Human Molecular Genetics

Human Molecular Genetics Advance Access originally published online on August 31, 2005
Human Molecular Genetics 2005 14(20):2991-3002; doi:10.1093/hmg/ddi329

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Intracellular entrapment of wild-type TSH receptor by oligomerization with mutants linked to dominant TSH resistance

Davide Calebiro^1,2, Tiziana de Filippis², Simona Lucchi², Cesare Covino³, Sara Panigone², Paolo Beck-Peccoz^1,4, David Dunlap³ and Luca Persani^1,2,*

¹Institute of Endocrine Sciences, University of Milan, Milan 20122, Italy, ²Laboratory of Endocrinological Research, Istituto Auxologico Italiano, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Via Zucchi 18, Cusano Milanino (MI) 20095, Italy, ³Advanced Light and Electron Microscopy Bio-Imaging Center (ALEMBIC), Istituto Scientifico San Raffaele, Milan 20132, Italy and ⁴Fondazione IRCCS Ospedale Maggiore Policlinico, Mangiagalli e Regina Elena, Milan 20122, Italy

^* To whom correspondence should be addressed. Tel: +39 02619112400; Fax: +39 02619113033; Email: luca.persani{at}unimi.it

Received May 25, 2005; Revised July 11, 2005; Accepted August 16, 2005

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS NOTE ADDED IN PROOF REFERENCES

 
TSH resistance is one of the causes of congenital hypothyroidism with thyroid gland in situ. We recently identified families with dominant transmission of partial TSH resistance due to heterozygous inactivating mutations in TSH receptor (TSHR) gene. Although we documented a poor routing of TSHR mutants to the cell membrane, the mechanism responsible for dominant inheritance of partial TSH resistance remained unexplained. We therefore co-transfected Cos-7 cells with wild-type TSHR and mutant receptors found in these patients. A variable impairment of cAMP response to bTSH stimulation was observed, suggesting that inactive TSHR mutants can exert a dominant negative effect on wild-type TSHR. We then generated chimeric constructs of wild-type or inactive TSHR mutants fused to different reporters. By fluorescence microscopy and immunoblotting, we documented an intracellular entrapment, mainly in the endoplasmic reticulum, and reduced maturation of wild-type TSHR in the presence of inactive TSHR mutants. Finally, fluorescence resonance energy transfer and co-immunoprecipitation experiments were performed to study the molecular interactions between wild-type and mutant TSHRs. The results are in agreement with the presence of oligomers formed by wild-type and mutant receptors in the endoplasmic reticulum. Such physical interaction represents the molecular basis for the dominant negative effect of inactive TSHR mutants. These findings provide an explanation for the dominant transmission of partial TSH resistance. This is the first report linking dominant negative mutations of a G protein-coupled receptor to an abnormal endocrine phenotype in heterozygous patients.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS NOTE ADDED IN PROOF REFERENCES

 
Resistance to TSH is a syndrome characterized by thyroid hyposensitivity to biologically active TSH and represents one of the causes of congenital hypothyroidism with thyroid gland in situ. Depending on the degree of thyroid refractoriness to TSH stimulation, subjects present elevated TSH and normal levels of serum thyroid hormone (partial TSH resistance), at the one extreme of the spectrum, or severe hypothyroidism (complete TSH resistance), at the other (1,2). After the cloning of TSH receptor (TSHR) gene (3–6), 23 different TSHR loss-of-function mutations have been documented to be responsible for TSH resistance (7–21). In the earlier studies, in which only probands with large TSH elevations were screened for mutations, the disease was linked to homozygous or compound heterozygous mutations and was described to follow a recessive pattern of inheritance (7–18). More recently, we looked for TSHR mutations in a population with less pronounced TSH elevations in the absence of autoimmune thyroid disease (19). All patients had neonatal to juvenile evidence of mild hypothyroidism with thyroid gland in situ and a familial history of hypothyroidism was documented in eight out of 10 cases. With this approach, we disclosed the existence of familial cases of partial TSH resistance associated with heterozygous inactivating mutations in the TSHR gene and a dominant mode of inheritance (Fig. 1). Interestingly, recent reviews of the literature on TSH resistance by us (1) and others (2) have highlighted the presence of elevated TSH levels in several heterozygous relatives of previously described cases (Table 1), suggesting that the prevalence of partial TSH resistance due to heterozygous TSHR mutations may be higher than previously thought. Even if TSHR mutants linked to dominant TSH resistance are characterized by reduced cell surface expression, the molecular mechanism underlying the dominant inheritance of partial TSH resistance remained unexplained (19).

View larger version (20K): [in this window] [in a new window]   Figure 1. Pedigrees of families with partial TSH resistance associated with heterozygous TSHR mutations (C41S, L467P and C600R). Probands are indicated by an arrow. Proband of family A was carrier of compound heterozygous mutations (C600R and P162A). Most patients were tested on multiple occasions and TSH values above the upper limit of normal range (0.24–4.0 mU/l) are underlined. The defect is dominantly inherited and co-segregates with simple heterozygous TSHR mutations in the three families.

