Sequential cleavage and excision of a segment of the thyrotropin receptor ectodomain

The thyrotropin (TSH) receptor belongs to a subfamily of G protein-coupled receptors, which also includes luteinizing hormone and follicle-stimulating hormone receptors, The TSH receptor (TSHR) differs from the latter by the presence of an additional specific segment in the C-terminal part of its ec todomain. We show here that this insertion is excised in the majority of re ceptor molecules. Preparation of specific monoclonal antibodies to this reg ion, microsequencing, enzyme-linked immunosorbent assay, and immunoblot stu dies have provided insight into the mechanisms of this excision, In the hum an thyroid gland, N termini of the transmembrane receptor beta subunit were found to be phenylalanine 366 and leucines 370 and 378, In transfected L c ells a variety of other more proximal N termini were found, probably corres ponding to incomplete excisions, The most extreme N terminus was observed t o lie at Ser-314, These observations suggest that after initial cleavage at Ser-314 the inserted fragment of TSHR is progressively clipped out by a se ries of cleavage reactions progressing up to amino acids 366-378, The impos sibility of recovering the excised fragment from purified receptor, cell me mbranes, or culture medium supports this interpretation, The cleavage enzym e has previously been shown to be inhibited by BB-2116, an inhibitor of mat rix metalloproteases, However, we show here that it is unaffected by tissue inhibitors of metalloproteases. The cleavage enzyme is very similar to TAC E (tumor necrosis factor Lu-converting enzyme) in both these characteristic s. However, incubation of the TSH receptor with the purified recombinant ca talytic domain of TACE, co transfection of cells with TACE and TSHR express ion vectors, and the use of mutated Chinese hamster ovary cells in which TA CE is inactive suggested that the TSHR cleavage enzyme is different from TA CE, TACE and TSHR cleavage enzyme may thus possibly be related but differen t members of the adamalysin family of metzincin metalloproteases.