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Molecular Endocrinology 20 (4): 893-903
Copyright © 2006 by The Endocrine Society

Repulsive Separation of the Cytoplasmic Ends of Transmembrane Helices 3 and 6 Is Linked to Receptor Activation in a Novel Thyrotropin Receptor Mutant (M626I)

Usanee Ringkananont1, Joost Van Durme1, Lucia Montanelli, Figen Ugrasbul, Y. Miles Yu, Roy E. Weiss, Samuel Refetoff and Helmut Grasberger

Departments of Medicine (U.R., R.E.W., S.R., H.G.), Pediatrics (S.R.), and Committee on Genetics (S.R.), The University of Chicago, Chicago, Illinois 60637; Institut de Recherche Interdisciplinaire en Biologie Humaine et Nucleaire (J.V.D., L.M.), Universite Libre de Bruxelles, B1070 Brussels, Belgium; and Children’s Mercy Hospital (F.U., Y.M.Y.), Kansas City, Missouri 64108

Address all correspondence and requests for reprints to: Helmut Grasberger, The University of Chicago, MC3090, 5841 South Maryland Avenue, Chicago, Illinois 60637. E-mail: hgrasber{at}uchicago.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Ligand-dependent activation of G protein-coupled receptors (GPCRs) involves repositioning of the juxtacytoplasmic ends of transmembrane helices TM3 and TM6. This concept, inferred from site-directed spin labeling studies, is supported by chemical cross-linking of the cytoplasmic ends of TM3 and TM6 blocking GPCR activation. Here we report a novel constitutive active mutation (M626I) in TM6 of the TSH receptor (TSHR), identified in affected members of a family with nonautoimmune hyperthyroidism. The specific constitutive activity of M626I, measured by its basal cAMP generation corrected for cell surface expression, was 13-fold higher than that of wild-type TSHR. Homology modeling of the TSHR serpentine domain based on the rhodopsin crystal structure suggests that M626 faces the side chain of I515 of TM3 near the membrane-cytoplasmic junction. Steric hindrance of the introduced isoleucine by I515 is consistent with the fact that shorter or more flexible side chains at position 626 did not increase constitutivity. Furthermore, a reciprocal mutation at position 515 (I515M), when introduced into the M626I background, acts as revertant mutation by allowing accommodation of the isoleucine sidechain at position 626 and fully restoring the constitutive activity to the level of wild-type TSHR. Thus, repulsive separation of the juxtacytoplasmic TM6 and TM3 in the M626I model conclusively demonstrates a direct link between the opening of this cytoplasmic face of the receptor structure and G protein coupling.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE HUMAN TSH receptor (TSHR) belongs to the class A rhodopsin-like family of G protein-coupled receptors (GPCRs). The C-terminal half of TSHR comprises the seven putative transmembrane helices (TMs) prototypical of all GPCRs. The large N-terminal ligand-binding domain of TSHR is composed of leucine-rich repeats, a feature shared with the other glycoprotein hormone receptors, FSH receptor and LH/choriogonadotropin receptor (LHCGR).

The currently favored model for activation of GPCRs proposes an equilibrium between discrete inactive and active states, characterized by different relative positions of some of the TMs. There is compelling evidence that the separation of the cytoplasmic ends of TM3 and TM6 takes center stage as a common switch in GPCR activation (1, 2, 3, 4, 5, 6). A translational movement of the cytoplasmic end of TM6, away from the C terminus of TM3, in conjunction with a rotational movement around the helical axis, has first been proposed based on site-directed spin labeling studies of the conformational changes in rhodopsin upon photoactivation (7). An association between such a conformational change and receptor activation has now been demonstrated for class A and class D GPCRs (8, 9, 10, 11, 12, 13) and thus appears to be a general feature of GPCR activation. Also in support of this concept, cross-linking of the cytoplasmic ends of TM3 and TM6 either via disulfide bonds or metal chelate bridges interferes with GPCR activation (6, 7, 14).

Agonist binding appears to trigger this conformational change by disruption of intramolecular interactions between specific residues of the helix bundle. This concept is mainly inferred from the analysis of mutant receptors with an increase in constitutive (ligand-independent) activity compared with their wild-type (wt) counterparts.

