title: Specific antibody to the thyrotropin receptor identifies multiple receptor forms in membranes of cells transfected with wild-type receptor complementary deoxyribonucleic acid: characterization of their relevance to receptor synthesis, processing, structure, and function.
abstract: An antibody to a peptide of the TSH receptor, residues 352-366 which are not present in gonadotropin receptors, specifically identifies three major forms of the receptor on Western blots of detergent-solubilized membrane preparations from Cos-7 cells transfected with full-length rat and human TSH receptor cDNA: 230, 180, and 95-100 kilodaltons (kDa), based on simultaneously run protein standards. The 95- to 100-kDa protein is absent in cells transfected with a mutant receptor with no signal peptide and is sensitive to endoglycosidase-F. Its size is consistent with the sum of amino acids predicted from its cDNA sequence (84 kDa after subtracting the signal peptide) plus its carbohydrate content (14 kDa estimated from glycosylation mutants). It alone is absent in two deletion mutants that have lost TSH binding and activity after transfection: M1 missing residues 37-121 and M2 missing residues 110-307. It, thus, appears to be the processed glycosylated functional receptor on the cell surface. The 230-kDa protein is a nonprocessed form of the receptor, as evidenced by its insensitivity to endoglycosidase-F and its continued presence in cells transfected with a mutant receptor with no signal peptide. It is the primary form identified in rat FRTL-5 thyroid cells that have a functioning TSH receptor; it is not present in rat FRT thyroid cells with no functioning TSH receptor or receptor RNA. It appears, therefore, to be a early synthetic form of the functional TSH receptor. The 180-kDa protein is endoglycosidase-F sensitive and appears to be a processed intermediate between the 230-kDa early synthetic form and the 95- to 100-kDa functional receptor, rather than a dimer of the latter. Thus, with decreases in size appropriate to a receptor monomer, it remains present in membranes from the M1 and M2 deletion mutants that contain the 230-kDa protein but are missing the 95- to 100-kDa receptor form in association with lost TSH binding and activity after transfection. Minor receptor forms (54 kDa in rat receptor transfectants, 54 and 48 kDa in human receptor transfectants) appear to be degraded forms of the processed and glycosylated 95- to 100-kDa receptor. The presence or absence of reducing agents in the detergent solubilization mixture does not change the pattern or amount of the receptor forms recognized by the antibody, including the 54-kDa form; however, boiling does.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Cell Regulation Section, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland 20892.
authors.forename: T
authors.initials: T
authors.lastname: Ban
authors.forename: S
authors.initials: S
authors.lastname: Kosugi
authors.forename: L D
authors.initials: LD
authors.lastname: Kohn
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D000918:Antibody Specificity; D015153:Blotting, Western; D002462:Cell Membrane / Q000737:chemistry*; D004247:DNA / Q000235:genetics*; D003902:Detergents; D006031:Glycosylation; D006358:Hot Temperature; D006801:Humans; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D051381:Rats; D011989:Receptors, Thyrotropin / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D013329:Structure-Activity Relationship; D014162:Transfection
chemical_list: D000906:Antibodies; D003902:Detergents; D011989:Receptors, Thyrotropin; D004247:DNA; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase