A Free Carboxylate Oxygen in the Side Chain of Position 674 in Transmembrane Domain 7 Is Necessary for TSH Receptor Activation

Susanne Neumann, G. Krause, S. Chey and Ralf Paschke

Third Medical Department (S.N., S.C., R.P.), University of Leipzig, D-04103 Leipzig, Germany; and Institute for Molecular Pharmacology (G.K.), D-10315 Berlin, Germany

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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
A specific H-bonding network formed between the central regions of transmembrane domain 6 and transmembrane domain 7 has been proposed to be critical for stabilizing the inactive state of glycoprotein hormone receptors. Many different constitutively activating TSH receptor point mutations have been identified in hyperfunctioning thyroid adenomas in the lower portion of transmembrane domain 6. Position D633 in transmembrane domain 6 of the human TSH receptor is the only one in which four different constitutively activating amino acid exchanges have been identified. Further in vitro substitutions led to constitutive activation of the TSH receptor (D633Y, F, C) as well as to the first inactivating TSH receptor mutation in transmembrane domain 6 without changes of membrane expression or TSH binding (D633R). Molecular modeling of this inactivating TSH receptor mutation revealed potential interaction partners of R633 in transmembrane domain 3 and/or transmembrane domain 7, presumably via hydrogen bonds that could be responsible for locking the TSH receptor in a completely inactive state. To further elucidate the H-bond network that most likely maintains the inactive state of the TSH receptor, we investigated these potential interactions by generating TSH receptor double mutants designed to break up possible H bonds. We excluded S508 in transmembrane domain 3 as a possible interaction partner of R633. In contrast, a partial response to TSH stimulation was rescued in a receptor construct with the double-substitution D633R/N674D. Our results therefore confirm the H bond between position 633 in transmembrane domain 6 and 674 in transmembrane domain 7 suggested by molecular modeling of the inactivating mutation D633R. Moreover, the mutagenesis results, together with a three-dimensional structure model, indicate that for TSH receptor activation and G protein-coupled signaling, at least one free available carboxylate oxygen is required as a hydrogen acceptor atom at position 674 in transmembrane domain 7.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE TSH RECEPTOR (TSHR) is a member of the superfamily of G protein-coupled receptors (GPCRs) (1, 2), which also includes the FSH receptor and the LH/CG receptor (LHR) in the subfamily of glycoprotein hormone receptors (3, 4). Sequence alignment of GPCRs has revealed a high degree of homology in their transmembrane domains (TMs), suggesting similarities in transmembrane structural features and signal transduction mechanisms (5). Activation of these receptors by their diverse agonists is associated with crucial conformational changes in the receptor molecule resulting in a movement of transmembrane helices relative to one another and subsequent G protein activation (6).

As for most GPCRs, the precise molecular mechanism of the TSHR activation is not known. The understanding of the intramolecular interactions and conformational changes underlying receptor activation is hindered by a lack of information on the three-dimensional high-resolution structure of the TSHR. Low-resolution structure data (6 Å) from cryomicroscopy data of frog rhodopsin suggested a tilt of the transmembrane segments. However, they could not provide structural information about individual side chains or even atoms (7, 8). Very recently the x-ray crystal structure of the bovine rhodopsin with a resolution of 2.8 Å provided new, important insights into side chain orientations of rhodopsin (9). In this model some helical portions of TM2, TM5, TM6, and TM7 are not organized in an ideal {alpha}-helical backbone structure, which may be due to particular amino acid residues. Noteworthy, in comparison to rhodopsin, glycoprotein hormone receptors have different residues in almost all respective critical positions; thus an identical side chain orientation is very unlikely.

Site-directed mutagenesis and the evaluation of its effects on receptor binding and signal transduction provide a valid means of obtaining insights into intramolecular changes of glycoprotein hormone receptors, as well as GPCRs in general, during activation (5). Moreover, molecular modeling is necessary to integrate experimental observations and biophysical and structural data into a mechanistic three-dimensional receptor model. Models also contribute to reduce the gap of resolution between the 6A and atom level. Homologous receptor models are used to indicate likely conformational differences in the new rhodopsin x-ray structure. They are also important in explaining relations between receptor structure and function. Many models for different GPCRs have been generated from a combination of structural data derived from rhodopsin and mutagenesis data on the receptors themselves (10).

Constitutively activating mutations have been identified in many GPCRs (11). In the TSHR gene, gain of function mutations cause autosomal dominant nonautoimmmune hyperthyroidism and toxic thyroid nodules (12). The identification and functional characterization of naturally occurring mutations in the TSHR provided the first information about mechanisms of receptor signaling. Therefore, naturally occurring mutations that activate the TSHR provide unique hints for further mutagenesis experiments, as has recently been shown (13, 14, 15). However, this approach, until now, has not been employed for TM6.

Figure 1Go shows a schematic representation of the TSHR with the different amino acid exchanges of somatic TSHR mutations in toxic thyroid nodules collected in a TSHR mutation database (16). Most of these activating mutations have been identified in exon 10 of the TSHR, in which TM6 represents a hot spot for activating mutations. However, none of the previous site-directed mutagenesis studies of the TSHR has focused on this domain, despite these indications for its significance in receptor structure and activation. The central part of TM6 and the intracellular half of TM7 are highly conserved among GPCRs. Molecular modeling of the LHR and other GPCRs has revealed consensus residues in TM6 that are most likely engaged in important interhelical interactions (11, 17). Conformational changes can induce relative movements of the helices—especially of helix 6—as reported for rhodopsin (18, 19). Recent data from the rat LHR have demonstrated a direct activation of GS{alpha} by TM6 (20). Therefore, mutations in TM6 of the LHR are likely to directly affect the coupling of the receptor to GS{alpha}. A model of the LHR revealed several pairs of strong interhelical side chain-side chain H bonds formed in the midregions of TM6 and TM7 (17). According to this LHR model, the homologous T632 and D633 residues of the TSHR could be involved in such interhelical H bonds in the TSHR. Moreover, the highly conserved residue C636 in TM6 could either be hidden in the inactive state or participate in stabilizing the inactive receptor state by inter- or intrahelical interactions. This cysteine was observed in several GPCRs to be accessible for sulfhydryl-reactive agents only in activated receptors and not in the inactive state (21, 22).



