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 present evidence suggests that Site 1 lies between amino acid residues 303 and 317, the mechanism and exact amino acid(s) involved in cleavage are unknown. Previous amino acid substitutions at Site 1 failed to abrogate cleavage. We, therefore, performed deletion mutations within this region. Cleavage of cell surface TSHR, detected by 125I-TSH cross-linking to intact cells, was not prevented by deletion of four individual segments within the Site 1 cleavage region (delta305-308, delta309-312, delta313-316, delta317-320). However, deletion of the entire region (delta305-320) reduced the extent of cleavage and shifted the cleavage site upstream of the glycan at amino acid residue N302. Elimination of this glycan (N302Q substitution) reversed the effect of deleting amino acid residues 305-320 on TSHR cleavage, suggesting that reduced cleavage at the new, upstream cleavage site was caused by steric hindrance by the glycan at N302. In summary, deletion, as opposed to mutagenesis, of the TSHR cleavage Site 1 region produces a spatial shift in TSHR cleavage Site 1 from downstream to upstream of the glycan at N302. 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.
Tanaka
K
K
Autoimmune Disease Unit, Cedars-Sinai Research Institute and School of Medicine, University of California, Los Angeles 90048, USA.
Chazenbalk
G D
GD
McLachlan
S M
SM
Rapoport
B
B
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D017353:Gene Deletion; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D011134:Polysaccharides / Q000235:genetics; D011989:Receptors, Thyrotropin / Q000235:genetics*
D011134:Polysaccharides; D011989:Receptors, Thyrotropin