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Endocrinology Vol. 146, No. 12 5197-5203
Copyright © 2005 by The Endocrine Society

A Hydrophobic Cluster in the Center of the Third Extracellular Loop Is Important for Thyrotropin Receptor Signaling

Maren Claus, Holger Jaeschke, Gunnar Kleinau, Susanne Neumann, Gerd Krause and Ralf Paschke

Medical Department (M.C., H.J., R.P.), University of Leipzig, D-04103 Leipzig, Germany; Forschungsinstitut fuer Molekulare Pharmakologie (G.Kl., G.Kr.), D-13125 Berlin, Germany; and National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases (S.N.), Bethesda, Maryland 20814

Address all correspondence and requests for reprints to: Ralf Paschke, M.D., Medical Department, University of Leipzig, Philipp-Rosenthal-Strasse 27, 04103 Leipzig, Germany. E-mail: pasr{at}medizin.uni-leipzig.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Previous reports on the follicle-stimulating hormone receptor and choriogonadotropic/LH receptor, which belong to the glycoprotein hormone receptor family, suggest that the extracellular loop (ECL) 3 could be a key domain for ligand binding and intramolecular receptor signaling. In contrast to ECLs 1 and 2 of glycoprotein hormone receptors, the ECL3 displays high sequence homology, particularly in the central portion of the loop. Therefore, we opted to identify amino acids with functional importance within ECL3 of the TSH receptor (TSHR). Single alanine substitutions of all residues in ECL3 were generated. Functional characterization revealed the importance of five amino acids in the central portion of ECL3 and K660 at the ECL3/transmembrane helix (TMH) 7 junction for TSHR signaling. Decrease of Gs activation and loss of Gq activation by substitutions of K660 demonstrates a role for this position for TSHR conformation and signal transduction. By molecular modeling, we predicted potential interaction partners of K660:E409 and D410 in the N terminus of TMH1 and D573 in the ECL2. Complementary double mutants did not reconstitute Gs/Gq-mediated signaling, suggesting that K660 is not directly involved in a structural unit between ECL3 and the N terminus of TMH1. These results support a TSHR model in which the side chain of K660 is orientated toward the backbone of ECL2. Moreover, our findings provide evidence that a hydrophobic cluster, comprising residues 652–656 of ECL3, strongly influences intramolecular signal transduction and G protein activation of the TSHR.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE TSH RECEPTOR (TSHR), together with the choriogonadotropic/LH receptor (CG/LHR) and the FSH receptor (FSHR), is a member of the superfamily of seven transmembrane-spanning receptors (1, 2) and belongs to the subfamily of glycoprotein hormone receptors (GPHRs) (3, 4). GPHRs are activated by high molecular weight ligands (TSH, CG, LH, FSH), and one characteristic feature is their large extracellular domain (ECD) (5, 6) consisting of at least 10 leucine-rich repeats (7, 8, 9, 10, 11, 12). This receptor domain is known for its for high affinity hormone binding (13, 14, 15). After interaction between the ligand and the ECD, the signal is predicted to pass on to the extracellular loops (ECLs), transmembrane helices (TMHs), and intracellular loops (ICLs) to induce an activated conformation of the receptor that allows coupling to G-proteins. Therefore, interactions between these receptor domains are likely to be involved in intramolecular signal transduction (7, 9, 10, 16, 17). However, until now, only very few details of this complex protein structure-function relationship are known (18, 19, 20).

An interaction between the ECD and the ECLs is suggested (16, 17, 21). Constitutively activating mutants I486F and I568T in ECL1 and 2 or the deletion del658–661 at the C terminus of ECL3 lose their activity after shortening of the ECD (16). These findings support a model in which activation of the cAMP pathway of the TSHR involves switching of the ectodomain from a tethered inverse agonist to an agonist (16). This also proposes that full stimulation of cAMP production by the TSHR involves more than the release of the ectodomain silencing effect on the serpentine domain. In a previous study, we demonstrated the dependence of constitutive activity of in vivo mutants in the ECLs on the intact function of the ECD. Our results suggest that an interplay between these receptor domains is required for TSHR activation and intramolecular signal transduction (22).

Naturally occurring TSHR mutants give valuable indications for specific functional features, which are important for the analysis of intra- and intermolecular structure-function relationships. The importance of ECLs in signal transduction is underlined by constitutively activating in vivo mutations. Two and one constitutively activating mutations were identified in ECL1 and ECL2, respectively (23, 24) (TSH Receptor mutation Database II, http://www.uni-leipzig.de/~innere/). Three different constitutively activating in vivo mutations in ECL3 (25, 26, 27, 28) point out functional importance of this loop. The relevance of ECL3 for TSHR signaling was also emphasized by analysis of a chimeric rat TSHR in which the entire ECL3 was replaced by corresponding residues of the ß2-adrenergic receptor. The affinity for TSH was increased in this chimeric receptor, whereas cell surface expression and cAMP and inositol phosphate (IP) production were strongly decreased, suggesting ECL3 is involved in signal transduction (29).