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Figure 1. Amino Acid Exchanges of Somatic TSHR Mutations in Toxic Thyroid Nodules

 
Substitutions of D633 in helix 6 are the most frequent naturally occurring constitutively activating TSHR mutations. Moreover, position D633 is the only one in which four different amino acid exchanges—alanine, histidine, tyrosine, and glutamate—were identified in hot thyroid nodules as shown in Fig. 1Go (23, 24, 25, 26). The D578 residue in the human LHR corresponds to D633 in the TSHR and in comparison with this residue it is the most frequently observed constitutively activating substitution in the LHR (17, 27). In contrast to the TSHR and the LHR, this aspartate residue in TM6 is most frequently phenylalanine (91%) in other GPCRs (11). These observations, as well as the results of the molecular modeling of the LHR, indicate that position D633 has a pivotal role in the intramolecular signal transduction and the specific interhelical contacts between TM6 (D633) and TM7 (N670, N674), which constrain the inactive glycoprotein hormone receptor state. Therefore, we tested the likely significance of D633 for interhelical interactions by site-directed mutagenesis and molecular modeling.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Selection Criteria for the Amino Acid Exchanges at D633
To investigate the significance of the D633 residue in the TM6 of the human TSHR for intramolecular interactions that are responsible for the change between the active and the inactive receptor state, this aspartate residue was replaced by different amino acids.

The central cavities of glycoprotein hormone receptors including the D633 residue are quite polar (17). Moreover, D has a negative charge, is strongly acidic, and is characterized by the lowest hydrophobicity of all amino acids. To induce significant physicochemical changes, amino acids with qualities strongly differing from D were selected. D was therefore replaced by the amino acids R, C, W, and F. R is also polar, but it is positively charged, strongly basic, and weakly hydrophobic. In contrast to D and R, C has no charge. However, this amino acid residue is weakly polar but also hydrophobic. In contrast to these residues W and F are apolar, strongly hydrophobic, and not charged. Moreover, both W and F introduce bulky aromatic side chains at position 633. In addition, D633 was deleted.

Functional Assessment of the TSHR Mutants at Position D633 in TM6
The effects of all mutations at position D633 on basal and TSH-stimulated cAMP and inositol phosphate (IP) production are summarized in Table 1Go. As a control for our assay system, D633H, a naturally occurring activating mutation at position 633 (25, 26), was also investigated. Apart from the D633R substitution, all other initial amino acid substitutions resulted in constitutive activation of the cAMP signaling cascade (Fig. 2Go). Substitution of D633 with F or W induced the highest constitutive activity despite lower cell surface expression. In comparison to the wild-type (wt) TSHR, most of the substitutions resulted in a decreased cell surface expression. Therefore, we reduced the cell surface expression of the wt TSHR and the TSHR mutants to the level of the D633W mutant, which showed the lowest cell surface expression (16 ± 1.2% of wt TSHR, set at 100%, Table 1Go) apart from the deletion of D633. The wt TSHR and the mutated TSHR constructs were cotransfected with various amounts of plasmids containing the human V2 receptor (V2R). The cell surface expressions of these cotransfected TSHR constructs in comparison to the wt TSHR transfected alone were as follows: wt TSHR set at 100%; wt TSHR/V2R, 14.7 ± 1.0%; D633C/V2R, 11.7 ± 1.4%; D633F/V2R, 12.7 ± 1.5%; and D633H/V2R, 15.2 ± 3.1%. Measurement of cAMP accumulation under these conditions in three independent experiments confirmed the constitutive activity of the TSHR mutants D633C (3.2 ± 0.3-fold over wt TSHR basal), D633F (7.6 ± 0.4-fold), D633W (10.3 ± 0.5-fold), and D633H (7.0 ± 1.0-fold) for identical cell surface expressions of these TSHR constructs. The TSH-stimulated cAMP accumulation (wt TSHR, 14.5 ± 1.8-fold over wt TSHR basal; D633C, 22.9 ± 1.2-fold; D633F, 14.4 ± 3.0-fold; D633W, 14.8 ± 1.9-fold; and D633H, 14.6 ± 1.4-fold) was comparable or only slightly increased compared with data determined for different expression levels (Table 1Go).


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Table 1. Functional Characterization of the TSHR Single and Double Mutants

 


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Figure 2. TSH-Stimulated cAMP Accumulation of COS-7 Cells Expressing wt and Mutant TSHRs

cAMP accumulation assays were performed with transiently transfected COS-7 cells. Cells were incubated with various concentrations of bTSH (0–100 mU/ml), and increases in cAMP levels were determined as described in Materials and Methods. Data are expressed as fold over basal wt TSHR and presented as the mean ± SEM of three independent experiments, each carried out in duplicate.

 
In addition to the GS-cAMP pathway, the activated TSHR is also coupled to Gq/11, resulting in the activation of the PLC-IP pathway (28). Cells expressing the D633C mutant did not show increased accumulation of IP in the absence of TSH. In contrast, the mutants D633H, D633W, and D633F led to a clear stimulation of basal IP accumulation in comparison to the wt TSHR. However, the basal IP stimulation for D633F was lower than for D633H or D633W (Table 1Go).

Cells transfected with the TSHR deleted at position 633 (D633{Delta}) were characterized by low cell surface expression compared with the wt TSHR (Table 1Go and Fig. 4AGo). However, D633{Delta} was clearly detectable within the cells measured by fluorescence-activated cell sorting (FACS) analysis on permeabilized cells (Fig. 4BGo) suggesting increased accumulation of this receptor mutant within the cells. This mutant showed hardly detectable binding for labeled TSH (Fig. 3Go). Neither the adenylate cyclase nor the PLC signaling cascade are activated by this TSHR mutant (Table 1Go).



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Figure 4. Expression of the wt TSHR and the D633{Delta} Mutant Measured by Flow Immunocytometry Using a Mouse Antihuman TSHR Antibody

A, Nonpermeabilized COS-7 cells assayed after transfection with the pSVL vector alone, the wt TSHR, and the D633{Delta} mutant. B, Saponin-permeabilized COS-7 cells transfected identically. Details are described in Materials and Methods.

 


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Figure 3. Competitive Binding of 125I-bTSH on COS-7 Cells Transfected with wt and Mutant TSHRs

To determine KD and Bmax values, transfected cells were subjected to displacement studies using 160.000–180.000 cpm 125I-bTSH per well and increasing concentrations of bTSH. All data are presented as mean ± SEM of two independent experiments, each performed in duplicate.

