We examined the relationship between TSH receptor (TSHR) cleavage into two
subunits and ligand-independent, constitutive activity characteristic of th
is 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, ELK369-371T-Y (TSHR-E1a2) and NPQE(372-375
)SAIF (TSHR-E1b), introduce major changes into the potential tethered ligan
d. Basal, steady state intracellular cAMP levels in cloned, stably transfec
ted Chinese hamster ovary cells were expressed as a function of the number
of receptors (cAMP/receptor). None of these three mutations decreased ligan
d-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-Delta 50AA) with deletion of a unique 50-amino acid insertio
n (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. Fin
ally, 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 leve
l 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.