Glycoprotein hormone receptors: link between receptor homodimerization and negative cooperativity
2005; Springer Nature; Volume: 24; Issue: 11 Linguagem: Inglês
10.1038/sj.emboj.7600686
ISSN1460-2075
AutoresEneko Urizar, Lucia Montanelli, Tiffany Loy, Marco Bonomi, Stéphane Swillens, Céline Galès, Michel Bouvier, Guillaume Smits, Gilbert Vassart, Sabine Costagliola,
Tópico(s)Regulation of Appetite and Obesity
ResumoArticle12 May 2005free access Glycoprotein hormone receptors: link between receptor homodimerization and negative cooperativity Eneko Urizar Eneko Urizar IRIBHM, Université Libre de Bruxelles, Campus Erasme, Brussels, Belgium Departamento de Neurofarmacología, Facultad de Farmacia, Universidad del País Vasco, Vitoria-Gasteiz, Spain Search for more papers by this author Lucia Montanelli Lucia Montanelli IRIBHM, Université Libre de Bruxelles, Campus Erasme, Brussels, Belgium Search for more papers by this author Tiffany Loy Tiffany Loy IRIBHM, Université Libre de Bruxelles, Campus Erasme, Brussels, Belgium Search for more papers by this author Marco Bonomi Marco Bonomi IRIBHM, Université Libre de Bruxelles, Campus Erasme, Brussels, Belgium Institute of Endocrine Sciences, Istituto Auxologico Italiano IRCCS and Ospedale Maggiore di Milano IRCCS, Italy Search for more papers by this author Stéphane Swillens Stéphane Swillens IRIBHM, Université Libre de Bruxelles, Campus Erasme, Brussels, Belgium Search for more papers by this author Céline Gales Céline Gales Department of Biochemistry, Université de Montréal, succursale Centre-Ville, Montréal, Québec, Canada Search for more papers by this author Michel Bouvier Michel Bouvier Department of Biochemistry, Université de Montréal, succursale Centre-Ville, Montréal, Québec, Canada Search for more papers by this author Guillaume Smits Guillaume Smits IRIBHM, Université Libre de Bruxelles, Campus Erasme, Brussels, Belgium Service de Génétique Médicale, Hôpital Erasme, Brussels, Belgium Search for more papers by this author Gilbert Vassart Gilbert Vassart IRIBHM, Université Libre de Bruxelles, Campus Erasme, Brussels, Belgium Service de Génétique Médicale, Hôpital Erasme, Brussels, Belgium Search for more papers by this author Sabine Costagliola Corresponding Author Sabine Costagliola IRIBHM, Université Libre de Bruxelles, Campus Erasme, Brussels, Belgium Search for more papers by this author Eneko Urizar Eneko Urizar IRIBHM, Université Libre de Bruxelles, Campus Erasme, Brussels, Belgium Departamento de Neurofarmacología, Facultad de Farmacia, Universidad del País Vasco, Vitoria-Gasteiz, Spain Search for more papers by this author Lucia Montanelli Lucia Montanelli IRIBHM, Université Libre de Bruxelles, Campus Erasme, Brussels, Belgium Search for more papers by this author Tiffany Loy Tiffany Loy IRIBHM, Université Libre de Bruxelles, Campus Erasme, Brussels, Belgium Search for more papers by this author Marco Bonomi Marco Bonomi IRIBHM, Université Libre de Bruxelles, Campus Erasme, Brussels, Belgium Institute of Endocrine Sciences, Istituto Auxologico Italiano IRCCS and Ospedale Maggiore di Milano IRCCS, Italy Search for more papers by this author Stéphane Swillens Stéphane Swillens IRIBHM, Université Libre de Bruxelles, Campus Erasme, Brussels, Belgium Search for more papers by this author Céline Gales Céline Gales Department of Biochemistry, Université de Montréal, succursale Centre-Ville, Montréal, Québec, Canada Search for more papers by this author Michel Bouvier Michel Bouvier Department of Biochemistry, Université de Montréal, succursale Centre-Ville, Montréal, Québec, Canada Search for more papers by this author Guillaume Smits Guillaume Smits IRIBHM, Université Libre de Bruxelles, Campus Erasme, Brussels, Belgium Service de Génétique Médicale, Hôpital Erasme, Brussels, Belgium Search for more papers by this author Gilbert Vassart Gilbert Vassart IRIBHM, Université Libre de Bruxelles, Campus Erasme, Brussels, Belgium Service de Génétique Médicale, Hôpital Erasme, Brussels, Belgium Search for more papers by this author Sabine Costagliola Corresponding Author Sabine Costagliola IRIBHM, Université Libre de Bruxelles, Campus Erasme, Brussels, Belgium Search for more papers by this author Author Information Eneko Urizar1,2, Lucia Montanelli1, Tiffany Loy1, Marco Bonomi1,3, Stéphane