Activation of the cAMP Pathway by the TSH Receptor Involves Switching of the Ectodomain from a Tethered Inverse Agonist to an Agonist

Virginie Vlaeminck-Guillem1,2, Su-Chin Ho1, Patrice Rodien, Gilbert Vassart and Sabine Costagliola

Institut de Recherche Interdisciplinaire en Biologie Humaine et Nucléaire, Université Libre de Bruxelles (V.V.-G., G.V., S.C.), Campus Erasme, B-1070 Bruxelles, Belgium; Department of Endocrinology, Singapore General Hospital (S.-C.H.), Republic of Singapore 179101; and Service de Médecine, Centre Hospitalo Universitaire d’Angers (P.R.), Angers 49033, France

Address all correspondence and requests for reprints to: Professor Gilbert Vassart, Institut de Recherche Interdisciplinaire en Biologie Humaine et Nucléaire, Université Libre de Bruxelles, Campus Erasme, 808 route de Lennik, B-1070 Bruxelles, Belgium. E-mail: gvassart{at}ulb.ac.be.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Several lines of evidence indicate that constraining intramolecular interactions between transmembrane domains are required to maintain G protein-coupled receptors in an inactive conformation in the absence of agonist. For the glycoprotein hormone receptors, which harbor a long amino-terminal ectodomain responsible for hormone binding, it has been suggested that the ectodomain could contribute to these negative constraints. To test this hypothesis, we expressed at the surface of COS-7 cells mutants of the TSH receptor in which variable portions of the amino-terminal ectodomain are replaced by a 19-residue tag from bovine rhodopsin. Whereas none of the rhodopsin-tagged truncated mutants could be activated by saturating concentrations of TSH, the constructs with the shortest amino-terminal extension displayed increased constitutive activity toward the cAMP pathway, when compared with the wild-type holoreceptor. The shortest truncated construct was strongly activated by the introduction of mutations in transmembrane segment VI (D633A), or in the third intracellular loop (A623I) of the receptor. The magnitude of the stimulation was similar to that observed when the same mutations were introduced in the intact wild-type receptor. On the contrary, the shortest truncated construct was unaffected by activating mutations affecting residues of the extracellular loop region (I486F, I568T) or the top of transmembrane segment VII (del658–661). Together, our results are compatible with a model in which activation of the cAMP pathway by the TSH receptor involves switching of the ectodomain from a tethered inverse agonist to a true agonist.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE SUPERFAMILY OF rhodopsin-like G protein-coupled receptors (GPCRs) includes over 500 members, which can further be classified into subfamilies according to the size and nature of their agonists. Although they are all characterized by a common fold including seven transmembrane {alpha}-helices and they all activate highly related trimeric G proteins, their natural ligands belong to a wide spectrum of molecules of diverse sizes and chemical nature [biogenic amines, nucleotides, neuropeptides, chemokines, glycoprotein hormones, lipids, etc. (for reviews, see Refs. 1 and 2)]. The structural similarity of the receptors (3) and their downstream G protein targets, as well as their evolution from a common ancestor, strongly suggest that similar mechanisms are involved in their activation. This, in turn, poses the intriguing question of how widely dissimilar agonists are capable of triggering a supposedly common activation phenomenon.

A currently favored model for activation of GPCRs holds that the receptors would explore a space of discrete states characterized by different conformations (4). According to this allosteric model, the active and inactive conformations of the receptors would be stabilized by agonists or inverse agonists, respectively (4, 5). Experimental evidences indicate that active and inactive conformations are characterized by different relative positions of some of the transmembrane helices, involving interactions between specific residues of the helical bundle (6, 7). The description of natural (8, 9, 10, 11, 12) or artificial mutations (13, 14) leading to agonist-independent activation of GPCRs provides a strong argument in favor of this model.

For small molecules such as the biogenic amines, there is strong evidence that the ligand interacts directly with specific residues of the transmembrane helices of the receptor (15, 16). For neuropeptides and small protein agonists such as neurokinins, it is believed that interaction involves both exoloops and the amino-terminal portion of the receptors, in association with residues of the transmembrane helices (17). The situation is less clear for receptors to glycoprotein hormones TSH, lutropin/CG and follitropin (FSH). In these cases, the agonists are bulky dimers of about 30 kDa made of a common {alpha}-subunit and hormone-specific ß-subunits (18, 19). The corresponding receptors contain a canonical serpentine portion, with seven transmembrane helices typical of rhodopsin-like GPCRs, and a large (350–400 residues) amino-terminal ectodomain containing leucine-rich repeats (20, 21, 22). The amino-terminal segments are responsible for high affinity binding of the hormones and recognition specificity: swapping amino-terminal domains between receptors results in parallel exchange of specificity (23, 24), and soluble ectodomains have been prepared that display binding affinity similar to that of the holoreceptors (25, 26, 27). How binding of the hormone to the ectodomain results in activation of the serpentine portion of the receptor is still unknown. It has been proposed that after high affinity binding of the hormone to the ectodomain, low affinity interaction between the hormone and the exoloops or, even, residues of the transmembrane helices would result in activation (28).

