On the functional importance of thyrotropin receptor intramolecular cleavage

We examined the relationship between TSH receptor (TSHR) cleavage into two subunits and ligand-independent, constitutive activity characteristic of this receptor. Because of homology to the thrombin receptor-tethered ligand, we focused initially on a region in the vicinity of the second, downstream cleavage site of the TSHR ectodomain. We introduced into the wild-type TSHR three mutations. One mutation, TSHR(GQE(367-369)NET) prevents cleavage at site 2. The other two mutations, ELK(369-371)T-Y (TSHR-E1a2) and NPQE(372-375)SAIF (TSHR-E1b), introduce major changes into the potential tethered ligand. Basal, steady state intracellular cAMP levels in cloned, stably transfected Chinese hamster ovary cells were expressed as a function of the number of receptors (cAMP/receptor). None of these three mutations decreased ligand-independent constitutive activity, thereby excluding the tethered ligand hypothesis as well as a requirement for cleavage at site 2 in this process. Turning to the more upstream site 1 in the TSHR ectodomain, we examined a receptor (TSHR-delta50AA) with deletion of a unique 50-amino acid insertion (residues 317-366) that appears to be involved in cleavage at this site. Constitutive cAMP production was similar to that of the wild-type TSHR. Finally, we studied a TSHR mutant that cleaves at neither site 1 (deletion of residues 317-366) nor site 2 (GQE(367-369)NET substitution) and, therefore, does not cleave into A and B subunits. Again, the basal, constitutive level of cAMP production was similar to that of the wild-type TSHR. In summary, contrary to the prevailing hypothesis based on several lines of evidence, TSHR cleavage into subunits is not associated with constitutive, ligand-independent activity.