 

View this table: [in this window] [in a new window]   Table 1. Elevated serum TSH levels in patients with heterozygous TSHR mutations reported by other authors

 
Loss-of-function mutations of membrane proteins, such as receptors and ion channels, have been traditionally thought to interfere directly with protein activity (e.g. ligand binding, signal coupling, ion transport). However, it is becoming evident that some mutations may exert their activity by causing protein misfolding, misassembly and/or aberrant oligomerization (22). Indeed, some of these proteins form supramolecular complexes during the biosynthetic process (23–26), which may explain the genetic dominance of mutations interfering with their intracellular trafficking (27–32). The recent finding that TSHR, like other G protein-coupled receptors (GPCRs), can oligomerize in living cells (33) led us to hypothesize that the formation of complexes between wild-type and mutant receptors could be responsible for partial TSH resistance in patients with heterozygous TSHR mutations. In this study, we utilized a combination of functional assays, imaging techniques and co-immunoprecipitation experiments to clarify the mechanism of genetic dominance of inactivating TSHR mutations. Evidence is presented that TSHR oligomerization occurs early along the biosynthetic pathway, is relevant for receptor maturation and is conserved between wild-type TSHR and inactive TSHR mutants. Such physical interaction between wild-type and mutant TSHRs results in intracellular entrapment of wild-type receptor, with a consequent partial impairment of cell responsiveness to TSH stimulation. Our data indicate negative dominance by direct interaction of mutant with wild-type receptor as an explanation for the dominant inheritance of partial TSH resistance.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS NOTE ADDED IN PROOF REFERENCES

 
Dominant negative effect of inactive TSHR mutants on wild-type receptor activity
To study in vitro the effect of the simultaneous expression of wild-type and mutant TSHRs that occur in patients with heterozygous TSHR mutations, we co-transfected wild-type and mutant (C41S, L467P, C600R) receptors in Cos-7 cells and measured maximum cAMP response to bTSH stimulation at different ratios of mutant to wild-type DNAs. In cells co-transfected with wild-type and mutant TSHRs, we observed a statistically significant impairment of cAMP production, when compared with cells co-expressing wild-type TSHR and a control protein (E. coli ß-galactosidase, LacZ). The negative effect was dose-dependent for all mutants (Fig. 2A). In the case of C600R mutant, a plateau was apparently reached at mutant to wild-type ratio of 1:1. In addition, cAMP response to increasing doses of bTSH was evaluated in cells co-transfected at a constant mutant to wild-type ratio. Again, the expression of mutant TSH receptors was associated with a reduced cAMP response to bTSH stimulation. The inhibitory effect was present both at basal conditions and at saturating bTSH concentrations (10–100 U/l) (Fig. 2B).

View larger version (50K): [in this window] [in a new window]   Figure 2. Dominant negative effect of inactive TSHR mutants. (A) cAMP production in response to 50 U/l bTSH in cells co-transfected with wild-type TSHR and inactive TSHR mutants (C41S, L467P and C600R) at different mutant to wild-type DNA ratios. Empty pTarget vector was used to keep the total amount of transfected DNA constant. A plasmid coding for ß-galactosidase (pT-LacZ) was used as negative control. The data represent the mean±SEM of three independent experiments in eight replicate measurements. Differences are statistically significant by two-way ANOVA (F=18.25, P<0.0001 for co-transfected plasmids; F=4.86, P<0.05 for amount of mutant TSHR or pT-LacZ plasmid), followed by Bonferroni's post hoc test versus cells transfected with pT-LacZ at same DNA ratio. *P<0.05; **P<0.01; ***P<0.001. (B) cAMP dose–response curve to increasing bTSH concentrations in cells co-transfected with wild-type TSHR and inactive TSHR mutants at a 3:1 mutant to wild-type DNA ratio. Inset shows basal cAMP values. Data are from a representative experiment.

 
Generation and characterization of chimeric fluorescent proteins of wild-type and mutant TSHRs
To investigate their subcellular localization by fluorescence microscopy, we generated fluorescent chimeras of wild-type and inactive TSHR mutants, by fusing fluorescent reporters (EGFP, ECFP or EYFP) to the COOH-terminal tail of receptors. Wild-type TSHR chimeras were clearly expressed at the cell membrane and to a lesser extent in intracytoplasmic compartments. In contrast, mutant receptor chimeras were retained in intracytoplasmic compartments and very poorly expressed on the plasma membrane (Fig. 3A). To exclude the possibility that the intracellular retention of mutant receptors could be simply due to higher expression levels, an immunoblot experiment was performed (Fig. 3B). In agreement with previous reports (33,34), two bands corresponding to precursor (i.e. partially glycosylated) and mature (i.e. fully glycosylated) receptors were present. The total level of protein expression was similar for all constructs, even if a relative shift from mature to precursor form was observed in the case of mutant receptors. An immunoblot experiment on protein extracts subjected to complete enzymatic deglycosylation (Fig. 3C) confirmed that the band of higher molecular weight corresponds to fully glycosylated TSHR, whereas the band of lower molecular weight corresponds to partially glycosylated TSHR (34). Wild-type TSHR chimeras maintained cAMP response to bTSH stimulation (440–549% of basal) and the negative effect of mutant receptor co-expression was observed also with chimeric constructs (data not shown).

View larger version (39K): [in this window] [in a new window]   Figure 3. Characterization of chimeric constructs of TSHRs and fluorescent reporters. (A) Cos-7 cells transfected with EGFP chimeras of wild-type or mutant TSHRs and visualized by fluorescence microscopy. Wild-type receptor is clearly expressed at the plasma membrane and in a perinuclear Golgi-like region. Mutant C41S receptors are poorly expressed at the plasma membrane and are retained in intracellular regions. Similar results were obtained with L467P or C600R mutants. (B) Immunoblot analysis of different EGFP chimeras using an anti-GFP antibody. Two bands consistent with the molecular weights of precursor (123 kDa, partially glycosylated TSHR+EGFP) and mature (140 kDa, fully glycosylated TSHR+EGFP) receptor forms were present. An inversion of the mature to precursor ratio was observed in the case of mutant receptors. (C) Deglycosylation of TSHR by PNGase F removing all N-linked oligosaccharides. Complete deglycosylation results in the shift of both bands into a single band of about 110 kDa. N.T., not treated cells.