The TSH receptor (TSHr) offers interesting characteristics with respect to the activation mechanisms: in addition to its natural agonist, it can be activated by autoantibodies in patients with Graves’ disease (29, 30, 31); it is also particularly easy to activate by mutations affecting the serpentine portion of the receptor (8, 9). The observation that mutations of specific residues of the ectodomain (serine 281) and the exoloops (isoleucine 486, isoleucine 568) constitutively activate the receptor led to the hypothesis that the ectodomain would exert an inhibitory constraint on the inherently noisy serpentine portion of the TSHr through interaction with the exoloops (32, 33, 34). This hypothesis is in agreement with the observation that mild treatment of cells expressing the TSHr with trypsin results in ligand-independent activation, while simultaneously removing an epitope of TSHr ectodomain (35). More recently, receptor mutants with truncations of their ectodomains have provided support to this model (36).

In the present study we have explored the role of the ectodomain of TSHr, both as a silencer and an activator of the serpentine portion of the receptor toward its main target, the G protein G{alpha}s. By comparing the constitutive activity of amino-terminally truncated receptors in which a spectrum of activating mutations have been engineered, we provide evidence that the ectodomain of TSHr functions as a molecular switch, displaying agonist or inverse agonist properties toward the serpentine domain, depending on whether it is bound to its ligand or not.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Validation of the Rhodopsin Tag (RT) Holoreceptor Construct [Signal Peptide (SP)-RT-TSHr]
To assay the effect of amino-terminal truncations of the TSHr on constitutive activity, a reliable method has to be available to normalize cAMP production to cell surface expression of the various constructs. Inclusion at the amino terminus of human TSHr constructs of a tag corresponding to the first 19 amino acids of bovine rhodopsin provides an efficient means, both to get cell surface expression of truncated mutant (Ref. 37 and see below) and to reliably assay surface expression with a tag-specific monoclonal antibody [Mab OR2-15A-6 (see Materials and Methods)]. The addition of the 19-residue RT at the amino terminus of the wild-type TSHr (immediately downstream of the SP; see Fig. 1Go) did not modify the functional and structural characteristics of the receptor when expressed in COS cells (Fig. 2Go). When compared with the wild-type receptor, both molecules were expressed at cell surface at a similar level, as assayed in flow cytometry with a Mab recognizing a conformational epitope of the amino-terminal ectodomain [BA8 (Ref. 38 and Fig. 2AGo)]. Both receptors also underwent cleavage of the connecting peptide between the ectodomain and the serpentine portion of the molecules (39, 40, 41), as evidenced in Western blot experiments with Mab 28, a Mab directed against a linear epitope at the N terminus of the human TSHr [hTSHr (Fig. 2BGo)]. The resulting {alpha}-subunit, released from the receptor by reduction of disulfide bonds, was, however, 40 kDa larger for the SP-RT-TSHr construct than for the WT-TSHr (Fig. 2BGo). This higher molecular mass resulted most probably from addition of extra N-linked glycan moieties on the two putative N-glycosylation sites present in the RT (see legend to Fig. 1Go). In addition, mature forms of single-chain (uncleaved) receptor were not detectable with the SP-RT-TSHr construct, suggesting that all molecules expressed at the cell surface were in the cleaved form (Fig. 2BGo). cAMP production in transfected COS cells, whether basal or after stimulation by TSH, was identical for SP-RT-TSHr and WT-TSHr. Figure 2CGo illustrates typical concentration-response curves. The corresponding 50% effective concentrations (EC50) for WT-TSHr and SP-RT-TSHr were 1.4 mIU/ml and 1.9 mIU/ml, respectively.



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Figure 1. Schematic Representation of the Amino-Terminally Truncated TSHr Mutants

(a), SP of the human TSHr, amino acid sequence: MRPADLLQLVLLLDLPRDLGG. (b), RT, amino acids sequence: MNGTEGPNFYVPFSNKTGVV. Two putative glycosylation sites are underlined. (c), First amino acid after RT.

