Functional characterization of five constitutively activating thyrotrophin receptor mutations

Area of Science:

• Endocrinology
• Molecular Biology
• Cell Signaling

Background:

• Gain of function mutations in the thyrotrophin receptor (TSHR) alter receptor function, impacting cAMP and inositol phosphate (IP) accumulation, cell surface expression, and TSH affinity.
• Understanding these mutations is crucial for elucidating TSHR signaling pathways and associated thyroid disorders.

Purpose of the Study:

• To compare the functional characteristics of five constitutively activating TSHR mutations, including four point mutations and one deletion mutant.
• To investigate the effects of these mutations on cAMP and IP accumulation, cell surface expression, and TSHR regulation.

Main Methods:

• Transiently expressed wild-type TSHR and mutant TSHRs (S505N, L629F, I630L, V656F, and deletion 613-621) in COS-7 cells.
• Assessed basal and stimulated cAMP and IP accumulation, cell surface expression (FACS, ELISA), and TSHR downregulation post-transfection.

Main Results:

• Despite varying specific constitutive activity and cell surface expression levels, basal cAMP accumulation was uniform across mutants.
• Point mutations did not constitutively activate the phospholipase C cascade, whereas the deletion mutant (613-621) exhibited constitutive IP pathway activity.
• All mutants showed TSH-stimulated receptor downregulation, independent of the phospholipase C pathway.

Conclusions:

• Uniform basal cAMP levels suggest regulation within the COS-7 overexpression system, likely due to receptor downregulation.
• The TSHR deletion mutant (613-621) displayed constitutive activity in both Galphas and Galphaq/11 pathways.
• The contrasting responses to TSH for cAMP and IP accumulation in the deletion mutant support the model of distinct TSHR active conformations.