| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Institut de Recherche Interdisciplinaire en Biologie Humaine et Nucléaire, Université Libre de Bruxelles (V.V.-G., G.V., S.C.), Campus Erasme, B-1070 Bruxelles, Belgium; Department of Endocrinology, Singapore General Hospital (S.-C.H.), Republic of Singapore 179101; and Service de Médecine, Centre Hospitalo Universitaire d’Angers (P.R.), Angers 49033, France
Address all correspondence and requests for reprints to: Professor Gilbert Vassart, Institut de Recherche Interdisciplinaire en Biologie Humaine et Nucléaire, Université Libre de Bruxelles, Campus Erasme, 808 route de Lennik, B-1070 Bruxelles, Belgium. E-mail: gvassart{at}ulb.ac.be.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
-helices and they all activate highly related trimeric G proteins, their natural ligands belong to a wide spectrum of molecules of diverse sizes and chemical nature [biogenic amines, nucleotides, neuropeptides, chemokines, glycoprotein hormones, lipids, etc. (for reviews, see Refs. 1 and 2)]. The structural similarity of the receptors (3) and their downstream G protein targets, as well as their evolution from a common ancestor, strongly suggest that similar mechanisms are involved in their activation. This, in turn, poses the intriguing question of how widely dissimilar agonists are capable of triggering a supposedly common activation phenomenon. A currently favored model for activation of GPCRs holds that the receptors would explore a space of discrete states characterized by different conformations (4). According to this allosteric model, the active and inactive conformations of the receptors would be stabilized by agonists or inverse agonists, respectively (4, 5). Experimental evidences indicate that active and inactive conformations are characterized by different relative positions of some of the transmembrane helices, involving interactions between specific residues of the helical bundle (6, 7). The description of natural (8, 9, 10, 11, 12) or artificial mutations (13, 14) leading to agonist-independent activation of GPCRs provides a strong argument in favor of this model.
For small molecules such as the biogenic amines, there is strong evidence that the ligand interacts directly with specific residues of the transmembrane helices of the receptor (15, 16). For neuropeptides and small protein agonists such as neurokinins, it is believed that interaction involves both exoloops and the amino-terminal portion of the receptors, in association with residues of the transmembrane helices (17). The situation is less clear for receptors to glycoprotein hormones TSH, lutropin/CG and follitropin (FSH). In these cases, the agonists are bulky dimers of about 30 kDa made of a common -subunit and hormone-specific ß-subunits (18, 19). The corresponding receptors contain a canonical serpentine portion, with seven transmembrane helices typical of rhodopsin-like GPCRs, and a large (350–400 residues) amino-terminal ectodomain containing leucine-rich repeats (20, 21, 22). The amino-terminal segments are responsible for high affinity binding of the hormones and recognition specificity: swapping amino-terminal domains between receptors results in parallel exchange of specificity (23, 24), and soluble ectodomains have been prepared that display binding affinity similar to that of the holoreceptors (25, 26, 27). How binding of the hormone to the ectodomain results in activation of the serpentine portion of the receptor is still unknown. It has been proposed that after high affinity binding of the hormone to the ectodomain, low affinity interaction between the hormone and the exoloops or, even, residues of the transmembrane helices would result in activation (28).
The TSH receptor (TSHr) offers interesting characteristics with respect to the activation mechanisms: in addition to its natural agonist, it can be activated by autoantibodies in patients with Graves’ disease (29, 30, 31); it is also particularly easy to activate by mutations affecting the serpentine portion of the receptor (8, 9). The observation that mutations of specific residues of the ectodomain (serine 281) and the exoloops (isoleucine 486, isoleucine 568) constitutively activate the receptor led to the hypothesis that the ectodomain would exert an inhibitory constraint on the inherently noisy serpentine portion of the TSHr through interaction with the exoloops (32, 33, 34). This hypothesis is in agreement with the observation that mild treatment of cells expressing the TSHr with trypsin results in ligand-independent activation, while simultaneously removing an epitope of TSHr ectodomain (35). More recently, receptor mutants with truncations of their ectodomains have provided support to this model (36).
