Scheme of initial TSHR activation.

<p>New functional-structural TSHR features at the extracellular site are described for the hinge region and the LRRD ain a schematic overview. Repeat 11 with the pivotal helix (green) is part of the LRRD that ends at Asn288 and therefore includes Cys283 and Cys284 of cysteine-box 2 (C-b2). These cysteines are linked by disulfide bridges to Cys398 and Cys408 of cysteine-box 3, respectively. Constitutively activating mutations at Pro280 and Ser281 (magenta) influence the conformation of this helix and underline the importance of this region for receptor function. Further positions of CAMs are located at the C-terminal part of C-b3, in close spatial proximity to Cb-2. Mutations at both fragments probably cause conformational changes at this region which induce receptor activation. Thus, these wild type amino acids constitute an activation-sensitive <i>intramolecular agonistic unit</i> (magenta, boxed). This unit is adjacent to TMH1 and is probably located between the ECLs as an interface between the extracellular and transmembrane regions. The transition between the extracellular region and TMH1 is made up of negatively charged amino acids, in which side chain substitutions lead to impaired signaling capacity. These are key to interactions with the serpentine domain spanning the membrane. Several residues identified as sensitive to hormone binding are enclosed in a red box (Glu297, Glu303, Asp382 and Asp386) and represent a spatial cluster that is linked by a disulfide bridge between Cys301 and Cys390. Asp382 (and Glu303) are important for super-agonistic effects of bTSH but are not important for hTSH. Sulfation at Tyr385 is obligatory for hormone binding and is accompanied by negatively charged amino acids close to Tyr385 (Asp386, Asp382). Two cleavage sites define the so-called cleavable peptide (C-peptide, ∼50 amino acids). Deletions at the transition between the C-peptide and the Cb-3 are reported to activate the TSHR (and also the FSHR) constitutively (magenta trapezoid). In contrast, single point mutations lead to partial inactivation of TSHR signaling at this region. The dashed arrows indicate the potential signal processing path for activation upon ligand binding from the hinge region and/or potentially via the LRRD. In either case, the extracellular modifications converge at the pivotal helix that links the <i>intramolecular agonistic unit</i> together (dashed box).</p

The FSHR LRRD and hinge region with bound FSH.

<p>The crystal structure of the FSHR LRRD/hinge region fragment/FSH complex is trimeric <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052920#pone.0052920-Jiang1" target="_blank">[54]</a> (insert, boxed window). A monomeric LRRD/FSH/hinge region complex was extracted from the trimeric arrangement to visualize specific important structural and functional features. The LRRD was previously assumed to end at LRR9 with an additional tenth β-strand <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052920#pone.0052920-Fan1" target="_blank">[21]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052920#pone.0052920-Sanders2" target="_blank">[23]</a>. The new structure shows an additional eleventh repeat and extends the LRRD through amino acid Asn280 (green backbone). This is followed by a short helical secondary structure, comprising the first two cysteines (Cys275, Cys276, yellow lines) of cysteine box 2 (Cb-2, N-terminal hinge region). These form disulfide bridges with cysteines (Cys346, Cys356, yellow) of cysteine-box 3 (Cb-3, C-terminal hinge region, chocolate brown), in which a short β-strand is additionally assembled parallel to the eleventh strand of the concave LRRD β-sheet. The N-terminal section of the hinge region after the LRRD is crystallized till residue Ser295. The inner cysteine bridge (Cys292-Cys338) between the two remaining extracellular cysteines connects this section with the C-terminal hinge region. It was already known from mutagenesis studies that the sulfated Tyr335 (at the C-terminal hinge region that is crystallized from position Thr331-Ile359) is mandatory for hormone binding and signaling and interacts tightly with amino acids of the hormone subunits, between the β-subunit loop 2 and the α-loop 1. The side chains of the hormone constitute a binding pocket for the sulfated tyrosine; light green sticks represent aromatic residues, green sticks hydrophobic residues and orange sticks hydrophilic residues. The adjacent negatively charged Asp334 interacts directly with the positively charged Arg53 of the hormone β-subunit. The C-terminus of the hinge region at Cys346 forms a β-strand parallel to the last repeat strand of the LRRD and ends with residues that are conserved among the GPHRs and that are known from studies on TSHR to be relevant to signaling <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052920#pone.0052920-Kleinau2" target="_blank">[11]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052920#pone.0052920-Mueller1" target="_blank">[44]</a>.</p

Amino acid sequences of hormone subtypes and specific receptor fragments.