Swillens1, Céline Gales4, Michel Bouvier4, Guillaume Smits1,5, Gilbert Vassart1,5 and Sabine Costagliola 1 1IRIBHM, Université Libre de Bruxelles, Campus Erasme, Brussels, Belgium 2Departamento de Neurofarmacología, Facultad de Farmacia, Universidad del País Vasco, Vitoria-Gasteiz, Spain 3Institute of Endocrine Sciences, Istituto Auxologico Italiano IRCCS and Ospedale Maggiore di Milano IRCCS, Italy 4Department of Biochemistry, Université de Montréal, succursale Centre-Ville, Montréal, Québec, Canada 5Service de Génétique Médicale, Hôpital Erasme, Brussels, Belgium *Corresponding author. IRIBHM, Université Libre de Bruxelles, Campus Erasme, 808 Route de Lennik, 1070 Bruxelles, Belgium. Tel.: +32 2 555 4169; Fax: +32 2 555 4212; E-mail: [email protected] The EMBO Journal (2005)24:1954-1964https://doi.org/10.1038/sj.emboj.7600686 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The monomeric model of rhodopsin-like G protein-coupled receptors (GPCRs) has progressively yielded the floor to the concept of GPCRs being oligo(di)mers, but the functional correlates of dimerization remain unclear. In this report, dimers of glycoprotein hormone receptors were demonstrated in living cells, with a combination of biophysical (bioluminescence resonance energy transfer and homogenous time resolved fluorescence/fluorescence resonance energy transfer), functional and biochemical approaches. Thyrotropin (TSHr) and lutropin (LH/CGr) receptors form homo- and heterodimers, via interactions involving primarily their heptahelical domains. The large hormone-binding ectodomains were dispensable for dimerization but modulated protomer interaction. Dimerization was not affected by agonist binding. Observed functional complementation indicates that TSHr dimers may function as a single functional unit. Finally, heterologous binding-competition studies, performed with heterodimers between TSHr and LH/CG–TSHr chimeras, demonstrated the unsuspected existence of strong negative cooperativity of hormone binding. Tracer desorption experiments indicated an allosteric behavior in TSHr and, to a lesser extent, in LH/CGr and FSHr homodimers. This study is the first report of homodimerization associated with negative cooperativity in rhodopsin-like GPCRs. As such, it may warrant revisitation of allosterism in the whole GPCR family. Introduction G protein-coupled receptors (GPCRs) constitute the largest and structurally best-conserved superfamily of signaling molecules (Kristiansen, 2004). Over the years, the initial concept 'one receptor/one G-protein/one regulatory cascade' has yielded the stage to a more complex picture (Park et al, 2004). The discovery that GPCRs belonging to family 3 (Bockaert and Pin, 1999) function as obligatory heterodimeric (GABAB, taste (T1R1−3) receptors) or homodimeric structures (metabotropic glutamate (mGluR) and calcium-sensing receptors; reviewed in Pin et al, 2003), and that family 1 receptors are also capable of homo or heterodimerization (Terrillon and Bouvier, 2004) has added additional complexity. A functional role of heterodimerization has been well demonstrated in GABAB and taste receptors, where it is involved in the routing of the molecules or selectivity for agonists (Margeta-Mitrovic et al, 2000; Nelson et al, 2001). In family 1 GPCRs, the situation is less clear: the obligatory character of their homodimeric nature in native systems is still debated, even for rhodopsin (Chabre et al, 2003), as is the physiological relevance of heterodimers (Terrillon and Bouvier, 2004). The glycoprotein hormones (thyrotropin (TSH), follitropin (FSH) and lutropin (LH/CG)) are dimeric 30 kDa proteins with important roles in the control of metabolism and reproduction. Their receptors (GpHr) belong to a subgroup of family 1 GPCRs (LGRs) characterized by a large ligand-binding domain containing leucine-rich repeats, responsible for recognition specificity (Smits et al, 2003; Fan and Hendrickson, 2005), and a heptahelical transmembrane domain typical of rhodopsin-like receptors (Vassart et al, 2004). It has been reported that receptors belonging to the GpHr family would dimerize (Osuga et al, 1997; Horvat et al, 2001; Ji et al, 2004; Fan and Hendrickson, 2005). However, in the LH/CGr, stimulation by the agonist has been reported to augment dimerization (Tao et al, 2004), whereas in the TSHr, activation was shown to promote dissociation (Latif et al, 2002). Determination of the monomeric/oligomeric state of transmembrane proteins in native systems is not a trivial task. After early experiments involving mainly co-immunoprecipitation of tagged receptors (Gomes et al, 2001), Bouvier and co-workers have taken advantage of a biophysical assay called bioluminescence resonance energy transfer (BRET) to assess GPCR oligomerization (Xu et al, 1999; Angers et al, 2000; reviewed in Bouvier, 2001). The intensity of the generated signal depends on the distance between the donor and the acceptor and their relative orientation (Wu and Brand, 1994). Although efficient and sensitive, BRET technology has two main limitations: (1) it requires transfection in recipient cells of chimeric constructs between RLuc or EYFP and the proteins of interest, which limits exploration of native systems; and (2) it does not discriminate between signals generated intracellularly or at the cell surface. Fluorescence resonance energy transfer (FRET) between a donor and an acceptor fluorescent construct allows visualization, at the subcellular level, of protein–protein interactions in living cells (Cubitt et al, 1995). It has been successfully used to monitor GPCR dimerization (Overton and Blumer, 2000), but suffers from the same limitation as BRET regarding the need for transfection of chimeric constructs. Moreover, it is sensitive to photobleaching of the donor fluorophore and to autofluorescence of the endogenous cell components or medium (Boute et al, 2002), and the artifactual direct excitation of the acceptor by the light source is an additional source of difficulties. Homogenous time resolved fluorescence (HTRF) is an interesting new FRET technology recently applied to the study of GPCR dimerization (Maurel et al, 2004). It is based on FRET between two differentially labeled antibodies (cryptate and cyanine derivatives) and allows detection of interaction between native transmembrane proteins, in intact cells, at the plasma membrane. With the aim of clarifying the situation regarding dimerization of GpHr, we have exploited the BRET and HTRF technologies to demonstrate unambiguously that TSHr form dimers stabilized by interactions between their heptahelical domains. These new findings blend the recent structural data generated by crystallization of FSHr–FSH complex (Fan and Hendrickson, 2005) and lead to the unexpected discovery that GpHr homodimers display negative cooperativity for the binding of their respective hormones. Our data provide a molecular basis to previous observations related to the allosteric behavior of GPCRs (Christopoulos and Kenakin, 2002) and suggest that negative cooperativity could play an important role in defining the characteristics of concentration–effects relations for agonists acting via GPCRs. Results Biophysical evidence for dimerization of the TSHr Evidence from BRET experiments. Homodimerization of the TSHr was investigated by semiquantitative BRET analysis. All TSHr-RLuc and TSHr-EYFP-tagged receptors were comparable to the wild-type (WT) receptor for both expression and functional parameters (not shown). Titration curves were performed by transfecting HEK 293T cells with constant TSHr-RLuc and increasing amounts of TSHr-EYFP constructs. A robust and very reproducible transfer was observed between TSHr fusion proteins (BRETmax: 0.106±0.018; BRET50: 0.004±0.002; n=12) (Figure 1A). However, transfection of the TSHr-RLuc construct with increasing amounts of the unrelated GABAB2 receptor tagged with EYFP (Gbr2-EYFP) also generated a titration curve, although with a significantly lower maximal net BRET and with a 10 times higher BRET50 (BRETmax: 0.031±0.008) (Figure 1A, inset). Therefore, we explored the specificity of the interactions by conducting competition experiments with untagged constructs. A constant ratio of TSHr-EYFP to TSHr-RLuc constructs and increasing amounts of WT TSHr or the chemokine receptor CCR5 were cotransfected and the net BRET signals were measured and plotted versus the expression of the competitor (see Materials and methods) (Figure 1B). The unlabeled TSHr readily competed the interaction, while CCR5 was totally ineffective (Figure 