In the present study we have explored the role of the ectodomain of TSHr, both as a silencer and an activator of the serpentine portion of the receptor toward its main target, the G protein Gs. By comparing the constitutive activity of amino-terminally truncated receptors in which a spectrum of activating mutations have been engineered, we provide evidence that the ectodomain of TSHr functions as a molecular switch, displaying agonist or inverse agonist properties toward the serpentine domain, depending on whether it is bound to its ligand or not.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
) did not modify the functional and structural characteristics of the receptor when expressed in COS cells (Fig. 2). When compared with the wild-type receptor, both molecules were expressed at cell surface at a similar level, as assayed in flow cytometry with a Mab recognizing a conformational epitope of the amino-terminal ectodomain [BA8 (Ref. 38 and Fig. 2A)]. Both receptors also underwent cleavage of the connecting peptide between the ectodomain and the serpentine portion of the molecules (39, 40, 41), as evidenced in Western blot experiments with Mab 28, a Mab directed against a linear epitope at the N terminus of the human TSHr [hTSHr (Fig. 2B)]. The resulting -subunit, released from the receptor by reduction of disulfide bonds, was, however, 40 kDa larger for the SP-RT-TSHr construct than for the WT-TSHr (Fig. 2B). This higher molecular mass resulted most probably from addition of extra N-linked glycan moieties on the two putative N-glycosylation sites present in the RT (see legend to Fig. 1). In addition, mature forms of single-chain (uncleaved) receptor were not detectable with the SP-RT-TSHr construct, suggesting that all molecules expressed at the cell surface were in the cleaved form (Fig. 2B). cAMP production in transfected COS cells, whether basal or after stimulation by TSH, was identical for SP-RT-TSHr and WT-TSHr. Figure 2C illustrates typical concentration-response curves. The corresponding 50% effective concentrations (EC50) for WT-TSHr and SP-RT-TSHr were 1.4 mIU/ml and 1.9 mIU/ml, respectively.
|
|
. They were made of the serpentine portion of the hTSHr and portions of the ectodomain extending variably toward the amino terminus. All constructs harbored a RT at their extreme N terminus. After transfection in COS cells, expression at the cell surface was measured with Mab OR2-15A-6 directed against the RT (Fig. 3A) and basal intracellular cAMP levels were assayed. Great differences were observed in the level of expression of individual mutants, ranging from 0 to 50% of the expression achieved by SP-RT-TSHr. Constructs extending upwards of codon 192 displayed low expression (QGYA, LDV, LKKL), and two of them (TQTL, LKFL) did not reach the cell surface at all. Inclusion of a SP in the construct was not necessary for expression of the three shortest mutants (KFLR, FNPC, YDY). In contrast, for the QGYA, LDV, LKKL, KNQK, and GFGQ mutants, expression was undetectable in the absence of a SP (data not shown). Basal intracellular cAMP was measured in COS cells transfected with each construct (Fig. 3B), and the specific constitutive activity (SCA, i.e. basal activity normalized to surface expression) was calculated as described in Materials and Methods (Fig. 3C). The linear relation observed between receptor expression, as measured by flow immunocytometry, and the intracellular levels of cAMP in transfected cells demonstrate the validity of the normalization method. Data are illustrated in Fig. 3D for the SP-RT-WT-TSHr and a construct (SPRT I568T) chosen for its good expression and strong constitutive activity.
|
), close to the maximal activity triggered by 100 mIU/ml of TSH (see Fig. 4). In contrast, truncated constructs including amino acids upstream of codon 286 (QGYA, LKKL, LDV) presented no significant increase in basal activity over that of SP-RT-TSHr (Fig. 3C). Even when exposed to very high concentrations of hormone (100 mIU/ml), none of the truncated receptors could be stimulated by TSH (Fig. 4).
|
)], and basal cAMP production was assayed in transiently transfected COS cells (Fig. 5, C and D). The SP-RT-TSHr construct was engineered to harbor the same mutations to provide pertinent controls that could be assayed at the cell surface with the same anti-RT Mab (Fig. 5B). When normalized to surface expression of the mutants, the D633A and A623I substitutions caused an increase in constitutive activity whether present in the holoreceptor (SP-RT-TSHr) or the RT-KFLR truncated mutant (Fig. 5C).
|
) and tested in COS cells as described above, no significant increase in constitutive activity could be observed over that of the wild-type RT-KFLR construct [P = 0.9491, P = 0.089, P = 0.7751, respectively (Fig. 5D)]. As expected, when present in the SP-RT-TSHr background, the same mutations caused strong increase in constitutive activity (Fig. 5D). |
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
) causes an increase in basal activity (5- to 6-fold; Fig. 3C) as compared with holoreceptor. They extend other results obtained with ectodomain-truncated TSHr mutants (36) and provide evidence that the amino-terminal portion of TSHr behaves as a tethered inverse agonist (Fig. 3). Whether this effect involves specific interactions, as suggested by the differential effects of the individual mutants (Fig. 3), or is secondary to a mere stabilization of the structure of the serpentine, is presently unknown. In this context, it is noteworthy that activating mutations have been shown to cause destabilization of GPCRs (4, 44). According to one lutropin receptor model, the glycoprotein hormone receptor ectodomain would bind the hormone with high affinity and present it to the serpentine for a low-affinity productive interaction resulting in activation (28). Interestingly, none of our truncated TSHr mutants could be stimulated by a saturating concentration of TSH, in sharp contrast with the above model [in which no direct evidence for expression of the truncated mutants at the cell surface was provided (28)].
