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Endocrinology Vol. 147, No. 4 1753-1760
Copyright © 2006 by The Endocrine Society

An Aromatic Environment in the Vicinity of Serine 281 Is a Structural Requirement for Thyrotropin Receptor Function

H. Jaeschke1, S. Neumann1, G. Kleinau, S. Mueller, M. Claus, G. Krause and R. Paschke

Third Medical Department, University of Leipzig (H.J., S.M., M.C., R.P.), 04103 Leipzig, Germany; National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health (S.N.), Bethesda, Maryland 20892; and Leibniz-Institute for Molecular Pharmacology (G.Kl., G.Kr.), 13125 Berlin, Germany

Address all correspondence and requests for reprints to: Dr. Ralf Paschke, Third Medical Department, University of Leipzig, Ph. Rosenthal Strasse 27, 04103 Leipzig, Germany. E-mail: ralf.paschke{at}medizin.uni-leipzig.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The majority of constitutively activating human TSH receptor (hTSHR) mutations are located in the transmembrane helices as well as in the extracellular (ECLs) and intracellular loops. S281 is one of two positions in the ectodomain in which activating hTSHR mutations have been identified in vivo (S281T, I, and N). To investigate the functional properties of this key residue in more detail, S281 was replaced by each of the other 19 amino acids. Many substitutions led to constitutive receptor activation, suggesting that S281 plays a pivotal role in maintaining the receptor in its inactive state. Strikingly, all substitutions with aromatic residues (S281W, F, Y, and H) show expression similar to that of wild-type hTSHR and are tolerated at this position because they maintain basal activity or express only slight constitutive activity. Three-dimensional modeling of the hTSHR suggested that S281 and surrounding residues are in close proximity to ECL1. To investigate the possible importance of an aromatic environment between the ectodomain in the vicinity of S281 and ECL1, aromatic residues Y279, Y476, H478, Y481, Y482, and H484 were replaced by alanine. Functional characterization showed impaired cell surface expression and signaling for Y279A and Y481A, in contrast to the other alanine mutants. However, substitutions of Y279 and Y481 with other aromatic residues exhibited surface expression and signaling comparable to wild-type hTSHR. Our results suggest that Y279 in the extracellular domain and probably Y481 in the ECL1 also are involved in an aromatic environment around S281 in the hTSHR, which is important for functional receptor conformation and intramolecular receptor signaling.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
TOGETHER WITH THE human chorionic gonadotropin/LH receptor (hCG/LHR), human FSH receptor (hFSHR), and leucine-rich repeat (LRR) containing glycoprotein receptors, the human TSH receptor (hTSHR) belongs to the subfamily of glycoprotein hormone receptors (GPHRs) (1, 2, 3). The TSHR couples preferentially to Gs, resulting in activation of the cAMP-protein kinase A cascade. At 10-fold higher TSH concentrations, TSHR also triggers the Gq-mediated activation of phospholipase C. However, cAMP production is the main pathway for the growth and function of thyroid epithelial cells (4).

Despite numerous identified hTSHR mutations and their functional in vitro characterization, the precise molecular mechanism of hTSHR activation is not understood (4, 5). After interaction between ligand and receptor, the signal is believed to pass the extracellular domain (ECD), extracellular loops (ECLs), transmembrane helices (TMHs), and intracellular loops to induce activated conformation of the receptor, which allows activation of G proteins. Therefore, interactions among these receptor domains are likely to be involved in intramolecular signal transduction (6, 7, 8, 9, 10). However, few details of this complex protein structure-function relationship are known (11, 12, 13).

Naturally occurring hTSHR mutations give valuable indications for amino acid positions that are probably involved in signal transduction and offer a target for the design of functional studies to understand the molecular mechanisms of receptor activation in more detail. The majority of constitutively activating hTSHR mutations is located in the TMHs and ECLs (14, 15, 16, 17, 18). Besides R310C, S281 is the only position in the ECD that harbors activating hTSHR mutations in vivo (S281T, I, and N) (19, 20, 21, 22) (Fig. 1Go). The conserved S281 of the hTSHR, like the corresponding positions S277 in the hCG/LHR and S273 in the hFSHR, is located in the hinge region between the LRR motif in the ECD and the TMHs. Several studies have shown that most mutations of this serine lead to constitutive activation (19, 20, 21). Substitutions of S277 in the hCG/LHR by all other 19 amino acids increased basal activity by varying degrees (23). In contrast to the hCG/LHR, only two mutations in the hTSHR, S281A and G, were functionally characterized in addition to the naturally occurring S281T, I, and N mutations (24). Substitution by A had no stimulatory effect on basal cAMP activity, whereas T, I, N, and G resulted in constitutive activity of the hTSHR, underlining the importance of this conserved serine for GPHR activation.


