From the Medizinische Klinik und Poliklinik III, Universität
Leipzig, 04103 Leipzig, Germany and Institut
für Pharmakologie, Freie Universität Berlin,
14195 Berlin, Germany
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ABSTRACT |
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Gain-of-function mutations of the thyrotropin receptor (TSHR) gene have been invoked as one of the major causes of toxic thyroid adenomas. In a toxic thyroid nodule, we recently identified a 9-amino acid deletion (amino acid positions 613-621) within the third intracellular (i3) loop of the TSHR resulting in constitutive receptor activity. This finding exemplifies a new mechanism of TSHR activation and raises new questions concerning the function of the i3 loop. Because the i3 loop is thought to be critical for receptor/G protein interaction in many receptors, we systematically reexamined the role of the TSHR's i3 loop for G protein coupling. Thus, various deletion mutants were generated and functionally characterized. We identified an optimal deletion length responsible for constitutive activity. If the number of deleted amino acids was reduced, elevated basal cAMP accumulation was found to be concomitantly diminished. Expansion of the deletion dramatically impaired cell surface expression of the receptor. Shifting the deletion toward the N terminus of the i3 loop resulted in unaltered strong constitutive receptor activity. In contrast, translocation of the deletion toward the C terminus led to significantly reduced basal cAMP formation, most probably due to destruction of a conserved cluster of amino acids. In this study, we show for the first time that amino acid deletions within the i3 loop of a G protein-coupled receptor result in constitutive receptor activity. In the TSHR, 75% of the i3 loop generally assumed to play an essential role in G protein coupling can be deleted without rendering the mutant receptor unresponsive to thyrotropin. These findings support a novel model explaining the molecular events accompanying receptor activation by agonist.
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INTRODUCTION |
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The glycoprotein hormone TSH1 is the major regulator of growth and differentiation of the thyroid gland (1). TSH acts by binding to its receptor at the basolateral membrane of thyroid follicular cells. The thyrotropin receptor (TSHR) belongs to the large superfamily of heptahelical G protein-coupled receptors (GPCRs) (1-3). In the human thyroid, the ligand-bound TSHR leads to stimulation of adenylyl cyclase and phospholipase C by interacting with Gs and Gq/11 (4). The cAMP regulatory cascade controls growth and differentiated function (thyroid hormone secretion, iodide trapping), whereas Ca2+ and diacylglycerol stimulate iodination and thyroid hormone synthesis (1).
Recent evidence suggests that agonist-dependent activation
of GPCRs elicits a crucial conformational change in the receptor molecule resulting in a movement of transmembrane helices relative to
one another and subsequent G protein activation (5, 6). The
reallocation of transmembrane domains (TMs) is brought about by
breaking and formation of interhelical noncovalent bonds between specific amino acid residues (7-11). Interestingly, these
intermolecular rearrangements can be mimicked by mutations that create
constitutively active receptors. Gain-of-function mutations have been
identified in many GPCRs (12). In the TSHR gene, gain-of-function
mutations cause autosomal dominant nonautoimmune hyperthyroidism and
toxic thyroid nodules (13, 14). Constitutively activating mutations have been predominantly identified in TMs but also in connecting intra-
and extracellular loops (15-22). Within the overall receptor structure, the third intracellular loop (i3) and TM6 appear to be hot
spots for gain-of-function mutations in different GPCRs. Several
reports suggest that these domains may be particularly important for
receptor/G protein interaction (12). In the adrenergic receptor system,
cytoplasmic domains which are in juxtaposition to the plasma membrane,
particularly near TMs 5, 6, and 7, have been implicated in G protein
coupling (23). Site-directed mutagenesis and substitution of amino acid
clusters with corresponding regions from 1- and
2-adrenergic receptors revealed selective effects of
different regions in the i3 loop of the TSHR on inositol phosphate (IP)
and cAMP signaling cascades. The N- and C-terminal i3 loop junctions
appear to be involved in phosphoinositide hydrolysis, and sequence
variation can result in loss- or gain-of-function (24-26). Mutations
of the conserved A623 in the C-terminal junction of the i3 loop to
lysine or glutamic acid was reported to result in loss of
TSH-stimulated IP formation leaving cAMP accumulation unaltered (27).
