Related Experiment Videos
Functional characterization of five constitutively activating thyrotrophin receptor mutations
1Medizinische Klinik und Poliklinik III, Universität Leipzig, Leipzig, Germany.
Objective:
Gain of function mutations of the thyrotrophin receptor (TSHR) affect several functional characteristics, such as cAMP and inositol phosphate (IP) accumulation, cell surface expression and TSH affinity. In this study we compared five constitutively activating TSHR mutations, four receptors with a point mutation (S505N, L629F, I630L, V656F) and a nine amino acid (aa) deletion mutant (aa positions 613-621) for these functional parameters in parallel transfection experiments.
Methods:
The wild-type TSHR (wt) and TSHRs containing the mutations S505N, L629F, I630L, V656F and the deletion 613-621 (all cloned in the expression vector pSVL) were transiently expressed in COS-7 cells in parallel experiments. Forty-eight hours after transfection the basal and stimulated cAMP and inositol phosphate accumulation as well as the cell surface expression (by FACS and ELISA), KD-values and TSHR down regulation by different stimuli were determined.
Results:
In contrast to the very different values for specific constitutive activity (sca) (ranging from 7.5 to 100.3-fold wt) and very different levels of receptor cell surface expression (11-94% wt level) the basal cAMP accumulation determined in transfected COS-7 cells was surprisingly uniform (6.5-8.0 over wt basal). None of the point mutated receptors constitutively activates the phospholipase C cascade. In contrast the deletion 613-621 mutant showed constitutive activity for the IP pathway with a twofold increase in basal IP accumulation compared to the wild type TSHR. All investigated TSHR-mutants showed a TSH-stimulated receptor down-regulation, which seems to be independent of the phospholipase C pathway.
Conclusions:
The uniform basal cAMP values in spite of the large variation in specific constitutive activity values suggest that the COS-7 cell overexpression system used for the in vitro characterization is partly regulated. This regulation is most likely due to receptor down regulation. The TSHR deletion mutant (613-621) showed a constitutive activity for both the Galphas and the Galphaq/11 pathways. The TSH-mediated IP-stimulation by this mutant contrasts with its unresponsiveness to TSH for cAMP accumulation and therefore supports the model of different active conformations of the TSHR.
Insights
Gain of function mutations in the thyrotrophin receptor (TSHR) were studied. A deletion mutant showed constitutive activity for both cAMP and inositol phosphate pathways, suggesting different TSHR active conformations.
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.