The Thyrotropin (TSH)-Releasing Hormone Receptor: Interaction with TSH and Autoantibodies*

Rapoport, Basil; Chazenbalk, Gregorio D.; Jaume, Juan Carlos; McLachlan, Sandra M.

doi:10.1210/edrv.19.6.0352

Cited by 239 publications

(153 citation statements)

References 331 publications

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“…4 in Ref. 1). Proof of at least one disulfide bond involving the four N-terminal cysteines was obtained with mouse mAb 3BD10, which has a conformational epitope encompassing residues 25-51 in the TSHR N terminus (10).…”

Section: Discussionmentioning

confidence: 99%

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A Full Biological Response to Autoantibodies in Graves' Disease Requires a Disulfide-bonded Loop in the Thyrotropin Receptor N Terminus Homologous to a Laminin Epidermal Growth Factor-like Domain

Chen

Tanaka

Chazenbalk

et al. 2001

Journal of Biological Chemistry

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We observed amino acid homology between the cysteine-rich N terminus of the thyrotropin receptor (TSHR) ectodomain and epidermal growth factor-like repeats in the laminin ␥1 chain. Thyroid-stimulating autoantibodies (TSAb), the cause of Graves' disease, interact with this region of the TSHR in a manner critically dependent on antigen conformation. We studied the role of the cluster of four cysteine (Cys) residues in this region of the TSHR on the functional response to TSAb in Graves' patients' sera. As a benchmark we also studied TSH binding and action. comprises, in part, the highly conformational epitope for TSAb at the critical N terminus of the TSHR. Amino acid homology, as well as cysteine pairing similar to the laminin ␥1 chain epidermal growth factor-like repeat 11, suggests conformational similarity between the two molecules and raises the possibility of molecular mimicry in the pathogenesis of Graves' disease. The thyrotropin receptor (TSHR)1 is the target of the immune system in Graves' disease, and autoantibodies directed against this antigen are the direct cause of clinical hyperthyroidism (reviewed in Ref. 1). Understanding the molecular interaction between TSHR autoantibodies and the TSHR is, therefore, important for advances in the diagnosis and therapy of Graves' disease, one of the most common organ-specific autoimmune diseases affecting humans. Studies of TSHR autoantibody-autoantigen interaction have been difficult because of the very low serum concentrations of the autoantibodies (2, 3) and because of the highly conformational nature of their epitopes. Although the binding sites for TSHR autoantibodies and TSH involve discontinuous segments throughout the TSHR ectodomain (4, 5), the N-terminal region of the ectodomain appears to be particularly important for the functional activity of TSHR autoantibodies (5-9).Comparison of the primary amino acid sequences of the large ectodomains of the glycoprotein hormone receptors reveals a moderately conserved, central leucine-rich repeat region flanked by segments of much lower homology. The poorly conserved TSHR N-terminal region extends from amino acid residues 22 to 56 (the signal peptide being residues 1-21). This short (35-residue) region contains four of the eleven Cys residues (Cys 24 , Cys 29 , Cys 31 , and Cys 41 ) in the large (397-residue) TSHR ectodomain, supporting the importance of secondary and tertiary structure in TSHR recognition by autoantibodies. Another indicator of the importance of conformation in the TSHR N terminus region is that TSHR autoantibodies and a mouse monoclonal antibody to this segment (3BD10; epitope within amino acid residues 25 to 51) interact reciprocally and exclusively with two different folded forms of the native molecule present in the same TSHR preparation (10).A number of lines of evidence suggest that the four N-terminal cysteines form disulfide bonds with one another rather than with other Cys residues in the TSHR. First, from the current three-dimensional model of the TSHR leucine-rich repeat area (11) b...

