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

Chen, Chun-Rong; McLachlan, Sandra M.; Rapoport, Basil

doi:10.1210/en.2008-0207

Cited by 22 publications

(19 citation statements)

References 37 publications

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“…19,20 The results showed that TSH-stimulated production of cAMP in L-02 cells and human primary hepatocytes cultured in the presence of CS-17 was significantly lower than that in cells cultured without CS-17 (P < 0.001) (Fig. 3A, upper).…”

Section: Resultsmentioning

confidence: 96%

A Novel Role for Thyroid-Stimulating Hormone: Up-Regulation of Hepatic 3-Hydroxy-3-Methyl-Glutaryl-Coenzyme A Reductase Expression Through the Cyclic Adenosine Monophosphate/Protein Kinase A/Cyclic Adenosine Monophosphate–Responsive Element Binding Protein Pathway

et al. 2010

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Elevated thyroid-stimulating hormone (TSH) and hypercholesterolemia commonly coexist, as typically seen in hypothyroidism, but there is no known mechanism directly linking the two. Here, we demonstrated that in liver cells, TSH promoted the expression of 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMGCR), a rate-limiting enzyme in cholesterol synthesis, by acting on the TSH receptor in hepatocyte membranes and stimulating the cyclic adenosine monophosphate / protein kinase A / cyclic adenosine monophosphate-responsive element binding protein (cAMP/PKA/CREB) signaling system. In thyroidectomized rats, the production of endogenous thyroid hormone was eliminated and endogenous TSH was suppressed through pituitary suppression with constant administration of exogenous thyroid hormone, and hepatic HMGCR expression was increased by administration of exogenous TSH. These results suggested that TSH could up-regulate hepatic HMGCR expression, which indicated a potential mechanism for hypercholesterolemia involving direct action of TSH on the liver. (HEPATOLOGY 2010;52:1401-1409 H ypothyroidism is well known to be associated with elevated serum TC, which can result in hypercholesterolemia. 1,2 The underlying mechanism is widely thought to be TH deficiency.However, elevation of serum TC has also been observed in patients with subclinical hypothyroidism (SCH), in which TSH is elevated but TH stays within its normal range. 1,3,4 Thus, the development of

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Section: Resultsmentioning

confidence: 96%

A Novel Role for Thyroid-Stimulating Hormone: Up-Regulation of Hepatic 3-Hydroxy-3-Methyl-Glutaryl-Coenzyme A Reductase Expression Through the Cyclic Adenosine Monophosphate/Protein Kinase A/Cyclic Adenosine Monophosphate–Responsive Element Binding Protein Pathway

et al. 2010

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“…As detected by ELISA, diethylaminoethyl (DEAE) chromatography accomplished sufficient separation for the HR from the bulk of medium proteins (primarily albumin) to permit subsequent anti-5His affinity purification followed by Superdex-75 gel filtration. However, because of total overlap between the TSHR ECD and medium proteins, we turned to affinity chromatography with CS-17, a murine mAb primarily to the LRD (30,31), linked to CNBrSepharose 4B (Sigma-Aldrich).…”

Section: Protein Purificationmentioning

confidence: 99%

“…Anion exchange chromatography at various pH levels failed to provide an initial, relatively selective capture of the ECD, with complete overlap in elution of the ECD as detected by ELISA and the bulk of serum proteins in the culture medium (data not shown). Finally, we generated an affinity column with TSHR mAb CS-17 whose epitope is primarily to the LRD (31). Fortunately, as determined by PAGE, this mAb captured the TSHR ECD from conditioned culture medium in a single step to more than 90% purity with a yield of 0.1-0.3 mg/L in different preparations, typically approximately 0.2 mg/L ( Figure 5).…”

Section: Purification Of the Tshr Ecd And Hrsmentioning

confidence: 99%

Deleting the Redundant TSH Receptor C-Peptide Region Permits Generation of the Conformationally Intact Extracellular Domain by Insect Cells

et al. 2015

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The TSH receptor (TSHR) extracellular domain (ECD) comprises a N-terminal leucine-rich repeat domain and an hinge region (HR), the latter contributing to ligand binding and critical for receptor activation. The crystal structure of the leucine-rich repeat domain component has been solved, but previous attempts to generate conformationally intact complete ECD or the isolated HR component for structural analysis have failed. The TSHR HR contains a C-peptide segment that is removed during spontaneous TSHR intramolecular cleavage into disulfide linked A- and B-subunits. We hypothesized that deletion of the redundant C-peptide would overcome the obstacle to generating conformationally intact TSHR ECD protein. Indeed, lacking the C-peptide region, the TSHR ECD (termed ECD-D1) and the isolated HR (termed HR-D1) were secreted into medium of insect cells infected with baculoviruses coding for these modified proteins. The identities of TSHR ECD-D1 and HR-D1 were confirmed by ELISA and immunoblotting using TSHR-specific monoclonal antibodies. The TSHR-ECD-D1 in conditioned medium was folded correctly, as demonstrated by its ability to inhibit radiolabeled TSH binding to the TSH holoreceptor. The TSHR ECD-D1 purification was accomplished in a single step using a TSHR monoclonal antibody affinity column, whereas the HR-D1 required a multistep protocol with a low yield. In conclusion, we report a novel approach to generate the TSHR ECD, as well as the isolated HR in insect cells, the former in sufficient amounts for structural studies. However, such studies will require previous complexing of the ECD with a ligand such as TSH or a thyroid-stimulating antibody.