 


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Figure 2. Validation of the RT Holoreceptor Construct (SP-RT-TSHr)

A, The expression of WT-TSHr and SP-RT-TSHr was measured by flow cytometry, at the cell surface of COS cells with BA8 antibody. B, The natural cleavage of the TSHr at the cell surface, producing the {alpha}-subunit, was evaluated by immunoblotting with antibody 28. Lane 1, SP-RT-TSHr; lane 2, WT-TSHr; lane 3, pcDNA3. Below two nonspecific (ns) bands generated with this antibody, we observed: {alpha}, {alpha}-subunit; hm, mature forms of uncleaved TSHr expressed at the cell surface; hi, immature forms of uncleaved TSHr trapped inside the cell. C, TSH-induced cAMP increase in WT-TSHr ({blacktriangleup}; EC50 = 1.4 mIU/ml) and in SPRT-TSHr ({blacksquare}; EC50 = 1.9 mIU/ml). Results were analyzed by nonlinear regression using the GraphPad Software, Inc. (San Diego, CA) Prism software.

 
Amino-Terminal Truncation Increases Constitutive Activity of the hTSHr Toward the cAMP Pathway
Truncated hTSHr constructs were generated by PCR, as described in Materials and Methods and summarized in Fig. 1Go. They were made of the serpentine portion of the hTSHr and portions of the ectodomain extending variably toward the amino terminus. All constructs harbored a RT at their extreme N terminus. After transfection in COS cells, expression at the cell surface was measured with Mab OR2-15A-6 directed against the RT (Fig. 3AGo) and basal intracellular cAMP levels were assayed. Great differences were observed in the level of expression of individual mutants, ranging from 0 to 50% of the expression achieved by SP-RT-TSHr. Constructs extending upwards of codon 192 displayed low expression (QGYA, LDV, LKKL), and two of them (TQTL, LKFL) did not reach the cell surface at all. Inclusion of a SP in the construct was not necessary for expression of the three shortest mutants (KFLR, FNPC, YDY). In contrast, for the QGYA, LDV, LKKL, KNQK, and GFGQ mutants, expression was undetectable in the absence of a SP (data not shown). Basal intracellular cAMP was measured in COS cells transfected with each construct (Fig. 3BGo), and the specific constitutive activity (SCA, i.e. basal activity normalized to surface expression) was calculated as described in Materials and Methods (Fig. 3CGo). The linear relation observed between receptor expression, as measured by flow immunocytometry, and the intracellular levels of cAMP in transfected cells demonstrate the validity of the normalization method. Data are illustrated in Fig. 3DGo for the SP-RT-WT-TSHr and a construct (SPRT I568T) chosen for its good expression and strong constitutive activity.



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Figure 3. Cell Surface Expression and Constitutive Activity of SP-RT-TSHr, SP-RT-TSHr-S281L, and RT-Truncated TSHr Constructs, after Transfection in COS Cells

A, Cell-surface expressions after transfection of empty pcDNA3 plasmid, SP-RT-wtTSHr, SPRT-wtTSHr-S281L, and RT-truncated TSHr constructs were measured by flow cytometry using a Mab directed against the RT. The data represent the mean and SD (expressed in arbitrary fluorescence units) of triplicate dishes from a representative experiment of three. B and C, Basal cAMP accumulation was measured for empty pcDNA3 plasmid, SP-RT-wtTSHr, SPRT-wtTSHR-S281L, and RT-truncated TSHr constructs. The data are presented as raw values [intracellular cAMP accumulation in picomoles per milliliter (B)] or normalized to cell-surface expression of the constructs and expressed relative to values obtained with the SP-RT-wtTSHr construct [SCA (C)]. D, The validity of the method used to normalize cAMP production to cell-surface expression of the constructs (see Materials and Methods) is illustrated in this panel. A clear linear relation is observed between receptor expression, as measured by flow immunocytometry (SPRT wild type and SPRT I568T), and the intracellular levels of cAMP achieved in transfected COS cells.

 
TSHr mutants with truncation extending to residue 286 (KNQK, GFGQ, YDY, FNPC, KFLR) showed a 5- to 6-fold increase of SCA, as compared with SP-RT-TSHr. In comparison, substitution of leucine for serine in position 281 of the holoreceptor caused a 20-fold increase in SCA (Fig. 3CGo), close to the maximal activity triggered by 100 mIU/ml of TSH (see Fig. 4Go). In contrast, truncated constructs including amino acids upstream of codon 286 (QGYA, LKKL, LDV) presented no significant increase in basal activity over that of SP-RT-TSHr (Fig. 3CGo). Even when exposed to very high concentrations of hormone (100 mIU/ml), none of the truncated receptors could be stimulated by TSH (Fig. 4Go).



