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)

Processing of the precursors of the human thyroid‐stimulating hormone receptor in various eukaryotic cells (human thyrocytes, transfected L cells and baculovirus‐infected insect cells)

“…Such a size is difficult to reconcile with the molecular mass (84.5 kDa) of the amino acid backbone. A monomer of 95-100kDa has been observed in transfected Cos-7 [38] and CHO cells [39]. The accumulation in transfected cells of the mannose-rich monomeric precursor may explain some of these observations.…”

“…Precursors of the TSH receptor of 230 and 180 kDa have been described in transfected Cos-7 and FRTL-5 cells [38]. Such a size is difficult to reconcile with the molecular mass (84.5 kDa) of the amino acid backbone.…”

The Signal Peptide of the G Protein-coupled Human Endothelin B Receptor Is Necessary for Translocation of the N-terminal Tail across the Endoplasmic Reticulum Membrane

“…An essential function can be concluded in the case of the rat thyrotropin receptor, where deletion of a sequence containing the putative signal peptide led to nonfunctional receptors (8,9). However, this may not always be the case: for unrelated membrane proteins such as the human sodium calcium exchanger (10) and the human UDP-glucuronosyltransferase 1A6 (11), it was shown that mutants lacking the signal peptide are processed correctly.…”

Type 2 Diabetes Mellitus and Glucagon Like Peptide-1 Receptor Signalling

“…Deleting the signal peptide sequence of the thyrotropin receptor abolished functionality [172,173]. However, the corticotropin-releasing factor receptor type 2a signal peptide although present, was found to be incapable of mediating ER targeting [174,175].…”