Inactivation of a Frameshift TSH Receptor Variant Val711Phefs*18 is Due to Acquisition of a Hydrophobic Degron

J Clin Endocrinol Metab. 2021 Jan 1;106(1):e265-e272. doi: 10.1210/clinem/dgaa772.

Abstract

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.

Keywords: TSH receptor; congenital hypothyroidism; protein stability; variant.

Publication types

  • Case Reports
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Substitution
  • Arginine / genetics
  • Child, Preschool
  • Congenital Hypothyroidism / diagnosis
  • Congenital Hypothyroidism / genetics*
  • Congenital Hypothyroidism / metabolism
  • DNA Mutational Analysis
  • Frameshift Mutation / genetics
  • HEK293 Cells
  • Histidine / genetics
  • Humans
  • Hydrophobic and Hydrophilic Interactions
  • Male
  • Parents
  • Pedigree
  • Phenylalanine / genetics
  • Protein Domains / genetics
  • Protein Stability
  • Proteolysis
  • Receptors, Thyrotropin / chemistry
  • Receptors, Thyrotropin / genetics*
  • Receptors, Thyrotropin / metabolism*
  • Valine / genetics

Substances

  • Receptors, Thyrotropin
  • Phenylalanine
  • Histidine
  • Arginine
  • Valine