Reassessment of the Location of the Thyrotropin Receptor 50 Amino Acid "Insertion" Provides Evidence in Favor of a Second Downstream Cleavage Site

Cleavage of thyrotropin receptors (TSHR) on the cell surface into disulfide-linked A and B subunits involves deletion of an intervening region that corresponds approximately to a 50 amino acid "insertion" in the TSHR relative to the noncleaving luteinizing hormone/choriogonadotropin receptor (LH/CGR). The location of this insertion is imprecise because of the relatively low homology between the two receptors in this region. We tested the hypothesis that the TSHR 50 amino acid insertion was further downstream than we previously concluded, a possibility that would relocate the crucial LH/CGR glycan at N₂₉₁ relative to the position of the TSHR insertion, and that would mitigate against the 50 amino acid insertion playing a role in TSHR intramolecular cleavage. Thus, we transferred the LH/CGR glycan at amino acid 291 from downstream (N₃₆₇) to upstream of the 50 amino acid insertion (N₃₁₇) in the TSHR, leaving this insertion intact. TSHR cleavage persisted. Moreover, deletion of amino acid residues 320-366 in addition to the upstream N₂₉₁ substitution (ALN_317-319NET) also did not prevent cleavage. On the other hand, deletion of three contiguous downstream residues (GQE_367-369) in the TSHR 50 amino acid insertion abolished receptor cleavage into subunits. In summary, the present data are consistent with our previous location of the TSHR 50 amino acid insertion and, therefore, do not undermine evidence for the involvement of this insertion in TSHR cleavage. In addition, the data regarding TSHR residues GQE_367-369 (far downstream of cleavage site 1) support the controversial possibility of a secondary cleavage site downstream of the insertion.