Thyrotropin Receptor Cleavage at Site 1 Involves Two Discontinuous Segments at Each End of the Unique 50-Amino Acid Insertion

Kunihiko Tanaka,Gregorio D Chazenbalk,Sandra M Mclachlan,Basil Rapoport

doi:10.1074/jbc.274.4.2093

Abstract

Among the glycoprotein hormone receptors, only the thyrotropin receptor (TSHR) cleaves (at two sites) into disulfide-linked A and B subunits. A 50-amino acid insertion unique to the TSHR ectodomain (residues 317-366) plays no role in ligand binding or signal transduction, but its deletion abrogates cleavage at Site 1, closely upstream of the insertion. We sought to define the region within the 50-amino acid tract involved in TSHR cleavage at Site 1. Mutation of small segments within this region previously failed to prevent cleavage at Site 1. We, therefore, divided the 50-amino acid insertion into quartiles and deleted each one individually (TSHR residues 317-327, 328-338, 339-350, and 351-362). As determined by covalent cross-linking of 125I-TSH to intact cells expressing the mutant receptors, none of these deletions prevented TSHR cleavage at Site 1. Neither did larger deletions of quartiles 1 + 2, 2 + 3, and 3 + 4. However, qualitative differences in the extent of receptor cleavage suggested that quartiles 1 and 4 were playing a greater role in cleavage at Site 1 than were the middle two quartiles. In support of this hypothesis, deletion of these two discontinuous segments almost completely eliminated TSHR cleavage at Site 1. In conclusion, intramolecular cleavage at Site 1 requires the presence of the N-terminal and C-terminal quartiles of the 50-amino acid insertion unique to the TSHR. Taken together with previous observations, our data suggest that this tract may provide a discontinuous binding site for a protease that clips the TSHR at Site 1.

Highlights

Intramolecular cleavage at Site 1 requires the presence of the N-terminal and C-terminal quartiles of the 50-amino acid insertion unique to the TSH receptors (TSHR)
A variable proportion of TSH receptors (TSHR) on the cell surface cleave into two subunits (A and B) that remain linked by disulfide bonds [2,3,4]
TSH Receptor Mutations—All the following deletion mutations were introduced into a TSHR unable to cleave at Site 2 (GQE367–369NET) [9]; amino acid residues 317–327, 328 –338, 339 –350, 351–362, 317–338, 339 –362, 328 –350, and 317–328 ϩ 351–362

Summary

Introduction

Intramolecular cleavage at Site 1 requires the presence of the N-terminal and C-terminal quartiles of the 50-amino acid insertion unique to the TSHR. Relative to the other glycoprotein hormone receptors, the TSHR contains an insertion of 50 amino acids in the vicinity of residues 317–366 (low homology makes the exact boundaries difficult to define). This insertion, bordered by cysteine residues and with characteristics of an hydrophilic external loop, can be deleted without affecting ligand and autoantibody binding and receptor activation [6]. Cleavage at Site 2, which lacks a specific motif, is not dependent on the 50-amino acid insertion but can be prevented by replacing TSHR amino acid residues 367–369 with the N-linked glycosylation motif at the corresponding location in the noncleaving lutropin/choriogonadotropin receptor [9]. Because there is no amino acid specificity at the cleavage site itself, our data suggest that the 50amino acid insertion in the TSHR may function as a discontinuous binding site for a protease that clips the TSHR at Site 1

Methods

Results

Conclusion