 
Mutant receptors hamper the cell surface expression of wild-type TSHR
A possible explanation for the dominant negative effect is that mutant receptors, crippled by a defect in membrane targeting, may trap wild-type receptors in intracytoplasmic compartments. To test this hypothesis, we co-expressed chimeras of wild-type and inactive TSHR mutants, linked to two different fluorescent reporters (ECFP or EYFP), in Cos-7 cells. Cells were then visualized by fluorescence microscopy. In cells co-expressing wild-type and inactive TSHR mutants, we observed a conspicuous retention of wild-type TSHR in intracellular compartments where it co-localized with mutant receptors (Fig. 4A). This effect was specific for TSHR, because inactive TSHR mutants did not alter the cell surface expression of two control proteins: a membrane-targeted EYFP (EYFPmem) (Fig. 4B) and a transmembrane serine/threonine kinase receptor, Bone Morphogenetic Protein Receptor 1 B (BMPR1B), fused to ECFP (Fig. 4C).

View larger version (30K): [in this window] [in a new window]   Figure 4. Effect of inactive TSHR mutants on the subcellular localization of wild-type TSHR. (A) Fluorescence microscope images of Cos-7 cells co-transfected with wild-type TSHR-EYFP chimera (red) and ECFP chimeras of wild-type TSHR or inactive TSHR mutants (blue). The co-expression of inactive TSHR mutants is associated with intracellular retention of wild-type TSHR. Wild-type and mutant TSHRs co-localize in intracytoplasmic compartments, as indicated by the presence of violet color in merge images. (B) Fluorescence microscope images of Cos-7 cells co-transfected with EYFPmem (red) and ECFP chimeras of wild-type TSHR or inactive TSHR mutants (blue). Inactive TSHR mutants do not alter the membrane expression of EYFPmem. (C) Fluorescence microscope images of Cos-7 cells co-transfected with BMPR1B-ECFP chimera (blue) and EYFP chimeras of wild-type TSHR or inactive TSHR mutants (red). Inactive TSHR mutants do not alter the membrane expression of BMPR1B-ECFP chimera.

 
Inactive TSHR mutants cause wild-type TSHR retention in the endoplasmic reticulum (ER)
Cos-7 cells were transfected with wild-type TSHR-EGFP chimera, C41S-EGFP chimera or wild-type TSHR-EGFP chimera together with untagged C41S mutant and stained in vivo for ER, Golgi or lysosomes (Fig. 5). In addition to being expressed at the plasma membrane, wild-type TSHR was found to co-localize with Golgi and to a lesser extent with ER staining. A poor co-localization was observed with lysosomal compartments. As previously shown, the co-expression of inactive TSHR mutants was associated with wild-type TSHR intracellular retention. In particular, TSHR was found to co-localize with ER and only partially with Golgi signals.

View larger version (20K): [in this window] [in a new window]   Figure 5. Modifications of wild-type TSHR subcellular localization in the presence of inactive TSHR mutants. Cos-7 cells were transfected with wild-type TSHR-EGFP chimera, C41S-EGFP chimera or wild-type TSHR-EGFP chimera+untagged C41S mutant. Cells were than stained in vivo for ER, Golgi or lysosomes, and imaged by fluorescence microscopy. EGFP chimeras are visualized in green, whereas ER, Golgi and lysosomes are visualized in red. Yellow indicates co-localization between EGFP chimeras and subcellular staining. Similar results were obtained with L467P or C600R mutants.

 
TSHR oligomerization occurs in intracellular membranes
To detect molecular interactions between wild-type and mutant TSHRs, fluorescence resonance energy transfer (FRET) between fluorescent TSHR chimeras was measured (Fig. 6). Cos-7 cells were co-transfected with wild-type TSHR-ECFP chimera (FRET donor) and EYFP chimeras of wild-type or mutant TSHRs (FRET acceptor). Time-lapse series of images of donor fluorescence were then acquired under continuous illumination, and decay constants of donor photobleaching (_bl) were determined by fitting to an exponential decay the intensity values of pixels in a selected region of interest (ROI). In the presence of FRET, an increase of mean _bl values is expected. Two mutations, one in the extracellular (C41S) and one in the transmembrane (L467P) domain, were tested. As mutant receptors were almost exclusively located intracellularly and caused wild-type TSHR entrapment mainly in the ER, FRET was analyzed in ROIs placed in the ER-Golgi. Parts B1–B5 in Figure 6 show the pixel-by-pixel distribution of _bl values in the intracellular ROIs from a representative experiment. A right-shift of _bl values in cells co-transfected with wild-type TSHR-ECFP chimera (FRET donor) and EYFP chimera of wild-type or inactive TSHR mutants (FRET acceptor) was indicative of FRET. This effect was dose-dependent with higher _bl values being observed at 1:4 donor to acceptor ratio, as compared to 1:1 ratio. In contrast, _bl values of cells co-transfected with wild-type TSHR-ECFP chimera and EYFPmem, used as control, were almost superimposable to those of cells expressing wild-type TSHR-ECFP chimera alone. Mean FRET efficiencies were then calculated from _bl data of independent experiments (Fig. 7). A significant increase in FRET efficiency was present in cells co-transfected with ECFP and EYFP chimeras of wild-type TSHR, as well as in those co-transfected with wild-type TSHR-ECFP chimera and EYFP chimeras of inactive TSHR mutants. Instead, no significant change in FRET efficiency occurred in cells co-transfected with wild-type TSHR and EYFPmem.