TSH-induced cAMP accumulation, however, was affected only by mutations
in the C-terminal region of the i3 loop (24-26). Characterization of
mutant receptors harboring deletions in the i3 loop confirmed the
importance of this particular region for receptor/G protein
interaction. In the glucagon (28), glucagon-like peptide-1 (29), and
the muscarinic acetylcholine receptor (30), trimmed i3 loops resulted
either in severely impaired or unaltered signaling abilities.
We recently identified a 9-amino acid deletion (amino acid positions 613-621) within the i3 loop of the TSHR in a toxic thyroid nodule (31). In contrast to functional properties of all previously described deletion mutants in the corresponding region of other GPCRs, the TSHR deletion mutant displayed constitutive activity. This finding prompted us to further evaluate the role of the TSHR i3 loop for G protein coupling. Thus, we generated various TSHR deletion mutants within the i3 loop varying in length and location and characterized their functional profile.
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EXPERIMENTAL PROCEDURES |
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Generation of Mutant TSHR Genes-- To characterize functional properties of different deletions within the i3 loop of the TSHR (see Fig. 1), mutations were created by employing standard PCR mutagenesis techniques (32) using the human TSHR expression plasmid, TSHR-pSVl (33), as a template. PCR fragments containing the mutations were digested and used to replace the corresponding Eco81l/Eco91l fragment in the TSHR-pSVl vector. 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 Pharmacia Biotech).
Cell Culture and Transient Expression of Mutant TSHRs-- COS-7 cells were grown in Dulbecco`s modified Eagle's medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin (Life Technologies, Inc.) at 37 °C in a humidified 7% CO2 incubator. For cAMP and radioligand binding assays, the cells were transfected using a DEAE-dextran method (34). In brief, 2 × 106 COS-7 cells were seeded into 100-mm dishes and transfected with different TSHR deletion constructs (5 µg/dish). Approximately 24 h after transfection, cells were split into 12-well plates at a density of 2 × 105 cells/well. Functional assays were performed 48 h after transfection. For the measurement of agonist-induced phosphoinositide hydrolysis, COS-7 cells were transfected in 12-well plates (3 × 105 cells/well) using LipofectAMINE (Life Technologies, Inc.) according the manufacturer's instructions. The cells were used for an IP accumulation assay 48 h after transfection.
Radioligand Binding Assays--
Transfected COS-7 cells were
washed once with Hank's solution without NaCl containing 280 mmol/liter sucrose, 0.2% bovine serum albumin (Sigma), and 2.5% low
fat milk (19). Thereafter, cells were incubated in the same medium in
the presence of 140,000-160,000 cpm of 125I-bovine
thyrotropin (bTSH) (25 µCi/µg, 40 units/mg, Brahms Diagnostica) and
the appropriate concentrations of nonlabeled bTSH (Sigma) at room
temperature for 4 h. Cells were subsequently solubilized with 1 N NaOH after two washes with Hank's solution. Bound
radioactivity was determined in a -counter. bTSH, TSHR
concentrations, and KD values are expressed as
milliunits/ml. Data were analyzed assuming a one-site binding model
using the fitting module of SigmaPlot 2.0 for Windows (35).
cAMP Accumulation Assays-- For cAMP assays cells were washed once in serum-free Dulbecco's modified Eagle's medium, followed by a preincubation with the same medium containing 1 mM 3-isobutyl-1-methylxanthine (Sigma) for 20 min at 37 °C in a humidified 7% CO2 incubator. Subsequently, cells were stimulated with appropriate concentrations of bTSH for 1 h. Reactions were terminated by aspiration of the medium and addition of 1 ml 0.1 N HCl. Supernatants were collected and dried. cAMP content of the cell extracts was determined with a commercial kit (Amersham Pharmacia Biotech) according to the manufacturer's instructions.