 
The most interesting TSHR mutant was D633R. COS-7 cells transfected with this TSHR mutant showed a decreased basal cAMP activity compared with the basal wt TSHR activity, despite a slightly increased cell surface expression (124%) of this mutant compared with the wt TSHR (set at 100%) (Fig. 2Go). Cells transfected with this D633R TSHR construct could not be stimulated with TSH, although this receptor mutant exhibits significant binding of labeled TSH (Figs. 2Go and 3Go). D633R reached only the basal level of the wt TSHR cAMP and IP accumulation (Table 1Go), meaning that this TSHR mutant is completely inactive.

Molecular Modeling of the Human TSHR
To investigate the alterations of TSHR activation caused by the substitution of aspartate with arginine, a computer model, based on the TSHR model published by Biebermann et al. (13), was developed. This model suggests the following potential interaction partners for R633 to maintain the inactive receptor conformation (Fig. 6AGo):



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Figure 6. Suggested Scenario for Interhelical Interactions between TM6 and TM7 of the TSHR Which Lead to TSHR Activation

A, wt TSHR: a hydrogen bond between D633 and N674 in TM6 and TM7 of the wt TSHR maintains the inactive basal state. Since N674 acts as hydrogen donator for D633, its free side chain carboxylate oxygen is available for the activation process involving TM7. During the activation process, a hydrogen bond is released, thus allowing movement of TM6. B, D633R mutant: N674 also forms a hydrogen bond with TM6. However, R633 enforces the carboxylate oxygen of the N674 side chain to act as hydrogen acceptor. A side chain carboxylate oxygen is no longer available in TM7 for the activation process. R633 locks the receptor between TM6 and TM7, thereby completely blocking TSHR activation. C, D633R/N674D double mutant: D674 forms a hydrogen bond with R633 one side chain oxygen. The other oxygen is available for the activation process and allows a partial rescue of the blocked TSHR activation. R633 still partially blocks (very likely by interaction with the TM6 backbone) the activation.

 
In the wt TSHR the aspartate residue in TM6 is involved in a network of hydrogen bonds between the polar patches of TM6 and TM7. D633 acts as H acceptor and has the ability to form an H bond primarily with N674 in TM7 or alternatively, but less likely, with N670 in TM7 (Fig. 6AGo). The side chains N674 and N670 are in close proximity within TM7. Within the hydrogen-bonding network constraining the inactive state, N674 itself forms further hydrogen bond(s) to N670 within TM7. It is assumed that weakening or loss of these H bonds may destabilize the contact surface and may reduce the packing specificity (17). D633 mutations that weaken these interhelical H bond interactions are expected to cause activation, whereas mutations that maintain the H bonds are expected to hinder activation of the TSHR.

Theoretically, and in contrast to all other amino acid substitutions at this position, there are several simultaneous possibilities for R633 to form H bonds with TM7 or TM3 on the basis of available hydrogens. For the prediction of a possible H bond pattern, it was necessary to determine whether the 633 residue must be charged in the TSHR or whether it can be uncharged as in the D578N mutant of the LHR (29). To further delineate the possible interaction partners of R633, which contribute to the formation of H bonds and preserve a completely inactive receptor conformation, the following single and double substitutions were generated. For the identification of functionally relevant interactions, the D633 residue was substituted with lysine (D633K). In addition, the introduction of this amino acid residue led to a shortening of the side chain at position 633 and therefore tested potential long-range interaction partners such as D460 in TM2 of the TSHR. Double mutants—R633 in combination with substitutions of N674 (D633R/N674D, D633R/N674A, D633R/N674S) and of N670 (D633R/N670A, D633R/N670S)—were generated with the aim of rescuing the receptor signaling by destroying possible H bonds suggested by computer modeling and thereby releasing the completely blocked inactive conformations. To evaluate the contribution of the second partner within the double mutants, the single mutations (N674D, N674A, N674S, N670A, N670S) were also tested. Moreover, the computer simulations also suggested Ser508 in TM3 as an alternative interaction site for R633 (Fig. 6AGo). Therefore, the double mutant D633R/S508G was created to abrogate a potential interaction of R633 with TM3.

Among the polar amino acid residues present in the cytoplasmatic half of TM3 are S505 and E506, which point toward the intracellular receptor part but are too far away from D633 for interhelical interactions in the native inactive TSHR state. Y510 (TM3) very likely interacts with a hydrophobic cluster between TM4 and TM5. T632 and C636 of TM6 and C672 of TM7 are clearly oriented toward TM7 and TM1, respectively. While C636 and C672 are involved in aromatic/hydrophobic interactions and are contributing to interhelical stabilization, T632 forms a hydrogen bond toward the TM6 backbone and thus appears very likely to stabilize TM6 in an intrahelical manner.

Experimental Testing of R633 Interactions Maintaining the Inactive State
Similarly to the D633R mutant cells transfected with the TSHR mutant, D633K showed a lower basal cAMP activity in comparison to the wt TSHR at comparable receptor density on the cell surface (Table 1Go). In contrast to D633R, cells transfected with the D633K construct showed a slight increase in cAMP production after stimulation with high TSH concentrations of 10 to 100 mU/ml TSH, which do not occur in physiological in vivo conditions. The substitution at position 633 with an uncharged asparagine did not result in a constitutive activation of the cAMP pathway (Table 1Go). Receptor density on the cell surface, dissociation constant (KD) values, and basal and stimulated cAMP levels were similar for D633N and the wt TSHR receptor (Table 1Go). The substitution of S508 in TM3 with G (S508G) had a significant effect on the cell surface expression (23% of the wt). The basal cAMP level was slightly below the wt TSHR level. However, the agonist-dependent cAMP activation corresponds to the wt TSHR. In contrast, the TSHR double mutant S508G/D633R, like D633R, was completely inactive for cAMP and IP production at a similar cell surface expression as the wt TSHR (Table 1Go).

Partial Rescue of TSHR Signaling by the Double Mutation D633R/N674D
COS-7-cells transfected with the substitutions N674D or N674S exhibited an increase in basal cAMP accumulation in comparison to the wt TSHR at a significantly reduced cell surface expression. Both constitutively active mutants N674D and N674S showed a reduced cAMP accumulation after bovine TSH (bTSH) stimulation compared with the wt TSHR. Cells transfected with the N674A TSHR mutant showed a strongly impaired TSHR function. The cAMP accumulation after bTSH stimulation was extremely reduced. Both double mutants D633R/N674A and D633R/N674S were inactive with respect to the adenylate cyclase pathway, despite a slightly increased cell surface expression compared with the wt TSHR (Table 1Go). The double mutants D633R/N670A and D633R/N670S were also completely inactive as regards the cAMP pathway. The TSHR substitution N670S resulted in constitutive activation as previously described (30) (Table 1Go).

