Abstract
The TSH receptor (TSHR) is a G protein-coupled receptor (GPCR) with a division of labor between agonist binding, by a large ectodomain, and activation of the G protein, by a canonical heptahelical serpentine domain. Strong experimental arguments suggested that activation of TSHR involves switching of the ectodomain into a tethered agonist of the serpentine portion, upon binding of the hormones to the ectodomain. The TSHR exists as functional oligo(di)mers at the cell surface. It displays strong negative cooperativity for binding of TSH, leading to the notion that a single protomer is activated under conditions of physiological agonist concentrations. From a topological point of view, negative cooperativity implies introduction of asymmetry upon exposure of the dimer to low concentration of the agonist. This poses the question of the overall stoechiometry between agonist(s), receptor(s) and the G protein(s), and about the conformational changes achieved in individual protomers upon activation. In the present study we took advantage of the wide panel of activating mutations available for the TSHR to explore the effect of constitutive, i.e. symmetrical, activation of the mutants on their allosteric behavior.
Our results demonstrate unambiguously an inverse relation between constitutive activity and negative cooperativity. The most constitutively activated mutants, while still dimerizing, as demonstrated by FRET, have almost completely lost allosteric behavior. As a consequence of this, for a given level of expression at the cell surface, constitutively active mutants display an increase of maximum TSH binding when compared to wild type TSHR. This observed effect may be linked to the fact that the constitutive mutant receptors could bind two TSH molecules per dimer with high affinity, instead of one for the wild type TSHR. In addition, this observation demonstrates that binding measurement is not a reliable method to quantify the expression of constitutively activated TSHR mutants.