Although mutations of critical residues outside of TM3 and TM6 can trigger a series of conformational changes that may lead to the relative movement of TM3 and TM6 (reviewed in Ref.4), most constitutive active mutations (CAMs) have been identified in the juxtacytoplasmic portions of TM6 and TM3, suggesting that they may directly affect critical TM3/TM6 interactions. The availability of atomic-resolution crystal structure models of the inactive form of bovine rhodopsin (15, 16) facilitates computational modeling of such constraints, yet only few have been characterized to date. For instance, although unlikely to be an universal feature of GPCRs, there is evidence for an ionic lock stabilizing the TM3/TM6 interaction at the cytoplasmic surface of certain GPCRs (12, 17). However, analysis of the rhodopsin structure predicts that the major forces stabilizing the juxtacytoplasmic portions of TM3 and TM6 are packing interactions within a hydrophobic layer composed of two methionine residues in TM6 faced by two leucine residues each in TM2 and TM3. This conserved arrangement is believed to prevent helix movement in the inactive receptor and facilitate the relative movement of TM3 and TM6 after photoactivation while retaining van der Waals contacts (16, 18).

In this report, we have used a combination of molecular modeling and site-directed mutagenesis experiments to characterize a novel CAM of TSHR (M626I) in the juxtacytoplasmic TM6, identified in a family with autosomal dominant, nonautoimmune hyperthyroidism. In the homology model of human TSHR, the side chain of M626 faces that of I515 in TM3. By identification of a reciprocal revertant mutant (I515M), we provide evidence for a shared microenvironment of M626 and I515. Isoleucine, but not smaller or more flexible aliphatic side chains at position 626, disrupts the juxtacytoplasmic TM3-TM6 interface secondary to steric hindrance by I515. The repulsive separation of the juxtacytoplasmic TM6 and TM3 in the M626I model indicates a direct link between opening of this cytoplasmic face of TSHR and Gs{alpha} protein coupling.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Detection of Novel TSHR Gene Mutation in Familial Nonautoimmune Hyperthyroidism
Five members of a nonconsanguineous family were found to have primary hyperthyroidism, apparently transmitted as an autosomal dominant trait (Fig. 1AGo). None of them had immunological or clinical signs of autoimmune thyroid disease. There was a variable age of onset and severity of the initial clinical presentation (see brief case history in Materials and Methods; results of thyroid function tests are listed in Fig. 1AGo).


Figure 1
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  		Fig. 1. Identification of a Novel Germline TSHR Mutation (M626I) in Familial Nonautoimmune Hyperthyroidism

A, Pedigree of the family with nonautoimmune hyperthyroidism segregating in an autosomal dominant fashion. Subjects are identified by generation (Roman numerals) and individual number (Arabic numerals). Results of thyroid function tests are aligned with each symbol, with values outside the normal range typed in boldface. TT4, Total serum T4; TT3, total serum T3; TrT3, total reverse T3; FT4I, free T4 index. B, Sequencing electropherogram of part of TSHR exon 10 of the propositus (III-3, left panel) compared with that of his euthyroid half-sister (III-1, right panel). The propositus has a heterozygous C>G transversion at the third position of codon 626. This mutation results in replacement of the normal methionine residue by isoleucine (M626I). The same heterozygous mutation was detected in all other hyperthyroid family members (III-2, III-4, II-4, I-1), but not in the two subjects with normal thyroid function tests (II-2, II-3). C, Stereo cartoon representation of the TMs in the homology-model of the human TSHR. TM3 is colored in red, TM6 is colored in blue, and all other TMs are colored in gray. The side chains of the investigated residues are represented as sticks (I515, M626, and D633) and are colored by atom type. The extracellular side is on top.

 
Sequencing of the TSHR gene revealed a novel G to C transversion at codon 626 in affected family members resulting in the replacement of a methionine residue by isoleucine (M626I) (Fig. 1BGo). M626 (M6.37 according to the standardized numbering system of Ballesteros and Weinstein (19) is completely conserved among glycoprotein hormone receptors (multiple sequence alignment available at http://gris.ulb.ac.be/) and located within the juxtacytoplasmic portion of TM6 in the TSHR model structure (Fig. 1CGo). M626 is part of a conserved hydrophobic layer arranged perpendicularly to the helix bundle and comprising residues of TM2, TM3, and TM6.

Functional Characterization of M626I as Constitutive Active Mutation
To evaluate the functional significance of the M626I mutation, we first tested the ability of the mutant protein to activate a cotransfected cAMP-responsive reporter gene (CRE-Luc). Under basal conditions (i.e. in the absence of TSH stimulation), cotransfection of wt TSHR expression vector into COS-7 cells produced approximately 4.5-fold stimulation of luciferase activity compared with cotransfection with empty vector (Fig. 2AGo). In contrast, coexpression of the M626I mutant resulted in 3-fold higher basal luciferase activity vs. wt receptor. The basal luciferase activation elicited by expression of M626I was only slightly lower than that produced by expression of D633A, a previously characterized mutation causing robust constitutive activation (20).