The most important finding was the partial rescue of the impaired signaling of D633R by the double mutant D633R/N674D, as demonstrated by TSH stimulation of the mutants. The cell surface expression of 117% for D633R/N674D was similar to the cell surface expression of the inactive D633R substitution (124%). The basal cAMP production for the D633R/N674D double mutant was below the wt TSHR level. However, after stimulation with 100 mU/ml bTSH, the D633R/N674D TSHR mutant reached 36% of the stimulated wt TSHR cAMP accumulation (Fig. 5Go and Table 1Go).



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Figure 5. TSH-Stimulated cAMP Accumulation: Comparison of the TSHR D633R Mutant with the Double Mutant D633R/N674D

cAMP accumulation assays were performed in COS-7 cells, transiently transfected with wt TSHR ({square}), D633R ({circ}), and D633R/N674D ({triangleup}) plasmids. Forty eight hours after transfection, cells were incubated with various concentrations of bTSH (0–100 mU/ml), and increases in cAMP levels were determined as described in Materials and Methods. Data are expressed as fold over basal wt TSHR and presented as the mean ± SEM of three independent experiments, each carried out in duplicate.

 
None of the mutations generated to identify potential interaction partners of R633 induced an increase of basal IP accumulation. When stimulated with bTSH, the TSHR mutant D633N only reached half of the stimulated wt TSHR IP accumulation. The single mutants S508G and N674D also showed an impaired bTSH stimulation of the IP pathway in comparison to the wt TSHR. None of the double substitutions, or any of the single substitutions D633K, N674A, and N674S, activated the PLC-IP pathway (Table 1Go).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The identification of a large number of different constitutively activating TSHR mutations led to the identification of two important domains for intramolecular TSHR signal transduction—the TM6 and the intracellular loop 3 (31). In the TSHR and the LHR an aspartate residue is located in the highly conserved TM6 at positions 633 and 578, respectively. The aspartate residue at this position in TM6 is exclusive for glycoprotein hormone receptors. In 91% of the members of the Rhodopsin family of GPCRs, this aspartate position is replaced by phenylalanine (11). In our receptor model this aspartate residue in the TSHR or in the LHR points into a space between the helices of TM6, TM7, and TM3, right in the center of the seven-helix bundle. This prominent location of D633 is self-explanatory for its likely interhelical contacts.

Recent in vivo findings for the TSHR and the LHR support the hypothesis that this aspartate residue in TM6 could play a pivotal role in the activation process of the glycoprotein hormone receptors. Four different naturally occurring constitutively activating mutations of the D633 residue of the TSHR are known (Fig. 1Go). Analogous to the TSHR D578 in the LHR is the only position with four different amino acid substitutions (D578G, D578Y, D578H, D578E) (27, 32, 33, 34, 35). Kosugi et al. (29) suggested that the D578 side chain could serve as a properly positioned hydrogen bond acceptor and could thus be important for stabilizing the inactive state of the LHR. Furthermore, Lin et al. (17) proposed that the H bond interactions between TM6 and TM7 could constrain the inactive receptor state and that release or weakening of the H bonds could cause reorientation of TM6 as an important step in LHR activation. Rearrangements of TM6 during the activation of the TSHR were concluded from mutations disturbing the positions of the tightly packed hydrophobic helices of TM5 and TM6 (13). An additional rearrangement of TM7 in the LHR was recently suggested by data obtained with different gain or loss of function mutants of the two highly conserved asparagines in TM7 (N670 and N674 in the TSHR) (36). Moreover, recent evidence demonstrates that the activation of GPCRs is accompanied by a rigid body movement of the intracellular portion of TM6 and especially of the TM6 region relative to the other TMs (18, 19, 37). Investigations, based on the protein structure of bovine and frog rhodopsin (38, 39), and experimental data, obtained for the LHR (17), suggest that the activation signal is propagated from the ligand binding site along the axis TM2–TM3—TM7 and further to TM6 toward the intracellular part of the receptor.

Based on the observation that D633 is the only amino acid with four different amino acid exchanges in vivo (Fig. 1Go), this study focused on the pivotal role of the D633 residue in the TSHR for interhelical interactions, receptor activation, and signaling. The further in vitro substitutions at this position led to constitutive activation (D633Y, F, C) as well as to inactivation of the TSHR without changes of membrane expression or binding (D633R). Substitution of D633 with F or W caused the highest constitutive activity of the cAMP pathway as well as constitutive activation of the IP pathway. These findings are in accordance with the previously reported data for the LHR (29). The high constitutive activity for the cAMP pathway is very likely due to the introduction of bulky hydrophobic side chains. Phenylalanine and tryptophan require not only a larger space at position 633 but also tend to escape from the strong hydrophilic proximity of TM7 toward hydrophobic patches either between TM6 (F634), TM3 (e.g. L512), and TM5 (e.g. F594) or between TM6 and TM7 (L677). Both possibilities result in a rearrangement of TM6 (e.g. by pushing the helices apart). A further likely consequence is a partial destabilization of the transmembrane segments and their packing. These strong conformational rearrangements obviously release conformational constraints, most likely enabling the receptor to interact with heterotrimeric Gq/11 proteins and thus initiating the constitutive activation of the IP pathway (Table 1Go). The same effect seems to be true for the D633H substitution. At physiological pH conditions, histidine has H acceptor and also H donator properties. However, histidine also has a bulky shape similar to phenylalanine. Our results demonstrate that the substitution of D633 with H results in a strong constitutive activity for both the cAMP and the IP signaling cascades. To our knowledge, this is the first time that constitutive activity for the IP pathway could also be demonstrated for the naturally occurring D633H TSHR mutation (40). Recently, in a patient with a Leydig cell tumor, the homologous D578H mutation in the LHR was identified, which was also constitutively active for both signaling pathways (33).

