Production of bioactive amino‐terminal domain of the thyrotropin receptor via insertion in the plasma membrane by a glycosylphosphatidylinositol anchor

Costagliola, Sabine; Khoo, D. H. C.; Vassart, Gilbert

doi:10.1016/s0014-5793(98)01177-6

Cited by 74 publications

(65 citation statements)

References 47 publications

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“…The reduction in cell surface expression could be due to an abnormal folding of the mutant receptors preventing the correct membrane targeting. The lower expression levels given at the analysis with the monoclonal antibody against a conformational epitope, such as BA8 (18), may also be due to the altered immunoreactivity of the mutant receptor. The presence of the V597F mutant on the plasmatic membrane is con®rmed by the conserved response to TSH stimulation.…”

Section: Discussionmentioning

confidence: 99%

“…For permeabilization, cells were incubated with PBS-paraformaldehyde 2% in ice for 10 min, washed once with PBS-BSA 0.1% and treated with 2% saponine at room temperature for 30 min. Permeabilized and non-permeabilized cells were labeled with 3G4 monoclonal antibody recognizing a linear epitope, and with BA8 antibody recognizing a conformational epitope, both within the ligand-binding domain of TSHR (18).…”

Section: Flow Cytometrymentioning

confidence: 99%

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A novel germline mutation in the TSH receptor gene causes non-autoimmune autosomal dominant hyperthyroidism

Alberti

Proverbio

Costagliola

et al. 2001

European Journal of Endocrinology

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Objective: Clinical and genetic investigations were undertaken in a case of familial hyperthyroidism, with onset of thyrotoxic symptoms varying between childhood/adolescence. Methods: Automatic sequence analysis was carried out of the TSH receptor (TSHR) gene. Functional studies were undertaken of mutant TSHR in transient expression experiments in COS-7 cells including the evaluation of cAMP accumulation and of protein expression by¯ow cytometry, as well as the calculation of speci®c constitutive activity (SCA). Results: In four affected cases, the age of onset of thyrotoxic manifestations of non-autoimmune origin varied between 5 and 18 years. The disease transmission was typically autosomal dominant. TSHR gene sequence revealed the presence of a germline heterozygous substitution at codon 597 leading to the novel mutation V597F. This residue is located in the 5th transmembrane domain of the receptor protein in a critical region for membrane targeting and signal transduction. Functional studies of the V597F mutant indicate an 11-fold increase in SCA, associated with a reduction in receptor protein expression on the cytoplasmic membrane. Conclusions: Description was made of a family with non-autoimmune autosomal dominant hyperthyroidism carrying a novel mutation of TSHR leading to the increment in speci®c constitutive activity. Factors that may in¯uence the clinical expression of TSHR germline mutations are discussed.

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Section: Discussionmentioning

confidence: 99%

Section: Flow Cytometrymentioning

confidence: 99%

A novel germline mutation in the TSH receptor gene causes non-autoimmune autosomal dominant hyperthyroidism

Alberti

Proverbio

Costagliola

et al. 2001

European Journal of Endocrinology

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“…Expression of WT, chimeric and mutant receptors (RT or not) was quantified by FACS with the mAb BA8 (anti-TSHr), 16B5 (anti-LH/CGr), 5B2 (anti-FSHr) or the mAb OR2-15A-6 (anti-RT) exactly as explained previously 58 .…”

Section: Methodsmentioning

confidence: 99%

Evidence for activity-regulated hormone-binding cooperativity across glycoprotein hormone receptor homomers

et al. 2012

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Glycoprotein hormone receptors show strong negative cooperativity. As a consequence, at physiological hormone concentrations, a single agonist binds to a receptor dimer. Here we present evidence that constitutively active receptors lose cooperative allosteric regulation in direct relation with their basal activity. The most constitutive mutants lost nearly all cooperativity and showed an increase of initial tracer binding, reflecting the ability of each protomer to bind with equal affinity. Allosteric interaction between the protomers takes place at the transmembrane domain. The allosteric message resulting from hormone binding to the ectodomain of one protomer travels 'downward' to its transmembrane domain, before affecting the transmembrane domain of the other protomer. This results in transmission of an 'upward' message lowering the binding affinity of the ectodomain of the second protomer. our results demonstrate a direct relation between the conformational changes associated with activation of the transmembrane domain and the allosteric behaviour of glycoprotein hormone receptors dimers.

