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

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.