In cells transfected with the D633{Delta} mutant, neither the adenylate cyclase nor the PLC cascade could be stimulated by TSH (Fig. 2Go and Table 1Go). This could be due to the strongly decreased cell surface expression of this receptor mutant (Fig. 4AGo). Detection of the D633{Delta} receptor within the cells suggested that this receptor mutant was synthesized (Fig. 4BGo). Obviously, this receptor variant does not reach the cell surface, most likely because of strong alterations of the receptor folding. In the vasopressin V2 receptor, a deletion of the V278 residue in TM6 was reported in a patient with diabetes insipidus (41). This V278{Delta} mutant totally abolished receptor-ligand binding and subsequent adenylyl cyclase stimulation (42). These identical phenotypes of two deletion mutants in TM6 in different GPCRs suggest that a deletion of a central transmembrane residue probably causes a strong perturbation of the helix packing within the shifted part by interrupting interactions of complementary side chains between the helices (e.g. TM6 and TM5).

Our first major experimental finding was that the substitution D633R holds the TSHR tightly in a completely inactive conformation since this receptor does not respond to TSH stimulation. This is the first mutation in TM6 that inactivates the TSHR without impairing cell surface expression or ligand binding. Others have proposed that the central aspartate in TM6 in the LHR (D633 in the wt TSHR) functions as an H acceptor. D633, together with T632, is therefore most likely involved in the H bonding network between the central portions of TM6 and the two asparagines (N670, N674 in the wt TSHR) of TM7 (17). This H bonding network has been proposed to be essential to preserve the inactive receptor conformation. Molecular dynamics simulations (MD) of our wt TSHR model suggested conformations in which D633 primarily forms an interhelical H bond with N674 (Fig. 7Go). In the hydrogen bonding network constraining the inactive state, N674 also forms an intrahelical H bond to N670 in TM7. In addition to establishing the strong interhelical H bond to TM7 during the MD runs, D633 was also observed to simultaneously form intrahelical H bonds with TM6. H bonds have been observed between D633 and the side chains of C636 and/or T632, but with somewhat weaker occurrence. Both of these residues are highly conserved within the glycoprotein receptors and have been reported to be affected by mutation T632 in the TSHR and mutation C581 in the LHR (TSHR C636) (12, 27).



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Figure 7. Model of the Transmembrane Core of the Human TSHR Including the Interaction between Position 633 in TM6 and Position 674 in TM7

Left panel, Molecular model of the TSHR‘s inactive state with extra- and intracellular loops, without the large N-terminal domain. The TM is based on the low-resolution structure of frog rhodopsin (8 ). The intracellular loops are based on the nuclear magnetic resonance structures of peptides from rhodopsin (50 ), while the extracellular loops are assembled from fragments occurring several times with a similar backbone in the protein database. Right panel, Inactive state of the TSHR (basal activity): the hydrogen donator N674 of TM7 interacts with D633 of TM6. N674 has a carboxylate oxygen (red) available for the activation process along TM7.

 
Based on the experimental inactivation of the TSHR by the D633R mutation, we performed further MD simulations with different "mutated" TSHR models. Possible interaction partners of position 633 were searched, which could be involved in the molecular mechanisms of TSHR activation or inactivation. We started with different side chain conformations for R633. They were generated by scanning a side chain rotamere library of an {alpha}-helix backbone. The replacement of the H acceptor aspartate with the much larger but flexible H-donating side chain arginine at position 633 causes a rigorous opposite orientation of the H bond network between TM6 and TM7. Clearly, the function of residue N674 in TM7 is determined by its function as an H-donating side chain toward the H-accepting TM6. This is only possible if the asparagine midgroup can rotate. Subsequently, the carboxylate oxygen atom of the N674 side chain located in TM7, which is not primarily involved in interhelical interactions in the wt TSHR, is occupied in the D633R mutant (Fig. 6Go, A and B). The R633-TSHR model suggested that arginine, due to five available donating hydrogens, is able to simultaneously form several H bonds between TM6 and TM7 or between TM6 and TM3, respectively (Fig. 6Go). In addition to N674, the model screening also revealed the backbone of TM6, S508 in TM3, or N670 as putative interaction partners for R633. These residues were investigated by a strategy of double mutations, designed to release the totally blocked inactive TSHR conformation and to rescue at least part of the TSHR activity by deleting the H bonds of R633 with its possible interaction partners suggested by computer modeling. With the double mutant S508G/D633R, we could point out that there is no H bond interaction of R633 with S508 in TM3, because this receptor remains in a completely inactive state (Table 1Go). The same applies to mutants of N670 in TM7, since the double mutant R633 combined with N670 was also inactive.

Our second major experimental finding is the partial rescue of the TSHR activity by the double mutant D633R/N674D (Fig. 5Go). Interestingly, aspartate at position 674 contains two oxygen atoms in the side chain, where according to our model one oxygen is involved in the H bond with R633. Therefore, the other oxygen is available in TM7 as an H acceptor for the activation process (Fig. 6CGo). Additional arginine double mutants containing serine or alanine in position 674, and thus missing a freely available carboxylate oxygen as a strong H bond acceptor, were not able to rescue any TSHR activity. Results for mutants like N674A (see Table 1Go) prompted us to assume (an) additional partner(s) for R633. Based on our experimental data, we suggest the following scenario. Since S508 and N670 are not involved in R633 interactions, it appears likely that the completely inactive D633R mutant locks the receptor by H bonds between N674 in TM7 and the backbone of TM6. N674 in TM7 forms H bonds with TM6 in the unstimulated wt (D633) (Figs. 6Go and 7Go) and the mutant (R633) TSHR as well. However, the rotatable amide group allows N674 in the wt TSHR (D633) to act as a hydrogen donator, whereas in the mutant TSHR (R633) it acts as a hydrogen acceptor. Thus in the wt TSHR there is a free carboxylate oxygen at position 674 available as an H acceptor (TM7), which mediates the TSHR activation by H bond release upon TSH stimulation (Figs. 6Go and 7Go). However, R633 enforces an inverted H bond with N674 and causes a reverse effect, since in N674 the only side chain oxygen is already involved in an H bond with R633 and no longer available in TM7 for the activation process. Moreover, the mutant receptor R633 presumably not only interacts with N674 but also with TM6. As a consequence, the receptor is constrained in an inactive conformation not responsive to TSH stimulation. Due to the rotatable midgroups of the two asparagines, it is conceivable that the activation signal is propagated along TM7 by a mechanism inducing an inversion of the H bond network between the highly conserved residues N670 and N674, thereby causing a release of interhelical H bonds between TM6 and TM7. The availability of a side chain carboxylate oxygen in position 674 is obviously essential to serve as H acceptor for this activation mechanism along TM7 for the glycoprotein hormone receptors. Interestingly, not only the cAMP pathway is affected by this phenomenon. Of all our single mutants, only those containing aspartate or asparagine either in position 633 or in position 674 are able to stimulate the IP pathway. Therefore, the activation of the IP pathway also appears to be initiated by the release of the H bonds between TM6 and TM7. Alternatively, there could be an intersection of both pathways at position N674 within the highly conserved (N/D)PxxY motif. This hypothesis is supported by the description of the IP sensitive mutation N391A (homologous to N674 in the TSHR) in the cholecystokinin B receptor, which also abolishes Gq protein activation without affecting binding or expression of the receptor (43).