Stimulation of Inositol Phosphate Formation-- Transfected COS-7 cells were incubated with 2 µCi/ml [myo-3H]inositol (18.6 Ci/mmol, Amersham Pharmacia Biotech) for 18 h. Thereafter, cells were washed once with serum-free Dulbecco's modified Eagle's medium without antibiotics containing 10 mM LiCl. TSH-induced increases in intracellular inositol phosphate levels were determined by anion exchange chromatography as described previously (36).
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RESULTS |
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A nine-amino acid deletion (613-621) within the i3 loop of the
TSHR (Fig. 1) resulted in constitutive
activation of the cAMP signaling cascade reflected by a 5-fold increase
in basal cAMP levels in conjunction with a markedly blunted maximal
cAMP response (approximately 60% of wt receptor level) after
stimulation with 100 milliunits/ml of bTSH (see Figs. 3 and 4). Taking
this naturally occurring deletion mutant as a point of reference, we
generated further mutants (Fig. 1) to clarify the relative importance
of distinct amino acids as well as of the extent and the location of
the deletion for constitutive TSHR activity. All mutant receptors with
deletions of more than one amino acid showed reduced cell surface
expression (Table I). This was
particularly prominent for the
609-621 and
609-624 deletion
constructs (Table I), indicating that deletions in the i3 loop can
severely interfere with correct folding and trafficking of the receptor
to the plasma membrane. The TSHR has been described to be
constitutively active, and elevated basal cAMP levels can be observed
after transient transfection of COS cells (37). We confirmed this
finding by comparing basal cAMP levels determined in COS-7 cells
transfected with identical amount of either the human V2 vasopressin or
the TSH receptor (see Fig. 3). Several TSHR deletion mutants
(
609-621,
613-624,
609-624, and
618-624) showed
decreased basal cAMP levels most likely due to decreased cell surface
expression levels.
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Deletion of Conserved and Nonconserved Regions within
613-621--
Sequence comparison within the family of glycoprotein
hormone receptors reveals that the
613-621 deletion can be
subdivided into a highly conserved C-terminal and a less conserved
N-terminal portion (Fig. 2). To test for
differential functions of these two portions with regard to TSHR
activation, we created the mutant receptors
618-621 and
613-617
with deletions of 4 conserved or 5 nonconserved amino acids,
respectively. Both mutant receptors remained constitutively active,
although at levels lower than the initial
613-621 mutant (Fig.
3). Receptor density
(Bmax), KD values, and cAMP
levels after maximal bTSH stimulation were comparable between
618-621 and
613-621 (Fig. 4;
Table I). When compared with the wt receptor, deletion of the less
conserved N-terminal amino acids (
613-617) had only minor effects
on maximal TSH-stimulated cAMP accumulation (Table I). In accord with
the latter findings, the
613-617 mutant showed a slightly increased cell surface expression compared with mutants
613-621 and
618-621 (Table I).
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N- and C-terminal Extension of the Deletion 613-621--
To
investigate the functional importance of the size of the deletion, we
generated three mutant receptors with expanded deletions. Only mutant
613-624, the smallest deletion within this series of mutants,
displayed expression levels of a similar order of magnitude as the
initial mutant
613-621 (Table I). TSH responsiveness in terms of
cAMP accumulation, however, of this C-terminally extended mutant was
markedly blunted (Fig. 3). On the contrary, mutant
609-621 located
in the central and N-terminal portion of the i3 loop, responded to TSH
challenge with an increase in cAMP levels comparable to
613-624
despite a profoundly diminished receptor density (Fig. 3, Table I).
Further extension of the deletion (
609-624) led to an accompanying
decrease in cell surface expression and hormonal responsiveness.
Shifting 613-621 within the i3 Loop--
Having shown that the
length of the deletion was a crucial parameter defining functional
consequences, we addressed the issue as to whether the location of the
deletion within the i3 loop would also be of importance. To this end,
we created two mutant receptors lacking 7 and 9 amino acids,
respectively, in regions adjacent to the initial
613-621 deletion.