Figure 1
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  		FIG. 1. Relative localization of mutated residues in TSHR. A schematic arrangement of the TSHR is shown, highlighting residues S281 and Y279 of the ECD and Y476, Y481, Y482, and H484 of the transition of TMH2 and ECL1. Residue numbers were determined by counting from the methionine start site of TSHR.

 
In our three-dimensional (3D) TSHR model, a spatial orientation between the LRR structure and the linked components of the cysteine box 2 (C-b2; hTSHR 280–301) and cysteine box 3 (C-b3; hTSHR 390–410) was demonstrated (10). The S281 region forms a loop/turn structure between the LRR and the C-b2 motive. The C-b3 motif is located near TMH1 and organizes the disulfide-linked C-b2, the C-terminal LRR motif, and subsequently also the S281 region close to the TMHs and ECLs. Mutagenesis studies on the hFSHR and hCG/LHR have shown that the second and third ECLs play important roles in GPHR signal transduction (12, 25, 26, 27, 28, 29, 30). In contrast to ECL2 and -3, little is known about the function of ECL1 in GPHRs (31, 32, 33, 34, 35). The functional characteristics of three hTSHR in vivo mutations, T477I (inactive) (35) and I486F and M (active) (16, 17, 18), indicate that ECL1 also plays an important role in the structure and signaling of the hTSHR. Moreover, position D474 in the hTSHR within the junction of TMH2 and ECL1 was intensively functionally characterized by in vitro studies, demonstrating that this position is crucial for hTSHR activation (36).

To functionally characterize hTSHR position S281 in more detail, first we substituted it with all other 19 amino acids. Analysis of these data in combination with molecular modeling predicted the importance of aromatic residues in the vicinity of S281 (Fig 1Go). Subsequent mutagenesis revealed that Y279 and probably Y481 are involved in this aromatic environment, which is important for receptor conformation and signal transduction of the TSHR.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Site-directed mutagenesis
Mutations were introduced into the human hTSHR via the QuikChange site-directed mutagenesis kit (Stratagene). hTSHR-pSVL (37) was used as a template. PCR products containing mutations were digested with BspTI and Eco91I (MBI Fermentas, Vilnius, Lithuania) and used to replace the analogous BspTI/Eco91I fragment in the wild-type (wt) hTSHR-pSVL vector. Sequences of mutated receptors were verified by dideoxy sequencing with Big Dye Terminator Cycle Sequencing chemistry (AB Advanced Biotechnologies, Inc., Columbia, MD). Sequencing reactions were analyzed on a Genetic analyzer ABI 310 (Applied Biosystems, Darmstadt, Germany).

Cell culture and transfection
COS-7 cells were grown in DMEM supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 10 µg/ml streptomycin (Invitrogen Life Technologies, Inc., Paisley, UK) at 37 C in a humidified 5% CO2 incubator. Cells were transiently transfected in 24-well plates (0.5 x 105 cells/well) with 0.5 µg DNA/well for cAMP accumulation and bTSH binding analysis. For determinations of inositol phosphate (IP) formation, cells were transiently transfected in 12-well plates (1 x 105 cells/well) with 1 µg DNA/well. COS-7 cells were transfected using FuGene 6 reagent (Roche, Basel, Switzerland).

FACS analysis
After transfection, cells were cultured for 48 h, harvested using 1 mM EDTA and 1 mM EGTA in PBS, and transferred to Falcon 2058 tubes (BD Biosciences, Franklin Lakes, NJ). Cells were washed once with PBS containing 0.1% BSA and 0.1% NaN3 (binding buffer), incubated for 1 h with a 1:200 dilution of mouse antihuman TSHR antibody (Serotec Ltd., Oxford, UK) in binding buffer, washed twice, and incubated for 1 h in the dark with a 1:200 dilution in binding buffer of a fluorescein-labeled F(ab')2 rabbit antimouse IgG (Serotec Ltd.). Before FACS analysis (FACSCalibur, BD Biosciences), cells were washed twice and fixed with 1% paraformaldehyde. Receptor expression was estimated by fluorescence intensity, and transfection efficiency was estimated from the percentage of fluorescent cells.

cAMP accumulation assay
Measurement of cAMP accumulation was performed 48 h after transfection as previously described (38).