1B). In similar experiments, increasing amounts of coexpressed untagged TSHr did not decrease the signal coming from the energy transfer between TSHr-RLuc and Gbr2-EYFP nor between Gbr1 and Gbr2 (not shown). These experiments demonstrate the specificity of the interaction between TSHr-RLuc and TSHr-EYFP and illustrate the danger of relying on single or even titration net BRET experiments, alone, to explore putative interaction by this technology. It is likely that the signal obtained by cotransfection of TSHr-RLuc and Gbr2-EYFP is due to the extremely high level of expression reached by Gbr2-EYFP as indicated by the high BRET50. Figure 1.Attach-BRET demonstrates specific homodimerization. (A, C, E) Titration curves. HEK 293T cells are cotransfected with a constant DNA amount of RLuc constructs and increasing DNA concentrations of EYFP-tagged receptors: TSHr (A), Gbr2 (A, inset), 7TM (C) or LH/CGr (E). The BRET, total luminescence and total fluorescence were measured 48 h post-transfection. BRET signals are plotted over the relative expression levels (total fluorescence over total luminescence) (see Materials and methods for details). (B, D, F) Competition experiments. HEK 293T cells were cotransfected with constant DNA amounts of –Rluc- and -EYFP-tagged constructs (concentrations determined to give a signal close to the BRET50) and increasing concentrations of the unlabeled SPRT-TSHr (▪; B, D) or SPRT-LH/CGr (▪; F) or CCR5 (□). The BRET signals are plotted over the relative level of expression of the unlabeled receptor determined by FACS. The results are expressed as mean±s.e.m. and the graphs show one representative experiment from a total of at least two separate experiments carried out with sextuplets samples. Download figure Download PowerPoint The ectodomain of TSHr is dispensable for dimerization. To test whether dimerization of the TSHr required interactions involving the ectodomain (ECD), we performed BRET titration curves with a construct devoid of N-terminal hormone-binding domain. This serpentine-alone construct (named 7TM) (Vlaeminck-Guillem et al, 2002) yielded also a saturation curve (BRETmax: 0.047±0.006; BRET50: 0.002±0.001) (Figure 1C) that was specific, since it was competed for by increasing amounts of the unlabeled tagged holo-TSHr (SPRT-TSHr) (Vlaeminck-Guillem et al, 2002), but not by CCR5 (Figure 1D). Dimerization of 7TM constructs with holo-TSHr shows characteristics intermediate between those of the two kinds of homodimers (7TM-RLuc and TSHr-EYFP (BRETmax 0.064±0.007) and TSHr-RLuc and 7TM-EYFP (BRETmax 0.061±0.014); Supplementary Figure S1). Dimerization in other members of the GpHr family. Homodimerization of the LH/CGr was demonstrated by saturable BRET between RLuc and EYFP constructs (BRETmax: 0.058±0.006; BRET50: 0.006±0.002) (Figure 1E). Competition experiments with unlabeled SPRT-LH/CGr or CCR5 demonstrated the specificity of the interaction (Figure 1F). The heterodimerization between the TSHr and the LH/CGr was also demonstrated (Supplementary Figure S1). Considering the high sequence identity within the serpentine portions of these paralogous molecules (more than 70%), this agrees with our conclusion that dimerization involves primarily interactions between the transmembrane segments of the receptors (see above). Unfortunately, no BRET study could be performed with FSHr, probably because of the extremely low level of expression of FSHr fusion constructs (not shown). HTRF: TSHr dimerizes at the plasma membrane. In an effort to explore specifically the status of TSHr inserted in the plasma membrane, we relied on HTRF assays (Maurel et al, 2004). Iri-Sab1 and Iri-Sab2 (Costagliola et al, 2004) were selected as the two monoclonal antibodies displaying the highest level of energy transfer (ΔF) (Supplementary Figure S2A). In order to differentiate between intra- and intermolecular FRET, we relied on the observation that TSHr from the rat is not recognized by Iri-Sab1, whereas a human TSHr construct with substitutions in the ECD (T48; Figure 2B, inset; Smits et al, 2003) does not contain the epitope of Iri-Sab2 (Costagliola et al, 2004). The rat TSHr and the T48 cDNA constructs were transfected in HEK 293T cells, alone or in combination, and HTRF was monitored. A FRET signal was only observed in cells