Context: Inactivating variants of thyrotropin (thyroid-stimulating hormone; TSH) receptor (TSHR) cause congenital hypothyroidism. More than 60 such variants have been reported so far, most of which were located in the extracellular or transmembrane domain. Objective: We report the identification and characterization of a frameshift TSHR variant in the intracytoplasmic C-tail region. Methods: Sequencing of TSHR was performed in a patient with congenital hypothyroidism. The functionality of the identified variants was assessed by expressing TSHR in HEK293 cells and measuring TSH-dependent activation of the cAMP-response element-luciferase reporter. A series of systematic mutagenesis experiments were performed to characterize the frameshifted amino acid sequence. Results: The proband was heterozygous for a known TSHR variant (p.Arg519His) and a novel frameshift TSHR variant (p.Val711Phefs∗18), which removed 54 C-terminal residues and added a 17-amino acid frameshifted sequence. The loss of function of Val711Phefs∗18-TSHR was confirmed in vitro, but the function of Val711∗-TSHR was found to be normal. Western blotting showed the low protein expression of Val711Phefs∗18-TSHR. Fusion of the frameshift sequence to green fluorescent protein or luciferase induced inactivation of them, indicating that the sequence acted as a degron. A systematic mutagenesis study revealed that the density of hydrophobic residues in the frameshift sequence determined the stability. Eight additional frameshift TSHR variants that covered all possible shifted frames in C-tail were created, and another frameshift variant (Thr748Profs∗27) with similar effect was found. Conclusions: We characterized a naturally occurring frameshift TSHR variant located in C-tail, and provided a unique evidence that hydrophobicity in the C-terminal region of the receptor affects protein stability.
ASJC Scopus subject areas
- Endocrinology, Diabetes and Metabolism
- Clinical Biochemistry
- Biochemistry, medical