Subunit structure of thyrotropin receptors expressed on the cell surface

We studied cell surface thyrotropin receptor (TSHR) by biotinylating protei ns on the surface of metabolically labeled, intact cells. In addition to TS HR cleaved into A and B subunits, mature single-chain receptors with comple x carbohydrate were also present on the cell surface, A low A/B subunit rat io indicated partial shedding of extracellular A subunits from transmembran e B subunits. TSHR cleavage at upstream site 1 (within amino acid residues 305-316) would generate a B subunit of 51-52 kDa. However, only smaller B s ubunits (40-46 kDa) were detected, corresponding to N termini from residues similar to 370 (site 2) extending downstream to the region of B subunit in sertion into the plasma membrane. The intervening C peptide region between sites 1 and 2 could not be purified from TSHR epitope-tagged (c-myc) within this region. However, the small proportion of B subunits recovered with a c-myc antibody were larger (45-52 kDa) than the majority of B subunits reco vered with a C-terminal antibody. In conclusion, our study provides the fir st characterization of cell surface TSHR including their A and B subunits. Single-chain, mature TSHR do exist on the cell surface. The C peptide lost during intramolecular cleavage disintegrates rapidly following cleavage at upstream site 1 of the single-chain TSHR into A and B subunits. N-terminal disintegration of the B subunit pauses at site 2, but then progresses downs tream to the vicinity of the plasma membrane, revealing a novel mechanism f or A subunit shedding.