<p><b>A</b>) Alignment of hormone subtype amino acid sequences with signal peptides (numbering with signal peptides throughout the manuscript). The α-subunits of hTSH, bTSH, hTSH analogue (TR1401, Trophogen Inc., Rockville, MD) and thyrostimulin are shown, together with the β-subunit sequences of hFSH, hLH and hCG. Residues are colored according to their biophysical properties: blue - positively charged, red - negatively charged, green - hydrophobic, orange - hydrophilic, magenta – histidine, brown – cysteine, black – proline, light green - aromatic residues). <b>B</b>) Comparison of the amino acid sequences of different GPHR subtypes and LGRs. Residues are presented from the cysteine-box 2 through the transmembrane helix 1. Specificities are highlighted, including the positions of CAMs and amino acids relevant to binding.</p

The TSHR LRRD and hinge region in complex with TSH.

<p><b>A-B</b>) Homology model of the extracellular TSHR region complexed with bTSH. The new LRRD (pale beige backbone ribbon) repeat LRR 11 (green) contains a helical secondary element with two consecutive cysteines (yellow) of cysteine-box 2. The hormone is visualized as a surface (α-subunit in orange, β-subunit in greenblue). The sulfated tyrosine 385 (TSHR number, red stick) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052920#pone.0052920-Costagliola1" target="_blank">[49]</a> location is comparable to that in the FSHR/FSH complex interacting with the hormone in a pocket constituted by hydrophilic (orange sticks), aromatic (light green sticks) and hydrophobic residues (green sticks). In contrast to FSH and human TSH, bovine TSH contains additional positively charged residues at the α-subunit (blue surface regions in A) and blue sticks at B)) which are known to increase hormone binding properties compared to hormone variants without those positive charges at the corresponding positions <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052920#pone.0052920-Grossmann1" target="_blank">[70]</a>. Our TSHR model suggests that these four side chains (at the α-subunit interact with negatively charged asparagines and glutamates (red sticks) in the TSHR hinge region (Asp382, Glu303) that have been shown experimentally to interplay with positively charged hormone side chains of bTSH <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052920#pone.0052920-Mueller2" target="_blank">[51]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052920#pone.0052920-Mueller3" target="_blank">[52]</a>. These interactions are responsible for the super-agonistic effects of bTSH compared to hTSH and are perfectly matched in this TSHR/TSH model based on the FSHR/FSH complex. Met300 and Leu299 (hydrophobic, green surface and sticks) and the negatively charged Glu297 (red stick in B) are already known to be sensitive to TSHR binding; these also have complementary partners on the hormone (details in B). Moreover, Cb-2 and C-b3 interact tightly. Amino acids at the N- and C- terminal sites (magenta) close to TMH1 (the serpentine domain) have been shown by mutation studies to activate TSHR constitutively. In Cb-2 (A), Pro280 and Ser281 at the end of helical repeat 11 region are in direct spatial proximity to amino acids Pro400–Pro407 of Cb-3. One difference between the β-subunits of bTSH and hTSH is not directly explained by the current TSHR/TSH model. The positively charged βArg89 of bTSH is absent in hTSH and substitution of this residue into hTSH leads to greatly enhanced binding of hTSH to TSHR <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052920#pone.0052920-Grossmann1" target="_blank">[70]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052920#pone.0052920-Szkudlinski1" target="_blank">[71]</a>. The TSHR monomer model reveals that this residue is located at the β-L3 loop and is not involved in any intra- or intermolecular interaction to the targeted receptor.</p

Comparison between Gαq, Gαs, Gαi and Gαt.