FSHR and CG/LHR mutagenesis studies have shown that the ECLs 2 and 3 play an important role in GPHR signal transduction (19, 30, 31, 32, 33, 34, 35). Alanine-scanning mutagenesis in the CG/LHR ECL3 revealed that with exception of P575 and V579 (equivalent to P652 and V656 in the TSHR), mutations caused only moderate effects on CG/LHR binding properties and signaling (36). In a comparable approach, different results were obtained for ECL3 of the FSHR. All mutations resulted in decrease or loss of Gs- and Gq-mediated signal transduction and influenced FSHR cell surface expression and ligand binding. Moreover, introduction of alanine at positions L583 or I584 (equivalent to L653 and I654 in the TSHR) caused improved affinity for FSH, whereas cAMP signaling was abolished (19, 31). The homologous positions of K660 in the TSHR were shown to be essential for signaling in the FSHR (K590) and CG/LHR (K583) (35, 37).

Therefore, the aim of our study was to identify important amino acids within ECL3 of the TSHR, which influence intra- and intermolecular signal transduction. We generated mutants wherein the residues N650–K660 were individually substituted with alanine (Fig. 1Go). Our findings provide evidence for a hydrophobic cluster in the center of ECL3, which severely impairs receptor-mediated IP production and to a lesser extent cAMP production. Moreover, molecular modeling and mutagenesis indicate that K660 is essential for receptor expression and TSH receptor signaling. We provide evidence that K660 is very likely orientated toward ECL2 and is engaged in hydrogen bonding interaction with the backbone carboxyl-oxygen of V566 rather than interacting with an acidic side chain in its spatial environment. This interaction is required for proper folding and signaling of the receptor.



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  		FIG. 1. TSHR mutations within the third ECL. The two dimensional structure of the TSHR is shown, highlighting ECL3 between amino acids 650 and 660 (black) and potential contact partners E409 and D410 at the N terminus of TMH1 and V566 and K573 in ECL2 (asterisks). Residue numbers are determined by counting from the methionine start site of the TSHR.

 

   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Site-directed mutagenesis
The TSHR double mutants were constructed by standard PCR mutagenesis techniques (38) using the human TSHR plasmid TSHR-pSVL as template (39). PCR fragments were digested with Eco81I and Eco91I (MBI Fermentas, Vilnius, Lithuania). The obtained fragments were used to replace the corresponding fragments in the wild-type (wt) TSHR-pSVL constructs. Mutated TSHR sequences were verified by dideoxy sequencing with dRhodamine Terminator Cycle Sequencing chemistry (ABI Advanced Biotechnologies, Inc., Columbia, MD). Sequencing reactions were analyzed on a Genetic analyzer ABI 310 (Applied Biosystems, Darmstadt, Germany).

Cell culture and transient expression of mutant TSHRs
COS-7 cells were grown in DMEM supplemented with 10% fetal calf serum, 100 U/ml penicillin, and 100 µg/ml streptomycin (Life Technologies, Inc., Paisley, UK) at 37 C in a humidified 5% CO2 incubator. Cells were transiently transfected in 12-well plates (1 x 105 cells per well) or 48-well plates (2.5 x 104 cells per well) with 1 µg respective 0.25 µg DNA per well using the GeneJammer Transfection Reagent (Stratagene, Amsterdam, The Netherlands).

Fluorescence-activated cell sorting (FACS) analyses
Transfected cells were detached from the dishes with 1 mM EDTA and 1 mM EGTA in PBS and transferred into Falcon 2054 tubes. Cells were washed once with PBS containing 0.1% BSA and 0.1% NaN3 and then incubated at 4 C for 1 h with a 1:200 dilution of a mouse antihuman TSHR antibody (2C11, 10 mg/liter; Serotec Ltd., Oxford, UK) in the same buffer.

Cells were washed twice and incubated at 4 C for 1 h with a 1:200 dilution of fluorescein-conjugated F(ab')2 rabbit antimouse IgG (Serotec Ltd.). Before FACS analysis (FACscan; Becton Dickinson and Co., Franklin Lakes, NJ) cells were washed twice and then fixed with 1% paraformaldehyde. Receptor expression was determined by the fluorescence intensity, whereas the percentage of signal-positive cells corresponds to the transfection efficiency.

cAMP accumulation assay
For cAMP assays cells were grown and transfected in 48-well plates. Forty-eight hours after transfection, cells were preincubated with serum-free DMEM containing 1 mM 3-isobutyl-1-methylxanthine (Sigma Chemical Co., St. Louis, MO) for 20 min at 37 C in a humidified 5% CO2 incubator. Subsequently, cells were stimulated with 100 mU/ml bovine TSH (bTSH) (Sigma Chemical Co.) for 1 h. Reactions were terminated by aspiration of the medium. The cells were washed once with ice-cold PBS and then lysed by addition of 0.1 N HCl. Supernatants were collected and dried. cAMP content of the cell extracts was determined using the cAMP AlphaScreen Assay (PerkinElmer Life Sciences, Zaventem, Belgium) according to the manufacturer’s instructions.