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Figure 4. cAMP Accumulation in COS Cells Transfected with RT-Truncated Constructs upon Stimulation by TSH

cAMP accumulation was measured after 1 h of incubation in presence (black bars) or not (white bars) of 100 mIU/ml TSH. The data are expressed as raw values (intracellular cAMP accumulation in picomoles per milliliter).

 
Activating Mutations in the Transmembrane Segments and the Intracellular Loop Increases Basal cAMP-Stimulating Activity of the RT-KFLR Truncated Construct
Amino acid substitutions at position D633 [in transmembrane segment VI; residue D6.44 in the numbering system of Ballesteros and Weinstein (42)] and A623 (at the junction between the third intracellular loop and transmembrane segment VI) have been shown to cause a strong increase in basal activity when introduced on the background of the holoreceptor (9, 33, 43). Two such substitutions (D633A and A623I) were introduced in the truncated construct with the shortest amino-terminal extension [RT-KFLR (Fig. 5AGo)], and basal cAMP production was assayed in transiently transfected COS cells (Fig. 5Go, C and D). The SP-RT-TSHr construct was engineered to harbor the same mutations to provide pertinent controls that could be assayed at the cell surface with the same anti-RT Mab (Fig. 5BGo). When normalized to surface expression of the mutants, the D633A and A623I substitutions caused an increase in constitutive activity whether present in the holoreceptor (SP-RT-TSHr) or the RT-KFLR truncated mutant (Fig. 5CGo).



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Figure 5. Cell-Surface Expression and Constitutive Activity of Wild-Type TSHr, RT-KFLR, RT-KFLR Mutants, and SP-RT-TSHr Mutants Transfected in COS Cells

A, Schematic representation of the activating mutations inserted in RT-KFLR and SP-RT-TSHr mutants. Only the serpentine portion of the TSH receptor is represented. B, Cell-surface expressions after transfection of COS cells with empty pcDNA3 plasmid, SP-RT-wtTSHr, and RT-KFLR were measured by flow cytometry using a Mab directed against the RT (RT-KFLR and SP-RT-wtTSHr). Expression of several RT-KFLR and SP-RT-TSHr mutants containing additional mutations in either transmembrane segments or intracellular loop region (A623I, D633A) or extracellular loops (I486F, I568T, and deletion 658–661) was also measured. The data are expressed as a percentage of SP-RT-WT (mean ± SD of three separate experiments). C and D, Basal cAMP accumulation was measured after transfection with empty pcDNA3 plasmid, SP-RT-wtTSHr, RT-KFLR, and control S281L mutant, as well as various RT-KFLR and SP-RT-TSHr mutants containing additional mutations in extracellular loops (I486F, I568T, and deletion 658–661) (left panels) or additional mutations in intracellular loops or transmembrane segments (A623I, D633A) (right panels). The data are expressed as raw values (cAMP accumulation, C) or normalized to cell-surface expression of the constructs and expressed relative to values obtained with the SP-RT-wtTSHr construct (relative SCA, D).

 
Activating Mutations in the Extracellular Loops Fail to Increase Basal cAMP-Stimulating Activity of the RT-KFLR Truncated Construct
Amino acid substitutions at positions I486 and I568 (in the first and second exoloops, respectively) and deletion of residues 658–661 (at the extreme top of transmembrane segment VII) are known to strongly activate the wild-type TSHr (33). When I486F, I568T, and del658–661 mutations were introduced in the RT-KFLR truncated construct (Fig. 5AGo) and tested in COS cells as described above, no significant increase in constitutive activity could be observed over that of the wild-type RT-KFLR construct [P = 0.9491, P = 0.089, P = 0.7751, respectively (Fig. 5DGo)]. As expected, when present in the SP-RT-TSHr background, the same mutations caused strong increase in constitutive activity (Fig. 5DGo).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Serpentine-Alone TSHr Constructs Display Increased Constitutive Activity Toward the cAMP Pathway and Cannot Be Stimulated by TSH
Serpentine-alone constructs of the TSHr provide an interesting opportunity to investigate the activation mechanisms of glycoprotein hormone receptors in the frame of the ternary complex model for GPCR activation. The inclusion at the N terminus of various TSHr constructs of a 19-residue RT (37) allows for efficient expression of amino-terminally truncated mutants at the surface of COS cells, while simultaneously providing a reliable tool to compare surface expression of individual constructs in a quantitative manner. In agreement with our prediction (25, 32, 34, 35), the present results clearly demonstrate that removal of the hormone-binding ectodomain (constructs KFLR to KNQK, Fig. 1Go) causes an increase in basal activity (5- to 6-fold; Fig. 3CGo) as compared with holoreceptor. They extend other results obtained with ectodomain-truncated TSHr mutants (36) and provide evidence that the amino-terminal portion of TSHr behaves as a tethered inverse agonist (Fig. 3Go). Whether this effect involves specific interactions, as suggested by the differential effects of the individual mutants (Fig. 3Go), or is secondary to a mere stabilization of the structure of the serpentine, is presently unknown. In this context, it is noteworthy that activating mutations have been shown to cause destabilization of GPCRs (4, 44). According to one lutropin receptor model, the glycoprotein hormone receptor ectodomain would bind the hormone with high affinity and present it to the serpentine for a low-affinity productive interaction resulting in activation (28). Interestingly, none of our truncated TSHr mutants could be stimulated by a saturating concentration of TSH, in sharp contrast with the above model [in which no direct evidence for expression of the truncated mutants at the cell surface was provided (28)].