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Section: Discussionmentioning

confidence: 99%

“…1). Understanding the molecular interaction between TSHR autoantibodies and the TSHR is, therefore, important for advances in the diagnosis and therapy of Graves' disease, one of the most common organ-specific autoimmune diseases affecting humans.…”

Section: The Thyrotropin Receptor (Tshr)mentioning

confidence: 99%

A Full Biological Response to Autoantibodies in Graves' Disease Requires a Disulfide-bonded Loop in the Thyrotropin Receptor N Terminus Homologous to a Laminin Epidermal Growth Factor-like Domain

Chen

Tanaka

Chazenbalk

et al. 2001

Journal of Biological Chemistry

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“…1). Remarkably, functional autoantibodies do not arise to the other, closely related members of the glycoprotein hormone receptor family.…”

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confidence: 99%

Subunit Structure of Thyrotropin Receptors Expressed on the Cell Surface

Tanaka

Chazenbalk

McLachlan

et al. 1999

Journal of Biological Chemistry

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We studied cell surface thyrotropin receptor (TSHR) by biotinylating proteins on the surface of metabolically labeled, intact cells. In addition to TSHR cleaved into A and B subunits, mature single-chain receptors with complex carbohydrate were also present on the cell surface. A low A/B subunit ratio indicated partial shedding of extracellular A subunits from transmembrane B subunits. TSHR cleavage at upstream site 1 (within amino acid residues 305-316) would generate a B subunit of 51-52 kDa. However, only smaller B subunits (40 -46 kDa) were detected, corresponding to N termini from residues ϳ370 (site 2) extending downstream to the region of B subunit insertion into the plasma membrane. The intervening C peptide region between sites 1 and 2 could not be purified from TSHR epitope-tagged (c-myc) within this region. However, the small proportion of B subunits recovered with a c-myc antibody were larger (45-52 kDa) than the majority of B subunits recovered with a C-terminal antibody. In conclusion, our study provides the first characterization of cell surface TSHR including their A and B subunits. Single-chain, mature TSHR do exist on the cell surface. The C peptide lost during intramolecular cleavage disintegrates rapidly following cleavage at upstream site 1 of the single-chain TSHR into A and B subunits. N-terminal disintegration of the B subunit pauses at site 2, but then progresses downstream to the vicinity of the plasma membrane, revealing a novel mechanism for A subunit shedding.Graves' disease, one of the most common autoimmune diseases affecting humans, is caused by autoantibodies that activate the thyrotropin (TSH) 1 receptor (TSHR) (reviewed in Ref. 1). Remarkably, functional autoantibodies do not arise to the other, closely related members of the glycoprotein hormone receptor family. A potential reason for this difference is the unique subunit structure of the TSH receptor. Thus, unlike the other glycoprotein hormone receptors, a variable proportion of TSHR on the cell surface cleaves into an extracellular A subunit and a largely transmembrane B subunit that remain linked by disulfide bonds (2-4). This cleavage introduces the potential for antigenic stimulation of the immune system, for two reasons. First, TSHR cleavage into A and B subunits results in the deletion of an intervening "C peptide" region (5, 6). Extracellular release of this polypeptide, either intact or in fragments, could initiate or propagate an immune response to the TSHR. Second, there is the propensity for the A subunit itself to be shed, at least in vitro (7,8). Determination of the molecular basis for TSH receptor cleavage and shedding is, therefore, of potential clinical importance in understanding the pathogenesis of Graves' disease.Many uncertainties remain regarding the process of TSHR cleavage and shedding, including the enzyme(s) involved (6, 7, 9) and the properties of the shed A subunits (7,8). The location and number of cleavage sites has also been controversial. Initial evidence for a cleavage site closely upstream o...