K660-double mutants and corresponding single mutants were assayed for cAMP activity as previously described (28).

Stimulation of IP formation
Forty hours after transfection, cells were incubated with 2 µCi/ml [myo-3H]inositol (18.6 Ci/mmol; Amersham Pharmacia Biotech, Braunschweig, Germany) for 8 h. Thereafter, cells were preincubated with serum-free DMEM without antibiotics containing 10 mM LiCl for 30 min. Stimulation was performed in the same medium containing 100 mU/ml bTSH (Sigma Chemical Co.) for 1 h. Intracellular IP levels were determined by anion exchange chromatography as described (40). IP values are expressed as the percentage of radioactivity incorporated from IP1–3 over the sum of radioactivity incorporated in IPs and phosphatidylinositoles.

Radioligand binding assay
To investigate TSH binding properties, cells were seeded in 48-well plates (0.25 x 105 cells per well). Competitive binding studies were performed as previously described (28). 125I-bTSH was received from BRAHMS Diagnostika (Henningsdorf, Germany). Data were analyzed assuming a one-site binding model using GraphPad Prism 2.01 for Windows.

Statistics
Statistical analysis was carried out by one-way ANOVA, followed by Dunnett’s multiple comparison test using GraphPad Prism 2.01 for Windows.

Structural bioinformatics and modeling studies
The packing of the seven-helix backbone of the rhodopsin x-ray structure [Protein Database (PDB) entry 1F88 (41), PDB entry 1GZM (42), PDB entry 1U19 (43)] was used as template for the transmembrane helices of the TSHR. The TSHR structure model was computed with special emphasis on the transmembrane and intracellular portions, without the C-terminal tail beyond the eighth helix and the N-terminal ECD but including the ECLs and ICLs. The starting conformation of the ECL2 was also adopted from the rhodopsin structure. For the remaining parts of the ECLs 1 and 3, conformational fragments of four to seven residues were retrieved from the three-dimensional PDB by means of FASTA (44). Overlapping fragments occurring more than once with a similar backbone conformation in the database were used for assembling the loops. In contrast to the rhodopsin structure, the helical transmembrane regions TMH2 and TMH5 of the TSHR model are constructed without a kink and bulge feature, respectively, because GPHR sequences are lacking kink or bulge causing sequences in these helices such as three consecutive threonines in TMH2 and proline in TMH5 region of rhodopsin.

All components were modeled with the biopolymer module of the SYBYL program package (TRIPOS Inc., St. Louis, MO). Conjugate gradient minimizations were performed until converging at a termination gradient of 0.05 kcal/mol·Å. For all energy and dynamics calculation, the AMBER 7.0 force field was used (45). Molecular dynamic simulations for the TSHR model were performed at 300 K for 2 nsec. The TSHR model was soaked with water resulting in a water-vacuum-water box (46). Initially, the atoms were kept fixed to relax the water during minimization. Later on, the entire system was considered. The geometrical quality of the resulting model was controlled using the program PROCHECK (47).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Systematic alanine scan
To identify positions in ECL3 with importance for TSHR signaling, we generated mutants, wherein the residues N650–K660 were substituted with alanine (Fig. 1Go). Because the K651E mutant displayed characteristics similar to the wt TSHR, the K651A mutant was not generated (Table 1Go). The functional characteristics of mutant TSHRs in comparison with the wt TSHR were studied by transient expression in COS-7 cells. Cells transfected with the empty pSVL vector were used as control. The effects of all mutations on bTSH binding, cell surface expression, basal and bTSH-stimulated cAMP, and IP accumulation are summarized in Table 1Go.


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  		TABLE 1. Functional characterization of alanine mutants in ECL3

 
Homologous competitive binding experiments and FACS analyses revealed that all mutant receptors showed TSH binding comparable with the wt TSHR and normal or only moderately decreased cell surface expression in the range of 66–108% of wt TSHR (Table 1Go).

ECL3 mutants displayed basal cAMP activity comparable with the wt TSHR (Table 1Go). In contrast, bTSH-stimulated cAMP signaling was decreased by alanine substitutions of residues P652–V656. Interestingly, the extent of signal reduction was most evident for mutants located in the central part of ECL3. Substitutions P652A, L653A, I654A, and T655A lowered bTSH-stimulated cAMP activity to about 60% of wt TSHR activity, whereas V656A reduced cAMP response to 40% of wt TSHR (Table 1Go). Moreover, mutants I654A and V656A displayed 3.5-fold higher EC50 values (Table 1Go). Stimulated cAMP production of ECL3 mutants N650A and K651E near TMH6 as well as S657A, N658A, and S659A near the ECL3/TMH7 junction was similar to that of the wt TSHR (Table 1Go). In contrast, introduction of alanine at position K660 caused a 60% reduction of cAMP formation compared with wt TSHR after stimulation with bTSH (Table 1Go).