Full Stimulation of cAMP Production by the TSHr Involves More than Release of the Silencing Effect of the Ectodomain on the Serpentine
The increase in basal activity of the mutants with the shorter amino-terminal extension does not reach the levels displayed by full stimulation of the holoreceptor by TSH or by S281L, a mutant with an activating amino acid substitution in the ectodomain (15- to 20-fold, as compared with 5- to 6-fold; see Fig. 3Go). This excludes a model in which normal activation of the receptor would simply result from the release of a silencing effect exerted by the ectodomain on the serpentine portion, via interaction with the extracellular loops. It indicates that the ectodomain, whether TSH liganded or mutated in position 281, would have a positive effect on activity of the serpentine portion.

A Structural Module Contributed by the Ectodomain and the Extracellular Loops of TSHr Constitutes a Molecular Switch Controlling the Activity of the Serpentine
Mutations of transmembrane helices or the third intracellular loop are equally effective, whether on a truncated construct or the holoreceptor background (Fig. 5Go). These results are consistent with those observed in other GPCR, such as the glucagon receptor (45). In contrast, activating mutations affecting residues of the extracellular loops (or located at the top of transmembrane segment VII) are without effects when engineered on the background of a serpentine-alone construct. This indicates that the ectodomain and the exoloops do cooperate in the building of a module capable of activating the serpentine. The portion of the ectodomain involved may contain the evolutionary conserved PSHCCAF segment located at the C-terminal border of the last leucine-rich motif of the receptor. Indeed, similar to the situation in the exoloops, mutation of the conserved serine residue in this segment (serine 281 in the TSHr) causes constitutive activation of all three glycoprotein hormone receptors (32, 34, 46, 47). Interestingly, investigation of the effects of various amino acid substitutions at the serine 281 position, or adjacent residues, revealed a direct relation between the expected loss of local structure and the magnitude of activation of basal activity (34, 47). Similarly, the nature of some of the activating mutations at (or close to) the extracellular loops (I486F, I486M, and, more so, del658–661) (33) are compatible with a gain of function that would be secondary to a loss of structure.

Comparison of the effects on basal cAMP-stimulating activity of the various truncated mutants suggests that the PXSHCCAF motif could be implicated in the silencing effect on the serpentine (compare activity of GQYA, LDV, and LKKL with that of KFLR, FNPC, YDY, GFGQ, and KNQK; Fig. 3Go). However, when the S281L mutation was introduced on the background of truncated mutants with wild-type constitutive activity (GQYA, LDV, LKKL; Fig. 3Go), no increase in constitutive activity could be observed (data not shown). With the limitation already mentioned that the silencing effect of the ectodomain observed with some mutants might involve nonspecific stabilizing interactions, these results suggest that different segments of the ectodomain would be implicated in the silencing and stimulatory interactions with the serpentine. An essentially intact ectodomain would be required to display stimulatory activity.

