Functional characterization of two different NAH-associated activating TSHR mutations

Megha Sunny; Blythe Wright; Nadia Schoenmakers; Erik Schoenmakers; Caroline Gorvin; Carla Moran; Robbie Kernan; Colm Costigan; Umaima Aboushofa; Vikashini Ravikumar

doi:10.1530/endoabs.117.OC4.1

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Endocrine Abstracts (2026) 117 OC4.1 | DOI: 10.1530/endoabs.117.OC4.1

SFEBES2026 Oral Communications Thyroid (6 abstracts)

Functional characterization of two different NAH-associated activating TSHR mutations

Megha Sunny ¹ , Blythe Wright ¹ , Nadia Schoenmakers ¹ , Erik Schoenmakers ¹ , Caroline Gorvin ² , Carla Moran ³ , Robbie Kernan ⁴ , Colm Costigan ⁵ , Umaima Aboushofa ⁶ & Vikashini Ravikumar ⁷

Author affiliations

¹Department of Metabolism and Systems Science, College of Medicine and Health, University of Birmingham Institute of Biomedical Research, University of Birmingham, Birmingham, United Kingdom; ²Department of Metabolism and Systems Science, College of Medicine and Health, University of Birmingham, Birmingham, United Kingdom; ³Dept of Endocrinology, Beacon Hospital, Dublin, St Vincent’s University Hospital, Dublin, UCD School of Medicine, Dublin, Ireland; ⁴, Nuala Murphy Department of Paediatric Endocrinology, CHI Temple St, Dublin, Ireland; ⁵Department of Paediatric Endocrinology, Children’s Health Ireland at Crumlin, Dublin, Ireland; ⁶Department of Paediatrics, Northern Lincolnshire & Goole NHS Foundation Trust, United Kingdom; ⁷Medical Research Council Metabolic Diseases Unit, Institute of Metabolic Science-Metabolic Research Laboratories, University of Cambridge, Cambridge, United Kingdom

Activating germline mutations of the thyroid-stimulating hormone receptor (TSHR) are rare, and result in congenital nonautoimmune hyperthyroidism (NAH). Here we report the first functional characterization studies of two NAH-associated TSHR mutations. Firstly, a novel, maternally inherited mutation (p.Leu512Val) identified in a clinically-euthyroid female with TRAb negative thyrotoxicosis and a strong family history of NAH. Secondly, a previously reported TSHR mutation (p. Thr490Arg), in a male, with similar biochemistry and a maternal history of NAH. Transfection studies were performed in heterologous cells to compare expression and function of wild-type (WT) and mutant TSHR’s. Western blotting, immunostaining and flow cytometric analysis demonstrated comparable total cellular and surface expression of WT and mutant receptors. TSHR is a G-protein coupled receptor activating both the Gs/adenylyl cyclase pathway (cAMP, predominant effect), as well as the Gq-phospholipase C pathway (Ca and PKC responsive). Compared with wild-type TSHR, two different cAMP responsive reporter assays, cAMP-RE-luc (indirect) and the Glosensor assay (direct), demonstrated elevated constitutive and TSH-stimulated Gs-coupled signalling for both mutant receptors. In contrast, a Nuclear Factor of Activated T-cells-RE reporter assay (Gq-pathway) did not show a difference. The effects of the TSHR mutations were analysed in silico using EM-structures (ref: https://doi.org/10.1038/s41586-022-05173-3 and https://doi.org/10.1038/s41586-022-05159-1). Substitution of L512, located in the middle of the transmembrane domain, with the smaller Valine is predicted to result in a more dense structure, resembling the active conformation. Replacement of Thr490, situated between the transmembrane and ligand binding domains, with the larger Arginine could, due to steric hindrance, alter the position of the ligand binding domain to a more upright position, similar to the ligand-bound active receptor. Collectively, our laboratory and in silico findings support increased constitutive Gs-signalling (cAMP), but not the Gq-signalling (Ca and PKC) pathway as a cause of NAH in individuals harbouring the T490R and L512V TSHR mutations.