Basal IP levels of all mutants were comparable with that of the wt TSHR (Table 1Go). As described for the cAMP signaling pathway, mutants N650A, K651E, and S659A displayed normal IP formation after stimulation with bTSH (Table 1Go). Ligand stimulated IP production was decreased to 50% of wt TSHR activity in COS-7 cells expressing TSHR mutants P652A, S657A, and N658A in the middle of ECL3. Furthermore, IP signaling was strongly decreased to 5–15% of wt TSHR by introduction of alanine at positions L653, I654, T655, and V656 in the center of ECL3 (Fig. 2BGo and Table 1Go). As observed for cAMP accumulation, mutant K660A appears to be an exception because basal and stimulated IP production is completely abolished in this mutant (Table 1Go).



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  		FIG. 2. Backbone visualization of the wt TSHR with proposed orientations and interaction partners of K660 (ECL3). Homology model of the TSHR serpentine domain (based on the x-ray structure of rhodopsin) with detailed top view of the ECL portions. Theoretical conformational orientations of the K660 side chain interacting with proposed amino acid partners are shown. Besides the three possible charged-charged interactions between the positively charged (blue) K660 (ECL3) and the negatively charged residues (red orange) E409 or D410 (N terminus of TMH1) (A) or D573 (ECL2) (B), also the interaction with the backbone of V566 in ECL2 (C) is possible. Experimental verification of interactions of charged amino acids by double mutants (Table 2Go) excludes an interaction of K660 with one of the acidic residues. Therefore, an orientation of K660 toward the backbone of ECL2 forming a hydrogen bond (blue dashed line) to a backbone carbonyl-oxygen of V566 as a requirement for TSHR signaling function is favored (C).

 
Potential interaction partner for K660 proposed by molecular modeling
Based on relaxed side chain conformations of the TSHR serpentine model, we considered all possible orientations of the K660 side chain using molecular dynamics simulation and determined potential interaction partners that might be able to form salt bridges or hydrogen bonds with K660. Theoretical movement of the K660 side chain at TMH7 has shown potential formation of salt bridges to side chains of residues located at ECL2 (D573) or at the N terminus of TMH1 (E409, D410) or a hydrogen bond to the carbonyl backbone of V566 at ECL2 (Fig. 2Go).

Investigation of potential interactions by site-directed mutagenesis
Single and double mutants of K660, D573, E409, and D410, which encode amino acids of opposite charge to that of wt TSHR, were functionally characterized. The effects of all mutations on cell surface expression, basal and bTSH-stimulated cAMP, and IP accumulation are summarized in Table 2Go.


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  		TABLE 2. Functional characterization of double mutants with K660 and corresponding single mutants

 
Cell surface expression of the single mutants K660E and K660D was decreased to 40–50% of the wt TSHR (set at 100%), and cAMP production was drastically reduced to 20% of wt TSHR (Table 2Go). Gq-mediated IP production was totally abolished by K660E and K660D. Mutation D410K showed comparable characteristics (Table 2Go). In contrast, cell surface expression of mutant E409K was comparable with wt TSHR. Nevertheless, basal and bTSH-stimulated cAMP signaling was strongly affected by this mutant, and IP production was totally abolished (Table 2Go). Mutant D573K in ECL2 displayed a strongly reduced cell surface expression (32% of wt). Despite the relatively high bTSH-induced cAMP response of D573K (70% of wt), nearly no IP accumulation was detectable (12% of wt TSHR) (Table 2Go).

Double mutants K660E/E409K, K660D/D410K, and K660D/D573K displayed reduced cell surface expression compared with wt TSHR (Table 2Go). For all double mutants, cAMP production was strongly impaired. Cells expressing the K660E/E409K construct showed 30% cAMP activity compared with wt TSHR. bTSH-induced cAMP production of K660D/D410K and K660D/D573K was strongly reduced. No basal or stimulated IP production was detectable in all double mutants.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
ECLs and ICLs are the most diverse structural domains of seven transmembrane-spanning receptors. Nevertheless, the ECL3 of the GPHRs displays high sequence homology in its central portion including residues P652–V656 (TSHR). Sequences of the C-terminal ECL3/TMH7 junction are conserved between TSHR and CG/LHR but differ for the FSHR. The N termini of FSHR and CG/LHR ECL3 are identical, whereas the TSHR contains different amino acids in this region (Fig. 3Go).



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  		FIG. 3. cAMP activity of ECL3 mutants. ECL3 and junctions to TMH6 and TMH7 of TSHR, FSHR, and CG/LHR are shown. Residue numbers are determined by counting from the methionine start site of the TSHR. Bold, Positions that significantly affect cAMP signaling after substitution for alanine; underlined, totally abolished cAMP activity after substitution for alanine. Mutation of L, I, and T of the CG/LHR does not significantly affect cAMP signaling (data for FSHR and CG/LHR were taken from Ref.31 ).