cotransfected with both cDNAs (Figure 2A) directly related to the level of expression of the individual constructs (Figure 2B). As an independent confirmation of dimerization, a single monoclonal (Iri-Sab2) was labeled with cryptate or XL665, and FRET was assayed in HEK 293T cells transfected with increasing amounts of WT TSHr (Figure 2C). A robust signal was obtained, the specificity of which is demonstrated by competition with the unlabeled mAb (Figure 2D). Together, the HTRF results confirm BRET data and demonstrate unambiguously that (at least some) TSHr are present at the plasma membrane as dimers. Figure 2.HTRF confirms BRET results: TSHr homodimerizes also in the plasma membrane. (A) HTRF between two different antibodies each recognizing only one of the two cotransfected receptors. HEK 293T cells are transfected with rat TSHr or TSHr T48 (TSHr harboring three point mutations in the ECD), or cotransfected with the two constructs (see cartoon in B, inset). Labeled mAbs Iri-Sab2-cryptate (donor, which recognizes only rat TSHr) and Iri-Sab1-XL (acceptor, recognizing only T48) are incubated with the cells. The FRET signals are plotted as ΔF value±s.e.m. (see Materials and methods) or (B) over the independent FACS level for each receptor when coexpressed. (C) WT TSHr HTRF using the same mAb as donor and acceptor. HEK 293T cells expressing increasing amounts of TSHr are incubated with mAbs Iri-Sab2-cryptate (donor) and Iri-Sab2-XL (acceptor). The histogram represents ΔF±s.e.m. (D) Competition with unlabeled mAbs. HEK 293T cells expressing WT hTSHr are incubated with mAbs Iri-Sab2-cryptate and Iri-Sab2-XL and increasing amounts of the nonlabeled Iri-Sab2 (▪) or 3G4 (□) (irrelevant mAb). The results are expressed as ΔF±s.e.m. plotted over the concentration of unlabeled mAb. All graphs are representative from at least two independent experiments performed in triplicate samples. Download figure Download PowerPoint Functional evidence for dimerization of TSHr Recovery of functional receptors in cells cotransfected with loss-of-function mutants showing no residual activity, when expressed alone, is a definite proof that dimers form in living cells. We wanted to assess this question with the TSHr by relying on two kinds of mutants. The first, 'KONAT', binds TSH with normal affinity but is totally unable to signal (Figure 3A). It combines mutation of residue N7.49 into alanine (Govaerts et al, 2001) and a substitution of three residues in the second intracellular loop of the TSHr (F525AM to S525PF) (Kosugi et al, 1994). The second is made of two different types of constructs, both unable to bind TSH, but able to couple to Gs and to signal efficiently. One is the 7TM construct (see above); the other is an LH/CGr–TSHr chimera (LT) responding only to hCG. When the individual mutants were expressed alone (Figure 3B) and challenged with increasing concentrations of recombinant hTSH, no response was observed (Figure 3A). But when the binding-deficient mutants (7TM or LT) were coexpressed with KONAT (Figure 3B), a clear response to the hormone was observed, although with a higher EC50 compared to the WT receptor (for KONAT and 7TM: 3.62±1.47; for KONAT and LT: 5.43±2.45; 0.24±0.11 for WT TSHr, in mIU/ml). These data confirm the ability of the TSHr to dimerize and demonstrate that the phenomenon has functional relevance. Figure 3.Functional complementation of cotransfected mutant TSHr. (A) Two deficient TSHr mutants are able to recover the response to the hormone. TSHr constructs deficient for hormone binding (7TM or LT), or unable to signal through Gs (KONAT), were engineered. HEK 293T cells were transfected with the different TSHr constructs individually ((*) pSVL; (▵) LT; (□) 7TM; (x) KONAT) or in combination ((▴) KONAT+LT; (▪) KONAT+7TM). After stimulation with increasing concentrations of rhTSH, intracellular cAMP values are determined. Results are expressed as fold stimulation (stimulated over basal activity); in the same experiments, the fold stimulation for the WT TSHr is 40 times the basal cAMP value (not shown). Each curve is representative of three independent experiments performed in duplicate samples. (B) Level of expression of the individual partners of dimers. FACS analysis was performed as described in Materials and methods