Functional characterization of five constitutively activating thyrotrophin receptor mutations.
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.
Wonerow
P
P
Medizinische Klinik und Poliklinik III, Universität Leipzig, Leipzig, Germany.
Chey
S
S
Führer
D
D
Holzapfel
H P
HP
Paschke
R
R
D000818:Animals; D019556:COS Cells / Q000378:metabolism; D000242:Cyclic AMP / Q000096:biosynthesis; D015536:Down-Regulation; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D017353:Gene Deletion; D007295:Inositol Phosphates / Q000378:metabolism; D017354:Point Mutation; D011989:Receptors, Thyrotropin / Q000032:analysis / Q000235:genetics*; D013268:Stimulation, Chemical; D013972:Thyrotropin; D014162:Transfection / Q000379:methods*
D007295:Inositol Phosphates; D011989:Receptors, Thyrotropin; D013972:Thyrotropin; D000242:Cyclic AMP