Full Stimulation of cAMP Production by the TSHr Involves More than Release of the Silencing Effect of the Ectodomain on the Serpentine
The increase in basal activity of the mutants with the shorter amino-terminal extension does not reach the levels displayed by full stimulation of the holoreceptor by TSH or by S281L, a mutant with an activating amino acid substitution in the ectodomain (15- to 20-fold, as compared with 5- to 6-fold; see Fig. 3). This excludes a model in which normal activation of the receptor would simply result from the release of a silencing effect exerted by the ectodomain on the serpentine portion, via interaction with the extracellular loops. It indicates that the ectodomain, whether TSH liganded or mutated in position 281, would have a positive effect on activity of the serpentine portion.
A Structural Module Contributed by the Ectodomain and the Extracellular Loops of TSHr Constitutes a Molecular Switch Controlling the Activity of the Serpentine
Mutations of transmembrane helices or the third intracellular loop are equally effective, whether on a truncated construct or the holoreceptor background (Fig. 5). These results are consistent with those observed in other GPCR, such as the glucagon receptor (45). In contrast, activating mutations affecting residues of the extracellular loops (or located at the top of transmembrane segment VII) are without effects when engineered on the background of a serpentine-alone construct. This indicates that the ectodomain and the exoloops do cooperate in the building of a module capable of activating the serpentine. The portion of the ectodomain involved may contain the evolutionary conserved PSHCCAF segment located at the C-terminal border of the last leucine-rich motif of the receptor. Indeed, similar to the situation in the exoloops, mutation of the conserved serine residue in this segment (serine 281 in the TSHr) causes constitutive activation of all three glycoprotein hormone receptors (32, 34, 46, 47). Interestingly, investigation of the effects of various amino acid substitutions at the serine 281 position, or adjacent residues, revealed a direct relation between the expected loss of local structure and the magnitude of activation of basal activity (34, 47). Similarly, the nature of some of the activating mutations at (or close to) the extracellular loops (I486F, I486M, and, more so, del658–661) (33) are compatible with a gain of function that would be secondary to a loss of structure.
Comparison of the effects on basal cAMP-stimulating activity of the various truncated mutants suggests that the PXSHCCAF motif could be implicated in the silencing effect on the serpentine (compare activity of GQYA, LDV, and LKKL with that of KFLR, FNPC, YDY, GFGQ, and KNQK; Fig. 3). However, when the S281L mutation was introduced on the background of truncated mutants with wild-type constitutive activity (GQYA, LDV, LKKL; Fig. 3), no increase in constitutive activity could be observed (data not shown). With the limitation already mentioned that the silencing effect of the ectodomain observed with some mutants might involve nonspecific stabilizing interactions, these results suggest that different segments of the ectodomain would be implicated in the silencing and stimulatory interactions with the serpentine. An essentially intact ectodomain would be required to display stimulatory activity.
In an attempt to integrate available information, we propose the following model for activation of the TSHr (see Fig. 6). In the absence of hormone, the ectodomain would exert a silencing effect on the serpentine portion of the receptor. Silencing would be incomplete, as the wild-type receptor displays readily measurable constitutive activity toward the cAMP pathway. Removal of the ectodomain would unmask the intrinsic basal activity of the serpentine, resulting in partial activation. Mutations of serine 281 in the ectodomain, or specific residues of the exoloops, would cause structural disorganization of a composite module made of segments of the ectodomain and the exoloops. The resulting structural change would trigger conversion of the inhibitory effect of the ectodomain into stimulation of the serpentine. According to this model, the ectodomain would behave as a tethered inverse agonist or an agonist, depending on whether it is wild type or mutated in position 281. Because the strongest activating mutations affecting S281 cause close to full activation of cAMP production by the receptor (Refs. 34 and 47 and Fig. 3), it is tempting to propose that the resulting structural changes would mimic those induced by hormone binding to the ectodomain. The model presents close similarity with the situation in rhodopsin. In this case, a covalently tethered ligand (retinal, residing within the transmembrane pocket) is also converted from inverse agonist to full agonist upon isomerization triggered by light (48, 49, 50). The model predicts that no direct interaction between the hormone and the exoloops or transmembrane helices is required for receptor activation, because the immediate agonist would be the ectodomain itself. This fits with the observation that stimulating autoantibodies from patients with Graves’ disease are capable of activating the receptor, while recognizing different epitopes of the ectodomain (29, 51, 52); it is not unexpected that different antibodies could cause similar loss of structure of the trigger module.