with three different mAbs for all the coexpression combinations: HEK 293T cells were transfected with the different TSHr constructs individually (pSVL; LT; 7TM; KONAT) or in combination (KONAT+LT; KONAT+7TM). BA8 recognizes the WT TSHr and the KONAT, 16B5 recognizes the LT chimera and anti-RT recognizes the 7TM in each of the cotransfections. Download figure Download PowerPoint Dimerization and allosterism Although they demonstrate the possibility for the protomers to signal 'in trans' within a dimer, the complementation experiments do not allow to discriminate formally between a model in which each protomer would, under normal circumstances (i.e. in a homodimer of WT subunits), bind and signal on its own, and another in which the dimer would behave as a single signaling unit. We decided to approach this question by performing radioligand-binding assays. We expressed the WT TSHr, alone or together with a chimeric receptor made of the ECD of the LH/CGr and the seven transmembrane domain of the TSHr. This chimera (LT; Figure 3) is unable to bind bTSH but binds hCG with nominal affinity (not shown). Using [125I]bTSH as tracer, we performed heterologous competition experiments with hCG as competitor. When the TSHr is expressed alone, hCG is a poor competitor of TSH binding (Figure 4A). However, when it is coexpressed with the LT construct, we detect a clear and important leftward shift in the ability of hCG to compete for binding, which could represent [125I]bTSH bound to heterodimers (Figure 4A). Indeed, the TSH tracer, which can bind only to the WT TSHr in the dimer, is displaced by hCG. concentrations that allow hCG to bind only to LT within the dimer. These observations are formally compatible with a situation where each dimer would contain a single binding pocket contributed by portions of the ECDs of each protomer (one orthosteric site per dimer), or with a model of two binding sites per dimer linked by a strong negative cooperativity (allosteric model). Figure 4.Binding assays demonstrate GpHr dimerization and allosteric modulation. (A) Heterologous binding competition in heterodimers: COS-7 cells expressing WT TSHr or chimeric LT receptors separately (▪) or together (□) are incubated with [125I]TSH and increasing amounts of hCG. Nonspecific binding (NSB) determined with mock-transfected cells was subtracted. No binding was detected in cells transfected with LT alone. The results are expressed as percentage of B0±s.e.m. and the curve is representative of four separate experiments performed in duplicate samples. (B, D) [125I]TSH binding desorption experiments. CHO cells stably expressing the WT TSHr (B) or porcine thyroid membranes (D) are incubated with [125I]TSH. After 2 h at room temperature, binding buffer alone (□) or buffer with 67 nM of bTSH (▪) or 200 nM of various mAbs (• and x) was added. Incubation was stopped by removing medium at various times after reagent addition (see Materials and methods for details). The data represent the ratio between the bound cpm at different desorption time (Bt) and the total bound cpm at time 0 (B0) (logarithmic scale) plotted over the desorption time in minutes; Bt/B0±s.e.m. The curves were fitted using nonlinear regression equations with Prism v4.0. Each curve is representative of 2–4 independent experiments performed in duplicate samples. (C) Desorption is linked to binding competition and not to activation. The same desorption experiments were carried out in CHO cells stably expressing TSHr with seven different purified mAbs exactly as explained for panels B and C. (E) Concentration–desorption curve for bTSH: determination of the affinity of the allosteric site. CHO cells expressing TSHr were incubated with [125I]TSH. After 2 h at room temperature, the desorption experiment was performed with increasing concentrations of ultrapurified bTSH that were added and incubated for 60 min. The curve is representative of three independent experiments performed in duplicate samples. (F) TSHr saturation curve. CHO cells expressing TSHr were incubated with increasing concentrations of [125I]TSH. Nonspecific binding was determined with mock-transfected CHO cells and was subtracted from the total binding. The results are expressed as specifically bound TSH in nM±s.e.m. plotted over the determined