 
The importance of ECL3 for ligand binding and signal transduction is well studied for the FSHR and the CG/LHR (19, 31, 32, 34, 35, 36). In vitro mutagenesis for these two receptors revealed that ECL3 is important for receptor activation; however, single amino acids contribute differently (19, 31, 32, 34, 35, 36, 37). Therefore, we studied the function of ECL3 of the TSHR in more detail. This idea was supported by the occurrence of three different constitutively activating in vivo mutations in ECL3 of the TSHR such as N650Y, V656F, and del658–661 (TSH Receptor Mutation Database II, http://www.uni-leipzig.de/~innere/).

The highly conserved position K660 at the ECL3/TMH7 junction has been described previously to be important for the signaling of the CG/LHR (K583) and the FSHR (K590) (19, 37). For this reason and based on the functional characteristics of the TSHR K660A mutant (Table 1Go), we assumed that this amino acid might also be a key position for Gs or Gq activation in TSHR. By molecular modeling, we determined three negatively charged potential interaction partners of the positively charged K660: E409 and D410 in the N terminus of TMH1 and D573 located in ECL2 (Fig. 2Go). However, switching the charges in the respective positions by amino acid substitutions and construction of corresponding double mutants did not rescue the loss of function observed by single substitutions of K660. All single mutants with charged amino acid substitutions led to a loss of function and/or receptor surface expression, which was further exacerbated in double mutants. Because the hypothesized reconstitution of Gs- or Gq-mediated signaling or even a reconstituted expression by the double mutants could not be verified, the likelihood is low that K660 is directly involved in side chain interactions within a structural unit between ECL3 and the N terminus of TMH1 (Fig. 2AGo) or ECL2 (Fig. 2BGo). We therefore favor an orientation of K660 toward the backbone of ECL2 forming a hydrogen bond to the backbone carboxyl-oxygen of V566 as a requirement for TSHR signaling (Fig. 2CGo). However, because contacts of the K660 side chain to the carbonyl backbone cannot be studied by standard mutagenesis approaches, this hypothetic interaction remains to be verified. The reduced cell surface expression of K660E and K660D suggests that the substitution of lysine for negatively charged amino acids might affect TSHR folding and subsequently trafficking of the molecule to the cell membrane. Therefore, strong impairment of Gs and Gq activation by these mutations is most likely caused by conformational changes and their influence on the interplay between TMH6 and TMH7. Our findings indicate that K660 is a critical position in intramolecular signaling of the TSHR.

The functional characterization of the TSHR ECL3 alanine substitutions revealed no significant effects on cell surface expression and TSH binding affinity (Table 1Go). In contrast, mutations at the CG/LHR positions K573, P575, I577, and N581 (TSHR: N650, P652, I654 and N658) resulted in strongly decreased receptor numbers, whereas expression of the other mutants was comparable with the wt CG/LHR (36) (Fig. 3Go). Moreover, alanine mutations in the FSHR ECL3 also caused a decrease of receptor molecules on the cell surface (31). Furthermore, mutation of the hydrophobic cluster in the FSHR ECL3 caused 3-fold increased FSH binding affinities (19), whereas TSHR mutants of the homologous hydrophobic residues showed no influence on TSH binding (Table 1Go). In summary, these data suggest that individual amino acids seem to play different roles in these three receptors regarding correct receptor folding.

For the TSHR ECL3, the constitutively activating in vivo mutations N650Y and V656F are known (TSH Receptor Mutation Database II, http://www.uni-leipzig.de/~innere/). However, mutants N650A and V656A display basal cAMP activity comparable with wt TSHR (Table 1Go), suggesting that an enlargement or more bulkiness of the side chain rather than a reduction causes constitutive activation. Substitutions of N650, K651, S657, N658, and S659 near the TSHR ECL3/TMH junctions caused the weakest effect on TSH-stimulated cAMP accumulation. In contrast, introduction of alanine for residues P, L, I, T, and V in the central ECL3 had the strongest effect on cAMP production (Table 1Go). However, contrary to the FSHR (19, 31, 32), TSHR mutants did not totally abolish cAMP formation but displayed about 50% decreased cAMP activity compared with the wt TSHR. In contrast to our findings (Table 1Go), alanine substitutions in the CG/LHR ECL3 caused only moderate effects on hCG-induced cAMP formation for most residues. However, mutations P575A and V579A in the CG/LHR (TSHR: P652A and V656A) were found to decrease stimulated cAMP production (31, 36). Therefore, for TSHR and FSHR but not for CG/LHR, a hydrophobic cluster in the center of ECL3 is most likely important for ligand-induced activation of the Gs-mediated cAMP-signaling pathway. This suggests that the CG/LHR ECL3 may act in a different manner in the process leading to Gs activation (Fig. 3Go). However, despite different functional characteristics of mutants in the central portion of ECL3, P652 and V656, which are adjacent to the described hydrophobic cluster, as well as K660 at the ECL3/TMH7 junction, are important for cAMP signaling in all three GPHRs (Fig. 3Go).