Stimulation of IP formation
Transfected COS-7 cells were incubated with 2 µCi [myo-3H]inositol (Amersham Biosciences, Braunschweig, Germany) for 8 h. Thereafter, cells were preincubated with serum-free DMEM containing 10 mM LiCl2 for 30 min. Evaluation of basal and bTSH-induced increases in intracellular IP levels was performed by anion exchange chromatography as previously described (39). IP values are expressed as the percentage of radioactivity incorporated from [3H]IP-1 to -3 over the sum of radioactivity incorporated in IPs and phosphatidylinositol.

Specific constitutive activity
COS-7 cells were transiently transfected in 48-well plates (0.5 x 105 cells/well) with increasing concentrations of wt and mutant DNA (50, 100, 150, 200, 250, and 300 ng/well). For radioligand binding assays, cells were incubated in the presence of 180,000–200,000 cpm [125I]bTSH (BRAHMS Diagnostica, Berlin, Germany) supplemented with 5 mIU/ml nonlabeled bTSH (Sigma-Aldrich Corp., St. Louis, MO). For cAMP assays, 48 h after transfection, cells were incubated with serum-free DMEM containing 1 mM 3-isobutyl-1-methylxanthine (Sigma-Aldrich Corp.) for 1 h. Cells were washed once with 1x PBS, then lysed using 0.1 N HCl. Supernatants were collected and dried. cAMP levels were determined using the cAMP AlphaScreen assay (PerkinElmer Life Sciences, Zaventem, Belgium) according to the instructions of the manufacturer. Basal cAMP formation as a function of receptor expression was analyzed according to Ballesteros et al. (40) using the fitting module of PRISM 2.01 for Windows (GraphPad, Inc., San Diego, CA).

Molecular modeling
The initial 3D structure of the TMHs of the hTSHR was established on the basis of the 3D structure of bovine rhodopsin (41) [Protein Data Base (Research Collaboratory for Structural Bioinformatics) entry codes 1F88, 1HZX, and 1L9H]. The construction of the transmembrane hTSHR model has been described previously (14). Modifications were necessary on TMH2 and TMH5. In rhodopsin’s TMH2, interactions of the side chains of two successive threonines with the helical backbone of the preceding residues caused a bulge in TMH2. In hTSHR, neither threonines nor prolines existed in TMH2, indicating a regular {alpha}-helix that extends to Y481. Consequently, residues D474, T477, H478, and Y481 are oriented toward the interior side of the receptor.

The construction of an ectodomain model for hTSHR has been described previously (10). The ectodomain and serpentine domain models were assembled manually or by constrained molecular dynamics simulations considering functional data and complementary side-chain properties with the biopolymer module of the SYBYL program package (TRIPOS, Inc., St. Louis, MO).

The assembled ecto-/serpentine domain model of hTSHR was embedded according to its extracellular, transmembrane, and intracellular portions in a triphase water-vacuum-water box. Initially, the ectodomain atoms were kept fixed to relax the water during minimization. Later, the entire system was considered without restraints. Minimizations were also performed until converging at a termination gradient of 0.05 kcal/mol·Å to a root mean square force of 0.05 kcal/mol·Å. Molecular dynamics simulations were performed at 300K for 1 nsec. For both, the AMBER 7.0 force field (42) was used, and the geometric quality of the model was controlled using PROCHECK software (43).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
All hTSHR mutants were functionally characterized in transiently transfected COS-7 cells by determination of cell surface expression, basal and bTSH-induced cAMP accumulation, and phosphoinositide hydrolysis. Specific constitutive activity was investigated for all S281 mutants. The data are summarized in Tables 1Go and 2Go. Cells transfected with a DNA construct encoding the wt hTSHR or the pSVL vector alone were used as controls.