Mutant
618-624 in which the deletion is shifted toward the C
terminus of the i3 loop was characterized by a receptor density (Table
I, Fig. 4) and a maximal cAMP response comparable to mutant
613-621, yet did not display constitutive activity (Figs. 3 and 4).
Like deletion mutant
613-624,
618-624 was also devoid of the
critical amino acids 622-624 in the conserved C-terminal part of the
i3 loop. On the contrary, deletion mutant
609-617 located in the
central and N-terminal portion of the i3 loop was effectively inserted into the plasma membrane and displayed prominent constitutive activity
(Table I, Fig. 4). Maximal hormone-stimulated cAMP formation reached
levels similar to the activated wt TSHR (Figs. 3 and 4).
Deletion of Single Amino Acids within 613-621--
To study
the effects of single amino acid deletions within the i3 loop on TSHR
function, we deleted individual amino acids in the conserved and
nonconserved portions of
613-621. When expressed in COS-7 cells,
mutants
615 and
616 located within the less conserved portion of
the i3 loop did not differ from the wt receptor in terms of receptor
density, KD values, basal and TSH-stimulated cAMP
levels (Table I, Fig. 3). A deletion of Lys618 situated in
the C-terminal highly conserved portion of
613-621 did not have
significant impact on the functional properties of the receptor (Table
I, Fig. 3). The small decrease of KD values was most
probably a consequence of a reduced receptor density (Table I).
Deletion of Asp619 resulted in profound constitutive
activity of the mutant receptor. This result was unexpected because a
mutant TSHR characterized by a deletion of Asp619 in
conjunction with a T620S substitution has previously been reported to
be functionally indistinguishable from the wt receptor (38).
Effect of Deletion Mutants on Phosphoinositide Hydrolysis--
In
addition to the Gs/adenylyl cyclase system the activated
human TSHR is also known to stimulate phospholipase C activity (39). To
address the question whether the various deletion mutations would
affect the IP signaling pathway, we tested all receptor mutants for
basal and hormone-induced IP production. None of the TSHR mutants
constitutively stimulated the IP signaling pathway (data not shown). As
shown in Table I, bTSH-induced IP accumulation closely correlated with
the relative maximal increase in cAMP levels. Deletion mutations
(609-621,
613-624, and
609-624) that were characterized by
low plasma membrane expression levels were unable to signal to
phospholipase C.
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DISCUSSION |
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A 9-amino acid deletion within the i3 loop of the TSHR
(613-621) leading to constitutive receptor activity prompted us to systematically analyze the effect of deletion mutations within the i3
loop on TSHR function. Our major finding is that various deletions in
the i3 loop result in ligand-independent receptor activation as long as
certain critical C-terminal amino acids are not affected. We show that
maximal constitutive activity correlates with an optimal length of the
deleted receptor portion. In contrast to previous assumptions, these
data entertain the notion that several clusters of amino acid in the
TSHR's i3 loop are not required for G-protein activation. However, the
i3 loop is an important structural determinant to safeguard receptor
folding, trafficking and activation by agonist.
First we asked the question whether the absence of distinct critical
amino acids within the original deletion would be responsible for
constitutive activity. Sequence comparison revealed that the four
C-terminal amino acids within 613-621 are highly conserved within
the family of glycoprotein hormone receptors (Fig. 2). Therefore, we
constructed two smaller deletions comprising either conserved
C-terminal or non-conserved N-terminal amino acids (
618-621 and
613-617, respectively). Both mutant receptors retained constitutive activity, albeit at reduced levels compared with
613-621. The observation that two separate portions of the original deletion each
caused constitutive activity, let us put forward the proposal that a
deletion per se was responsible for ligand-independent receptor activation. A reduction in the size of the deletion led to a
decreased constitutive receptor activity irrespective of the exact
position of the amino acids deleted.