Substitutions of residues in the hydrophobic cluster of ECL3 caused also the strongest reduction of Gq-mediated IP production (Table 1Go). In the FSHR (19, 31, 32), only N-terminal substitutions V581A and P582A (TSHR: K651 and P652) showed a detectable but strongly decreased IP accumulation after stimulation with FSH (19, 31, 32). This indicates that the entire ECL3 of the FSHR seems to be necessary for Gq activation. This is contrary to our findings that TSHR-mediated IP formation is affected only by mutations in the central ECL3 and at position K660. These findings suggest that the distribution of residues involved in activation of Gq-mediated IP production is different in these receptors.

Despite high-sequence homology within ECL3 (Fig. 3Go), comparison of results from alanine scanning mutagenesis of these closely related GPHRs reveals significant functional differences. Individual residues seem to play different roles in these receptors regarding receptor folding and cAMP and IP signaling. Our results underline, however, that the highly conserved K660 at the junction between ECL3 and TMH7 is essential in similar manner for both signaling pathways in all three GPHRs. Moreover, we provide evidence for a hydrophobic cluster, comprising residues 652–656 in the central part of ECL3, which is important for intramolecular signal transduction and G protein activation by the TSHR.


   Acknowledgments
 
We thank Mrs. Eileen Bösenberg for her excellent technical assistance.


   Footnotes
 
This work was supported by Deutsche Forschungsgemeinschaft Grants Pa 423/12-1 and Kr 1272/1-1) and by Medical Faculty, University of Leipzig Project formel1-53.

First Published Online September 8, 2005

Abbreviations: bTSH, Bovine TSH; CG/LHR, choriogonadotropic/LH receptor; ECD, extracellular domain; ECL, extracellular loop; FACS, fluorescence-activated cell sorting; FSHR, FSH receptor; GPHR, glycoprotein hormone receptor; ICL, intracellular loop; IP, inositol phosphate; TMH, transmembrane helix; TSHR, TSH receptor; wt, wild type. CG/LHR, Choriogonadotrophic/LH receptor; ECD, extracellular domain; ECL, extracellular loop; FACS, fluorescence-activated cell sorting; FSHR, FSH receptor; GPHR, glycoprotein hormone receptor; ICL, intracellular loop; IP, inositol phosphate; TMH, transmembrane helix; TSHR, thyroid-stimulating hormone receptor; wt, wild type.

Received June 14, 2005.