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  		TABLE 1. Functional characterization of mutants at amino acid position 281 in the extracellular domain

 

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  		TABLE 2. Functional analysis of substitutions of aromatic residues within the TMH2/ECL1 transition

 
Functional assessment of the hTSHR mutants at position S281
Basal and bTSH-stimulated cAMP accumulation.
Specific constitutive activity was determined to evaluate all S281 mutants independently from their cell surface expression. Fifteen mutants at amino acid position 281 (S281L, D, I, V, M, E, N, T, Q, P, G, W, C, H, and F) revealed constitutive activity with varying degrees from 3.6- to 86.4-fold over wt hTSHR basal (Table 1Go and Fig. 2Go). Replacement of S281 with the weak hydrophobic and small A or the slight hydrophilic Y with an aromatic side chain maintained a basal cAMP activity comparable to wt hTSHR. S281F, H, and W were characterized by only slight constitutive activity, suggesting that aromatic side chains are well tolerated in the vicinity of S281 (Table 1Go and Fig. 2Go). In contrast, amino acids with positively charged side chains (S281K and R) led to the loss of basal and bTSH-induced cAMP production, possibly due to strongly impaired cell surface expression (Table 1Go). Interestingly, despite very low cell surface expression, S281D was constitutively active (72.4-fold over wt hTSHR basal value; Table 1Go and Fig. 2Go).


Figure 2
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  		FIG. 2. Substitution of S281 of the hTSHR to all amino acid residues led to various degrees of constitutive activity. Specific constitutive activity is expressed as basal cAMP formation as a function of receptor expression determined by [125I]bTSH binding. Slopes were calculated by linear regression analysis. Mutants S281R and S281K were excluded from the calculation because of their strongly impaired cell surface expression, which results in loss of [125I]bTSH binding. All data are presented as the mean ± SD of two independent experiments, each performed in duplicate.

 
Effects of the mutants on phosphoinositide hydrolysis.
Except for S281I, V, and M, basal IP accumulation for all other mutants was not increased compared with wt hTSHR (Table 1Go). Noteworthy, mutant S281I showed the highest basal IP production despite a cell surface expression of only 22.9% compared with the wt hTSHR. However, maximum bTSH-induced IP accumulation was reduced. As a likely consequence of their strongly reduced cell surface expression, mutants S281K, R, D, and L revealed no activation of Gq-mediated signaling in the presence of bTSH.

Molecular modeling of hTSHR
According to our previous ectodomain model, the LRR domain (hTSHR 37–279), the back to back following C-b2 (hTSHR 280–301), and the linked C-b3 (hTSHR 390–410) are positioned in close proximity (10). In detail, amino acids of the C-b3 are the extreme N-terminal border of TMH1 of glycoprotein hormone receptors. It is known that the two highly conserved cysteines close to TMH1 (C398 and C408; first amino acid of TMH1 is D410) are linked by disulfide bridges to the highly conserved cysteines of C-b2 (C283 and C284) (44, 45). Consequently, C-b2 (hinge region) and C-b3 must be structurally close to establish these disulfide bridges. Serine 281 in the TSHR is directly neighboring cysteines 283 and 284 of C-b2; therefore, it is spatially close to TMH1. Due to 100% sequence homology of 278 SYPSHC 283 to the malonyl coenzyme structure (Protein Data Base entry code 1MLA), the S281 region (hTSHR 279–281) is expected to form a loop/turn conformation that is supported by the S281 side chain. This joined model of C-b2 and C-b3, the leucine-rich repeat motif, and the serpentine domain favors the orientation of C-b2 and the connected S281 region toward TMH1. Based on the structure of rhodopsin, the TMH2 is close to TMH1; therefore, S281 is very likely located between TMH1 and the TMH2/ECL1 junction (10).

In the hTSHR serpentine model, the TMH2 {alpha}-helix extends by one turn toward the junction with ECL1. Subsequently, D474, H478, and Y481 are located at the TMH2/ECL1 junction in helical phase pointing to the interior side of the receptor (Figs. 1Go and 3Go). Moreover, the ring systems of Y481 and H478 of the TMH2/ECL1 junction have the ability to interact with the aromatic system of Y279 from the S281 region (Fig. 3Go) in the joined model. Models of S281 mutants indicate that the small alanine does not interfere with the loop/turn conformation or with the environment. Moreover, they indicate that polar side chains at position 281 and especially those hydrophobic side chains with additional branched bulky extensions at the Cß atom (such as V, I, and T) probably cause clashes with residues of the ECL1. Only those residues with an angled side chain and a shallow aromatic ring system, such as tyrosine (S281Y), are spatially tolerated and can also interact with the ring systems of Y481. The remaining part of ECL1 is located in close proximity to the S281 region.