Figure 2
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  		Fig. 2. bTSH Concentration-Response Curves of wt and Mutant TSHRs

Activation of CRE-Luc by cotransfected wt or mutant TSHRs in COS-7 cells. Shown is the basal and stimulated luciferase activity using increasing concentrations of bTSH. Luciferase activities were normalized for Renilla luciferase activity driven from cotransfected pRL-Tk internal control vector. Bars represent means ± SD from three independent experiments performed in duplicate transfections. pSVL, transfection with empty expression vector; RLU, relative luciferase units (basal activity in pSVL transfected cells set to 1). A, For comparison with M626I, a previously described TSHR mutant (D633A) with robust constitutive activity was analyzed in the same experiments. B, Concentration-response curves of control mutants at position 626. C, Effect of the M515I mutation in the context of the M616I CAM. D, Effect of M515I in the background of the D633A control CAM.

 
In stimulation experiments with increasing doses of bovine TSH (bTSH), the reporter activity in cells transfected with the mutant readily increased in response to the lowest bTSH dose tested (5 x 10–4 U/liter) but reached a plateau at lower bTSH concentrations compared with wt TSHR expressing cells (Fig. 2AGo). The sensitive response of both mutants to bTSH is consistent with the increase in apparent affinity with no significant modification of hormone potency observed in most CAMs (21), including D633A (20). The lower maximum attainable activity of the mutants TSHRs compared with wt TSHR could be due to the lower cell surface expression of the former. The cell-surface expression was analyzed by fluorescence-activated cell sorting (FACS) using the monoclonal antibody BA8 directed against an epitope in the N-terminal TSHR ectodomain, which is unlikely to be altered by mutations in the transmembrane region. In agreement with the lower maximum bTSH-stimulated luciferase activity, M626I was only expressed at 28% of wt TSHR level (Fig. 3AGo). Surface expression of D633A was even lower compatible with previous reported data (22). In parallel, we measured the specific cAMP accumulation in transfected cells under basal conditions (Fig. 3BGo). The specific constitutive activity (SCA) of a mutant receptor molecule is represented by the specific cAMP accumulation normalized for the cell-surface expression of the receptor. This analysis revealed that M626I is approximately 13 times more constitutively active than wt TSHR (Fig. 3CGo).


Figure 3
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  		Fig. 3. Cell Surface Expression, Basal cAMP Generation, and Specific Constitutive Activity of Single and Double TSHR Mutants

A, Cell-surface expression of wt TSHR and the indicated single and double mutants in transiently transfected COS-7 cells was analyzed by FACS using the BA8 monoclonal antibody, which recognizes an epitope in the N-terminal receptor ectodomain. Results represent means ± SD of cell fluo-rescence from one representative experiment of three experiments. B, Levels of cAMP observed in cells transfected with empty pSVL vector, wt TSHR, or the indicated mutants. Results represent means ± SD of one representative experiment of three experiments. C, SCAs were determined by normalization of basal, background-subtracted cAMP levels to the cell-surface expression derived from the background-subtracted FACS signals. The background cAMP level and FACS signal was measured in cells transfected with empty pSVL vector. The average SCA values were rescaled setting SCA for wt TSHR to 1.

 
Molecular Modeling Predicts a Shared Microenvironment of M626 and I515 of TM3 and a Steric Conflict Caused by M626I
The molecular model of the TSHR serpentine structure based on the crystal structure of bovine rhodopsinsuggests that the side chain of M626 faces fully I515 (I3.46) in the adjacent TM3, with both residues being close to the membrane-cytoplasmic junction. In fact, these residues make a hydrophobic contact (C-C distance ~4 Å) through one of their side chain carbons (Fig. 4AGo). The residues are well packed, and no atomic clashes are observed in this microdomain. In contrast, modeling the replacement of M626 by isoleucine yields a preferred side chain conformation (Fig. 4BGo, cyan-colored side chain) not observed in the normal rotamer distribution. The optimal rotamer conformation for M626I is also depicted in Fig. 4BGo (magenta-colored side chain). This optimal rotamer of isoleucine does not fit into the microenvironment between TM3 and TM6 causing a steric conflict with the side chain of I515. The optimal distance between side chain carbon atoms in proteins is approximately 4 Å, with a minimum distance observed in the crystalline state of approximately 3 Å (23). The distance between the optimal rotamer of M626I and I515 (Fig. 4BGo) is only 2.7 Å (Table 1Go), indicating a severe atomic overlap. This mutation-induced clash was also predicted by the rotamer analysis module of the online Glycoprotein-Hormone Receptor Information System (GRIS; http://gris.ulb.ac.be/). As a consequence of the steric conflict with the side chain of I515, the cytoplasmic ends of TM3 and TM6 cannot approach each other as closely as in the wt receptor when the isoleucine at position 626 is to adopt its ideal rotamer (indicated by the magenta-colored side chain in Fig. 4BGo). The inferred separation of the cytoplasmic ends of TM3 and TM6 would render this part of the receptor more open and accessible to G proteins, thus providing an explanation for the observed constitutive activity on a structural level.