Alternatively, we examined the functional consequences of N- and
C-terminal extensions of the original deletion 613-621. Expanded
deletions led to a marked decrease in cell surface expression of the
mutated receptors. These results indicate that the length of the loop
connecting TM5 and TM6 cannot be reduced below a critical size, which
would interfere with correct insertion of the receptor into the cell
membrane. It is worth mentioning, however, that even large deletions
did not completely abrogate hormone-stimulable cAMP formation and cell
surface expression. Interestingly, mutants
609-621 and
613-624
showed a similar cAMP response, although the
613-624 construct was
expressed at 5-6-fold higher levels indicating that the
613-624
deletion targeted amino acids critically required for productive
Gs coupling. Indeed, amino acids 621-625 form a conserved
B-X-X-B-B motif with B denoting basic and X nonbasic amino acids (Figs.
1 and 2). All glycoprotein hormone receptors share this amino acid
cluster at corresponding positions within the i3 loop (40). The
substitution of Ala623 within this amino acid cluster with
different amino acids leads to constitutive activation of the cAMP
cascade (41) or to a selective loss of IP signaling (27) of the TSHR
further supporting the involvement of this region in G protein
activation. Moreover, similar motifs were identified as structural
determinants for Gi and Gs coupling in other
heptahelical receptors (30, 42).
To assess the importance of the location of deletions within the i3
loop for TSHR function, the original deletion was shifted N- and
C-terminally by three amino acids. The N-terminally shifted deletion
mutant (609-617) showed strong constitutive activity and a maximal
stimulation comparable to the wt receptor. A deletion within the i3
loop of the glucagon receptor comparable in length and location to
609-617, resulted in an attenuated glucagon-induced cAMP response
(28). This N-terminal deletion mutant showed a very low cell surface
expression and consequently, low agonist-induced cAMP accumulation.
These results are at odds with our results obtained with deletion
mutant
609-617 of the TSHR. However, receptor regions involved in G
protein coupling vary in location and sequence between different GPCRs
(3), and therefore, similar structural modifications at corresponding
locations do not necessarily have to yield identical functional effects
in different GPCRs.
In contrast to the latter results, a C-terminally shifted deletion
mutant (618-624) showed no constitutive activity. Basal values for
cAMP accumulation were lower than for wt TSHR, and the maximal response
was strongly attenuated. Interestingly, this effect is in good
agreement with the functional properties of a comparable deletion
mutant generated in the glucagon receptor (28). In the case of the
TSHR, the decreased cAMP response was not caused by a decrease in cell
surface expression because expression levels were comparable to the
original deletion
613-621 and also to the shorter deletion mutants
613-617 and
618-621 which all displayed constitutive activity.
A conspicuous difference between these four deletion mutants is the
destruction of the B-X-X-B-B motif in
618-624 indicating the
necessity of this amino acid cluster for effective Gs
coupling. It should not go unnoticed, however, that although the
destruction of the conserved motif attenuated constitutive activity,
the ability of the receptor to couple to Gs was not utterly
precluded as shown by reproducible increases in intracellular cAMP
levels after TSH stimulation.
As point mutations within the original deletion 613-621 have been
reported (18, 41) to result in constitutive activity, we analyzed the
influence of distinct single amino acid deletions within the i3 loop on
TSHR function. For this purpose, we generated four mutant receptors
with deletion of Pro615, Gly616,
Lys618, and Asp619, respectively. Only the
619 mutant showed constitutive activity. A D619G substitution also
leads to constitutive activity of the TSHR (18) indicating an important
role of this amino acid for receptor activation. Decreasing the length
of the i3 loop by one amino acid does not by itself lead to
constitutive activity as exemplified by the deletion mutants
Pro615, Gly616, and Lys618.
Therefore, deletions of critical amino acids may have an effect similar
to activating substitutions of these residues in that critical
interhelical, intrahelical, or intraloop bonds are disrupted, thereby
releasing a structural constraint in the receptor and exposing critical
activating receptor domains for interaction with G proteins.
Interestingly, the deletion of Asp619 in conjunction with a
T620S substitution has been reported not to lead to constitutive
activity (38).
