A functional site on the human TSH receptor: a potential therapeutic target in Graves’ disease

Johnstone, Alan P.; Cridland, Jeremy C.; Costa, Clive Da; Nussey, Stephen; Shepherd, Phillip

doi:10.1046/j.1365-2265.2003.01864.x

Cited by 13 publications

(5 citation statements)

References 21 publications

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“…The epitopes for two mAb are far removed from the LRD mutations, recognizing C-terminal portions of the hinge region close to its insertion into the plasma membrane. Thus, the epitopes for mAb 4C1 and 2C11 include residues 381–384 and 355–358, respectively [21] . In general, as measured by the latter two mAb, the level of cell surface expression progressively diminished in proportion to the number of TSHR mutations ( Fig.…”

Section: Resultsmentioning

confidence: 99%

Novel Information on the Epitope of an Inverse Agonist Monoclonal Antibody Provides Insight into the Structure of the TSH Receptor

et al. 2012

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The TSH receptor (TSHR) comprises an extracellular leucine-rich domain (LRD) linked by a hinge region to the transmembrane domain (TMD). Insight into the orientation of these components to each other is required for understanding how ligands activate the receptor. We previously identified residue E251 at the LRD-hinge junction as contributing to coupling TSH binding with receptor activation. However, a single residue cannot stabilize the LRD-hinge unit. Therefore, based on the LRD crystal structure we selected for study four other potential LRD-hinge interface charged residues. Alanine substitutions of individual residues K244, E247, K250 and R255 (as well as previously known E251A) did not affect TSH binding or function. However, the cumulative mutation of these residues in varying permutations, primarily K250A and R255A when associated with E251A, partially uncoupled TSH binding and function. These data suggest that these three residues, spatially very close to each other at the LRD base, interact with the hinge region. Unexpectedly and most important, monoclonal antibody CS-17, a TSHR inverse agonist whose epitope straddles the LRD-hinge, was found to interact with residues K244 and E247 at the base of the convex LRD surface. These observations, together with the functional data, exclude residues K244 and E247 from the TSHR LRD-hinge interface. Further, for CS-17 accessibility to K244 and E247, the concave surface of the TSHR LRD must be tilted forwards towards the hinge region and plasma membrane. Overall, these data provide insight into the mechanism by which ligands either activate the TSHR or suppress its constitutive activity.

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Section: Resultsmentioning

confidence: 99%

Novel Information on the Epitope of an Inverse Agonist Monoclonal Antibody Provides Insight into the Structure of the TSH Receptor

et al. 2012

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“…Of the six individual mutations, only CS-17 binding to Y195H was clearly diminished relative to control mAb 4C1 whose epitope includes further downstream acid residues 381-384 (11). On examining combinations of mutations in various permutations (not all shown in Fig.…”

Section: Identification Of Amino Acid Residues In the Cs-17 Epitopementioning

confidence: 97%

Identification of Key Amino Acid Residues in a Thyrotropin Receptor Monoclonal Antibody Epitope Provides Insight into Its Inverse Agonist and Antagonist Properties

2008

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CS-17 is a murine monoclonal antibody to the human TSH receptor (TSHR) with both inverse agonist and antagonist properties. Thus, in the absence of ligand, CS-17 reduces constitutive TSHR cAMP generation and also competes for TSH binding to the receptor. The present data indicate that for both of these functions, the monovalent CS-17 Fab (50 kDa) behaves identically to the intact, divalent IgG molecule (150 kDa). The surprising observation that CS-17 competes for TSH binding to the human but not porcine TSHR enabled identification of a number of amino acids in its epitope. Replacement of only three human TSHR residues (Y195, Q235, and S243) with the homologous porcine TSHR residues totally abolishes CS-17 binding as detected by flow cytometry. TSH binding is unaffected. Of these residues, Y195 is most important, with Q235 and S243 contributing to CS-17 binding to a much lesser degree. The functional effects of CS-17 IgG and Fab on constitutive cAMP generation by porcinized human TSHR confirm the CS-17 binding data. The location of TSHR amino acid residues Y195, Q235, and S243 deduced from the crystal structure of the FSH receptor leucine-rich domain provides valuable insight into the CS-17 and TSH binding sites. Whereas hormone ligands bind primarily to the concave surface of the leucine-rich domains, a major portion of the CS-17 epitope lies on the opposite convex surface with a minor component in close proximity to known TSH binding residues.

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“…Each of these approaches has limitations. In the present study, we used the latter because of the lesser sensitivity of flow cytometry (at least in our hands), but primarily because some of our hinge region mutations influenced binding by the most effective and commonly used monoclonal antibodies such as 2C11 (38 Another methodological approach requiring comment is the use of multiple, mild Ala mutations vs. individual mutations either to Ala, which may have little effect, or to residues with greatly different properties that may alter protein folding with possible allosteric effects. Our present findings demonstrate the value of this approach.…”

Section: Tshr Combining the K3r1 Quadruple And The De392-5 Triple Mutmentioning

confidence: 99%

The Thyrotropin Receptor Hinge Region as a Surrogate Ligand: Identification of Loci Contributing to the Coupling of Thyrotropin Binding and Receptor Activation

et al. 2012

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The TSH receptor (TSHR) hinge region, the least well understood component, bridges the leucine-rich repeat and transmembrane domains. We report data on clusters of hinge charged residues the mutation of which to Ala is compatible with cell surface expression and normal, or near normal, TSH binding affinity yet with a relative reduction in receptor activation. Mutation to Ala of E409 at the junction with the transmembrane domain was the most potent in uncoupling TSH binding and signal transduction (~22-fold less sensitive than the wild-type TSHR) and was unique among the residues studied in reducing both the amplitude and the sensitivity of the ligand-induced signal. Unexpectedly, a dual E409A/D410A mutation partially corrected the major suppressive effect of TSHR-E409A. The combined Ala substitution of a cluster of positively charged hinge residues (K287, K290, K291, R293; termed "K3R1") synergistically reduced sensitivity to TSH stimulation approximately 21-fold without altering the TSH binding affinity. Simultaneous Ala substitutions of a cluster of acidic hinge residues D392, E394, and D395 (termed "DE392-5A") partially uncoupled TSH binding from signal transduction (4.4-fold reduction in sensitivity), less than for E409A and K3R1A. Remarkably, the combination of the K3R1A and DE392-5A mutations was not additive but ameliorated the major uncoupling effect of K3R1A. This lack of additivity suggests that these two clusters contribute to a common signaling pathway. In summary, we identify several TSHR hinge residues involved in signal transmission. Our data support the concept that the hinge regions of the TSHR (and other glycoprotein hormone receptors) act as surrogate ligands for receptor activation.

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A functional site on the human TSH receptor: a potential therapeutic target in Graves’ disease

Abstract: Residues 381-384 of the human TSH-receptor are important in the physiological and pathological stimulation of the thyroid. This opens the possibility of more specific therapy of some Graves' disease patients by agents directed against this epitope.

Cited by 13 publications

References 21 publications

Novel Information on the Epitope of an Inverse Agonist Monoclonal Antibody Provides Insight into the Structure of the TSH Receptor

Novel Information on the Epitope of an Inverse Agonist Monoclonal Antibody Provides Insight into the Structure of the TSH Receptor

Identification of Key Amino Acid Residues in a Thyrotropin Receptor Monoclonal Antibody Epitope Provides Insight into Its Inverse Agonist and Antagonist Properties

The Thyrotropin Receptor Hinge Region as a Surrogate Ligand: Identification of Loci Contributing to the Coupling of Thyrotropin Binding and Receptor Activation

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