Accepted for publication August 30, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Gudermann T, Nurnberg B, Schultz G 1995 Receptors and G proteins as primary components of transmembrane signal transduction. Part 1. G-protein-coupled receptors: structure and function. J Appl Cryst 73:51–63
  2. Nagayama Y, Rapoport B 1992 The thyrotropin receptor 25 years after its discovery: new insight after its molecular cloning. Mol Endocrinol 6:145–156[Abstract]
  3. Segaloff DL, Ascoli M 1993 The lutropin/choriogonadotropin receptor. 4 years later. Endocr Rev 14:324–347[CrossRef][Medline]
  4. Simoni M, Gromoll J, Nieschlag E 1997 The follicle-stimulating hormone receptor: biochemistry, molecular biology, physiology, and pathophysiology. Endocr Rev 18:739–773[Abstract/Free Full Text]
  5. Rapoport B, Chazenbalk GD, Jaume JC, McLachlan SM 1998 The thyrotropin (TSH) receptor: interaction with TSH and autoantibodies. Endocr Rev 19:673–716[Abstract/Free Full Text]
  6. Vassart G, Desarnaud F, Duprez L, Eggerickx D, Labbe O, Libert F, Mollereau C, Parma J, Paschke R, Tonacchera M, Vanderhaeghen P, Van Sande J, Dumont J, Parmentier M 1995 The G protein-coupled receptor family and one of its members, the TSH receptor. Ann N Y Acad Sci 766:23–30[Medline]
  7. Kleinau G, Jaschke H, Neumann S, Lattig J, Paschke R, Krause G 2004 Identification of a novel epitope in the thyroid-stimulating hormone receptor ectodomain acting as intramolecular signaling interface. J Biol Chem 279:51590–51600[Abstract/Free Full Text]
  8. Bhowmick N, Narayan P, Puett D 1999 Identification of ionizable amino acid residues on the extracellular domain of the lutropin receptor involved in ligand binding. Endocrinology 140:4558–4563[Abstract/Free Full Text]
  9. Sangkuhl K, Schulz A, Schultz G, Schoneberg T 2002 Structural requirements for mutational lutropin/choriogonadotropin receptor activation. J Biol Chem 277:47748–47755[Abstract/Free Full Text]
  10. Smits G, Campillo M, Govaerts C, Janssens V, Richter C, Vassart G, Pardo L, Costagliola S 2003 Glycoprotein hormone receptors: determinants in leucine-rich repeats responsible for ligand specificity. EMBO J 22:2692–2703[CrossRef][Medline]
  11. Song YS, Ji I, Beauchamp J, Isaacs NW, Ji TH 2001 Hormone interactions to Leu-rich repeats in the gonadotropin receptors: II. Analysis of Leu-rich repeat 4 of human luteinizing hormone/chorionic gonadotropin receptor. J Biol Chem 276:3436–3442[Abstract/Free Full Text]
  12. Vischer HF, Granneman JC, Noordam MJ, Mosselman S, Bogerd J 2003 Ligand selectivity of gonadotropin receptors. Role of the ß-strands of extracellular leucine-rich repeats 3 and 6 of the human luteinizing hormone receptor. J Biol Chem 278:15505–15513[Abstract/Free Full Text]
  13. Fan QR, Hendrickson WA 2005 Structure of human follicle-stimulating hormone in complex with its receptor. Nature 433:269–277[CrossRef][Medline]
  14. Seetharamaiah GS, Kurosky A, Desai RK, Dallas JS, Prabhakar BS 1994 A recombinant extracellular domain of the thyrotropin (TSH) receptor binds TSH in the absence of membranes. Endocrinology 134:549–554[Abstract]
  15. Shi Y, Zou M, Parhar RS, Farid NR 1993 High-affinity binding of thyrotropin to the extracellular domain of its receptor transfected in Chinese hamster ovary cells. Thyroid 3:129–133[Medline]
  16. Vlaeminck-Guillem V, Ho SC, Rodien P, Vassart G, Costagliola S 2002 Activation of the cAMP pathway by the TSH receptor involves switching of the ectodomain from a tethered inverse agonist to an agonist. Mol Endocrinol 16:736–746[Abstract/Free Full Text]
  17. Zhang M, Tong KP, Fremont V, Chen J, Narayan P, Puett D, Weintraub BD, Szkudlinski MW 2000 The extracellular domain suppresses constitutive activity of the transmembrane domain of the human TSH receptor: implications for hormone-receptor interaction and antagonist design. Endocrinology 141:3514–3517[Abstract/Free Full Text]
  18. Nishi S, Nakabayashi K, Kobilka B, Hsueh AJ 2002 The ectodomain of the luteinizing hormone receptor interacts with exoloop 2 to constrain the transmembrane region: studies using chimeric human and fly receptors. J Biol Chem 277:3958–3964[Abstract/Free Full Text]
  19. Sohn J, Ryu K, Sievert G, Jeoung M, Ji I, Ji TH 2002 Follicle-stimulating hormone interacts with exoloop 3 of the receptor. J Biol Chem 277:50165–50175[Abstract/Free Full Text]
  20. Costagliola S, Panneels V, Bonomi M, Koch J, Many MC, Smits G, Vassart G 2002 Tyrosine sulfation is required for agonist recognition by glycoprotein hormone receptors. EMBO J 21:504–513[CrossRef][Medline]
  21. Vassart G, Pardo L, Costagliola S 2004 A molecular dissection of the glycoprotein hormone receptors. Trends Biochem Sci 29:119–126[CrossRef][Medline]
  22. Neumann S, Claus M, Paschke R 2005 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 152:625–634[Abstract/Free Full Text]
  23. Parma J, Van Sande J, Swillens S, Tonacchera M, Dumont J, Vassart G 1995 Somatic mutations causing constitutive activity of the thyrotropin receptor are the major cause of hyperfunctioning thyroid adenomas: identification of additional mutations activating both the cyclic adenosine 3',5'-monophosphate and inositol phosphate-Ca2+ cascades. Mol Endocrinol 9:725–733[Abstract]
  24. Tonacchera M, Agretti P, Pinchera A, Rosellini V, Perri A, Collecchi P, Vitti P, Chiovato L 2000 Congenital hypothyroidism with impaired thyroid response to thyrotropin (TSH) and absent circulating thyroglobulin: evidence for a new inactivating mutation of the TSH receptor gene. J Clin Endocrinol Metab 85:1001–1008[Abstract/Free Full Text]