Figure 4
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  		Fig. 4. Modeling of Single and Double Mutants at Positions 515 and 626

In all panels, TM3 is colored in red, TM6 in blue, and, where shown, TM2 in orange. The side chains of relevant residues are represented as stick models colored by atom type. Yellow dotted lines indicate hydrophobic contacts between side chain carbon atoms (C-C distances listed in Table 1Go). A, Situation in wt TSHR showing contacts between M626 of TM6 and I515 of TM3 (C-C distance ~4 Å) and contact between I515 and L452 in TM2. B, Discrepancy between the modeled rotamer conformation of M626I (cyan) and the optimal rotamer conformation of M626I (magenta). C, Double reciprocal mutant M626I/I515M. The more flexible methionine at position 515 allows accommodation of the isoleucine side chain at position 626. D, M626A. E, M626L. F, M626V. G, Hydrophobic contacts of the methionine side chain at position 515 in the I515M mutant receptor.

 

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  		Table 1. Distances of Side Chain C-C Contacts and Total Energy after Energy Minimization

 
Isoleucine But Not Other Aliphatic Side Chains at Position 626 Invokes Constitutive Activity Consistent with Steric Hindrance by I515
We used two different approaches to test our prediction that forced opening of the cytoplasmic face of the receptor structure is responsible for the increased constitutive activity of M626I. First, asking whether the physicochemical properties of the isoleucine side chain are responsible for the constitutive activity of M626I, we substituted M626 by other aliphatic residues harboring side chains of distinct size and flexibility. We found that replacement of M626 with leucine (M626L) did not alter the SCA (0.96) compared with wt TSHR, whereas substitution by alanine (M626A) or valine (M626V) produced only weak constitutive activation compared with M626I (SCA of 3.0 and 2.4 for M626A and M626V, respectively) (Fig. 3CGo). The three artificial mutants of M626 showed no loss of sensitivity to bTSH (Fig. 2BGo), and were expressed at similar level, between 53 and 60% of wt TSHR (Fig. 3AGo). Molecular modeling revealed that the preferred rotamer of leucine at position 626 does not make interactive clashes as observed for the predicted rotamer of isoleucine (Fig. 4EGo). The small side chain of alanine also fits within the TM3/TM6 microdomain (Fig. 4DGo). Although Cß-branched like isoleucine, the side chain of valine is one carbon atom shorter and thereby can be accommodated at position 626 without causing steric conflicts (Fig. 4FGo). Thus, consistent with the restricted mutational space at position 626 for constitutive activation, none of the three artificial mutants was predicted to cause an atomic clash with I515 in the computational model that would lead to an opening of the cytoplasmic face of the receptor as inferred for the M626I mutant.

A Reciprocal Mutation in TM3 (I515M) Can Fully Restore the Constitutive Activity of M626I to the Level of wt TSHR
The experimental and molecular modeling data so far suggested that the constitutive activity of mutants at position 626 correlates with a specific steric hindrance by I515. In a second experimental approach to demonstrate the role of hydrophobic packing of the M626/I515 microenvironment in constraining basal receptor activity, we tested the effect of the reciprocal mutation I515M on constitutive activity in the background of M626I. We hypothesized that a reciprocal double mutant (I515M/M626I) would restore the hydrophobic packing to the situation in the wt receptor and, thus, lower the basal activity compared with M626I. Indeed, in the TSHR homology model (Fig. 4CGo), replacement of the bulky and conformationally restricted I515 by a more flexible methionine would create sufficient space to accommodate an isoleucine at position 626. As an additional control, the effect of M515I was also analyzed in the background of D633A (double mutant I515M/D633A). Because D633 lies in the central portion of TM6 and faces TM7, it is not part of the immediate I515 environment (compare Fig. 1CGo).

