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 Gαs-cAMP/protein kinase A/ERK and Gαq-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 Gαs and Gαq effectors. Importantly, some TSHR-blocking and TSHR-neutral antibodies were also able to generate signals, influencing primarily the Gαq effectors and induced cell proliferation. Most strikingly, antibodies that used the Gαq 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. Antibodies to the TSH receptor produce unique signaling imprints which differ from its ligand, indicating that these antibodies have variable effects on thyroid cell biology.