View larger version (43K): [in this window] [in a new window]   Figure 6. FRET analysis of TSHR oligomerization. Cells were transfected with FRET donor (wild-type TSHR-ECFP chimera) alone or FRET donor and acceptor (TSHR-EYFP chimeras) at a 1:1 or 1:4 donor to acceptor DNA ratio and imaged by fluorescence microscopy. Results were compared with those obtained in the presence of EYFPmem, used as control. Time-lapse series were acquired at 480±40 nm (ECFP emission) under continuous illumination at 436±20 nm (ECFP excitation). (A1–A5) First images of time-lapse series of a typical experiment. (B1–B5) Distribution of bl values in intracellular ROIs (see white circle in panels A1–A5). A right-shift of bl is present in cells co-transfected with EYFP chimeras of wild-type TSHR or inactive TSHR mutants, but not in cells co-transfected with EYFPmem, indicating that wild-type TSHR can form both homo-oligomers and hetero-oligomers with inactive TSHR mutants. Dotted line represents distribution of bl values in cells transfected with FRET donor alone. Transfection conditions were as follows: 1, WT TSHR-ECFP; 2, WT TSHR-ECFP+EYFPmem; 3, WT TSHR-ECFP+WT TSHR-EYFP; 4, WT TSHR-ECFP+C41S-EYFP; 5, WT TSHR-ECFP+L467P-EYFP.

 

View larger version (21K): [in this window] [in a new window]   Figure 7. Mean FRET efficiencies in ER-Golgi region. Mean FRET efficiency was calculated as the percentage of change in the average time constant of donor photobleaching measured in specimens co-transfected with FRET donor and acceptor (donor+acceptor), with respect to that measured in specimens transfected with donor alone (donor), via the following equation: E=[1–donor/donor+acceptor]x100. For each condition, mean FRET efficiencies were calculated from at least 20 time-lapse series acquired in four independent experiments. Differences are statistically significant by one-way ANOVA (F=24.43, P<0.0001), followed by Bonferroni's post hoc test versus cells transfected with donor alone. *P<0.05; **P<0.001.

 
Wild-type TSHR entrapment by C41S mutant in stable cell lines
To further characterize the mechanisms of wild-type TSHR entrapment and dominant negative action by mutant receptors, stable cell lines co-expressing myc-tagged wild-type TSHR and EGFP chimeras of wild-type TSHR or C41S mutant were generated. Although myc-tagged wild-type TSHR was mainly located at the cell membrane (Fig. 8A), the co-expression of C41S mutant was associated with its intracellular retention (Fig. 8B). To estimate the degree of intracellular retention of wild-type TSHR, the ratio between membrane and intracellular fluorescence (f_R) of myc-tagged wild-type TSHR was calculated (Fig. 8C). In the absence of mutants, wild-type TSHR was prevalently expressed at the plasma membrane (f_R=3.0), whereas the co-expression of C41S mutant resulted in a 7.5-fold reduction of membrane to intracellular fluorescence ratio (f_R=0.4). To evaluate possible changes in wild-type TSHR post-translational modifications in the presence of inactive TSHR mutants, protein extracts from the same cell lines were blotted using an anti-myc antibody (Fig 8D). A significant reduction of mature TSHR was observed in cells co-transfected with C41S-EGFP chimera. Basal and TSH-stimulated cAMP production in the stable cell line co-expressing myc-tagged wild-type TSHR and C41S-EGFP chimera was then compared with that of a stable cell line expressing myc-tagged wild-type TSHR alone. The expression levels of myc-tagged wild-type TSHR were similar in the two cell lines (Fig. 8E), with density values calculated by densitometric analysis of 3519 for WT TSHR-myc and 3294 for WT TSHR-myc+C41S-EGFP. However, cells co-expressing C41S-EGFP chimera showed 4.1- and 3.8-fold lower constitutive and stimulated cAMP levels, respectively, when compared with cells expressing myc-tagged wild-type TSHR alone (Fig. 8F).

View larger version (34K): [in this window] [in a new window]   Figure 8. Intracellular entrapment of wild-type TSHR by C41S mutant in stable cell lines. Confocal images of stable cell lines co-expressing myc-tagged wild-type TSHR (red) and EGFP chimeras (green) of wild-type TSHR (A) or C41S mutant (B). Cells were treated with a protein synthesis inhibitor (cycloheximide), followed by immunofluorescence with an anti-myc antibody. EGFP chimeras were visualized directly, without staining. Nuclei were stained with DAPI (blue). Yellow indicates co-localization between myc-tagged wild-type TSHR and EGFP chimeras. myc-tagged wild-type TSHR and EGFP chimera of wild-type TSHR are mainly expressed at the cell membrane. Conversely, the EGFP chimera of C41S mutant is mainly localized intracellularly and causes intracellular entrapment of myc-tagged wild-type TSHR. (C) Ratio of membrane to intracellular fluorescence (fR) measured in myc-tagged wild-type TSHR channel. The co-expression of C41S mutant is associated with a 7.5-fold drop in fR (0.4 versus 3.0). (D) Immunoblot analysis of same stable cell lines with an anti-myc antibody. A reduction of the band corresponding to mature (i.e. fully glycosylated) receptor and a parallel increase of the band corresponding to precursor receptor is present in cells co-expressing C41S mutant. Similar amounts of high-molecular-weight (HMW) complexes of TSHR were seen in both TSHR-expressing cell lines. (E) Immunoblot analysis of stable cell lines with an anti-myc antibody showing similar levels of expression of myc-tagged wild-type TSHR in the cell line co-expressing C41S-EGFP chimera and in a control cell line expressing myc-tagged wild-type TSHR alone. (F) Basal and TSH-stimulated (50 U/l) cAMP production. Levels of cAMP were normalized for the total amount of myc-tagged wild-type TSHR in each condition (see E), estimated by densitometric analysis.

