Desensitization and internalization of constitutively activated TSH receptor mutants

By previous investigations we characterized the TSHR as a member of the class A of GPCRs, the same class as the β2-adrenergic receptor (β2AR). The β-arrestin dependent endocytosis of the β2-adrenergic receptor has been demonstrated by confocal fluorescence microscopy. Furthermore, it could be shown that a constitutively activated β2AR is also constitutively desensitized and down-regulated. To clarify whether constitutively activated TSHR mutants are also constitutively desensitized, we investigated the TSHR activity in association with β-arrestins in cotransfection experiments in HEK 293 cells using a cAMP accumulation assay. To examine whether constitutively activated TSHR mutants are internalized in a different way as the wt-TSHR, we investigated the TSHR trafficking in association with β-arrestins in cotransfection experiments in HEK 293 cells using confocal laser scanning microscopy and cell surface ELISA. We found, that both β-arrestins are able to internalize the wt-TSHR and the constitutively activated TSHR mutants in HEK 293 cells. However, an increased basal desensitization and internalization of constitutively activated TSHR mutants N670S, S505N and F631L cotransfected with β-arrestins could not be found. After TSH-stimulation the constitutively activated mutants showed the same time-course for internalization as the wt-TSHR. In summary contrary to data obtained for the β2AR the constitutive activation of the TSHR does not influence the desensitization and the time-course for internalization of the receptor.