THYROTROPIN RECEPTOR CLEAVAGE AT SITE-1 DOES NOT INVOLVE A SPECIFIC AMINO-ACID MOTIF BUT INSTEAD DEPENDS ON THE PRESENCE OF THE UNIQUE, 50 AMINO-ACID INSERTION

Thyrotropin (TSH) receptor (TSHR) A and B subunits are formed by intra molecular cleavage of the single chain receptor at two separate sites, The region in volved in cleavage at Site 2 has been identified, but p revious mutagenesis studies failed to identify Site 1. We now report f ortuitous observations on the effect of trypsin on the TSHR that local izes a small region harboring Site 1. Thus, as detected by immunoblott ing and by I-125-TSH cross-linking to TSHR expressed on the surface of intact CHO cells, trypsin clipped a small polypeptide fragment bearin g a glycan moiety from the C terminus of the A subunit. Based on the T SHR primary structure, this small fragment (1-2 kDa) contains Asn-302. This information, together with estimation of the size of the deglyco sylated A subunit relative to a series of C-terminal truncated TSHR ec todomain variants, places cleavage Site 1 in the vicinity of, or close ly upstream to, residue 317. Remarkably, mutagenesis of every amino ac id residue between residues 298-316 (present study) and 317-362 (previ ous data) did not prevent cleavage at Site 1. However, cleavage at thi s site was abrogated by deletion of a 50-amino acid segment (residues 317-366) unique to the TSHR in the glycoprotein hormone receptor famil y. In summary, these data provide novel insight into TSHR intramolecul ar cleavage, Cleavage at Site 1 does not depend on a specific amino ac id motif and differs from cleavage at Site 2 by involvement of a mecha nism requiring the presence of the enigmatic TSHR 50-amino acid ''inse rtion.''