 
C41S mutant co-immunoprecipitates with wild-type TSHR
To confirm the results of FRET experiments, we performed co-immunoprecipitation experiments on stable cell lines expressing TSHRs with different C-terminal tags (myc or EGFP). After precipitation with an anti-myc antibody and blotting with an anti-GFP antibody, GFP-positive bands were found only for cells co-expressing myc-tagged wild-type TSHR and EGFP chimeras of either wild-type or C41S mutant TSHR (Fig. 9).

View larger version (21K): [in this window] [in a new window]   Figure 9. Co-immunoprecipitation of wild-type and C41S mutant TSHRs. Stable cell lines expressing myc-tagged wild-type TSHR and/or EGFP chimeras of wild-type or C41S mutant TSHRs were precipitated (IP) with an anti-myc (or anti-GFP) antibody and blotted (WB) with an anti-GFP antibody. Bands corresponding to EGFP chimeras were present only in immunoprecipitates from cell lines co-expressing myc-tagged wild-type TSHR. C+, HEK293 cells expressing the EGFP chimera of wild-type TSHR, precipitated and blotted with anti-GFP antibody (positive control). C–, negative controls.

 

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS NOTE ADDED IN PROOF REFERENCES

 
The aim of this study was to clarify the molecular basis for dominant inheritance of TSH resistance linked to heterozygous inactivating mutations in TSHR gene. The present findings support a model in which mutant receptors exert a direct dominant negative effect on the activity of wild-type TSHR.

The co-expression of wild-type and mutant TSHRs was associated with a reduction of cAMP production. The inhibitory effect of mutant receptors was dose-dependent and statistically significant for two out of three mutants at mutant to wild-type ratio of 1 : 1, and for all mutants at 3 : 1 ratio. The negative effect of C600R mutant apparently reached a plateau at 1 : 1 ratio. Differences among mutants remain unexplained presently, but may be related to specific mutation effects. The dominant negative action of mutant receptors was observed both at basal conditions (ligand-independent activity) and at maximal TSHR stimulation. This finding is consistent with the idea that the expression of mutant receptors is associated with a reduction of the number of wild-type receptors present at the cell membrane level.

Several data, including the reduction of cAMP production and of wild-type TSHR post-translational modifications in the presence of inactive mutants, as well as the co-localization of wild-type and mutant receptors in intracytoplasmic compartments, suggest that inactive mutants exert their dominant effect by hampering wild-type receptor intracellular trafficking and cell surface expression. Intracellular retention was specific for TSHR as it did not involve another protein targeted to the membrane via a transmembrane domain (i.e. BMPR1B). These results are consistent with previous reports, indicating that naturally occurring mutants of other receptors (V2R, CCR5, GnRHR, Frizzled4, LHR) (27–32) can exert a dominant negative effect by causing wild-type receptor retention in intracellular compartments. A similar mechanism has been advocated very recently to explain autosomal dominant retinitis pigmentosa in the case of rhodopsin mutants (35). With the sole exception of rhodopsin and TSHR, no recognizable in vivo defect has been associated so far with heterozygous loss-of-function GPCR mutations. This is one of the first reports linking dominant negative mutations of a GPCR to an abnormal in vivo phenotype.

Several bodies of evidence implicate the ER as the main controller of protein folding and assembly (36). Our data indicate that inactive TSHR mutants can interfere with wild-type TSHR intracellular sorting, reducing its cell surface expression and causing its retention in the ER. As TSHR passage through the Golgi apparatus is associated with post-translational modifications, mainly represented by glycosylation, any obstacle to receptor exit from the ER is expected to result in an alteration of receptor glycosylation pattern. Indeed, retained TSHR mutants are prevalently present in the precursor (partially glycosylated) form and their co-expression was found to cause a significant reduction in the relative abundance of mature (fully glycosylated) wild-type TSHR in immunoblot experiments. This finding further supports the idea that the diversion of wild-type TSHR to intracytoplasmic compartments, which occurs in the presence of inactive TSHR mutants, is an early event, occurring before post-translational modifications in the Golgi apparatus.

Transient transfection experiments may be affected by possible drawbacks. On one hand, they are associated with transgene overexpression. On the other hand, transiently co-transfected cells are characterized by heterogeneous expression of the different constructs. Indeed, a portion of these cells might be expected to express prevalently wild-type TSHR which may attenuate the dominant negative effect of mutants on cAMP production. For these reasons, the observations on transiently transfected cells were extended to stable cell lines. Stable cell lines are associated with lower transgene expression when compared with transiently transfected cells (32) and contain a homogeneous cell population, which made them a more reliable system in which to verify our hypothesis. In HEK293 stable cell lines, wild-type TSHR was mainly expressed at the cell membrane, whereas a 7.5-fold reduction of membrane to intracellular ratio of wild-type receptor was observed in the presence of C41S mutant. The observation of wild-type TSHR entrapment by mutant receptors in stable cell lines also leads one to conclude that the described phenomenon is not merely a consequence of overexpression. Furthermore, in the stable cell line co-expressing C41S mutant, constitutive and TSH-stimulated cAMP productions were impaired by 4.1- and 3.8-fold, respectively. This impairment is larger than that determined in transiently transfected cells and more consistent with the reduction of wild-type TSHR membrane expression observed by fluorescence microscopy.

Receptor oligomerization is an emerging aspect of GPCR biology. The functional role of this phenomenon is still largely unclear, but may be relevant for receptor activation, ligand selectivity and signal specificity (37,38). In addition, oligomerization was shown to be an early post-translational event for some GPCRs (23–26,35), indicating that it may be required for complete receptor maturation and cell surface expression. Receptor complexes were also reported in the case of TSHRs (33). Even if the phenomenon itself and its functional relevance have been poorly characterized so far, the formation of complexes between wild-type and mutant TSHRs could constitute the molecular basis for the observed dominant negative effect. We therefore studied TSHR supramolecular interactions in native membranes by FRET. Resonance energy transfer techniques, including bioluminescence resonance energy transfer (BRET) and FRET, have been extensively used to study GPCR oligomerization (39). Although BRET can be used to study protein–protein interactions in populations of living cells, it cannot be applied to single-cell experiments and cannot provide information about the subcellular localization of energy transfer. In contrast, FRET microscopy techniques can be conveniently applied to study protein–protein interactions in single cells or even in selected subcellular regions. FRET was evaluated by means of the technique of donor photobleaching which has the main advantage of not being interfered by differences in absolute intensity values (40,41). By FRET analysis, we have documented for the first time that wild-type TSHR homo-oligomerization is an early event, already detectable in the ER-Golgi compartment. This finding supports the idea that TSHR oligomerization may be important for complete folding, ER export and cell surface targeting of TSHR. In addition, inactive TSHR mutants maintained the capacity to interact with wild-type receptor, thus providing the molecular basis for their dominant negative effect. It is worth noting that higher values of donor photobleaching time constant and so FRET efficiencies were observed under conditions that favored the surrounding of donor by acceptor fluorophores (1:4 ratio). The presence of such a dose-dependent effect and the absence of FRET in the case of the negative control (i.e. wild-type TSHR-ECFP and EYFPmem) are supporting the specificity of our determinations. Furthermore, FRET results were confirmed by co-immunoprecipitation experiments on stable cell lines co-expressing wild-type and C41S mutant TSHRs. FRET can only reveal the close proximity of two molecules, but co-immunoprecipitation can directly demonstrate a physical interaction. The combination of FRET and co-immunoprecipitation results is a strong argument for the existence of wild-type/mutant TSHRs oligomers.

Although this study was focused on selected mutations from our heterozygous patients with partial TSH resistance (19), negative dominance may be common to other TSHR mutants (see Table 1). Indeed, the mechanism of direct negative dominance we are reporting here may be shared by other TSHR mutants associated with poor cell surface expression. However, TSHR mutations associated with conserved membrane expression may interfere through additional mechanisms. For instance, reduced TSH sensitivity could result from membrane expression of hetero-oligomers endowed with altered functional activity. Although additional mechanisms may be demonstrated to be involved in the future, the present findings provide a possible explanation for the partial TSH resistance observed in patients with heterozygous TSHR inactivating mutations. We believe that the dominant negative effect observed in vitro may also play a relevant role in vivo. Indeed, several endogenous (e.g. modifier genes) and exogenous (e.g. dietary iodine supply) factors enter into play and influence the in vivo expression of the defect. Even in the same heterozygous individual, the levels of TSH fluctuate and may be normal in some instances (see Fig. 1), indicating that undetermined mechanisms are involved in the disease expression. Importantly, thyroid function requirements are more pronounced in the neonatal period and infancy, and this represents an additional factor affecting the phenotype of congenital thyroid diseases, as previously reported for heterozygous nonsense mutations of DUOX2 gene (42).

In conclusion, inactive TSHR mutants are retained in the ER, probably as a consequence of protein misfolding, but maintain the capability of associating with wild-type TSHR. As a result, wild-type TSHR is entrapped intracellularly, providing a molecular basis for the dominant forms of partial TSH resistance associated with heterozygous mutations in TSHR gene. To our knowledge, this is the first report linking the dominant negative effect of a mutant GPCR to an abnormal endocrine phenotype (i.e. partial TSH resistance) in simple heterozygous patients.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS NOTE ADDED IN PROOF REFERENCES

 
Plasmids
pTarget-based constructs (pT-WT-TSHR, pT-C41S-TSHR, pT-L467P-TSHR and pT-C600R) were obtained as previously described (19). Chimeras of wild-type and mutant TSHRs fused to ECFP, EYFP or EGFP reporters were generated as follows: TSHR sequences were amplified from pTarget-based constructs, using a forward primer containing EcoRI recognition sequence (5'-CTTCGAATTCTAGCCCCGAGTCCCGTGGAA-3', EcoRI site is in bold) and a reverse primer that eliminated the stop codon and introduced a BamHI site (5'-AGGGATCCTTCAAAACCGTTTGCATATACTC-3', BamHI site is in bold); PCR fragments were then double digested with EcoRI/BamHI and cloned in frame with fluorescent reporters into EcoRI/BamHI sites of pEGFP-N1, pEYFP-N1 or pECFP-N1 expression vectors (BD Biosciences, Milan, Italy). A similar procedure was employed for the production of the control construct pECFP-BMPR1B (Di Pasquale et al., manuscript in preparation). pCDNA4-MYC/HYS-WT-TSHR plasmid was obtained as follows: a DNA fragment corresponding to wild-type TSHR cDNA lacking the stop codon was obtained by BamHI digestion followed by HindIII partial digestion of pECFP-WT-TSHR; this DNA fragment was subsequently cloned in frame with myc sequence into HindIII/BamHI sites of pCDNA4/myc-His version C expression vector (Invitrogen, San Giuliano Milanese, Italy). All constructs were verified by direct sequencing. pEYFPmem was purchased from BD Biosciences.

