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Role of complex asparagine-linked oligosaccharides in the expression of a functional thyrotropin receptor.

ABSTRACT: To evaluate the functional role of complex asparagine-linked oligosaccharides of the human thyrotropin receptor (TSHR), a Chinese hamster ovary cell line (JP09) and a K562 cell line (K562-TSHR) expressing this receptor were treated with deoxymannojirimycin (dMM), a mannosidase I inhibitor. dMM blocks the formation of complex-type structures and leads to the formation of high-mannose-type structures. Treatment of cells with dMM led to a decrease in the number of thyrotropin (TSH)-binding sites at the cell surface. Detection of the TSHR at the cell surface using a monoclonal antibody directed against the A subunit showed that this decrease was not due to a decrease in the number of TSHRs expressed at the cell surface. However the recognition of TSHR by a monoclonal antibody directed against the C peptide was greatly decreased. On immunoblotting, after deglycosylation using peptide N-glycanase F, the A subunit was visualized as a doublet (36 and 41 kDa). In control cells the species of higher molecular mass was more abundant whereas after dMM treatment the species of lower molecular mass became more abundant. This difference in molecular mass between the two peptides is compatible with the removal of the C peptide. In conclusion, the results show that inhibition of complex-type structure formation leads to (i) an incapacity for TSHR to bind TSH, without affecting its intracellular transport and (ii) an increase of TSHR susceptibility to proteases that remove the C peptide. We then hypothesized that removal of the C peptide could contribute to the formation of a non-functional TSHR.

SUBMITTER: Siffroi-Fernandez S

PROVIDER: S-EPMC1221660 | biostudies-other | 2001 Mar

REPOSITORIES: biostudies-other

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Project description:The thyroid-stimulating hormone receptor (TSHR) is a member of the glycoprotein hormone receptors, a sub-group of class A G-protein-coupled receptors (GPCRs). TSHR and its endogenous ligand thyrotropin (TSH) are of essential importance for growth and function of the thyroid gland and proper function of the TSH/TSHR system is pivotal for production and release of thyroid hormones. This receptor is also important with respect to pathophysiology, such as autoimmune (including ophthalmopathy) or non-autoimmune thyroid dysfunctions and cancer development. Pharmacological interventions directly targeting the TSHR should provide benefits to disease treatment compared to currently available therapies of dysfunctions associated with the TSHR or the thyroid gland. Upon TSHR activation, the molecular events conveying conformational changes from the extra- to the intracellular side of the cell across the membrane comprise reception, conversion, and amplification of the signal. These steps are highly dependent on structural features of this receptor and its intermolecular interaction partners, e.g., TSH, antibodies, small molecules, G-proteins, or arrestin. For better understanding of signal transduction, pathogenic mechanisms such as autoantibody action and mutational modifications or for developing new pharmacological strategies, it is essential to combine available structural data with functional information to generate homology models of the entire receptor. Although so far these insights are fragmental, in the past few decades essential contributions have been made to investigate in-depth the involved determinants, such as by structure determination via X-ray crystallography. This review summarizes available knowledge (as of December 2016) concerning the TSHR protein structure, associated functional aspects, and based on these insights we suggest several receptor complex models. Moreover, distinct TSHR properties will be highlighted in comparison to other class A GPCRs to understand the molecular activation mechanisms of this receptor comprehensively. Finally, limitations of current knowledge and lack of information are discussed highlighting the need for intensified efforts toward TSHR structure elucidation.

Project description:Evidence from clinical and experimental data suggests that thyrotropin receptor (TSHR) signaling is involved in energy expenditure through its impact on white adipose tissue (WAT) and brown adipose tissue (BAT). TSHR expression increases during mesenchymal stem cell (MSC) differentiation into fat. We hypothesize that TSHR activation [TSHR*, elevated thyroid-stimulating hormone, thyroid-stimulating antibodies (TSAB), or activating mutation] influences MSC differentiation, which contributes to body composition changes seen in hypothyroidism or Graves' disease (GD). The role of TSHR activation on adipogenesis was first investigated using ex vivo samples. Neck fat (all euthyroid at surgery) was obtained from GD (n = 11, TSAB positive), toxic multinodular goiter (TMNG, TSAB negative) (n = 6), and control patients with benign euthyroid disease (n = 11, TSAB negative). The effect of TSHR activation was then analyzed using human primary abdominal subcutaneous preadipocytes (n = 16). Cells were cultured in complete medium (CM) or adipogenic medium [ADM, containing thiazolidinedione (TZD), PPARγ agonist, which is able to induce BAT formation] with or without TSHR activation (gain-of-function mutant) for 3 weeks. Adipogenesis was evaluated using oil red O (ORO), counting adipogenic foci, qPCR measurement of terminal differentiation marker (LPL). BAT [PGC-1α, uncoupling protein 1 (UCP1), and ZIC1], pre-BAT (PRDM16), BRITE- (CITED1), or WAT (LEPTIN) markers were analyzed by semiquantitative PCR or qPCR. In ex vivo analysis, there were no differences in the expression of UCP1, PGC-1α, and ZIC1. BRITE marker CITED1 levels were highest in GD followed by TMNG and control (p for trend = 0.009). This was associated with higher WAT marker LEPTIN level in GD than the other two groups (p < 0.001). In primary cell culture, TSHR activation substantially enhanced adipogenesis with 1.4 ± 0.07 (ORO), 8.6 ± 1.8 (foci), and 5.5 ± 1.6 (LPL) fold increases compared with controls. Surprisingly, TSHR activation in CM also significantly increased pre-BAT marker PRDM16; furthermore, TZD-ADM induced adipogenesis showed substantially increased BAT markers, PGC-1α and UCP1. Our study revealed that TSHR activation plays an important role in the adipogenesis process and BRITE/pre-BAT formation, which leads to WAT or BAT phenotype. It may contribute to weight loss as heat during hyperthyroidism and later transforms into WAT posttreatment of GD when patients gain excess weight.

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Role of complex asparagine-linked oligosaccharides in the expression of a functional thyrotropin receptor.

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