Figure 3
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  		FIG. 3. 3D model of the hTSHR, including S281, the TMH2/ECL1 transition, and C-b2 and C-b3. The TMH2/ECL1 junction with amino acids of functional importance (boxed annotations) and a potential orientation of the S281 region are shown in detail. C-b3 organizes the disulfide-linked C-b2 and the S281 loop/turn region (hTSHR 279–281) closely to the serpentine domain and favors an orientation of S281 toward ECL1. The residues D474, T477, Y481, and H484 of the TMH2 are oriented toward the interior side of the receptor. Aromatic residues Y279 and Y481 are spatially closest to S281. Amino acid coloring: aromatic side chains, magenta; hydrophobic side chains, green; cysteines, yellow; negatively charged side chains, red.

 
Functional assessment of the hTSHR mutants at Y279 (ECD), Y476, H478, Y481, Y482, and H484 (ECL1)
Basal and bTSH-stimulated cAMP accumulation.
None of these mutants showed constitutive cAMP accumulation (Table 2Go). All alanine substitutions, except Y476A, had decreased basal cAMP activity. Four mutations, Y476A, H478A, Y482A, and H484A, were characterized by cell surface expression in the range of 85.4–103.1% relative to that of wt hTSHR and showed activation for the Gs-adenylate cyclase pathway comparable with that of wt hTSHR after stimulation with bTSH (Table 2Go). Cells transfected with Y279A and Y481A displayed a strongly impaired cell surface expression, and the cAMP response was abolished (Table 2Go). In contrast, replacement of Y279 with the large aromatic amino acid W showed only a slight effect regarding cell surface expression (83.5%), and the ability to produce cAMP was comparable to that of wt hTSHR. Similar characteristics were determined for Y481 when substituted by the nonpolar aromatic residue F (Table 2Go).

Effects of the mutants on phosphoinositide hydrolysis.
As shown for cAMP production, no remarkable differences for mutants Y476A and H478A regarding phosphoinositide hydrolysis were determined when compared with wt hTSHR (Table 2Go). Cells transfected with Y279A and N, and Y481A and N did not show bTSH-induced IP accumulation (Table 2Go). Similar to cAMP production, substitutions to F at position Y481 and to W at position Y279 were well tolerated and activated Gq like wt hTSHR. Y482A and H484A, located in the C-terminal region of ECL1 showed wt properties regarding cAMP accumulation and cell surface expression. Interestingly, a strongly decreased Gq activation was determined for both mutants, whereas Y482A revealed the strongest selective effect between Gs and Gq activation (Table 2Go). Additional studies are necessary to identify the mechanism that leads to selective G protein activation.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The receptor region around S281 is conserved among glycoprotein hormone receptors, and constitutive activation of the hTSHR by mutations at S281 demonstrates a pivotal role for this residue in intramolecular signal transduction. The functional importance of this microdomain is supported by the vicinity of S281 to the C-b2 epitope, in which mutations at positions P280, C283, and C284 also lead to constitutive activity (24, 46), demonstrating that the C-b2 epitope 279 YPSHCC 284 can act as an intramolecular switch for receptor activation (24).

In the present study we investigated all possible natural amino acid substitutions at position S281 and their effects on receptor activation. The determination of specific constitutive activity allowed us to compare all mutants of S281 independently from their cell surface expression. Our data suggest that two distinct types of amino acids at position 281 lead to strong constitutive activity of the hTSHR. The first group includes hydrophobic amino acids V, I, L, and M and the hydrophilic amino acid T, whereas V, I, and T are further characterized by CH3 group extensions at the side-chain Cß atom. Interestingly, the hydrophobic substitutions S281V, I, and M also revealed constitutive activity for the Gq-mediated IP pathway. Similar results could be obtained for the hCG/LHR at position S277 (hTSHR S281) (23, 47). Amino acids I, V, and L displayed, as in the hTSHR at S281, the highest constitutive activity. One explanation might be steric impairment by the bulky branched hydrophobic side chains close to the backbone of position 281 causing a conformational change in the inactive loop/turn conformation of the S281 region. The second group consists of amino acids with a long side chain, containing a proton acceptor. Constitutive activity of these mutants might be caused by a repulsion with a not yet identified residue in close proximity to position 281. Introduction of negatively charged residues at S281 led to effects on basal signaling similar to substitutions by branched hydrophobic amino acids. S281E showed constitutive activity comparable to S277E in hCG/LHR (47). Strikingly, functional characterization of mutation S277D in hCG/LHR was not possible, because no cell surface expression could be determined (47). Mutation S281D in the hTSHR is also characterized by a low cell surface expression, but exhibits high constitutive activity. This finding underlines that differences in receptor activation by mutants at the same position exist between GPHRs and supports the necessity to study each receptor of this family separately.