  25. Fuhrer D, Holzapfel HP, Wonerow P, Scherbaum WA, Paschke R 1997 Somatic mutations in the thyrotropin receptor gene and not in the Gs {alpha} protein gene in 31 toxic thyroid nodules. J Clin Endocrinol Metab 82:3885–3891[Abstract/Free Full Text]
  26. Tonacchera M, Van Sande J, Cetani F, Swillens S, Schvartz C, Winiszewski P, Portmann L, Dumont JE, Vassart G, Parma J 1996 Functional characteristics of three new germline mutations of the thyrotropin receptor gene causing autosomal dominant toxic thyroid hyperplasia. J Clin Endocrinol Metab 81:547–554[Abstract]
  27. Trultzsch B, Nebel T, Paschke R 1999 The TSH receptor mutation database. Eur J Endocrinol 140:VII
  28. Wonerow P, Chey S, Fuhrer D, Holzapfel HP, Paschke R 2000 Functional characterization of five constitutively activating thyrotrophin receptor mutations. Clin Endocrinol (Oxf) 53:461–468[CrossRef][Medline]
  29. Kosugi S, Mori T 1994 The third exoplasmic loop of the thyrotropin receptor is partially involved in signal transduction. FEBS Lett 349:89–92[CrossRef][Medline]
  30. Sudo S, Kumagai J, Nishi S, Layfield S, Ferraro T, Bathgate RA, Hsueh AJ 2003 H3 relaxin is a specific ligand for LGR7 and activates the receptor by interacting with both the ectodomain and the exoloop 2. J Biol Chem 278:7855–7862[Abstract/Free Full Text]
  31. Ryu K, Gilchrist RL, Tung CS, Ji I, Ji TH 1998 High affinity hormone binding to the extracellular N-terminal exodomain of the follicle-stimulating hormone receptor is critically modulated by exoloop 3. J Biol Chem 273:28953–28958[Abstract/Free Full Text]
  32. Ryu K, Lee H, Kim S, Beauchamp J, Tung CS, Isaacs NW, Ji I, Ji TH 1998 Modulation of high affinity hormone binding. Human choriogonadotropin binding to the exodomain of the receptor is influenced by exoloop 2 of the receptor. J Biol Chem 273:6285–6291[Abstract/Free Full Text]
  33. Li S, Liu X, Min L, Ascoli M 2001 Mutations of the second extracellular loop of the human lutropin receptor emphasize the importance of receptor activation and de-emphasize the importance of receptor phosphorylation in agonist-induced internalization. J Biol Chem 276:7968–7973[Abstract/Free Full Text]
  34. Fernandez LM, Puett D 1996 Additions and Corrections to Lys583 in the third extracellular loop of the lutropin/choriogonadotropin receptor is critical for signaling. J Biol Chem 271:13925B–13926B
  35. Fernandez LM, Puett D 1996 Lys583 in the third extracellular loop of the lutropin/choriogonadotropin receptor is critical for signaling. J Biol Chem 271:925–930[Abstract/Free Full Text]
  36. Ryu KS, Gilchrist RL, Ji I, Kim SJ, Ji TH 1996 Exoloop 3 of the luteinizing hormone/choriogonadotropin receptor. Lys583 is essential and irreplaceable for human choriogonadotropin (hCG)-dependent receptor activation but not for high affinity hCG binding. J Biol Chem 271:7301–7304[Abstract/Free Full Text]
  37. Gilchrist RL, Ryu KS, Ji I, Ji TH 1996 The luteinizing hormone/chorionic gonadotropin receptor has distinct transmembrane conductors for cAMP and inositol phosphate signals. J Biol Chem 271:19283–19287[Abstract/Free Full Text]
  38. Higuchi R 1989 Using PCR to engineer DNA. In: Erlich HA, ed. PCR technology. New York: Stockton Press; 61–70
  39. Libert F, Lefort A, Gerard C, Parmentier M, Perret J, Ludgate M, Dumont JE, Vassart G 1989 Cloning, sequencing and expression of the human thyrotropin (TSH) receptor: evidence for binding of autoantibodies. Biochem Biophys Res Commun 165:1250–1255[CrossRef][Medline]
  40. Berridge MJ 1983 Rapid accumulation of inositol trisphosphate reveals that agonists hydrolyse polyphosphoinositides instead of phosphatidylinositol. Biochem J 212:849–858[Medline]
  41. Palczewski K, Kumasaka T, Hori T, Behnke CA, Motoshima H, Fox BA, Le Trong I, Teller DC, Okada T, Stenkamp RE, Yamamoto M, Miyano M 2000 Crystal structure of rhodopsin: a G protein-coupled receptor. Science 289:739–745[Abstract/Free Full Text]
  42. Li J, Edwards PC, Burghammer M, Villa C, Schertler GF 2004 Structure of bovine rhodopsin in a trigonal crystal form. J Mol Biol 343:1409–1438[CrossRef][Medline]
  43. Okada T, Sugihara M, Bondar AN, Elstner M, Entel P, Buss V 2004 The retinal conformation and its environment in rhodopsin in light of a new 2.2 A crystal structure. J Mol Biol 342:571–583[CrossRef][Medline]
  44. Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, Weissig H, Shindyalov IN, Bourne PE 2000 The Protein Data Bank. Nucleic Acids Res 28:235–242[Abstract/Free Full Text]
  45. Case DA, Pearlman DA, Caldwell JW, Cheatham Spaceiiiqq TE, Wang J, Ross WS, Simmerling CL, Darden TA, Merz KM, Stanton RV, Cheng AL, Vincent JJ, Crowley M, Tsui V, Gohlke H, Radmer RJ, Duan Y, Pitera J, Massova I, Seibel GL, Singh UC, Weiner PK, Kollman PA 2002 AMBER 7. San Francisco: University of California
  46. ter Laak AM, Kuhne R 1999 Bacteriorhodopsin in a periodic boundary water-vacuum-water box as an example towards stable molecular dynamics simulations of G-protein coupled receptors. Receptors Channels 6:295–308[Medline]
  47. Laskowski RA, MacArthur MW, Moss DS, Thornton JM 1993 Main-chain bond lengths and bond angles in protein structures. J Appl Cryst 26:283–291[CrossRef]
  48. Cetani F, Tonacchera M, Vassart G 1996 Differential effects of NaCl concentration on the constitutive activity of the thyrotropin and the luteinizing hormone/chorionic gonadotropin receptors. FEBS Lett 378:27–31[CrossRef][Medline]



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