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“…The isolated TSHR ectodomain tethered to the cell surface by a GPI anchor (TSHR ECD-GPI) contains the entire TSHbinding site and is reported to bind TSH with an affinity comparable with the TSH holoreceptor (15,19). For this reason, …”

Section: Dissociation Of Prebound Tsh From the Isolated Tshr Ectodomamentioning

confidence: 99%

Evidence That the Thyroid-stimulating Hormone (TSH) Receptor Transmembrane Domain Influences Kinetics of TSH Binding to the Receptor Ectodomain

Chen

McLachlan

Rapoport

2011

Journal of Biological Chemistry

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Thyroid-stimulating hormone (TSH)-induced reduction in ligand binding affinity (negative cooperativity) requires TSH receptor (TSHR) homodimerization, the latter involving primarily the transmembrane domain (TMD) but with the extracellular domain (ECD) also contributing to this association. To test the role of the TMD in negative cooperativity, we studied the TSHR ECD tethered to the cell surface by a glycosylphosphatidylinositol (GPI) anchor that multimerizes despite the absence of the TMD. Using the infinite ligand dilution approach, we confirmed that TSH increased the rate of dissociation (k off ) of prebound 125 I-TSH from CHO cells expressing the TSH holoreceptor. Such negative cooperativity did not occur with TSHR ECD-GPI-expressing cells. However, even in the absence of added TSH, 125 I-TSH dissociated much more rapidly from the TSHR ECD-GPI than from the TSH holoreceptor. This phenomenon, suggesting a lower TSH affinity for the former, was surprising because both the TSHR ECD and TSH holoreceptor contain the entire TSH-binding site, and the TSH binding affinities for both receptor forms should, theoretically, be identical. In ligand competition studies, we observed that the TSH binding affinity for the TSHR ECD-GPI was significantly lower than that for the TSH holoreceptor. Further evidence for a difference in ligand binding kinetics for the TSH holoreceptor and TSHR ECD-GPI was obtained upon comparison of the TSH K d values for these two receptor forms at 4°C versus room temperature. Our data provide the first evidence that the wild-type TSHR TMD influences ligand binding affinity for the ECD, possibly by altering the conformation of the closely associated hinge region that contributes to the TSH-binding site.The term "negative cooperativity" was coined in 1973 by de Meyts et al.(1) to interpret curvilinear Scatchard plots (2) observed with insulin and other receptors, i.e. occupancy of a receptor by a ligand at one site leads to an allosteric effect at another binding site, causing a reduction in binding affinity. Early studies prior to the molecular cloning of the thyroidstimulating hormone (TSH) 2 receptor (TSHR) cDNA suggested that ligand-induced negative cooperativity did not occur with this receptor (3, 4). Nearly 2 decades later, it was observed that expressing increasing numbers of recombinant TSHR on the surface of transfected cells was associated with a reduction in TSH binding affinity (5). This phenomenon suggested that enhanced interactions at higher receptor densities led to a ligand-independent form of negative cooperativity. In recent years, TSHRs were found to form homodimers or multimers on the cell surface (6, 7), like other members of the glycoprotein hormone receptor family (reviewed in Refs. 8 and 9). Using chimeric receptor molecules combined with the classical infinite ligand dilution approach of de Meyts et al., Urizar et al. (7) provided convincing evidence for ligand-induced negative cooperativity in which TSH binding to one protomer increased the rate of dissociation (k off )...

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Production of bioactive amino‐terminal domain of the thyrotropin receptor via insertion in the plasma membrane by a glycosylphosphatidylinositol anchor

Cited by 74 publications

References 47 publications

A novel germline mutation in the TSH receptor gene causes non-autoimmune autosomal dominant hyperthyroidism

A novel germline mutation in the TSH receptor gene causes non-autoimmune autosomal dominant hyperthyroidism

Evidence for activity-regulated hormone-binding cooperativity across glycoprotein hormone receptor homomers

Evidence That the Thyroid-stimulating Hormone (TSH) Receptor Transmembrane Domain Influences Kinetics of TSH Binding to the Receptor Ectodomain

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