In an attempt to integrate available information, we propose the following model for activation of the TSHr (see Fig. 6Go). In the absence of hormone, the ectodomain would exert a silencing effect on the serpentine portion of the receptor. Silencing would be incomplete, as the wild-type receptor displays readily measurable constitutive activity toward the cAMP pathway. Removal of the ectodomain would unmask the intrinsic basal activity of the serpentine, resulting in partial activation. Mutations of serine 281 in the ectodomain, or specific residues of the exoloops, would cause structural disorganization of a composite module made of segments of the ectodomain and the exoloops. The resulting structural change would trigger conversion of the inhibitory effect of the ectodomain into stimulation of the serpentine. According to this model, the ectodomain would behave as a tethered inverse agonist or an agonist, depending on whether it is wild type or mutated in position 281. Because the strongest activating mutations affecting S281 cause close to full activation of cAMP production by the receptor (Refs. 34 and 47 and Fig. 3Go), it is tempting to propose that the resulting structural changes would mimic those induced by hormone binding to the ectodomain. The model presents close similarity with the situation in rhodopsin. In this case, a covalently tethered ligand (retinal, residing within the transmembrane pocket) is also converted from inverse agonist to full agonist upon isomerization triggered by light (48, 49, 50). The model predicts that no direct interaction between the hormone and the exoloops or transmembrane helices is required for receptor activation, because the immediate agonist would be the ectodomain itself. This fits with the observation that stimulating autoantibodies from patients with Graves’ disease are capable of activating the receptor, while recognizing different epitopes of the ectodomain (29, 51, 52); it is not unexpected that different antibodies could cause similar loss of structure of the trigger module.



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Figure 6. Putative Model of the Intramolecular Interactions Involved in the Activation of Gs by TSHr

A, The basal state of the receptor is characterized by an inhibitory interaction between the ectodomain and the serpentine domain. The ectodomain would function as a tethered inverse agonist. B, Removal of the ectodomain releases the serpentine domain from the inhibitory interaction, resulting in partial activation. C, Mutation of serine 281 into leucine switches the ectodomain from an inverse agonist into a full agonist of the serpentine domain. D, Binding of TSH to the ectodomain is proposed to have a similar effect, converting it into a full agonist of the serpentine portion. It must be stressed that the scheme is purely illustrative. It emphasizes that, according to our model, activation does not require a direct interaction between the hormone and the serpentine domain. Such an interaction, however, is by no means excluded by our experiments.

 
Besides, our results introduce a clear hierarchy of molecular events in the activation of glycoprotein hormone receptors. Events specific for the receptor family do require integrity of the module identified here at the interface between the serpentine and the ectodomain. On the contrary, molecular events common to activation of most if not all rhodopsin-like GPCRs are unaffected by removal of the ectodomain. These involve modifications of structures in the intracellular loops and/or reorganization of interhelical interactions of the serpentine within the membrane, as exemplified by the strong activation achieved by mutations of Ala623 and Asp633 (Ref. 43 and Fig. 5Go).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Reagents
Plasmid pBluescript SK+ and pcDNA3 were obtained from Stratagene (La Jolla, CA) and Invitrogen (San Diego, CA), respectively. Restriction enzymes were from Life Technologies, Inc. (Rockville, MD) and New England Biolabs, Inc. (Beverly, MA). Platinum Pfx DNA polymerase was obtained from Life Technologies and Pfu Turbo polymerase from Stratagene. Vector pRK5 containing the 5' region of bovine rhodopsin was a generous gift from Randall R. Reed (Johns Hopkins University School of Medicine, Baltimore, MD) (37). Mab OR2-15A-6 was kindly provided by P. Hargrave (Department of Ophthalmology, University of Florida, Gainesville, FL) (53). Mab(s) BA8 and 28 were obtained by genetic immunization with the cDNA coding for the human TSHr and were directed against a conformational epitope and a linear epitope (residues 31–50) of the N-terminal portion of the TSHr, respectively (34, 38).

Construction of N Terminus Rhodopsin-Tagged Truncated hTSHr
A restriction fragment (HindIII/EcoRI) corresponding to the 5' untranslated region of the bovine rhodopsin gene and extending to codon 19 (RT) was excised from the pRK5 vector (37) and cloned in pBluescript SK+ (RT-SK+: RT-SK+). A cDNA fragment of the human TSHr extending from codon 21 (codon 1 corresponding to the initiation methionine) to the PstI site at codon 90 was PCR amplified [2.5 U Platinum Pfx DNA polymerase, 0.2 mM dNTP, 3 µM of each primer (the forward primer introduced a 5' EcoRI site), and 200 ng of hTSHr-pcDNA3 as template]. The PCR product, the hTSHr cDNA in pcDNA3, and the RT-SK+ constructs were digested with EcoRI/PstI, PstI/XbaI, and EcoRI/XbaI, respectively, and a trimolecular ligation was performed (RT-hTSHr-pcDNA3: RT-hTSHR-pcDNA3).