Moreover, the interactions between positions 633 and 674 and the importance of a free H acceptor in position 674 (Fig. 7Go) could be of relevance for all glycoprotein hormone receptors since nearly all glycoprotein hormone receptors contain aspartate in position 633 and asparagine in position 674. Only the FSH receptor-TSHR of Drosophila melanogaster (EMBL accession no. No. AAB07030) differs from all other glycoprotein hormone receptors by amino acid exchanges in both positions (asparagine in position 633 and aspartate in position 674, according to TSHR cDNA) (44). Our results and the suggested scenario for the intramolecular TSHR signaling are also complementary to recently reported mutations in TM7 of the rat LHR. Transferred to our TSHR model, the gain of function induced by the point mutations N593R and N597Q (equivalent to N670, N674 in the TSHR) in the rat LHR (36) is most likely caused by the introduction of the larger hydrophilic side chains. They allow a spontaneous rearrangement of TM6 by inducing larger distances while still maintaining a hydrogen bond network between TM7 and TM6. Moreover, at the mutated side chain in position 674 in TM7 (N597Q rat LHR), there is an H acceptor atom available for the activation process. Mutants of the rat LHR with loss of function are reported for N597R (position 674 TSHR) and N593A, Q (position 670 TSHR). These results fit perfectly with our hypothesis postulating an essential H acceptor at this position together with an H bond network along the TM7 segment. Only the reported loss of function for the rat LHR N593S mutant (position 670 in the TSHR) differs from our findings and the findings of others (30) showing a constitutive activity for N670S in the TSHR. This disagreement might illustrate the slightly different activation process of the rat LHR compared with the human TSHR, which remains to be elucidated.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Site-Directed Mutagenesis
The TSHR mutations were created by standard PCR mutagenesis techniques (45) using the human TSHR plasmid TSHR-pSVL (46) as template. For the construction of all double mutants, the TSHR fragment with the D633R mutation was used as a template for the PCR. Except for the S508G mutation and the double mutant S508G/D633R, the PCR fragments containing the mutation were digested and used to replace the corresponding Eco81I/Eco91I (MBI Fermentas, Vilnius, Lithuania) fragment in the TSHR-pSVL vector. For the S508G mutant and the double mutant S508G ID633R, the PCR fragment was incompletely digested with ScaI (there is an additional ScaI site within pSVL) and subsequently with Eco91I. The mutated TSHR constructs were generated by replacing the ScaI/Eco91I fragment in the wild-type TSHR cloned in pSVL with the corresponding mutated fragment. Mutated TSHR sequences were confirmed 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 Transfection
COS-7 cells were grown in DMEM supplemented with 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin (Life technologies, Paisley, UK) at 37 C in a humidified 7% CO2 incubator. Cells were transiently transfected in 12-well plates (1 x 105 cells per well) with 1 µg DNA/well using the FuGENE6 reagent (Roche, Basel, Switzerland).

Radioligand Binding Assay
Competitive binding studies were performed as previously described (15). Data were analyzed assuming a one-site binding model using the fitting module of SigmaPlot 2.0 for Windows (47).

FACS Analysis
Forty eight hours after transfection nonpermeabilized cells were detached from the dishes using 1 mM EDTA and 1 mM EGTA in PBS and transferred in Falcon 2052 tubes. Before incubation with the primary antibody, cells were washed once with PBS containing 0.1% BSA and 0.1% NaN3.

For permeabilized cell assays, cells were first fixed with 1% paraformaldehyde for 10 min on ice following an incubation with PBS containing 0.1% BSA, 0.1% NaN3, and 0.2% Saponin for 30 min. Saponin was supplemented in all subsequent buffers.

Afterward, cells were incubated with a mouse antihuman TSHR antibody (2C11, Serotec, Oxford, U.K.; 10 µg/ml) in the same buffer. Tubes were washed and incubated for 1 h on ice in the dark with fluorescein-conjugated F(ab')2 rabbit antimouse IgG (Serotec, dilution 1:1000). Before FACS analysis (FACscan Becton Dickinson and Co., Franklin Lakes, NJ), cells were washed twice and 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
Forty eight hours after transfection, measurement of cAMP accumulation was performed as previously described (15). Moreover, TSHR single mutants at position 633 were expressed at the same level, to additionally determine the cAMP accumulation at comparable cell surface expressions. To decrease the cell surface expression of the TSHR constructs expressed at a higher level (wt TSHR, D633C, D633H, D633F) to the level of the D633W mutant, we cotransfected these constructs with various amounts of plasmids containing the human V2 vasopressin receptor (48).

Stimulation of IP Formation
Transfected COS-7 cells were incubated with 2 µCi/ml of [myo-3H] inositol (18.6 Ci/mmol), Amersham Pharmacia Biotech, Braunschweig, Germany) for 6 h. Thereafter, cells were preincubated with serum free DMEM without antibiotics containing 10 mM LiCl for 30 min. Stimulation with TSH was performed with the same medium supplemented with 100 mU/ml TSH for 1 h. Basal and TSH-induced increases in intracellular IP levels were determined by anion exchange chromatography as described (49). IP values are expressed as the percentage of radioactivity incorporated from (3)[H]-inositol phosphates (IP1–3) over the sum of radioactivity incorporated in IPs and phosphatidylinositols.

Molecular Modeling
The approach used to construct the TSHR model was the same as previously described (13). Packing of the transmembrane helices was based on electron density maps of frog rhodopsin (8). The TSHR structure model was computed with special emphasis on the transmembrane and intracellular portions, without the large amino-terminal domain but including the extra- and intracellular loops. The starting conformation of the intracellular loops i1, i2, and the first portion of the C-terminal tail comprising the putative i4 loop of the TSHR were adopted from the nuclear magnetic resonance structure of the rhodopsin cytosolic loop peptide complex (50) as described elsewhere for the V2 receptor (51). For the remaining parts of the intracellular loops as well as for the extracellular loops, fragments of four to seven residues were selected and tested against the three-dimensional protein database. Only fragments occurring more than once with a similar backbone conformation in the database were used for assembling the loops. All model components were assembled with the biopolymer module of the SYBYL program package (TRIPOS Inc., St. Louis, MO) using the AMBER 5.0 force field (52). MD simulations were performed at 300 K for 200 psec, where only the helix stability was maintained by restraints for hydrogen bonds of the TM backbones.


