Current standards, variations, and pitfalls for the determination of constitutive TSHR activity in vitro

Constitutively activating mutations of the TSHR are the major cause for nonautoimmune hyperthyroidism, which is based on ligand independent, permanent receptor activation. Several reports have highlighted the difficulties to determine whether a TSHR mutation is constitutively active or not especially for borderline cases with only a slight increase of the basal cAMP activity. Current methods to precisely classify such mutants as constitutively active or not, are limited. In some cases, in vitro characterization of TSHR mutants has led to false positive conclusions regarding constitutive TSHR activity and subsequently the molecular origin of hyperthyroidism. For characterization of constitutive TSHR activity, a particular point to consider is that basal receptor activity tightly correlates with the receptor number expressed on the cell surface. Therefore, a comparison of the receptors basal activity in relation to the wild type is only possible with determination of the receptor cell surface expression. Thus, the experimental approaches to determine constitutive TSHR activity should consider the receptor's cell surface expression. We here provide a description of three methods for the determination of constitutive TSHR activity: (A) the evaluation of constitutive TSHR activity under conditions of equal receptor expression; (B) computation of the specific constitutive activity; and (C) the linear regression analysis (LRA). To date, LRA is the best experimental approach to characterize the mutant's basal activity as a function of TSHR cell surface expression. This approach utilizes a parallel measurement of basal cAMP values and receptor cell surface expression and therefore provides a more reliable decision with respect to the presence or absence of constitutive activity.