In vitro pharmacological profile of the TSH receptor-stimulating autoantibodies, M22 and K1-18.

Activation of thyroid-stimulating hormone receptor (TSHR) by autoantibodies is the primary cause of the autoimmune diseases Graves' hyperthyroidism and Graves' ophthalmopathy. Thus, recent pharmaceutical research has focused on blocking pathogenic TSHR activation. While this has potential to offer first-in-class disease-modifying medications to treat Graves' disease, the success of targeting drugs requires understanding of how TSHR-stimulating autoantibodies activate the receptor and mediate signalling. This study comprehensively characterized in vitro pharmacological profiles of two human TSHR-stimulating autoantibodies, M22 and K1-18, relative to bovine TSH (bTSH) in a HEK293A cell background expressing recombinant human TSHR. M22 and K1-18 exhibited subtle, G protein-specific, differences in activating Gα proteins, with both antibodies generally less potent and/or efficacious than bTSH. While both antibodies were equivalent in activation of most G proteins, K1-18 was less potent than M22 in activation of Gαs-short and Gαz. Interestingly, bTSH, M22 and K1-18 more potently activated Gα15 compared to other Gα proteins. In addition, M22 and K1-18 activated Gαs-dependent cAMP production and Gαq-coupled signalling (IP1 accumulation and iCa2+ mobilisation), recruited β-arrestin 1 and 2, and stimulated receptor internalization to early endosomes. K1-18 was less potent than bTSH in all these assays, while M22 was less potent than bTSH in iCa2+ mobilisation and β-arrestin recruitment. While M22 and K1-18 largely mirror the pharmacology of bTSH, quantification of revealed relative to bTSH they exhibit bias towards Gα12 activation, and away from β-arrestin 2 recruitment and iCa2+ mobilization relative to Gs and cAMP. Thus, these findings offer insight into the action of TSHR-stimulating antibodies.