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.