The various single and double mutants with I515M were expressed in the 30–105% range (Fig. 3AGo). In all cases, mutants with I515M had lower SCA than the corresponding receptors without I515M (Fig. 3CGo). However, compared with the parent receptors, the additional I515M mutation reduced the SCA of wt TSHR and D633A to 32% and 31%, respectively, but diminished the SCA of M626I to 8%. The SCA value for the I515M/M626I double mutant indicated that the reciprocal mutation I515M neutralized the constitutivity invoked by M626I (SCA = 1.05). All the mutants were able to respond to bTSH, with close correlation of the maximum attainable activity (at 50 U/liter bTSH) with the cell-surface expression determined by FACS (Fig. 2Go, C and D). Collectively, these findings demonstrated that, in the absence of agonist, M626 (TM6) and I515 (TM3) mediate hydrophobic interhelical contacts at the membrane-cytoplasmic junction, thereby preventing agonist-independent conversion of the receptor into an open, more active conformation.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
We present here the detailed analysis of a novel TSHR CAM, M626I, causing familial nonautoimmune hyperthyroidism. M626I is located in the juxtacytoplasmic TM6, which together with the cytoplasmic TM3 constitutes a mutational hotspot for CAMs. Still, the structural defects caused by mutations in this region and, therefore, the mechanisms underlying the constitutive activity, are largely unknown. There is, however, accumulating evidence from studies on various GPCRs that the ligand-induced activation of GPCRs involves the movement of TM6 and TM3 relative to each other (4, 6, 7, 8, 14), which most likely constitutes a conserved switch for receptor activation (24). We provide evidence using mutagenesis experiments and molecular modeling, that steric hindrance of the introduced isoleucine side chain with I515 of TM3 constitutes the molecular basis for the defect in M616I. Repulsion of the cytoplasmic ends of TM6 and TM3 appears to be sufficient to cause constitutive receptor activation, consistent with increased accessibility of the resulting open cytoplasmic face of the receptor structure to docking G proteins.

Probing the structural constraints of M626 by replacement with other hydrophobic residues of distinct side chain size and flexibility revealed that there is selectivity as to which newly introduced residues can confer constitutive activity. Of the tested amino acids at position 626, only isoleucine caused a robust increase in constitutivity. The specific side chain requirements at position 626 of the TSHR for increasing basal activity are reminiscent of findings for some, but not all, constitutive active GPCR mutants (25, 26, 27, 28, 29). In these cases, as with M626 of the TSHR, only a subset of possible substitutions of a given residue results in constitutive activation to variable degree. It has been suggested that, in addition to the disruption of interhelical bonds stabilizing the inactive state, constitutive activity of these mutants requires the formation of new bonds involving the introduced residue, which can stabilize an activated state of the receptor (30). As a logical extension of that idea, one may hypothesize that such mutant receptors are in a locked on state of intermediate to full activation, which would be relatively resistant to further activation by the addition of agonist. Indeed, such a phenomenon has been recently described for specific CAMs (31, 32). However, not only was M626I fully responsive to bTSH (Fig. 2AGo), but all of our experimental results can be accounted for in the absence of stabilizing interactions of the introduced isoleucine.

Rather, the results obtained with artificial mutants at position 626, together with the molecular modeling data, indicated that steric hindrance of M626I caused by the side chain of I515 in TM3 was the likely basis of the constitutive activity. This concept was further corroborated by the analysis of the reciprocal double mutant (I515M/M626I), predicted to act as revertant mutant by eliminating the steric clash of I515 with M626I. Indeed, the I515M mutant fully restored the basal activity to the level of wt TSHR when introduced in the background of M626I (Fig. 3CGo). Although to a lesser degree, I515M also reduced the basal activity as a single mutant (wt TSHR background) or in combination with D633A. The latter finding can, however, be explained by analysis of the I515 and I515M models, which reveals that a methionine at position 515 makes more hydrophobic contacts with neighboring residues than an isoleucine. The side chain of I515 is predicted to make one contact each with the side chains of M626 and L452 (TM2) (Fig. 4AGo). In contrast, a methionine at position 515 is predicted to have a total of 5 hydrophobic interactions, two contacts with M626 and one contact each with I622 (TM6), L452 (TM2), and R519 (TM3) (Fig. 4GGo). The three additional contacts made by I515M, but not I515, conceivably stabilize the inactive conformation by improving interhelical packing. Thus, apart from enabling the accommodation of the isoleucine side chain of M626I, the I515M mutant also increases the stability of the inactive form of the receptor independently of the presence of the M626I mutation. The specific constitutive activity of the double mutants implied that these two mechanisms act in an additive fashion, accounting for a relative more pronounced effect of M515I on basal activity of M626I as compared with wt TSHR or D633.

Our results are consistent with the idea that the introduced steric conflict causes repulsion of TM3 and TM6, shifting the cytoplasmic face of the receptor in a more active conformation. Still, based on strong evidence for a direct role of TM6 in Gs{alpha} coupling, an alternative scenario could be imagined where M626I directly enhances docking of G proteins by being part of the interacting molecular surface. In this respect, it is of interest that the peptide KMAILIFT corresponding to the N-terminal region of TM6 of LHCGR (residues 570–577; corresponding 625–632 region of TSHR: RMAVLIFT) stimulates adenyl cyclase activity by direct activation of Gs{alpha} (33). Introducing the natural I575L CAM increased the activity of this TM6 peptide. On the contrary, a LHCGR TM6 peptide with M571I substitution, a natural CAM (34, 35) equivalent to M626I of TSHR, did not have increased activity compared with the wt peptide (36). To explain this discrepancy, it has been speculated that, in the context of the holoreceptor, M571 may not directly interact with Gs{alpha}, and inhibit the activity of the receptor by stabilizing TM6 in an inactive conformation through interhelical interactions. The results reported here for TSHR provide experimental evidence for this hypothesis. Specifically, we demonstrated that M626 (equivalent to M571 of LHCGR) mediates the interaction of the juxtacytoplasmic portions of TM6 and TM3 by forming a microenvironment with I515, a residue completely conserved in glycoprotein hormone receptors. Forced separation of TM3 and TM6 by steric clash with I515 is the basis for the defect in M626I, whereas a direct effect of the mutated residue on Gs{alpha} docking, although not excluded by our data, would be inconsistent with the peptide experiments obtained with the highly conserved LHCGR peptide.

It should be noted that our model for the structural defect in M626I differs substantially from one previously proposed for the equivalent M571I LHCGR mutant based on a lower resolution 2D electron density map of bovine rhodopsin. Lin et al. (37) predicted that isoleucine at position 571 may cause steric overlap with residues in TM5, a clash unavoidable by local side chain adjustments while maintaining the tight packing of the wt receptor. However, we believe that the TM3/TM6 interaction in TSHR characterized here is conserved in the LHCGR (and other GPCRs) and that the discrepancy in molecular modeling results is due to the lower quality of the template structure used for this LHCGR model. In fact, in a more recent modeling of LHCGR by Fanelli et al. (38), M571 interacts with I460(3.46) the equivalent of I515 in TSHR. I460 of LHCGR also contacts TM2 via M398(2.43), similarly to our TSHR model, where I515 interacts with L452(2.42) (Fig. 4AGo). The repulsive scenario demonstrated here for the M626I mutation of TSHR should therefore also apply to the M571I mutation of LHCGR. This would be consistent with the results of full molecular dynamics simulations indicating that the minimized model of M571I LHCGR displays increased solvent-accessibility of residues in the interface between the cytoplasmic extensions of TM3 and TM6 (38).

In the crystal structure of rhodopsin, the residues corresponding to M626 and I515 are part of a hydrophobic layer composed of residues of TM2, TM3, and TM6 and arranged perpendicularly to the helical axes (16). This layer is stacked between the chromophore-binding site in the central region of the helix bundle and the cytoplasmic regions involved in G protein coupling. For the light energy transfer from photoisomerization of the chromophore to the conformational change of TM3 and TM6 at the cytoplasmic surface, these hydrophobic interactions must rearrange to permit coupling with G proteins (18). Similarly, the steric conflict induced by M626I in TSHR forces direct separation of the cytoplasmic ends of TM3 and TM6 and a concomitant rearrangement of the hydrophobic interhelical contacts. Apart from the repulsing scenario described here, other mutations may indirectly trigger an equivalent repositioning of TM3 and TM6. For instance, the disruption of a hydrophobic contact between TM3 and TM5 in the center of the TM region by the V509A TSHR CAM is predicted to also lead to a separation of the cytoplasmic ends of TM3 and TM6 (39). In the related LHCGR, replacing M398(2.43), equivalent to L242 of in TSHR (Fig. 4AGo), with side chains of smaller dimensions increases the solvent accessible area between TM3 and TM6 concomitant with increased basal activity, whereas replacement with larger side chains has the opposite effect (38).