<p>Corresponding residues of Gαs, Gαi and Gαt at positions where Gαq is suggested to interact with the TSHR in our homology model. These residues were revealed by a sequence alignment of the alpha subunits.</p

Homology complex model of the TSHR/Gq heterotrimer with focus on the interface between helix 8 and the transition of ICL3/transmembrane helix 6 with Gαq.

<p>Our new and recently published data of intracellular key-players for the TSHR and G-protein interaction are summarized and mapped on to the 3D complex model. Several mutations in the intracellular region of the TSHR are known to prevent Gs and Gq signaling simultaneously. All mutants show decreased cAMP production by TSH in conjunction with decreased activation of the IP pathway. The following wild type amino acids should therefore be considered as commonly sensitive for regulation of the receptor/G-protein interplay: ICL1 - I438, S442, R450; ICL2 - M527, R528, D530; ICL3/TMH6 - I622. Colour codes: purple - selectively impaired Gq activation by mutation; red - inactivating mutants for Gs and Gq coupling and cyan - known constitutively activating mutants. Dashed lines indicate potential H-bonds.</p

Homology model of the complex of TSHR/Gq with focus on the interface between ICL1 and Gq heterotrimer.

<p>The TSHR model suggests that in ICL1 (red) and in the transitions with the adjacent transmembrane helices (pale pink) the signaling sensitive amino acids (H443, R450) directly contact Gβγ (blue) and Gαq (C-term α5-helix: green), respectively. Dashed lines represent potential H-bonds. Others may indirectly affect Gq coupling (e.g. T441) via conformational changes of ICL1.</p

Alanine mutagenesis and functional characterization of amino acids in the ICL1 and transitions to the helices 1 and 2.

<p>COS-7 cells were transfected with wt TSHR or various mutant TSHRs. The vector pcDNA3.1(−) / hygromycin was used as a control. The TSHR is characterized by an elevated cAMP level compared to the control vector alone <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009745#pone.0009745-Cetani1" target="_blank">[76]</a>. Therefore, cAMP accumulation is expressed relative to wt TSHR basal level. TSH-mediated levels of cAMP and IP accumulation were determined after treatment of cells with 100 mU/ml bTSH. Expression of wt and mutant TSHRs were quantified on a FACS flow cytometer. Data are given as mean ± standard deviation (SD) of at least three independent experiments (n = 3), each carried out in duplicate. Constitutive activity by linear regression analyses was determined for mutant L439A. ^aP<0.001, ^bP = 0.001 to 0.01, ^cP = 0.01 to 0.05.</p

Potential direct intermolecular interaction partners between TSHR and Gq.

<p>The identification of potential interaction partners between TSHR and Gq was carried out using the molecular homology model of the entire receptor/Gq complex (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009745#pone-0009745-g003" target="_blank">Figure 3</a>) in combination with functional data (GPHR information resources: <a href="http://www.ssfa-gphr.de" target="_blank">http://www.ssfa-gphr.de</a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009745#pone.0009745-Kleinau1" target="_blank">[44]</a> and <a href="http://gris.ulb.ac.be" target="_blank">http://gris.ulb.ac.be</a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009745#pone.0009745-VanDurme1" target="_blank">[45]</a>).</p

Side-chain variations and functional characterization of H443 and R450 in ICL1 and transition to helix 2 and R687 in helix 8.

<p>COS-7 cells were transfected with wt TSHR or various mutant TSHRs. The vector pcDNA3.1(−) /hygromycin was used as a control. The TSHR is characterized by an elevated cAMP level compared to the control vector alone <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009745#pone.0009745-Cetani1" target="_blank">[76]</a>. Therefore, cAMP accumulation is expressed relative to wt TSHR basal level. TSH-mediated levels of cAMP and IP accumulation were determined after treatment of cells with 100 mU/ml bTSH. Expression of wt and mutant TSHRs were quantified on a FACS flow cytometer. Data are given as mean ± standard deviation (SD) of at least three independent experiments (n = 3), each carried out in duplicate. ^aP<0.001, ^bP = 0.001 to 0.01, ^cP = 0.01 to 0.05.</p