title: Subunit structure of thyrotropin receptors expressed on the cell surface.
abstract: 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.
authors.affiliation: Autoimmune Disease Unit, Cedars-Sinai Research Institute and School of Medicine, UCLA, Los Angeles, California 90048, USA.
authors.forename: K
authors.initials: K
authors.lastname: Tanaka
authors.forename: G D
authors.initials: GD
authors.lastname: Chazenbalk
authors.forename: S M
authors.initials: SM
authors.lastname: McLachlan
authors.forename: B
authors.initials: B
authors.lastname: Rapoport
mesh_terms: 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*
chemical_list: D000911:Antibodies, Monoclonal; D017077:Culture Media, Conditioned; D010446:Peptide Fragments; D011956:Receptors, Cell Surface; D011989:Receptors, Thyrotropin