In conclusion, this study explores how a defect in packing of the juxtacytoplasmic hydrophobic layer triggers a pathophysiologically relevant increase in basal TSHR activity. Repulsion of the cytoplasmic portions of TM6 and TM3 in the M626I CAM provides direct evidence for a link between the opening of this cytoplasmic face of the receptor and the accessibility of the docking surface to Gs{alpha}.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Human Study Subjects
Two subjects (III-2 and III-3, Fig. 1AGo) were independently referred to one of the coauthors (S.R.) for genetic investigation by their respective physicians (Y.M.Y. and F.U.). Subject III-2 was born at 39-wk gestation; birth weight of 3060 g to nonconsanguineous parents. At age 10 months, she was found to have tachycardia and increased length (>95th percentile). There was no history of hyperthyroidism in the mother. Total and free serum thyroid hormone levels were elevated and serum TSH was not detectable. Thyroid scan revealed a eutopic gland with an increased uptake of 62% (normal 10–35%) at 24 h. Thyroid antibodies including thyroglobulin (TG) antibodies (TGab), thyroperoxidase antibodies (TPOab) and thyroid-stimulating Igs (TSIs) were negative. Thyrotoxicosis was difficult to control with methimazole. Subject III-3 was born prematurely (34 wk gestation; birth weight of 2150 g) to nonconsanguineous parents. At 15 months of age, he presented with excessive sweating and tachypnea. Physical examination revealed a well-developed child, rather tall and heavy for age (>95th percentile for height and weight). No ophthalmopathy or dermopathy were noted. Tests of thyroid function showed marked elevation of total and free serum thyroid hormone levels and suppressed serum TSH (results of thyroid function tests are listed in Fig. 1AGo). TSI, TGab, and TPOab were undetectable. On thyroid ultrasound, the gland was mildly and symmetrically enlarged without nodules. A 99mpertechnetate scan revealed homogenous uptake in a eutopic thyroid gland.

It became apparent that the two children had a common father but different mothers (Fig. 1AGo). Furthermore, the father recalled signs and symptoms of persistent symptoms of thyrotoxicosis but was never diagnosed before. The paternal grandmother had a history of hyperthyroidism and underwent a subtotal thyroidectomy. When tested by us, both were found to be still thyrotoxic with suppressed TSH levels, but had no detectable thyroid autoantibodies. Nonautoimmune hyperthyroidism was also found in a sister (III-4), who was born during the course of this investigation. All mothers and a half-sister (III-1) had normal tests of thyroid function. All clinical investigation and genetic analyses were approved by the Institutional Review Board of the University of Chicago, and written informed consent was obtained from all participating subjects.

Thyroid Function Tests
Total T4, total T3, and TSH were measured by chemiluminescence immunometric assays using the Elecsys Automated System (Hitachi Boehringer Mannheim, Germany). 3,3',5'-L-Triiodothyronine (reverse T3; rT3) was measured using a commercial RIA (Adaltis Italia S.p.A, Italy) and serum TG by an in-house RIA. The serum free T4 index was calculated as the product of the serum TT4 concentration and the normalized resin T4 uptake ratio. TGab and TPOab were measured by passive hemagglutination (Fujirebio, Inc., Tokyo, Japan). Tests for TSIs were performed at Nichols Institute (Quest Diagnostics, San Juan Capistrano, CA).

Mutation Screening of the TSHR Gene
Blood samples were taken from eight available family members. Genomic DNA was extracted from peripheral mononuclear cells using a commercial kit (Blood Amp kit, QIAGEN Inc., Valencia, CA). All coding regions of the TSHR gene were amplified using primers and thermocycler settings similar to those previously described (40). Sequencing reactions for both DNA strands were performed with BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, CA) and analyzed with an automatic sequencer (Applied Biosystems; 3730xl).

Plasmid Constructs
Plasmids encoding the various TSHR single or double mutants (M626I, M626A, M626V, M626L, I515M, M626I/I515M, D633A/I515M) were constructed by site-directed mutagenesis (QuikChange II, Stratagene, La Jolla, CA) of the wt TSHR sequence cloned in plasmid pSVL (41). The sequences of all constructs were confirmed.

