue in Transmembrane Helix III Mol. Endocrinol., August 1, 2000; 14(8): 1272 - 1282. [Abstract] [Full Text]
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 R. M. Touyz, L. Jiang, and M. Ram Sairam Follicle-Stimulating Hormone Mediated Calcium Signaling by the Alternatively Spliced Growth Factor Type I Receptor Biol Reprod, April 1, 2000; 62(4): 1067 - 1074. [Abstract] [Full Text]
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