A SacII restriction site was introduced in the SP of the human TSHr at codon 16 by the QuikChange site mutagenesis method (Stratagene), starting with TSHr in pBluescript SK+ and two synthetic oligonucleotide primers containing the desired mutation (34, 54). The resulting mutated hTSHr construct was excised with KpnI/XbaI and cloned in pcDNA3 (hTSHr-pcDNA3-SacII).

A cDNA fragment of the RT-hTSHr construct including the AflIII site at codon 232 was generated with a forward primer introducing a SacII site in the 5' position, together with codons 17–20 of the hTSHr. This PCR product, the hTSHR-pcDNA3, and the hTSHR-pcDNA3-SacII constructs were digested with SacII/AflIII, AflIII/XbaI and SacII/XbaI, respectively, and a trimolecular ligation was performed (SP-RT-hTSHR-pcDNA3: SP-RT-TSHR-pcDNA3).

The TSHr constructs with truncations extending to various positions in the ectodomain were PCR amplified with Platinum Pfx DNA polymerase and the hTSHr-pcDNA3 as template, as described above (the oligonucleotide sequences used for PCR amplification of the various sequences are available upon request). The purified PCR products were digested with EcoRI/BamHI and ligated into the RT-TSHr-pcDNA3 vector (resulting in serpentine-KFLR, -FNPC, -YDY constructs) or SP-RT-TSHr-pcDNA3 vector (resulting in serpentine-GFGQ, -KNQK, -LKKL, -LDV, -QGYA, -LKFL, -TQTL constructs). All constructs were amplified in DH5{alpha}F' competent cells, and recombinant DNA was purified from selected clones and sequenced for confirmation of the nucleotide sequences. All the chimeras are summarized in Fig. 1Go.

Mutants of the SP-RT-TSHr and RT-KFLR
Artificial mutation S281L (47, 55) or mutations reported elsewhere in patients with autonomous thyroid adenomas or hereditary hyperthyroidism (I486F, I568T, del658–661, D633A, A623I; Refs. 9, 33 , and 43) were introduced in the hTSHr by the QuikChange site mutagenesis method, starting with TSHr in pBluescript SK+, as previously described (34, 54). Mutant RT-KFLRs and SPRT-TSHrs were obtained by subcloning the appropriate portions of TSHr mutants and sequenced for confirmation of the nucleotide sequence of the PCR-generated areas.

Transfection Experiments
COS-7 cells were used for transient expression allowing functional assays. They were transfected by the diethylaminoethyl-dextran method followed by a dimethylsulfoxide shock as described previously (43, 56). Two days after transfection, cells were used for cAMP determinations and flow immunocytofluorometry. Triplicate dishes were used for each assay. Each experiment was repeated at least twice. Cells transfected with the empty pcDNA3 vector were always run as controls. To demonstrate that constitutive activity toward the cAMP pathway is linearly related to the number of receptors present at the cell surface, variable amounts of SP-RT TSHr-pcDNA3 or SP-RT I568T-pcDNA3 constructs (complemented to 500 ng of DNA with empty pcDNA3 vector) were tranfected in COS cells.

Quantification of Cell Surface Expression of TSHr Constructs by Flow Immunocytometry
Cells were prepared as previously described (43). After detachment, they were centrifuged at 500 x g at 4 C for 3 min and the supernatant was discarded. They were then incubated for 30 min at room temperature in 100 µl 0.1% PBS-BSA containing either the BA8 Mab (38) or the OR2-15A-6 Mab directed against the N terminus of bovine rhodopsin (53). Cells were then washed with 4 ml 0.1% PBS-BSA and centrifuged as described above. They were incubated on ice for 30 min, in the dark, with fluorescein-conjugated {gamma}-chain-specific goat antimouse IgG (Sigma, St. Louis, MO) in the same buffer. Propidium iodide (10 µg/ml) was used for detection of damaged cells that were excluded from the analysis. Cells were washed and resuspended in 250 µl 0.1% PBS-BSA. The fluorescence of 10,000 cells per tube was assayed by a FACScan flow cytofluorometer (Becton Dickinson and Co., Eerenbodegem, Belgium).

