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 ( LHR) into a cleaved, two-subunit molecule. For this purpose, we generated a series of LHR mutants and chimeric LH-TSH receptors. Cleavage of mature, ligand binding receptors on the cell surface was determined by covalent 125I-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 has only one N-linked glycan. Elimination by mutagenesis of the most strategic N-linked glycan (LHR-N317Q) generated only a trace amount of LHR cleavage. Removal of the other N-linked glycans had no additive effect. A much greater degree of cleavage ( approximately 50%) was evident in a chimeric LH-TSHR in which the juxtamembrane segment of the LHR (domain E; amino acids 317-367) was replaced with the corresponding domain of the TSHR (residues 363-418). Similarly cleaving LHR were created using a much smaller component within 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 suggesting limited (not absent) amino-acid specificity in a region important for TSHR cleavage (GQE367-369). The data also support the concept of a separate and distinct downstream cleavage site 2 in the TSHR.[1]References
- Insight into thyrotropin receptor cleavage by engineering the single polypeptide chain luteinizing hormone receptor into a cleaving, two subunit receptor. Chazenbalk, G.D., McLachlan, S.M., Chen, C.R., Rapoport, B. Eur. J. Biochem. (2001) [Pubmed]
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