Cell cultures and transfections
Cos-7 and HEK293 cells were grown in Dulbecco's modified Eagle's medium (DMEM; Invitrogen) supplemented with 10% FCS and an ampicillin–streptomycin mixture. Transfection of Cos-7 cells was performed using Lipofectamine Reagent (Invitrogen). Cells were then grown for 48 h before cAMP determination or microscope imaging.

Generation of stable cell lines
Stable HEK293 clones were obtained by standard procedures. Briefly, 2x10⁶ cells were plated in 100 mm Petri dishes and either single transfected with pCDNA4-MYC/HYS-WT-TSHR, pEGFP-WT-TSHR, pEGFP-C41S-TSHR or co-transfected with pCDNA4-MYC/HYS-WT-TSHR+pEGFP-WT-TSHR or pCDNA4-MYC/HYS-WT-TSHR+pEGFP-C41S-TSHR. After selection with 500 µg/ml of the appropriate antibiotic, i.e. Geneticin (Invitrogen) for pEGFP-N1-based plasmids and/or Zeocin (Invitrogen) for pCDNA4-MYC/HYS-WT-TSHR plasmid, resistant cells were cloned by limiting-dilution. The expression of myc- and EGFP-tagged TSHRs was then verified by RT-PCR, western blot, immunofluorescence and fluorescence microscopy.

cAMP assay
Cells were plated at the density of 2x10⁵ in 35 mm Petri dishes. For transient expression experiments, transfection was done with 350 ng of total DNA. Transfection efficiency was checked by FACS on cells transfected with GFP. Efficiency of transfection was reproducible among different experiments and ranged between 58 and 70% at 48 h. Forty-eight hours after transfection or 24 h after plating for stable cell lines, culture medium was replaced with Krebs-Ringer Bicarbonate buffer supplemented with 750 mM 3-isobutyl-1-methylxanthine (IBMX) (KRB-IBMX buffer; Mediomics LLC, St Louis, MO, USA) and the indicated concentration of bovine TSH (bTSH; Sigma-Aldrich, Milan, Italy). Cells were then incubated at 37°C for 1 h, washed with PBS and incubated with 1 ml ethanol 100% at –20°C for 1 h. Ethanol samples were then collected in 1.5 ml tubes and vacuum-dried. cAMP assay was performed using Bridge-It cAMP Designer Fluorescence Assay (Mediomics LLC) in 96-well, black, round-bottom plates, according to manufacturer's instructions. Fluorescence measurements were taken with Fluoroskan Ascent FL multiplate reader (Thermo Labsystems, Helsinki, Finland).

Microscopy visualization of fluorescent chimeras
Cos-7 cells were plated at 40 000 cells/well on sterile coverslips placed in 35 mm Petri dishes and transfected with 1 µg of DNA. Forty-eight hours after transfection, cells were washed with PBS, fixed with PBS containing 3.7% paraformaldehyde (Sigma-Aldrich) for 10 min at 37°C and rinsed twice with PBS. Coverslips were then removed and mounted with glycerol on microscope slides. Images were recorded with a C4742-98 ORCA II cooled CCD camera (Hamamatsu Photonics, Hamamatsu City, Japan) mounted on an Axiovert S100TV (Zeiss, Jena, Germany) equipped with filters for ECFP (ex 436/20, dm 460, em 480/40), EGFP (ex 480/40, dm 505, em 535/50) and EYFP (ex 500/20, dm 515, em 535/30) (Chroma Technology Corp., Rockingham, VT, USA).

For ER, lysosome and Golgi staining, 48 h after transfection cells were washed with HBSS and incubated for 30 min with ER-Tracker Blue-White, LysoTracker Red or BODIPY TR C5-ceramide (Invitrogen) in HBSS. Cells were then washed with fresh HBSS and imaged in 500 µl HBSS at room temperature.

Stable cell lines were plated at 40 000 cells/well on sterile coverslips placed in 35 mm Petri dishes, incubated with DMEM containing 50 mg/ml of cycloheximide (Sigma-Aldrich) for 4 h and fixed with 3.7% paraformaldehyde (Sigma-Aldrich) in PBS. Cells were then blocked with 10% goat serum (Invitrogen), permeabilized with 1% Triton X-100 (Sigma-Aldrich), incubated with anti-myc antibody (Invitrogen) 1:500 in PBS+10% goat serum for 2 h at room temperature, incubated with secondary antibody (Alexa Fluor 546 goat anti-mouse IgG, Invitrogen) 1:2000 in PBS+10% goat serum for 1 h at room temperature, stained with DAPI (Sigma-Aldrich) and mounted with SlowFade Light Antifade Kit (Invitrogen). Slides were visualized with Leica TCS SP2 confocal microscope (Leica Microsystems, Mannheim, Germany). Mean membrane to intracellular fluorescence ratio (f_R) was then calculated from 20 cells per each condition after determination of fluorescence density in two separate regions corresponding to the plasma membrane and to the entire intracellular area, excluding nucleus.

FRET analysis
Cos-7 cells were seeded at a density of 40 000 cells/well on sterile coverslips placed in 35 mm Petri dishes. Twenty-four hours after plating, expression vectors encoding fluorescent chimeras were co-transfected at 1:1 (1+1 µg DNA) or 1:4 (1+4 µg DNA) donor (ECFP) to acceptor (EYFP) ratios. Forty-eight hours after transfection, cells were rinsed with PBS, fixed with paraformaldehyde 3.7% in PBS at 37°C for 10 min and washed twice with PBS. Coverslips were then removed, mounted with glycerol on microscope slides and sealed. FRET was then measured as previously described (40,41). Briefly, the specimen was irradiated at the wavelength of 436±20 nm and a time-lapse series of images of donor fluorescence was recorded at the wavelength of 480±40 nm during continuous illumination. From the first image of the series, a binary mask was prepared to select pixels in the ROI. The time series data for each pixel within an ROI were fit to an exponential decay function to determine decay constants of photobleaching. When FRET occurs between donor and acceptor fluorophores, the time constant for donor photobleaching increases (43). Thus, the efficiency (E) of FRET was calculated as the percentage of change in the average time constant of donor photobleaching measured in specimens co-transfected with donor and acceptor fluorophore (_{donor+acceptor}), with respect to that measured in specimens transfected with donor alone (_donor), via the following equation: E=[1–_donor/_{donor+acceptor}]x100. One of the advantages of this method for measuring FRET is that the measurements do not depend on absolute values of fluorescence. The photobleaching time constants were found to have skewed distributions, which became normal after logarithmic transformation. Therefore, data were analyzed using the natural logarithms of the photobleaching time constants, and efficiencies and statistics were derived by retransformation of the pertinent values.

Immunoblot analysis
Stable clones were plated in 100 mm Petri dishes. Upon reaching confluence, cells were washed twice with PBS, treated with 1 ml RIPA buffer, containing 10 mM Tris–HCl pH 7.2, 150 mM NaCl, 1% Triton X-100, 1% DOC, 0.1% SDS, 5 mM EDTA, 100 µM leupeptin, 1 µM pepstatin (all from Sigma-Aldrich), and scraped with a policeman. Lysates were then transferred to 2 ml tubes and sonicated. Protein concentration was determined by BCA assay (Pierce Biotechnology, Inc., Rockford, IL, USA). Twenty micrograms of each sample was then used for immunoblot experiments. Samples were electrophoresed on a 7.5% polyacrylamide gel and electro-transferred to a nitrocellulose membrane. Membrane was then blocked with TBS-T+5% milk, incubated with anti-myc antibody (Invitrogen) 1:7500 or anti-GFP antibody (BD Biosciences) 1:1000 in TBS-T+0.5% milk O/N at 4°C, washed with TBS-T, incubated with secondary antibody (HRP-conjugated antimouse IgG; Chemicon International, Inc., Temecula, CA, USA) 1:5000 in TBS-T+0.5% milk at room temperature for 1 h and thoroughly washed with TBS-T. Detection was performed using ECL plus kit (Amersham Biosciences Europe GmbH, Freiburg, Germany). Deglycosylation of TSHR was performed by treatment with 1 mU N-glycosydase F (PNGase F; Sigma-Aldrich) as described by Oda et al. (34).

Co-immunoprecipitation
For co-immunoprecipitation experiments, stable clones were plated in 100 mm Petri dishes. Upon reaching confluence, cells were washed twice with PBS, treated with 1 ml lysis buffer (20 mM Tris–HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100) containing a cocktail of protease inhibitors (Complete Mini, Roche Applied Science, Monza, Italy) and scraped with a policeman. Lysates were than sonicated, centrifuged at maximum speed for 15 min at 4°C, and supernatants were transferred to a new tube. Protein concentration was determined by BCA assay (Pierce Biotechnology, Inc.). Then, 2.5 mg of proteins from each lysate were pre-cleared with protein G for 1 h at 4°C, followed by O/N incubation with protein G and 1 µg/ml anti-myc antibody (Invitrogen). Immunoprecipitates were collected by centrifugation, washed four times with lysis buffer and eluted with SDS-PAGE sample buffer. Samples were then electrophoresed on a 7.5% polyacrylamide gel and electro-transferred to a nitrocellulose membrane. Membrane was blocked with TBS-T+5% milk, incubated with anti-GFP antibody (BD Biosciences) 1:1000 in TBS-T+5% milk O/N at 4°C, washed with TBS-T, incubated with secondary antibody (HRP-conjugated antimouse IgG, Chemicon International, Inc.) 1:5000 in TBS-T+5% milk at room temperature for 1 h and thoroughly washed with TBS-T. Detection was performed using ECL plus kit (Amersham Biosciences Europe GmbH).

Digital image processing
Digital processing and analyses were performed with Image Pro Plus 4.5 (Media Cybernetics, Silver Spring, MD, USA) using standard and custom (www.mediacy.com/apps/fretefficiency.pdf) routines.

Statistics
When indicated, one-way or two-way ANOVA followed by Bonferroni's post hoc test was used to estimate the significance of differences between means.

ACKNOWLEDGEMENTS
The authors thank E. Di Pasquale for the production of pECFP-BMPR1B plasmid. This work was partially supported by Funds of the Italian Ministry of Health (Ricerca Finalizzata 2003, Project: TECNOL 08M301), by Funds of Italian Ministry of Education, University and Research (PRIN 2004, Project no.: 2004052155_005) and by Research Funds of IRCCS Istituto Auxologico Italiano (Project: ALGIP, 05C901) to L.P.

Conflict of Interest statement. None declared.

	NOTE ADDED IN PROOF

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS NOTE ADDED IN PROOF REFERENCES

 
TSHR oligomerization has been very recently demonstrated also by means of BRET technology [Ref. Urizar, E., Montanelli, L., Loy, T., Bonomi, M., Swillens, S., Gales, C., Bouvier, M., Smits, G., Vassart, G. and Costagliola, S. (2005) Glycoprotein hormone receptors: link between receptor homodimerization and negative cooperativity. EMBO J. 24: 1954–1964].

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