|
). |
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Construction of N Terminus Rhodopsin-Tagged Truncated hTSHr
A restriction fragment (HindIII/EcoRI) corresponding to the 5' untranslated region of the bovine rhodopsin gene and extending to codon 19 (RT) was excised from the pRK5 vector (37) and cloned in pBluescript SK+ (RT-SK+: RT-SK+). A cDNA fragment of the human TSHr extending from codon 21 (codon 1 corresponding to the initiation methionine) to the PstI site at codon 90 was PCR amplified [2.5 U Platinum Pfx DNA polymerase, 0.2 mM dNTP, 3 µM of each primer (the forward primer introduced a 5' EcoRI site), and 200 ng of hTSHr-pcDNA3 as template]. The PCR product, the hTSHr cDNA in pcDNA3, and the RT-SK+ constructs were digested with EcoRI/PstI, PstI/XbaI, and EcoRI/XbaI, respectively, and a trimolecular ligation was performed (RT-hTSHr-pcDNA3: RT-hTSHR-pcDNA3).
A SacII restriction site was introduced in the SP of the human TSHr at codon 16 by the QuikChange site mutagenesis method (Stratagene), starting with TSHr in pBluescript SK+ and two synthetic oligonucleotide primers containing the desired mutation (34, 54). The resulting mutated hTSHr construct was excised with KpnI/XbaI and cloned in pcDNA3 (hTSHr-pcDNA3-SacII).
A cDNA fragment of the RT-hTSHr construct including the AflIII site at codon 232 was generated with a forward primer introducing a SacII site in the 5' position, together with codons 17–20 of the hTSHr. This PCR product, the hTSHR-pcDNA3, and the hTSHR-pcDNA3-SacII constructs were digested with SacII/AflIII, AflIII/XbaI and SacII/XbaI, respectively, and a trimolecular ligation was performed (SP-RT-hTSHR-pcDNA3: SP-RT-TSHR-pcDNA3).
The TSHr constructs with truncations extending to various positions in the ectodomain were PCR amplified with Platinum Pfx DNA polymerase and the hTSHr-pcDNA3 as template, as described above (the oligonucleotide sequences used for PCR amplification of the various sequences are available upon request). The purified PCR products were digested with EcoRI/BamHI and ligated into the RT-TSHr-pcDNA3 vector (resulting in serpentine-KFLR, -FNPC, -YDY constructs) or SP-RT-TSHr-pcDNA3 vector (resulting in serpentine-GFGQ, -KNQK, -LKKL, -LDV, -QGYA, -LKFL, -TQTL constructs). All constructs were amplified in DH5F' competent cells, and recombinant DNA was purified from selected clones and sequenced for confirmation of the nucleotide sequences. All the chimeras are summarized in Fig. 1.
Mutants of the SP-RT-TSHr and RT-KFLR
Artificial mutation S281L (47, 55) or mutations reported elsewhere in patients with autonomous thyroid adenomas or hereditary hyperthyroidism (I486F, I568T, del658–661, D633A, A623I; Refs. 9, 33 , and 43) were introduced in the hTSHr by the QuikChange site mutagenesis method, starting with TSHr in pBluescript SK+, as previously described (34, 54). Mutant RT-KFLRs and SPRT-TSHrs were obtained by subcloning the appropriate portions of TSHr mutants and sequenced for confirmation of the nucleotide sequence of the PCR-generated areas.
Transfection Experiments
COS-7 cells were used for transient expression allowing functional assays. They were transfected by the diethylaminoethyl-dextran method followed by a dimethylsulfoxide shock as described previously (43, 56). Two days after transfection, cells were used for cAMP determinations and flow immunocytofluorometry. Triplicate dishes were used for each assay. Each experiment was repeated at least twice. Cells transfected with the empty pcDNA3 vector were always run as controls. To demonstrate that constitutive activity toward the cAMP pathway is linearly related to the number of receptors present at the cell surface, variable amounts of SP-RT TSHr-pcDNA3 or SP-RT I568T-pcDNA3 constructs (complemented to 500 ng of DNA with empty pcDNA3 vector) were tranfected in COS cells.