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“…It is established that the C-b2 and 3 potentially interact via disulfide bonds (21)(22)(23) and that mutations disrupting the disulfide bridge lead to constitutive activation (24,25). In a previous study we identified new residues within C-b3 that are also involved in receptor stabilization and intramolecular signal transduction based on a suggested homologous molecular model for the C-b2/C-b3 unit (26).…”

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Significance of Ectodomain Cysteine Boxes 2 and 3 for the Activation Mechanism of the Thyroid-stimulating Hormone Receptor

et al. 2006

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Recently, we identified constitutively activating mutations at positions Asp-403, Glu-404, and Asn-406 in the third extracellular cysteine box (C-b3) of the thyroid-stimulating hormone receptor. We hypothesized that this region could act as a molecular interface between the extracellular and serpentine domain. In this study we present a model for properties of potential interaction partners for this region. Moreover, we show that Pro-400 and Pro-407 adjacent to this epitope are also important for stabilizing the partially active, basal conformation of the wild-type (WT) thyroid-stimulating hormone receptor. Furthermore, the mutation K291A in the second extracellular cysteine box (C-b2) was identified as a new constitutively activating mutation that releases the basal conformation of the WT receptor like the known tryptic cleavage in its close vicinity. Taken together, we provide an activation scenario at the C-b2/C-b3 unit. Three anchor fragments (anchors I-III) most likely constrain the basal conformation. The three anchor fragments are tightly packed. A disulfide bridge holds the C-b2/C-b3 portions in close positions. Independent of the type of conformational interference such as side chain modifications, tryptic cleavage, or hormone stimulation that act on the constrained C-b2/C-b3 WT conformation, it will always release one of the anchor fragments. Subsequently, this results in a conformational displacement of the C-b2/C-b3 portions relative to each other, inducing receptor activation.The human anterior pituitary releases the structurally related glycoprotein hormones (GPHs), 3 thyroid-stimulating hormone (TSH), follicle-stimulating hormone, luteinizing hormone, and chorionic gonadotropin. These large hormones (ϳ30 kDa) interact with the N-terminal extracellular domain (ECD) of their specific receptors (1, 2), TSHR, follicle-stimulating hormone receptor, and luteinizing hormone/choriogonadotropin receptor, which are members of the seven-transmembrane-spanning receptor family (3-7).The large N-terminal extracellular domain is a common structural characteristic of all GPH receptors. The N-terminal ECD of the TSHR (Met-1-Asp-410) can be subdivided into (all numbers are hTSHR specific): (i) the extreme N-terminal tail (including the signal peptide), (ii) the cysteine box 1 (C-b1; Cys-24-Cys-41), (iii) the 11 leucine-rich repeats (LRR; Pro-37-Tyr-279) and (iv) the hinge region (Pro-280-Asp-410), which can be subdivided further into (v) the central cysteine box 2 (C-b2; Pro-280-Cys-301), (vi) the cysteine box 2/3 linker region (C-bl; Asn-302-Ile-389), and (vii) the cysteine box 3 (C-b3; Cys-390-Asp-410) located close to transmembrane helix 1.In addition to the activation mediated by TSH, the receptor can be activated by constitutively activating mutants (CAMs) (8, 9), by mutants leading to promiscuous hormone interactions (10), tryptic action (11), and deletions in the ECD and serpentine domain (12)(13)(14). Several pathogenic gain of function mutants in the ECD of the TSHR characterized by increased constitutive ...

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The Thyrotropin (TSH)-Releasing Hormone Receptor: Interaction with TSH and Autoantibodies*

Cited by 239 publications

References 331 publications

A Full Biological Response to Autoantibodies in Graves' Disease Requires a Disulfide-bonded Loop in the Thyrotropin Receptor N Terminus Homologous to a Laminin Epidermal Growth Factor-like Domain

A Full Biological Response to Autoantibodies in Graves' Disease Requires a Disulfide-bonded Loop in the Thyrotropin Receptor N Terminus Homologous to a Laminin Epidermal Growth Factor-like Domain

Subunit Structure of Thyrotropin Receptors Expressed on the Cell Surface

Significance of Ectodomain Cysteine Boxes 2 and 3 for the Activation Mechanism of the Thyroid-stimulating Hormone Receptor

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