Evidence that cleavage of the thyrotropin receptor involves a "molecular ruler" mechanism: Deletion of amino acid residues 305-320 causes a spatial shift in cleavage site 1 independent of amino acid motif
K. Tanaka et al., Evidence that cleavage of the thyrotropin receptor involves a "molecular ruler" mechanism: Deletion of amino acid residues 305-320 causes a spatial shift in cleavage site 1 independent of amino acid motif, ENDOCRINOL, 141(10), 2000, pp. 3573-3577
Some TSH receptors (TSHR) on the cell surface cleave into A and B subunits.
Cleavage at upstream Site 1 is followed by the proteolytic excision of an
intervening C peptide region terminating at a downstream Site 2. Although p
resent evidence suggests that Site 1 lies between amino acid residues 303 a
nd 317, the mechanism and exact amino acid(s) involved in cleavage are unkn
own. Previous amino acid substitutions at Site 1 failed to abrogate cleavag
e. We, therefore, performed deletion mutations within this region. Cleavage
of cell surface TSHR, detected by I-125-TSH cross-linking to intact cells,
was not prevented by deletion of four individual segments within the Site
1 cleavage region (Delta(305-308), Delta(309-312), Delta(313-316), Delta(31
7-320)). However, deletion of the entire region (Delta(305-320)) reduced th
e extent of cleavage and shifted the cleavage site upstream of the glycan a
t amino acid residue N-302. Elimination of this glycan (N(302)Q substitutio
n) reversed the effect of deleting amino acid residues 305-320 on TSHR clea
vage, suggesting that reduced cleavage at the new, upstream cleavage site w
as caused by steric hindrance by the glycan at N-302. In summary, deletion,
as opposed to mutagenesis, of the TSHR cleavage Site 1 region produces a s
patial shift in TSHR cleavage Site 1 from downstream to upstream of the gly
can at N-302. These observations provide strong evidence that TSHR cleavage
at this site does not occur at a particular amino acid motif and suggests
that cleavage involves a "molecular ruler" mechanism involving cleavage at
a fixed distance from a protease attachment site.