cAMP Determination
For cAMP determinations, culture medium was removed 48 h after transfection and replaced by Krebs-Ringer-HEPES buffer for 30 min. Thereafter, cells were incubated for 60 min in fresh Krebs-Ringer-HEPES buffer supplemented with 25 µM of the phosphodiesterase inhibitor Rolipram (Laboratory Logeais, Paris, France) and various concentrations of bovine TSH (Sigma). At the end of a 1-h incubation, the medium was discarded and replaced with 0.1 M HCl. The cell extracts were dried in a vacuum concentrator, resuspended in water, and diluted appropriately for cAMP measurements by RIA according to the method of Brooker et al. (57). Duplicate samples were assayed in all experiments; results are expressed as picomoles cAMP per milliliter. Basal cAMP was normalized to cell-surface expression for each construct. To this end, receptor-dependent cAMP accumulation (i.e. cAMP in receptor-transfected cells—cAMP in pcDNA3-transfected cells) was divided by the receptor-dependent fluorescence measured by flow immunocytofluorometry (fluorescence of receptor-transfected cells—fluorescence of the pcDNA3-transfected cells). The values were then normalized to the basal activity of the wild-type TSHR, arbitrarily set to 1. The validity of this method of normalization was assessed by the observation that, for the range of constitutive activity investigated, a linear relation exists between cAMP accumulation and receptor expression at the cell surface (see Fig. 3DGo).

Normalized constitutive activities of the mutant TSHrs and wild-type TSHr were compared using the nonparametric unpaired Wilcoxon test. Differences were considered statistically significant when P < 0.05. We used the Stata 7.0 package (Stata Corp., College Station, TX).

Western Blot of SP-RT TSHr
Preparation of receptor.
Six dishes, each containing 300,000 cells transfected with pcDNA3, TSHr-pcDNA3, or SP-RT-TSHr pcDNA3 constructs, were treated with 5 mM EDTA and 5 mM EGTA in PBS, and the cells were spun down at 280 x g. The cell pellet was suspended and homogenized in a Potter-Elvehjem glass homogenizer with a teflon pestle (Fisher Bioblock Scientific, Tournai, Belgium) in 1,250 µl of lysis buffer [100 mM (NH4)2SO4, 20 mM TRIS at pH 7.5, and 10% glycerol] containing protease inhibitors (Complete, Roche Molecular Biochemicals, Somerville, NJ). The lysate was then centrifuged at 500 x g for 10 min and the supernatant recovered for further ultracentrifugation at 30,000 x g for 30 min. Two hundred microliters of lysis buffer containing 1% N-dodecyl-ß-D-maltoside (Anatrace, Maumee, OH) was added to the pellet and the suspension incubated for another 30 min at 4 C under constant rotation to allow thorough mixing. Final centrifugation was carried out at 100,000 x g for 1 h and the supernatant was stored at -80 C for further use. All procedures described were performed at 4 C.

SDS-PAGE and Immunoblotting
Three microliters of Laemli sample buffer (5x) containing SDS (10%) and ß-mercaptoethanol (1 M) as a reducing agent were added to 10 µl receptor protein, prepared as described above, and denatured at 40 C for 1 h. The sample was then run on 7% acrylamide gel and probed with Mab 28 (culture supernatant diluted 1:50), which recognizes a linear epitope at the N-terminal of the ectodomain from amino acid residues 31–50. The proteins were visualized with an antimouse IgG horseradish peroxidase conjugate and the ECL Plus Western blotting detection system (Amersham Pharmacia Biotech Benelux, The Netherlands).


    ACKNOWLEDGMENTS
 
We thank V. Janssens for expert technical assistance and C. Govaerts for schematic representation of TSHr. Vector pRK5 containing the 5' region of bovine rhodopsin was a generous gift from Randall R. Reed (Johns Hopkins University School of Medicine, Baltimore, MD). Mab OR2-15A-6 was kindly provided by P. Hargrave (Department of Ophthalmology, University of Florida, Gainesville, FL). This study was supported by the Belgian State, Prime Minister’s office, Service for Sciences, Technology and Culture. This work was also supported by grants from the Fonds de la Recherche Scientifique Médicale, Fonds National de la Recherche Scientifique, Association Recherche Biomédicale et Diagnostic, and BRAHMS Diagnostics. S.C. is Chercheur Qualifié at the Fonds National de la Recherche Scientifique. V.V.-G. is a recipient of a fund of the Société Française d’Endocrinologie.


    FOOTNOTES
 
1 V.V.-G. and S.-C.H. contributed equally to this paper. Back

2 Present address for V.V.-G.: Clinique Marc Linquette, Centre Hospitalo Universitaire de Lille, 59037 Lille, France. Back

Abbreviations: EC50, Fifty-percent effective concentration; GPCR, G protein-coupled receptor; hTSHr, human TSHr; Mab, monoclonal antibody; RT, rhodopsin tag; SCA, specific constitutive activity; SP, signal peptide; TSHr, TSH receptor.

Received for publication August 17, 2001. Accepted for publication December 17, 2001.


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