free radioligand, and fitted with Prism v4.0 using a nonlinear regression model, leaving the program to select between one or two binding sites, and affinity constants were calculated. The curve represents one experiment out of four separate experiments performed in duplicate samples. Scatchard plot is shown in the inset for the same experiment. (G) [125I]FSH binding desorption in FT. [125I]FSH was desorbed with buffer alone (□) or with 330 nM of FSH (▪) in cells expressing FT as explained. Results are expressed as for previous panels and they represent independent experiments performed twice with duplicate samples. (H) [125I]hLH binding desorption in LH/CGr. [125I]hLH was desorbed with buffer alone (x) or with 163 nM of hCG (□) or 32 nM hLH (▪) in cells expressing LH/CGr as explained. Results are expressed as for previous panels and they represent independent experiments performed twice with duplicate samples. Download figure Download PowerPoint To discriminate between these two models, we performed desorption experiments (Christopoulos et al, 1997). Whereas dilution with buffer alone resulted in negligible desorption of [125I]bTSH over a 180 min period, desorption was observed in the presence of excess ligand (t1/2: 46.28±2.66 min) (Figure 4B). This demonstrates cooperativity between two symmetrical binding sites. This effect is independent of the level of receptor expression (Supplementary Figure S2C). Similar experiments were performed with a panel of mAbs, as desorbing agents, directed against TSHr and displaying a variety of epitopes and functional characteristics. The ability to displace the tracer correlated with that of competing directly with TSH binding (1H7, Iri-Sab2 and 23.1), and not with agonistic activity (Iri-Sab1 and Iri-Sab2) (Costagliola et al, 2004) (Figure 4B and C). In agreement with the idea that the desorption is not related to the activity, when the desorption was performed with an inactive mutant receptor (KONAT; see Figure 3A), we detected a significant desorption (Supplementary Figure S2F). Interestingly, when one of the mAbs was used as tracer (1H7) instead of TSH, it was desorbed by itself, by Iri-Sab2 and by TSH (Supplementary Figure S2D). The same type of experiments, performed with membranes from porcine thyroid glands, demonstrated that negative cooperativity is a characteristic of native TSHr, at their normal site and level of expression (Figure 4D). The detected lower efficient desorption is probably due to differences in the interspecies binding properties (Supplementary Figure S2G). When similar experiments were performed in intact CHO cells expressing the LH/CGr, or on membranes, we observed a desorption for radioiodinated hLH (Figure 4H). However, we did not detect any desorption of [125I]hCG in the presence of excess unlabeled hCG (Supplementary Figure S3D). This fits with the results obtained in direct competition of [125I]hCG binding involving cells coexpressing the LT and TSHr and illustrates the parallelism between both types of experiments. Indeed, when [125I]hCG was used as the tracer and bTSH as competitor, no change in displacement ability was observed between single-transfection (LT alone) and double-transfection experiments (LT and TSHr) (not shown). This asymmetrical behavior of the dimer TSHr–LT toward the two hormones remains to be clarified. Interestingly, whereas hCG was unable to desorb [125I]hCG from LH/CGr homodimers, it was effective in desorption of radiolabeled TSH from cells coexpressing WT TSHr and the LT chimera (Supplementary Figure S2B). To determine the affinity of the allosteric site of the TSHr for TSH, we performed desorption experiments, for a fixed time (60 min, close to the determined half-life), with increasing concentrations of TSH. This yielded an affinity of 6.48±2.50 nM (Figure 4E). We performed saturation experiments with CHO cells stably expressing the TSHr (Figure 4F). Fitting the resulting curve according to a nonlinear regression model yielded two binding sites with the following dissociation constants: Kd1, 0.169±0.087; Kd2, 20.020±4.369 nM (Figure 4F). Even though the exact Kd computed value for the low-affinity binding site lies outside the concentration range explored experimentally,
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