Characterization of Thyrotropin Receptor Antibody-Induced Signaling Cascades

Abstract

The TSH receptor (TSHR) is constitutively active and is further enhanced by TSH ligand binding or by stimulating TSHR antibodies (TSHR-Abs) as seen in Graves' disease. TSH is known to activate the thyroid epithelial cell via both Galphas-cAMP/protein kinase A/ERK and Galphaq-Akt/protein kinase C coupled signaling networks. The recent development of monoclonal antibodies to the TSHR has enabled us to investigate the hypothesis that different TSHR-Abs may have unique signaling imprints that differ from TSH ligand itself. We have, therefore, performed sequential studies, using rat thyrocytes (FRTL-5, passages 5-20) as targets, to examine the signaling pathways activated by a series of monoclonal TSHR-Abs in comparison with TSH itself. Activation of key signaling molecules was estimated by specific immunoblots and/or enzyme immunoassays. Continuing constitutive TSHR activity in thyroid cells, deprived of TSH and serum for 48 h, was demonstrated by pathway-specific chemical inhibition. Under our experimental conditions, TSH ligand and TSHR-stimulating antibodies activated both Galphas and Galphaq effectors. Importantly, some TSHR-blocking and TSHR-neutral antibodies were also able to generate signals, influencing primarily the Galphaq effectors and induced cell proliferation. Most strikingly, antibodies that used the Galphaq cascades used c-Raf-ERK-p90RSK as a unique signaling cascade not activated by TSH. Our study demonstrated that individual TSHR-Abs had unique molecular signatures which resulted in sequential preferences. Because downstream thyroid cell signaling by the TSHR is both ligand dependent and independent, this may explain why TSHR-Abs are able to have variable influences on thyroid cell biology.

Accuracy of receptor-based methods for detection of thyrotropin-receptor autoantibodies: a new automated third-generation immunoassay shows higher analytical and clinical sensitivity for the differential diagnosis of hyperthyroidism

“…This finding is probably because of the presence of TSH blocking or TSH-binding neutral antibodies that inhibit the binding of M22 mAb [38].…”

Thyroid Cancer in Patients with Hyperthyroidism

“…Moreover, TSAbs might play a role in stimulating thyroid cancer growth, invasiveness [ 9 , 60 ] and angiogenesis by upregulating vascular endothelial growth factor, placenta growth factor, and their receptors. TSAbs use the same signaling pathways that are used by TSH for cell activation and growth [ 61 ] . Taking into consideration that chronic TSH stimulation aff ects the prognosis of thyroid cancer it could be postulated that the TSH mimicking eff ect of TSAbs could explain the increased aggressiveness of thyroid cancer in Graves' patients.…”

TSH receptor antibody titers measured with a third-generation assay did not reflect the activity of Graves’ ophthalmopathy in untreated Japanese Graves’ disease patients

“…TBII should include various kind of TRAb, because TBII was measured by the inhibition activity of TSH binding to TSH receptor. In contrast, the third-generation TRAb assay uses monoclonal antibody, and has high affinity for TSH receptor, and stimulates not only cAMP production but also Akt and PKC pathways [15]. Higher levels of expression of TSH receptor have been observed in orbital tissue from GO as compared to controls and gross correlation between the presence of TRAb and severity of GO [11].…”