GPCRs are assumed to exist in equilibrium between inactive and active states, and only the active state effectively interacts with G proteins. At the structural level, the molecular events accompanying the functional transition from inactive to active states are largely unknown. Recent studies with spin-labeled rhodopsin (6) or rhodopsin molecules carrying engineered metal-ion-binding sites (5) emphasized the importance of rigid body movements of helices relative to one another during the activation process. Furthermore, a model of the LHR suggested possible structural and functional effects of constitutively activating mutations (43). A tightly packed hydrophobic cluster between the intracellular halves of TM5 and TM6 is postulated to be essential for receptor quiescence. According to the model, activating mutations would then disrupt the hydrophobic packing and disturb the relative positioning of TM5 and TM6 in the plasma membrane (43). Our results with various deletion mutants of the TSHR are fully compatible with such models and may provide further insight into the molecular mechanism of GPCR activation.
Based on our results we propose a novel model of TSHR activation. We show that shortening of the TSHR's i3 loop rather than deletion of distinct amino acids is responsible for constitutive activity. This assumption is based on the fact that several deletion mutations which do not overlap in the deleted sequence display constitutive activity. Therefore, it is very likely that a similar mechanism accounts for shifting the receptor into the active conformation. Additionally, the extent of constitutive activity appears to be dependent on the length of the deletion. It can not be excluded, however, that a loss of one or more distinct amino acids within a TSHR deletion mutant is responsible for constitutive activation.
The i3 loop has previously been thought to directly mediate receptor/G protein interaction. Surprisingly, none of the deletion mutants examined showed a complete abolishment of Gs coupling. It is likely that a decrease in the length of the i3 loop will affect the conformation of the adjacent transmembrane domains. The N-terminal part of TM6 appears to be most important for LHR activation. There is evidence that a peptide consisting of the wt sequence of the lower portion of TM6 of the LHR can activate adenylyl cyclase (44). Within this receptor region there is more than 90% amino acid identity among glycoprotein hormone receptors. Thus, the N-terminal portion of TM6 is capable of activating Gs, if freed from all conformational constraints imposed by neighboring receptor sequences. Agonist binding (Fig. 5A), activating point mutations, and a shortening of the i3 loop (Fig. 5C) may all lead to a relative movement of TM5 to TM6, most likely allowing a relative movement of TM6 toward the cytoplasm, thus enabling critical transmembrane sequences to interact with Gs. A similar hypothesis has been derived from alanine-insertion studies (Fig. 5B) with the m2 muscarinic receptor (45). The mechanism of TSHR activation proposed in Fig. 5 is, however, limited to the mutations made and tested in this study. Since at present no data are available whether insertion mutations within TM6 of a glycoprotein hormone receptor may display constitutive activity as observed with the m2 muscarinic receptor a direct comparison of receptor activation between these two GPCRs remains speculative.
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In conclusion, we performed a thorough analysis of a naturally occurring mutant of the human TSHR and developed a novel model describing TSHR activation at the molecular level. Additional studies with the TSHR will have to show whether this model is correct and whether it can be extended to all glycoprotein hormone receptors or even to other GPCRs more distantly related.
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ACKNOWLEDGEMENTS |
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We express our gratitude to Brahms Diagnostica (Berlin) for providing 125I-bTSH and to Dr. G. Vassart for supplying the plasmid TSHR-pSVl.
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FOOTNOTES |
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* This work was supported by grants from the Deutsche Forschungsgemeinschaft and Fonds der Chemischen Industrie.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Prof. Dr. Ralf Paschke, III. Medizinische Klinik und Poliklinik, Universität Leipzig, Philipp-Rosenthal-Straße 27, D-04103 Leipzig, Germany. Tel.: 49-341-97-13200; Fax: 49-341-97-13209.
1 The abbreviations used are: TSH, thyrotropin; TSHR, thyrotropin receptor; bTSH, bovine thyrotropin; GPCR, G protein-coupled receptor; i3, third intracellular loop; IP, inositol phosphate; LHR, lutropin/choriogonadotropin receptor; PCR, polymerase chain reaction; TM, transmembrane domains; wt, wild-type.
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REFERENCES |
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