Quantification of Cell Surface Expression of TSHr Constructs by Flow Immunocytometry
Cells were prepared as previously described (43). After detachment, they were centrifuged at 500 x g at 4 C for 3 min and the supernatant was discarded. They were then incubated for 30 min at room temperature in 100 µl 0.1% PBS-BSA containing either the BA8 Mab (38) or the OR2-15A-6 Mab directed against the N terminus of bovine rhodopsin (53). Cells were then washed with 4 ml 0.1% PBS-BSA and centrifuged as described above. They were incubated on ice for 30 min, in the dark, with fluorescein-conjugated 
-chain-specific goat antimouse IgG (Sigma, St. Louis, MO) in the same buffer. Propidium iodide (10 µg/ml) was used for detection of damaged cells that were excluded from the analysis. Cells were washed and resuspended in 250 µl 0.1% PBS-BSA. The fluorescence of 10,000 cells per tube was assayed by a FACScan flow cytofluorometer (Becton Dickinson and Co., Eerenbodegem, Belgium).
cAMP Determination
For cAMP determinations, culture medium was removed 48 h after transfection and replaced by Krebs-Ringer-HEPES buffer for 30 min. Thereafter, cells were incubated for 60 min in fresh Krebs-Ringer-HEPES buffer supplemented with 25 µM of the phosphodiesterase inhibitor Rolipram (Laboratory Logeais, Paris, France) and various concentrations of bovine TSH (Sigma). At the end of a 1-h incubation, the medium was discarded and replaced with 0.1 M HCl. The cell extracts were dried in a vacuum concentrator, resuspended in water, and diluted appropriately for cAMP measurements by RIA according to the method of Brooker et al. (57). Duplicate samples were assayed in all experiments; results are expressed as picomoles cAMP per milliliter. Basal cAMP was normalized to cell-surface expression for each construct. To this end, receptor-dependent cAMP accumulation (i.e. cAMP in receptor-transfected cells—cAMP in pcDNA3-transfected cells) was divided by the receptor-dependent fluorescence measured by flow immunocytofluorometry (fluorescence of receptor-transfected cells—fluorescence of the pcDNA3-transfected cells). The values were then normalized to the basal activity of the wild-type TSHR, arbitrarily set to 1. The validity of this method of normalization was assessed by the observation that, for the range of constitutive activity investigated, a linear relation exists between cAMP accumulation and receptor expression at the cell surface (see Fig. 3D).
Normalized constitutive activities of the mutant TSHrs and wild-type TSHr were compared using the nonparametric unpaired Wilcoxon test. Differences were considered statistically significant when P < 0.05. We used the Stata 7.0 package (Stata Corp., College Station, TX).
Western Blot of SP-RT TSHr
Preparation of receptor.
Six dishes, each containing 300,000 cells transfected with pcDNA3, TSHr-pcDNA3, or SP-RT-TSHr pcDNA3 constructs, were treated with 5 mM EDTA and 5 mM EGTA in PBS, and the cells were spun down at 280 x g. The cell pellet was suspended and homogenized in a Potter-Elvehjem glass homogenizer with a teflon pestle (Fisher Bioblock Scientific, Tournai, Belgium) in 1,250 µl of lysis buffer [100 mM (NH4)2SO4, 20 mM TRIS at pH 7.5, and 10% glycerol] containing protease inhibitors (Complete, Roche Molecular Biochemicals, Somerville, NJ). The lysate was then centrifuged at 500 x g for 10 min and the supernatant recovered for further ultracentrifugation at 30,000 x g for 30 min. Two hundred microliters of lysis buffer containing 1% N-dodecyl-ß-D-maltoside (Anatrace, Maumee, OH) was added to the pellet and the suspension incubated for another 30 min at 4 C under constant rotation to allow thorough mixing. Final centrifugation was carried out at 100,000 x g for 1 h and the supernatant was stored at -80 C for further use. All procedures described were performed at 4 C.
SDS-PAGE and Immunoblotting
Three microliters of Laemli sample buffer (5x) containing SDS (10%) and ß-mercaptoethanol (1 M) as a reducing agent were added to 10 µl receptor protein, prepared as described above, and denatured at 40 C for 1 h. The sample was then run on 7% acrylamide gel and probed with Mab 28 (culture supernatant diluted 1:50), which recognizes a linear epitope at the N-terminal of the ectodomain from amino acid residues 31–50. The proteins were visualized with an antimouse IgG horseradish peroxidase conjugate and the ECL Plus Western blotting detection system (Amersham Pharmacia Biotech Benelux, The Netherlands).