Cell Culture, Transient Transfection, and Luciferase Reporter Assay
COS-7 cells were maintained in DMEM (Invitrogen Life Technologies, Carlsbad, CA) supplemented with 2 mM L-glutamine, 4.5 g/liter D-glucose, 50 U/ml penicillin, 50 µg/ml streptomycin, and 5% fetal bovine serum under 5% CO2/95% air at 37 C. For reporter assays, cells grown in 12-well plates to 70–80% confluence, were cotransfected per well with 0.5 µg of a CRE plasmid (CRE-Luc; CLONTECH Laboratories, Inc., Palo Alto, CA), 3 ng pRL-Tk internal control vector (Promega Corp., Madison, WI) and 100 ng of effector plasmid/empty vector using FuGENE 6 reagent (Roche Applied Science, Indianapolis, IN). Twenty-four hours after transfection, the medium was replaced by fresh medium without or with various doses of bTSH (Sigma, St. Louis, MO). Cells were harvested 18 h later and analyzed sequentially for firefly and Renilla luciferase activities (Dual-Luciferase Reporter Assay System; Promega). The ratios between the measured firefly and Renilla luciferase activities were expressed relative to the ratios obtained in cells transfected with reporter and empty pSVL vector only.

Flow Immunocytofluorometry
Cell surface expression of wt and mutant TSHR proteins expressed in COS-7 cells was quantified by flow immunocytofluorometry (FACS) with the monoclonal antibody BA8, directed against an epitope of the N-terminal TSHR ectodomain (42), as described in detail previously (20). For each experiment, cells transfected with empty pSVL vector and wt TSHR construct were run as negative and positive controls, respectively. The specific FACS values correspond to the raw FACS signals corrected for background fluorescence determined in cells transfected with empty pSVL vector.

cAMP Determination
Accumulation of cAMP was determined 48 h after transfection as described previously (20). To obtain the specific cAMP accumulation, the cAMP accumulation in pSVL-transfected cells was subtracted from the cAMP generation in receptor-transfected cells.

SCA
Under the experimental conditions of this study, there is a linear relationship between specific basal cAMP accumulation and cell surface FACS signal of wt or constitutive active TSHRs (43), including low expressed CAMs, such as D633A (20). Basal cAMP accumulation was therefore normalized to cell surface expression for each of the constructs. The ratio of specific basal cAMP generation to the specific FACS value reflects the SCA. SCA values are expressed relative to the SCA of wt TSHR (arbitrarily set to 1).

Molecular Modeling and Image Representation
The structure of bovine rhodopsin (16) was employed as template for the TSHR models. Alignment of the sequences of bovine rhodopsin and the human TSHR was based on the alignment of the most conserved residues in each TM: N432 in TM1, L456 in TM2, R519 in TM3, W546 in TM4, Y601 in TM5, P639 in TM6 and P675 in TM7.

All models of the human TSHR were built using the BLDPIR command of the WHAT IF program (44), based on a backbone-dependent and position-specific rotamer library. To remove bumps and correct the covalent geometry, the structure was energy-minimized with YASARA by applying the Yamber2 force field (44), using a 7.86-Å force cutoff. After removal of conformational stress by a short steepest descent minimization, the procedure continued by simulated annealing (time step 2 fsec, atom velocities scaled down by 0.9 every 10th step) until convergence was reached, i.e. no energy improvement was found for 200 steps. The internal consistency of the TSHR models was checked by WHAT IF showing good overall structure scores for all models (see validation report published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org). The optimal rotamer conformation of the M626I mutant was calculated using the DGROTA option of WHAT IF. Molecular graphics were created with YASARA (45) and PovRay (http://www.povray.org).

Atom Nomenclature
For each residue, starting from the C{alpha} atom (part of the protein backbone) and going along the side chain, atoms names are designated according to the Greek alphabet. In the case of valine and leucine, which have two C{gamma} atoms and two C{delta} atoms, respectively, these terminal atoms are additionally numbered 1 and 2. Nomenclature example for the side chain of leucine: C{alpha} -> Cß -> C{gamma} -> C{delta}1 and C{delta}2.


   ACKNOWLEDGMENTS
 
We are grateful to Gilbert Vassart and Sabine Costagliola for their friendly support and helpful advice.


   FOOTNOTES
 
This work was supported by National Institutes of Health (DK17050, DK58281, DK20595, and RR00055).

Disclosure: The authors have nothing to declare related to this study.

First Published Online December 8, 2005

1 U.R. and J.V.D. should be considered co-first authors. Back

Abbreviations: bTSH, Bovine TSH; CAM, constitutively activating mutation; CRE-luc, cotransfected cAMP-responsive reporter gene; FACS, fluorescence-activated cell sorting; GPCR, G protein-coupled receptor; LHCGR, LH/choriogonadotropin receptor; SCA, specific constitutive activity; TG, thyroglobulin; TGab, TG antibodies; TM, transmembrane helix; TPOab, thyroperoxidase antibodies; TSHR, TSH receptor; TSIs, thyroid-stimulating Igs; wt, wild type.

Received for publication August 24, 2005. Accepted for publication November 28, 2005.


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