Demonstration of reduced in vivo surface expression of activating mutant thyrotrophin receptors in thyroid sections

Abstract

Objective: Thyroid function and growth are controlled by TSH. Hyperthyroidism can be due to Graves' Disease (GD), in which thyroid-stimulating antibodies mimic TSH, or gain-of-function mutations in the TSH receptor (TSHR). These activating mutations have poor surface expression when assessed in non-thyroidal cells in vitro but nothing is known of their in vivo behaviour. Several TSHR antibodies have been produced but none has been applied to thyroid paraffin sections. This study aimed to develop a technique suitable for use on paraffin sections and apply it to investigate TSHR expression in thyroids harbouring activating TSHR germline mutations compared with normal and GD thyroids. Design and methods: Immunocytochemistry coupled with antigen retrieval, using a spectrum of antibodies to the TSHR, was applied to paraffin sections of GD thyroid tissue. Subsequently, TSHR immunoreactivity was examined in three normal thyroids, three patients with GD and three patients with familial hyperthyroidism, due to different gain-of-function TSHR germline mutations, using the optimised protocol. Results: Two antibodies, A10 and T3-495, to the extracellular domain (ECD) and membrane spanning region (MSR) of the TSHR respectively, produced specific basolateral staining of thyroid follicular cells. In normal and GD thyroids, basolateral staining with T3-495 was generally more intense than with A10, suggesting a possible surfeit of MSR over ECD. Graves' Disease thyroids have more abundant TSHR than normal glands. In contrast, thyroids harbouring gain-of-function mutations have the lowest expression in vivo, mirroring in vitro findings. Conclusions: The development of an immunocytochemical method applicable to paraffin sections has demonstrated that different molecular mechanisms causing hyperthyroidism result in the lowest (mutation) and highest (autoimmunity) levels of receptor at the thyrocyte surface.

Adipose thyrotrophin receptor expression is elevated in Graves' and thyroid eye diseases ex vivo and indicates adipogenesis in progress in vivo

“…Furthermore, the results we obtained from preadipocytes in culture showing increased TSHR transcripts (the current study) and TSHR immunoreactivity (Crisp et al 2000) imply that transcript also correlates with protein in cultured primary cells. The higher levels of TSHR transcript in GD thyroid tissue compared with MNG mirrors our previous demonstration of receptor protein in these glands using ICC (Sequeira et al 2002).…”

Biological Activity of Activating Thyroid-Stimulating Hormone Receptor Mutants Depends on the Cellular Context

“…cAMP and inositol phosphate production, for the mutants in comparison with the WT TSHR (3,4,6,11). This system, however, cannot be relied upon to generate data representative of effects in normal human thyroid cells because 1) it involves gross artefactual overexpression of the receptors, whereas expression of native TSHR in thyroid follicular cells, and particularly mutant forms, is remarkably low (12,13); 2) signaling in COS-7 cells may not accurately reflect the situation in thyroid follicular cells due to the specificity of cell signaling (14); and 3) investigation of direct biological effects on growth, differentiation, and function is not possible in a cell system, in which gene expression is only transient.…”

A Novel Thyrotropin Receptor Germline Mutation (Asp617Tyr) Causing Hereditary Hyperthyroidism

“…Comparison of the specific constitutive activity index (basal cAMP/receptor number) showed that the activities of Asp619Gly and Ala623Val TSHRs in the third intracellular loop were approximately one sixth of those of Thr632Ile and Cys672Tyr TSHRs in TM6 and TM7, respectively [35]. The constitutively activating TSHR mutations generally exhibit reduced surface expression as compared with wt TSHR, because of interference with correct folding and trafficking of the receptor to the plasma membrane [36]. Because the expression levels and basal cAMP levels of Asp619Gly TSHR are almost equivalent between stably transfected CHO cells and transiently transfected COS-7 cells [30], it seems that Asp617Tyr TSHR is a relatively weak stimulator of cAMP production.…”