    ACKNOWLEDGMENTS
 
We would like to thank Mrs. Eileen Bösenberg for her technical assistance and Th. Gudermann for discussions. We thank BRAHMS Diagnostica (Berlin, Germany) for providing 125I-bTSH. We thank Dr. Vassart for supplying the plasmid TSHR-pSVL.


    FOOTNOTES
 
This work was supported by the Deutsche Forschungsgemeinschaft (Pa 423/3-2) and the Bundesministerium für Bildung und Forschung (BMB+F), Interdisciplinary Center for Clinical Research (IZKF) at the University of Leipzig (01KS 9504, project B10 and B14).

Abbreviations: bTSH, bovine TSH; FACS, fluorescence-activated cell sorting; GPCR, G protein-coupled receptor; IP, inositol phosphate; LHR, LH receptor; MD, molecular dynamics simulations; TSHR, TSH receptor; TM, transmembrane domain; V2R, human V2 receptor; wt, wild type

Received for publication October 23, 2000. Accepted for publication April 4, 2001.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Gudermann T, Nurnberg B, Schultz G 1995 Receptors and G proteins as primary components of transmembrane signal transduction. I. G-protein-coupled receptors: structure and function. J Mol Med 73:51–63[Medline]
  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[Abstract]
  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. Baldwin JM 1994 Structure and function of receptors coupled to G proteins. Curr Opin Cell Biol 6:180–190[Medline]
  6. Gudermann T, Schoneberg T, Schultz G 1997 Functional and structural complexity of signal transduction via G-protein-coupled receptors. Annu Rev Neurosci 20:399–427[CrossRef][Medline]
  7. Shenker A 1995 G protein-coupled receptor structure and function: the impact of disease-causing mutations. Baillieres Clin Endocrinol Metab 9:427–451[Medline]
  8. Unger VM, Hargrave PA, Baldwin JM, Schertler GF 1997 Arrangement of rhodopsin transmembrane {alpha}-helices. Nature 389:203–206[CrossRef][Medline]
  9. Palczewski K, Kumasaka T, Hori T, et al. 2000 Crystal structure of rhodopsin: a G protein-coupled receptor. Science 289:739–745[Abstract/Free Full Text]
  10. Strader CD, Fong TM, Tota MR, Underwood D, Dixon RA 1994 Structure and function of G protein-coupled receptors. Annu Rev Biochem 63:101–132[CrossRef][Medline]
  11. Van Rhee AM, Jacobson KA 1996 Molecular architecture of G protein-coupled receptors. Drug Dev Res 1–38
  12. Paschke R, Ludgate M 1997 The thyrotropin receptor in thyroid diseases. N Engl J Med 337:1675–1681[Free Full Text]
  13. Biebermann H, Schoneberg T, Schulz A, et al. 1998 A conserved tyrosine residue (Y601) in transmembrane domain 5 of the human thyrotropin receptor serves as a molecular switch to determine G- protein coupling. FASEB J 12:1461–1471[Abstract/Free Full Text]
  14. Kosugi S, Okajima F, Ban T, Hidaka A, Shenker A, Kohn LD 1992 Mutation of alanine 623 in the third cytoplasmic loop of the rat thyrotropin (TSH) receptor results in a loss in the phosphoinositide but not cAMP signal induced by TSH and receptor autoantibodies. J Biol Chem 267:24153–24156[Abstract/Free Full Text]
  15. Wonerow P, Schoneberg T, Schultz G, Gudermann T, Paschke R 1998 Deletions in the third intracellular loop of the thyrotropin receptor. A new mechanism for constitutive activation. J Biol Chem 273:7900–7905[Abstract/Free Full Text]
  16. Trulzsch B, Nebel T, Paschke R 1999 The thyrotropin receptor mutation database. Thyroid 9:521–522[Medline]
  17. Lin Z, Shenker A, Pearlstein R 1997 A model of the lutropin/choriogonadotropin receptor: insights into the structural and functional effects of constitutively activating mutations. Protein Eng 10:501–510[Abstract]
  18. Dunham TD, Farrens DL 1999 Conformational changes in rhodopsin. Movement of helix f detected by site-specific chemical labeling and fluorescence spectroscopy. J Biol Chem 274:1683–1690[Abstract/Free Full Text]
  19. Farrens DL, Altenbach C, Yang K, Hubbell WL, Khorana HG 1996 Requirement of rigid-body motion of transmembrane helices for light activation of rhodopsin. Science 274:768–770[Abstract/Free Full Text]
  20. Abell AN, Segaloff DL 1997 Evidence for the direct involvement of transmembrane region 6 of the lutropin/choriogonadotropin receptor in activating Gs. J Biol Chem 272:14586–14591[Abstract/Free Full Text]
  21. Javitch JA, Fu D, Liapakis G, Chen J 1997 Constitutive activation of the ß2 adrenergic receptor alters the orientation of its sixth membrane-spanning segment. J Biol Chem 272:18546–18549[Abstract/Free Full Text]
  22. Rasmussen SG, Jensen AD, Liapakis G, Ghanouni P, Javitch JA, Gether U 1999 Mutation of a highly conserved aspartic acid in the ß2 adrenergic receptor: constitutive activation, structural instability, and conformational rearrangement of transmembrane segment 6. Mol Pharmacol 56:175–184[Abstract/Free Full Text]
  23. Kosugi S, Shenker A, Mori T 1994 Constitutive activation of cyclic AMP but not phosphatidylinositol signaling caused by mutations in the 6th transmembrane helix of the human thyrotropin receptor. FEBS Lett 356:291–294[CrossRef][Medline]
  24. Porcellini A, Ciullo I, Laviola L, Amabile G, Fenzi G, Avvedimento VE 1994 Novel mutations of thyrotropin receptor gene in thyroid hyperfunctioning adenomas. Rapid identification by fine needle aspiration biopsy. J Clin Endocrinol Metab 79:657–661[Abstract]
  25. Russo D, Arturi F, Suarez HG, et al. 1996 Thyrotropin receptor gene alterations in thyroid hyperfunctioning adenomas. J Clin Endocrinol Metab 81:1548–1551[Abstract]