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
2 Present address for V.V.-G.: Clinique Marc Linquette, Centre Hospitalo Universitaire de Lille, 59037 Lille, France.
Abbreviations: EC50, Fifty-percent effective concentration; GPCR, G protein-coupled receptor; hTSHr, human TSHr; Mab, monoclonal antibody; RT, rhodopsin tag; SCA, specific constitutive activity; SP, signal peptide; TSHr, TSH receptor.
Received for publication August 17, 2001. Accepted for publication December 17, 2001.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
2-adrenergic receptor. J Biol Chem 268:16483–164871B-adrenergic receptor by all amino acid substitutions at a single site. Evidence for a region which constrains receptor activation. J Biol Chem 267:1430–1433 genes as a cause of toxic thyroid adenomas. J Clin Endocrinol Metab 82:2695–2701
isoleucine) in the extracellular domain of the thyrotropin receptor. J Clin Invest 100:1634–1639[Medline]
This article has been cited by other articles:
 |
|
 |
|
  S. Neumann, W. Huang, S. Titus, G. Krause, G. Kleinau, A. T. Alberobello, W. Zheng, N. T. Southall, J. Inglese, C. P. Austin, et al. Small-molecule agonists for the thyrotropin receptor stimulate thyroid function in human thyrocytes and mice PNAS, July 28, 2009; 106(30): 12471 - 12476. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. G. Baratta, I. Porreca, and R. Di Lauro Oncogenic Ras Blocks the cAMP Pathway and Dedifferentiates Thyroid Cells Via an Impairment of Pax8 Transcriptional Activity Mol. Endocrinol., June 1, 2009; 23(6): 838 - 848. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Kleinau and G. Krause Thyrotropin and Homologous Glycoprotein Hormone Receptors: Structural and Functional Aspects of Extracellular Signaling Mechanisms Endocr. Rev., April 1, 2009; 30(2): 133 - 151. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Rigutto, C. Hoste, H. Grasberger, M. Milenkovic, D. Communi, J. E. Dumont, B. Corvilain, F. Miot, and X. De Deken Activation of Dual Oxidases Duox1 and Duox2: DIFFERENTIAL REGULATION MEDIATED BY cAMP-DEPENDENT PROTEIN KINASE AND PROTEIN KINASE C-DEPENDENT PHOSPHORYLATION J. Biol. Chem., March 13, 2009; 284(11): 6725 - 6734. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Agrawal and R. R. Dighe Critical Involvement of the Hinge Region of the Follicle-stimulating Hormone Receptor in the Activation of the Receptor J. Biol. Chem., January 30, 2009; 284(5): 2636 - 2647. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Bruysters, M. Verhoef-Post, and A. P. N. Themmen Asp330 and Tyr331 in the C-terminal Cysteine-rich Region of the Luteinizing Hormone Receptor Are Key Residues in Hormone-induced Receptor Activation J. Biol. Chem., September 19, 2008; 283(38): 25821 - 25828. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Kleinau, H. Jaeschke, S. Mueller, B. M. Raaka, S. Neumann, R. Paschke, and G. Krause Evidence for cooperative signal triggering at the extracellular loops of the TSH receptor FASEB J, August 1, 2008; 22(8): 2798 - 2808. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C.-R. Chen, S. M. McLachlan, and B. Rapoport Identification of Key Amino Acid Residues in a Thyrotropin Receptor Monoclonal Antibody Epitope Provides Insight into Its Inverse Agonist and Antagonist Properties Endocrinology, July 1, 2008; 149(7): 3427 - 3434. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Mizutori, C.-R. Chen, S. M. McLachlan, and B. Rapoport The Thyrotropin Receptor Hinge Region Is Not Simply a Scaffold for the Leucine-Rich Domain but Contributes to Ligand Binding and Signal Transduction Mol. Endocrinol., May 1, 2008; 22(5): 1171 - 1182. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Feng, T. Muller, D. Mizrachi, F. Fanelli, and D. L. Segaloff An Intracellular Loop (IL2) Residue Confers Different Basal Constitutive Activities to the Human Lutropin Receptor and Human Thyrotropin Receptor through Structural Communication between IL2 and Helix 6, via Helix 3 Endocrinology, April 1, 2008; 149(4): 1705 - 1717. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Ando, R. Latif, and T. F Davies Antibody-induced modulation of TSH receptor post-translational processing J. Endocrinol., October 1, 2007; 195(1): 179 - 186. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Grasberger, J. Van Sande, A. Hag-Dahood Mahameed, Y. Tenenbaum-Rakover, and S. Refetoff A Familial Thyrotropin (TSH) Receptor Mutation Provides in Vivo Evidence that the Inositol Phosphates/Ca2+ Cascade Mediates TSH Action on Thyroid Hormone Synthesis J. Clin. Endocrinol. Metab., July 1, 2007; 92(7): 2816 - 2820. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C.-R. Chen, S. M. McLachlan, and B. Rapoport Suppression of Thyrotropin Receptor Constitutive Activity by a Monoclonal Antibody with Inverse Agonist Activity Endocrinology, May 1, 2007; 148(5): 2375 - 2382. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Nurwakagari, A. Breit, C. Hess, H. Salman-Livny, D. Ben-Menahem, and T. Gudermann A conformational contribution of the luteinizing hormone-receptor ectodomain to receptor activation J. Mol. Endocrinol., February 1, 2007; 38(2): 259 - 275. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S.-C. Ho, S.-S. Goh, I. H C Kee, P. K H Chow, C.-P. Yeo, and D. H C Khoo Effects of genetic immunization of Swiss outbred mice with human thyroid stimulating hormone receptor cDNA plasmids harboring gain-of-function mutations J. Mol. Endocrinol., February 1, 2007; 38(2): 277 - 288. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Rossi, A. Dimida, M. T. Dell'anno, M. L. Trincavelli, P. Agretti, F. Giorgi, G. U. Corsini, A. Pinchera, P. Vitti, M. Tonacchera, et al. The Thyroid Disruptor 1,1,1-Trichloro-2,2-Bis(p-Chlorophenyl)-Ethane Appears to Be an Uncompetitive Inverse Agonist for the Thyrotropin Receptor J. Pharmacol. Exp. Ther., January 1, 2007; 320(1): 465 - 474. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Bonomi, M. Busnelli, L. Persani, G. Vassart, and S. Costagliola Structural Differences in the Hinge Region of the Glycoprotein Hormone Receptors: Evidence from the Sulfated Tyrosine Residues Mol. Endocrinol., December 1, 2006; 20(12): 3351 - 3363. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Mueller, G. Kleinau, H. Jaeschke, S. Neumann, G. Krause, and R. Paschke Significance of Ectodomain Cysteine Boxes 2 and 3 for the Activation Mechanism of the Thyroid-stimulating Hormone Receptor J. Biol. Chem., October 20, 2006; 281(42): 31638 - 31646. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Van Durme, F. Horn, S. Costagliola, G. Vriend, and G. Vassart GRIS: Glycoprotein-Hormone Receptor Information System Mol. Endocrinol., September 1, 2006; 20(9): 2247 - 2255. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
U. Ringkananont, J. Van Durme, L. Montanelli, F. Ugrasbul, Y. M. Yu, R. E. Weiss, S. Refetoff, and H. Grasberger Repulsive Separation of the Cytoplasmic Ends of Transmembrane Helices 3 and 6 Is Linked to Receptor Activation in a Novel Thyrotropin Receptor Mutant (M626I) Mol. Endocrinol., April 1, 2006; 20(4): 893 - 903. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Jaeschke, S. Neumann, G. Kleinau, S. Mueller, M. Claus, G. Krause, and R. Paschke An Aromatic Environment in the Vicinity of Serine 281 Is a Structural Requirement for Thyrotropin Receptor Function Endocrinology, April 1, 2006; 147(4): 1753 - 1760. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Claus, H. Jaeschke, G. Kleinau, S. Neumann, G. Krause, and R. Paschke A Hydrophobic Cluster in the Center of the Third Extracellular Loop Is Important for Thyrotropin Receptor Signaling Endocrinology, December 1, 2005; 146(12): 5197 - 5203. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Costagliola, E. Urizar, F. Mendive, and G. Vassart Specificity and promiscuity of gonadotropin receptors Reproduction, September 1, 2005; 130(3): 275 - 281. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Karges, G. Krause, J. Homoki, K.-M. Debatin, N. de Roux, and W. Karges TSH receptor mutation V509A causes familial hyperthyroidism by release of interhelical constraints between transmembrane helices TMH3 and TMH5 J. Endocrinol., August 1, 2005; 186(2): 377 - 385. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Karges, S. Gidenne, C. Aumas, F. Haddad, P. A. Kelly, E. Milgrom, and N. de Roux Zero-Length Cross-Linking Reveals that Tight Interactions between the Extracellular and Transmembrane Domains of the Luteinizing Hormone Receptor Persist during Receptor Activation Mol. Endocrinol., August 1, 2005; 19(8): 2086 - 2098. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Urizar, S. Claeysen, X. Deupi, C. Govaerts, S. Costagliola, G. Vassart, and L. Pardo An Activation Switch in the Rhodopsin Family of G Protein-coupled Receptors: THE THYROTROPIN RECEPTOR J. Biol. Chem., April 29, 2005; 280(17): 17135 - 17141. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Neumann, M. Claus, and R. Paschke Interactions between the extracellular domain and the extracellular loops as well as the 6th transmembrane domain are necessary for TSH receptor activation Eur. J. Endocrinol., April 1, 2005; 152(4): 625 - 634. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Kleinau, H. Jaschke, S. Neumann, J. Lattig, R. Paschke, and G. Krause Identification of a Novel Epitope in the Thyroid-stimulating Hormone Receptor Ectodomain Acting as Intramolecular Signaling Interface J. Biol. Chem., December 3, 2004; 279(49): 51590 - 51600. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Joubert, B. Hanson, G. Barthet, M. Sebben, S. Claeysen, W. Hong, P. Marin, A. Dumuis, and J. Bockaert New sorting nexin (SNX27) and NHERF specifically interact with the 5-HT4(a) receptor splice variant: roles in receptor targeting J. Cell Sci., October 15, 2004; 117(22): 5367 - 5379. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. R. Farid and M. W. Szkudlinski Minireview: Structural and Functional Evolution of the Thyrotropin Receptor Endocrinology, September 1, 2004; 145(9): 4048 - 4057. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Montanelli, J. J. J. Van Durme, G. Smits, M. Bonomi, P. Rodien, E. J. Devor, K. Moffat-Wilson, L. Pardo, G. Vassart, and S. Costagliola Modulation of Ligand Selectivity Associated with Activation of the Transmembrane Region of the Human Follitropin Receptor Mol. Endocrinol., August 1, 2004; 18(8): 2061 - 2073. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Vassart and S. Costagliola A Physiological Role for the Posttranslational Cleavage of the Thyrotropin Receptor? Endocrinology, January 1, 2004; 145(1): 1 - 3. [Full Text] [PDF]
|
|
|
|
|
|
C.-R. Chen, G. D. Chazenbalk, S. M. McLachlan, and B. Rapoport Evidence that the C Terminus of the A Subunit Suppresses Thyrotropin Receptor Constitutive Activity Endocrinology, September 1, 2003; 144(9): 3821 - 3827. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Vasseur, P. Rodien, I. Beau, A. Desroches, C. Gerard, L. de Poncheville, S. Chaplot, F. Savagner, A. Croue, E. Mathieu, et al. A Chorionic Gonadotropin-Sensitive Mutation in the Follicle-Stimulating Hormone Receptor as a Cause of Familial Gestational Spontaneous Ovarian Hyperstimulation Syndrome N. Engl. J. Med., August 21, 2003; 349(8): 753 - 759. [Full Text] [PDF]
|
|
|
|
|
|
G. Smits, O. Olatunbosun, A. Delbaere, R. Pierson, G. Vassart, and S. Costagliola Ovarian Hyperstimulation Syndrome Due to a Mutation in the Follicle-Stimulating Hormone Receptor N. Engl. J. Med., August 21, 2003; 349(8): 760 - 766. [Full Text] [PDF]
|
|
|
|
|
|
C.-R. Chen, G. D. Chazenbalk, S. M. McLachlan, and B. Rapoport Targeted Restoration of Cleavage in a Noncleaving Thyrotropin Receptor Demonstrates that Cleavage Is Insufficient to Enhance Ligand-Independent Activity Endocrinology, April 1, 2003; 144(4): 1324 - 1330. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Latif, P. Graves, and T. F. Davies Ligand-dependent Inhibition of Oligomerization at the Human Thyrotropin Receptor J. Biol. Chem., November 15, 2002; 277(47): 45059 - 45067. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
; EC50 = 1.4 mIU/ml) and in SPRT-TSHr (
; EC50 = 1.9 mIU/ml). Results were analyzed by nonlinear regression using the GraphPad Software, Inc. (San Diego, CA) Prism software.