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“…Confirmation using a second control mAb that deletion of TSHR N-terminal disulfide-linked loop 1 reduces the number of cell surface receptors detected by M22. The experiments were identical to those described in Figure 2 using CHO cells stably expressing TSHR-ECD-GPI and D22-30 TSHR-ECD-GPI except for the use of control murine mAb CS-17 (A) whose epitope lies within the junctional region between the TSHR LRD and hinge regions, far downstream of the N-terminal cysteine cluster (24,25). (B) Flow cytometry with M22 using aliquots in the same experiment of cells used for CS-17.…”

Section: Functional Studies With the Tsh Holoreceptormentioning

confidence: 99%

Insight into Thyroid-Stimulating Autoantibody Interaction with the Thyrotropin Receptor N-Terminus Based on Mutagenesis and Re-Evaluation of Ambiguity in This Region of the Receptor Crystal Structure

Hamidi

Chen

McLachlan

et al. 2011

Thyroid

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Background: Thyroid-stimulating autoantibodies (TSAb) bind to the thyrotropin receptor (TSHR) extracellular domain, or ectodomain (ECD), comprising a leucine-rich repeat domain (LRD) linked by a hinge region to the transmembrane domain (TMD). The LRD (residues 22-260; signal peptide 1-21) contains two disulfide-bonded loops at its N-terminus. In the crystal structure of the isolated LRD complexed with human TSAb monoclonal antibody (mAb) M22, N-terminal disulfide loop 1 (residues 22-30) could not be determined because of crystal disorder. Nevertheless, present crystal structure data are interpreted to exclude a role for the LRD N-terminal disulfide loops in the TSAb epitope(s), contradicting prior functional evidence of a role for these loops in TSAb function. Materials and Methods: To re-examine this issue we studied two cell types expressing the TSHR with the extreme Nterminal loop 1 (residues 22-30) deleted: the TSHR ECD lacking the TMD and tethered to the plasma membrane by a glycosyl-phosphatidylinositol (GPI) anchor, and the TSH holoreceptor containing the TMD. Because TSAb including M22 ''see'' the holoreceptor poorly relative to the TSHR ECD-GPI, we used the latter to examine the effect of deleting residues 22-30 on M22 binding by flow cytometry and the holoreceptor to test the effect of this deletion on the functional response to M22. Results: Deletion of TSHR N-terminal loop 1 (residues 22-30) reduced the number of TSHR-ECD-GPI recognized by M22 relative to two TSHR mAb with epitopes far downstream of the LRD N-terminal loops. Relative to control mAb 2C11, M22 recognized only 60.4% of cell surface receptors (p ¼ 0.02). In contrast to M22 binding to TSHR-ECD-GPI, in functional studies with the TSH holoreceptor, M22 stimulation of cAMP generation was unaltered by the loop 1 deletion. Conclusions: Our data support the concept that TSAb interact with the cysteine-rich N-terminus of the TSHR. Comparison of crystal structures of the same TSHR LRD in complex with TSAb M22 or blocking antibody K1-70 helps reconcile contradictory viewpoints. A difference between M22 interaction with the identical TSHR N-terminus expressed on the TSHR-ECD-GPI and holoreceptor suggests that crystallization of the TSHR LRD-M22 complex may not provide a complete understanding of the functional TSAb epitope(s) in Graves' disease.

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

Cited by 22 publications

References 37 publications

A Novel Role for Thyroid-Stimulating Hormone: Up-Regulation of Hepatic 3-Hydroxy-3-Methyl-Glutaryl-Coenzyme A Reductase Expression Through the Cyclic Adenosine Monophosphate/Protein Kinase A/Cyclic Adenosine Monophosphate–Responsive Element Binding Protein Pathway

A Novel Role for Thyroid-Stimulating Hormone: Up-Regulation of Hepatic 3-Hydroxy-3-Methyl-Glutaryl-Coenzyme A Reductase Expression Through the Cyclic Adenosine Monophosphate/Protein Kinase A/Cyclic Adenosine Monophosphate–Responsive Element Binding Protein Pathway

Deleting the Redundant TSH Receptor C-Peptide Region Permits Generation of the Conformationally Intact Extracellular Domain by Insect Cells

Insight into Thyroid-Stimulating Autoantibody Interaction with the Thyrotropin Receptor N-Terminus Based on Mutagenesis and Re-Evaluation of Ambiguity in This Region of the Receptor Crystal Structure

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