Functional characterization of five constitutively activating thyrotrophinreceptor mutations

OBJECTIVE Gain of function mutations of the thyrotrophin receptor (TSHR) af fect several functional characteristics, such as cAMP and inositol phosphat e (IP) accumulation, cell surface expression and TSH affinity. In this stud y 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, L 629F, I630L, V656F and the deletion 613-621 (all cloned in the expression v ector pSVL) were transiently expressed in COS-7 cells in parallel experimen ts. 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), K-D-values and TSHR down regulation by different stimuli w ere 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 level s of receptor cell surface expression (11-94% wt level) the basal cAMP accu mulation determined in transfected COS-7 cells was surprisingly uniform (6. 5-8.0 over wt basal). None of the point mutated receptors constitutively ac tivates the phospholipase C cascade. In contrast the deletion 613-621 mutan t showed constitutive activity for the IP pathway with a twofold increase i n basal IP accumulation compared to the wild type TSHR. All investigated TS HR-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 i n specific constitutive activity values suggest that the COS-7 cell overexp ression system used for the in vitro characterization is partly regulated. This regulation is most likely due to receptor down regulation. The TSHR de letion mutant (613-621) showed a constitutive activity for both the G(alpha s) and the G(alpha q/11) pathways. The TSH-mediated IP-stimulation by this mutant contrasts with its unresponsiveness to TSH for cAMP accumulation an d therefore supports the model of different active conformations of the TSH R.