Jacqueline Van Sande

SPECIFIC ACTIVATION OF THE THYROTROPIN RECEPTOR BY TRYPSIN

The identification of 16 different activating mutations in the TSH receptor, found in patients suffering from toxic autonomous adenomas or congenital hyperthyroidism, leads to the concept that this receptor is in a constrained conformation in its wild-type form. We used mild trypsin treatment of CHO-K1 cells or COS-7 cells, stably or transiently transfected with the human TSH receptor, respectively, and measured its consequences on the TSH receptor coupled cascades, i.e. cyclic AMP and inositol-phosphates accumulation. A 2-min, 0.01% trypsin treatment increased stably cyclic AMP but not inositol-phosphates formation. This was not observed after chymotrypsin, thrombin and endoproteinase glu C treatment. The TSH action on cyclic AMP was decreased by only 25%. The effect was also observed in cells expressing the dog TSH receptor. It was not observed in MSH receptor, LH receptor expressing or mock transfected cells (vector alone). It is therefore specific for the TSH receptor, for its action on the Gs/adenylate cyclase cascade, and for the proteolytic cleavage caused by trypsin. Using monoclonal (A. Johnstone and P. Shepherd, personal communication) and polyclonal antibodies directed against the extracellular domain of the TSH receptor, it was shown that treatment by trypsin removes or destroys a VFFEEQ epitope (residues 354-359) from the receptor. The effect mimics the action of TSH as it activates Gs alpha and enhances the action of forskolin. It is not reversible in 1 h. The results support the concept that activation of the receptor (by hormone, autoantibodies, mutations or mild proteolysis) might involve the relief of a built-in negative constrain. They suggest that the C-terminal portion of the large extracellular domain plays a role in the maintenance of this constrain.

Negative control of norepinephrine on the thyroid cyclic AMP system

Negative control on the thyroid cyclic AMP system has been studied. The increase of cyclic AMP levels induced by TSH in dog thyroid slices and its consequent secretion were inhibited by norepinephrine. This effect was different from the previously described activation of cyclic AMP disposal by acetylcholine: it was not calcium-dependent, was observed in the presence of isobutyl methylxanthine and was not inhibited by atropine. The inhibitory action of norepinephrine was abolished by phentolamine but not by L-propranolol. Clonidine and epinephrine also markedly inhibited the elevation of cyclic AMP levels, but phenylephrine did not. The inhibitory effect of norepinephrine and clonidine was abolished by yohimbine but not by prazosin. These results suggest that the effect of adrenergic agents on dog thyroid follicular cells is mediated by alpha 2-receptors. Similar results were obtained on dog thyroid adenylate cyclase activity: norepinephrine diminished the activation of adenylate cyclase induced by TSH, in a sodium-dependent process. This inhibition was abolished by phentolamine and yohimbine, but not by L-propranolol and and prazosin. This shows that the negative alpha 2-adrenergic effect bears directly on adenylate cyclase.

Thyroid Gene Expression in Familial Nonautoimmune Hyperthyroidism Shows Common Characteristics with Hyperfunctioning Autonomous Adenomas

CONTEXT Dominant activating mutations of the TSH receptor are the cause of familial nonautoimmune hyperthyroidism (FNAH) (inherited mutations affecting the whole gland since embryogenesis) and the majority of hyperfunctioning autonomous adenomas (AAs) (somatic mutations affecting only one cell later in the adulthood). OBJECTIVE The objective of the study was defining the functional and molecular phenotypes of FNAH and comparing them with the ones of AA. DESIGN Functional phenotypes were determined in vitro and molecular phenotypes by hybridization on microarray slides. PATIENTS Nine patients with FNAH were investigated, six for functional in vitro study of the tissue and five for gene expression. RESULTS Iodide metabolism, H(2)O(2), cAMP, and inositol phosphate generation in FNAH slices stimulated or not with TSH were normal. The mitogenic response of cultured FNAH thyrocytes to TSH was normal but more sensitive to the hormone. Gene expression profiles of FNAH and AAs showed that among 474 genes significantly regulated in FNAH, 93% were similarly regulated in AAs. Besides, 783 genes were regulated only in AAs. Bioinformatic analysis pointed out common down-regulations of genes involved in immune response, cell/cell and cell/matrix adhesions, and apoptosis. Pathways up-regulated only in AAs mainly involve diverse biosyntheses. These results are consonant with the larger growth of AAs than FNAH tissues. CONCLUSIONS Whether hereditary or somatic after birth, activating mutations of the TSH receptor have the same qualitative consequences on the thyroid cell phenotype, but somatic mutations in AAs have a much stronger effect than FNAH mutations. Both are variants of one disease: genetic hyperthyroidism.