  26. Russo D, Tumino S, Arturi F, et al. 1997 Detection of an activating mutation of the thyrotropin receptor in a case of an autonomously hyperfunctioning thyroid insular carcinoma. J Clin Endocrinol Metab 82:735–738[Abstract/Free Full Text]
  27. Wu SM, Leschek EW, Rennert OM, Chan WY 2000 Luteinizing hormone receptor mutations in disorders of sexual development and cancer. Front Biosci 5:D343–D352
  28. Van Sande J, Raspé E, Perret J, et al. 1990 Thyrotropin activates both the cyclic AMP and the PIP2 cascades in CHO cells expressing the human cDNA of TSH receptor. Mol Cell Endocrinol 74:R1–R6
  29. Kosugi S, Mori T, Shenker A 1996 The role of Asp578 in maintaining the inactive conformation of the human lutropin/choriogonadotropin receptor. J Biol Chem 271:31813–31817[Abstract/Free Full Text]
  30. Tonacchera M, Van Sande J, Cetani F, et al. 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]
  31. Farid NR, Kascur V, Balazs C 2000 The human thyrotropin receptor is highly mutable: a review of gain-of-function mutations. Eur J Endocrinol 143:25–30[Medline]
  32. Laue L, Chan WY, Hsueh AJ, et al. 1995 Genetic heterogeneity of constitutively activating mutations of the human luteinizing hormone receptor in familial male-limited precocious puberty. Proc Natl Acad Sci USA 92:1906–1910[Abstract]
  33. Liu G, Duranteau L, Carel JC, Monroe J, Doyle DA, Shenker A 1999 Leydig-cell tumors caused by an activating mutation of the gene encoding the luteinizing hormone receptor. N Engl J Med 341:1731–1736[Free Full Text]
  34. Themmen APN, Brunner HG 1996 Luteinizing hormone receptor mutations and sex differentiation. Eur J Endocrinol 134:533–540[Medline]
  35. Wu SM, Leschek EW, Brain C, Chan WY 1999 A novel luteinizing hormone receptor mutation in a patient with familial male-limited precocious puberty: effect of the size of a critical amino acid on receptor activity. Mol Genet Metab 66:68–73[CrossRef][Medline]
  36. Angelova K, Narayan P, Simon JP, Puett D 2000 Functional role of transmembrane helix 7 in the activation of the heptahelical lutropin receptor. Mol Endocrinol 14:459–471[Abstract/Free Full Text]
  37. Sheikh SP, Zvyaga TA, Lichtarge O, Sakmar TP, Bourne HR 1996 Rhodopsin activation blocked by metal-ion-binding sites linking transmembrane helices C and F. Nature 383:347–350[CrossRef][Medline]
  38. Schertler GF, Villa C, Henderson R 1993 Projection structure of rhodopsin. Nature 362:770–772[CrossRef][Medline]
  39. Schertler GF, Hargrave PA 1995 Projection structure of frog rhodopsin in two crystal forms. Proc Natl Acad Sci USA 92:11578–11582[Abstract]
  40. Parma J, Duprez L, Van Sande J, et al. 1997 Diversity and prevalence of somatic mutations in the thyrotropin receptor and Gs{alpha} genes as a cause of toxic thyroid adenomas. J Clin Endocrinol Metab 82:2695–2701[Abstract/Free Full Text]
  41. Tsukaguchi H, Matsubara H, Aritaki S, Kimura T, Abe S, Inada M 1993 Two novel mutations in the vasopressin V2 receptor gene in unrelated Japanese kindreds with nephrogenic diabetes insipidus. Biochem Biophys Res Commun 197:1000–1010[CrossRef][Medline]
  42. Tsukaguchi H, Matsubara H, Mori Y, et al. 1995 Two vasopressin type 2 receptor gene mutations R143P and delta V278 in patients with nephrogenic diabetes insipidus impair ligand binding of the receptor. Biochem Biophys Res Commun 211:967–977[CrossRef][Medline]
  43. Gales C, Kowalski-Chauvel A, Dufour MN, et al. 2000 Mutation of Asn 391 within the conserved NPXXY motif of the cholecystokinin B receptor abolishes Gq protein activation without affecting its association with the receptor. J Biol Chem 275:17321–17327[Abstract/Free Full Text]
  44. Hauser F, Nothacker HP, Grimmelikhuijzen CJ 1997 Molecular cloning, genomic organization, and developmental regulation of a novel receptor from Drosophila melanogaster structurally related to members of the thyroid-stimulating hormone, follicle-stimulating hormone, luteinizing hormone/choriogonadotropin receptor family from mammals. J Biol Chem 272:1002–1010[Abstract/Free Full Text]
  45. Higuchi R 1989 Using PCR to engineer DNA. In: Ehrlich HA, ed. PCR technology. New York: Stockton Press; 61–70
  46. Libert F, Lefort A, Gérard C, et al. 1989 Cloning, sequencing and expression of the human thyrotropin (TSH) receptor: evidence for binding of autoantibodies. Biochem Biophys Res Commun 165:1250–1255[Medline]
  47. Swillens S 1995 Interpretation of binding curves obtained with high receptor concentrations: practical aid for computer analysis. Mol Pharmacol 47:1197–1203[Abstract]
  48. Schöneberg T, Yun J, Wenkert D, Wess J 1996 Functional rescue of mutant V2 vasopressin receptors causing nephrogenic diabetes insipidus by a co-expressed receptor polypeptide. EMBO J 15:1283–1291[Abstract]
  49. Berridge MJ 1983 Rapid accumulation of inositol trisphosphate reveals that agonists hydrolyse polyphosphoinositides instead of phosphatidylinositol. Biochem J 212:849–858[Medline]
  50. Yeagle PL, Alderfer JL, Albert AD 1997 Three-dimensional structure of the cytoplasmic face of the G protein receptor rhodopsin. Biochemistry 36:9649–9654[CrossRef][Medline]
  51. Krause G, Hermosilla R, Oksche A, Rutz C, Rosenthal W, Schulein R 2000 Molecular and conformational features of a transport-relevant domain in the C-terminal tail of the vasopressin V(2) receptor. Mol Pharmacol 57:232–242[Abstract/Free Full Text]
  52. Weiner S, Kollmann P, Nguyen D, Case T 1986 An all atom forcefield for simulation of proteins und nucleic acids. J Comp Chem 7:230–252
  53. 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]