Surprisingly, not only S281 substitutions by small residues A and G were well tolerated at this position, but also bulky aromatic amino acids W, F, Y, and H showed high expression levels and only slightly increased basal activity. The small side chain of alanine at position 281 does not yield constitutive activity, indicating that it does neither interfere with the inactive turn/loop conformation of the S281 region or with the environment. In contrast, the small side chain of threonine, which contains a branched CH3 group at Cß atom, causes constitutive activity at position 281. In contrast, the effect of aromatic amino acids at position 281 on constitutive activity was also much less pronounced compared with the activating mutations described previously. Substitution of S277 in hCG/LHR (hTSHR S281) by aromatic amino acids W, F, Y, and H likewise showed only slightly increased basal activity, as observed for hTSHR (23). Hence, aromatic residues at positions S281 and S277 are more compatible with the wt function in both receptors. Our 3D model suggests that the side chains of aromatic residues provide an angled orientation and that they lack a bulky branch at the Cß atom. Moreover, the large ring systems are directed in a pocket that accepts aromatic amino acids well. These data support the idea that the native side chain S281 itself is facilitating the turn/loop conformation instead of directly interacting with a partner at the serpentine domain. However, the S281 microdomain is embedded in an aromatic environment.

Consequently, functional characterization of S281 and the derived hypothesis of its aromatic environment enabled refinement of our 3D model of the ECD (10). We hypothesized that a toleration of aromatic residues at position 281 could be best achieved by additional aromatic residues in it is immediate vicinity. Our model positioned the extracellular C-b2 region in the neighborhood of the serpentine domain. A search of the serpentine domain within the TMH2/ECL1 junction identified five aromatic residues (Y476, H478 Y481, Y482, and H484) as potential counterparts for the aromatic 281 environment, but none of those is suggested to be a directly interacting partner of S281. Our data revealed that Y476, H478, Y482, and H484 are not involved in such aromatic vicinity. However, substitutions Y481A and N, and Y279A and N resulted in loss of cell surface expression and receptor signaling. In contrast, mutations Y481F and Y279W were well tolerated at this position, suggesting that the aromatic ring structure of Y481 and Y279 is essential for receptor stability and function. Aromatic residues 279 and 481 are 100% conserved within all GPHRs, suggesting a common mechanism. Due to the spatial proximity of Y481 to Y279 and S281 in our 3D model, these findings indicate that position Y481 may be part of the aromatic environment of S281. However, our data cannot exclude the possibility that only the ring system of Y279 in the vicinity of S281 is necessary.

Taken together, our study provides evidence for 1) a tight conformational packing of the S281 turn/loop region, which tolerates only very small or aromatic side chains at position 281; and 2) an aromatic vicinity of S281 region, which is necessary for receptor trafficking and signaling. Our refined TSHR model and mutagenesis data at the TMH2/ECL1 junction suggest spatial proximity of the microdomain S281 to the junction of TMH2 and ECL1.


   Acknowledgments
 
We thank Mrs. Eileen Bösenberg for her excellent technical assistance. We also thank Dr. Bruce Raaka for critical reading of the manuscript.


   Footnotes
 
This work was supported by grants from Deutsche Forschungsgemeinschaft (PA 423/12-1 and KR 1273/1) and the Bundesministerium für Bildung und Forschung, Interdisciplinary Center for Clinical Research (Project B19), at University of Leipzig.

All authors have nothing to declare.

First Published Online January 12, 2006

1 H.J. and S.N. contributed equally to this work. Back

Abbreviations: bTSH, Bovine TSH; C-b1, cysteine box 1; C-b2, cysteine box 2; C-b3, cysteine box 3; 3D, three-dimensional; ECD, extracellular domain; ECL, extracellular loop; IP, inositol phosphate; GPHR, glycoprotein hormone receptor; hCG/LHR, human chorionic gonadotropin/LH receptor; hFSHR, human FSH receptor; hTSHR, human TSH receptor; LRR, leucine-rich repeat; TMH, transmembrane helix; wt, wild type.

Received September 6, 2005.

Accepted for publication January 4, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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