Subunit structure of thyrotropin receptors expressed on the cell surface.
We studied cell surface thyrotropin receptor (TSHR) by biotinylating proteins on the surface of metabolically labeled, intact cells. In addition to TSHR cleaved into A and B subunits, mature single-chain receptors with complex carbohydrate were also present on the cell surface. A low A/B subunit ratio indicated partial shedding of extracellular A subunits from transmembrane 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 subunits (40-46 kDa) were detected, corresponding to N termini from residues approximately 370 (site 2) extending downstream to the region of B subunit insertion 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 recovered with a C-terminal antibody. In conclusion, our study provides the first 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 downstream to the vicinity of the plasma membrane, revealing a novel mechanism for A subunit shedding.
Tanaka
K
K
Autoimmune Disease Unit, Cedars-Sinai Research Institute and School of Medicine, UCLA, Los Angeles, California 90048, USA.
Chazenbalk
G D
GD
McLachlan
S M
SM
Rapoport
B
B
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001665:Binding Sites; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism*; D006224:Cricetinae; D017077:Culture Media, Conditioned / Q000737:chemistry; D015870:Gene Expression; D006031:Glycosylation; D006801:Humans; D006868:Hydrolysis; D051379:Mice; D010446:Peptide Fragments / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D011989:Receptors, Thyrotropin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D017077:Culture Media, Conditioned; D010446:Peptide Fragments; D011956:Receptors, Cell Surface; D011989:Receptors, Thyrotropin