An Inactivating Mutation within the First Extracellular Loop of the Thyrotropin Receptor Impedes Normal Posttranslational Maturation of the Extracellular Domain

Abstract

The TSH receptor (TSHR), a member of the large family of G protein-coupled receptors, controls both function and growth of thyroid cells; hence, mutations of this receptor result in thyroid dysfunction. Here, we took advantage of the description of a new inactivating TSHR mutation (Q489H) in two brothers with hypothyroidism, to precise maturation, intracellular trafficking, exporting pathways, and activation mechanisms of this receptor. Functional characterization of the Q489H-TSHR in transiently transfected HEK293 cells showed cell surface expression, normal TSH binding affinity, and its inability to generate intracellular cAMP in response to TSH stimulation. Western blot analysis of the whole membrane proteins or proteins expressed at the cell surface showed that Q489H-TSHR expressed in HEK293 transfected cells are restricted to mannose-rich uncleaved receptor. Analysis of the export pathway toward cell surface indicated that both Q489H and wild-type receptors followed the same intracellular route to cell surface throughout endoplasmic reticulum and Golgi apparatus. This study shows that Q489H substitution impedes complete glycosylation of TSHR extracellular domain within the Golgi apparatus and that Q489H-TSHR expressed at the cell surface is unable to undergo intramolecular cleavage as well as to switch toward an active conformation under TSH stimulation. Altogether, our results show that 1) Q489H substitution within the first extracellular loop induces a misfolding of TSHR, blocking it into an inactive conformation and impeding complete glycosylation and intramolecular cleavage, and 2) a misfolded G protein-coupled receptor can bypass endoplasmic reticulum or Golgi apparatus quality control and reach the cell surface as an immature receptor.

Current loss-of-function mutations in the thyrotropin receptor gene: when to investigate, clinical effects, and treatment

“…To date, more than 60 mutations in the TSHR gene have been described in association with different degrees of TSH resistance (Table 2, continued 1, continued 2,) (4,5,6,7,8,9,10,11,12,13,14,15,16,17,18, 19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36, 37,38,39,40,41,42). The mutations are all point mutations, such as base substitutions and small deletions/insertions that cause missense (the most frequent), nonsense and frame shift mutations.…”

High Frequency of D727E Polymorphisms in Exon 10 of the TSHR Gene in Brazilian Patients with Congenital Hypothyroidism

“…Exons 1 through 9, and part of exon 10 codify the extracellular domain of the receptor, while the transmembrane and intracellular domains are codified by Exon 10. Thyroid metabolism is stimulated by TSHR through protein G 8,9 .…”

Molecular description of non-autoimmune hyperthyroidism at a neonate caused by a new thyrotropin receptor germline mutation

“…These activating mutations are indicators that the extracellular loops acting as important determinants for receptor activation. In support to this assumption also few naturally occurring inactivating mutations were published previously: ECL1 - W488R [27], Q489H [28]; ECL3 - L653V [29]. …”