Insight into thyrotropin receptor cleavage by engineering the single polypeptide chain luteinizing hormone receptor into a cleaving, two subunit receptor

To gain insight into the thyrotropin hormone (TSH) receptor (TSHR) cleavage , we sought to convert the noncleaving luteinizing hormone (LH) receptor (L HR) into a cleaved, two-subunit molecule. For this purpose, we generated a series of LHR mutants and chimeric LH-TSH receptors. Cleavage of mature, li gand binding receptors on the cell surface was determined by covalent I-125 -labeled hCG crosslinking to intact, stably transfected mammalian cells. We first targeted a cluster of three N-linked glycans in the LHR (N295, N303, N317) in a region corresponding to the primary TSHR cleavage site, which h as only one N-linked glycan. Elimination by mutagenesis of the most strateg ic N-linked glycan (LHR-N317Q) generated only a trace amount of LHR cleavag e. Removal of the other N-linked glycans had no additive effect. A much gre ater degree of cleavage (approximate to 50%) was evident in a chimeric LH-T SHR in which the juxtamembrane segment of the LHR (domain E; amino acids 31 7-367) was replaced with the corresponding domain of the TSHR (residues 363 -418). Similarly cleaving LHR were created using a much smaller component w ithin this region, namely LHR-NET317-319 replaced with TSHR-GQE367-369, or by substitution of the same three amino-acid residues with AAA (LHR-NET317- 319AAA). In summary, our data alter current concepts regarding TSHR cleavage by sugg esting limited (not absent) aminoacid specificity in a region important for TSHR cleavage (GQE367-369). The data also support the concept of a separat e and distinct downstream cleavage site 2 in the TSHR.