TSH receptor (TSHR) cleavage into two subunits (A and B) was explored using two new mammalian cell lines expressing the recombinant receptor; 1) TSHR-10,000 CHO cells overexpressing the TSHR; 2) TSHRmyc cells with a c-myc epitope inserted at residues 338-349. Immunoprecipitation or immunoblotting of TSHR-10,000 cells with mAb to either the A subunit or the B subunit revealed multiple forms of the TSHR: 1) uncleaved receptors of approximately 115 kDa and approximately 100 kDa with complex carbohydrate and high mannose carbohydrate, respectively; 2) two subunit TSHR with an approximately 62 kDa A subunit containing complex carbohydrate. The A subunit was approximately 35 kDa after enzymatic deglycosylation (predicted C-terminus near residue 330). The nonglycosylated B subunit was evident primarily as an approximately 42 kDa band (predicted N terminus near residue 380). The sum of the A and B subunit polypeptide backbones was smaller than the predicted size of the TSHR, a polypeptide backbone (84.5 kDa), raising the possibility that an approximately 5-kDa polypeptide fragment was excised during intramolecular cleavage. This hypothesis was supported by data obtained with the TSHRmyc cells. Thus, mAb to the c-myc epitope and to amino acid residues 22-35 (mAb A10) were equally effective in detecting the single chain forms of the TSHR in these cells. However, the 35 kDa, deglycosylated A subunit was clearly visible on immunoprecipitation with mAb A10 to the TSHR amino terminus, but not with the anti-myc mAb, indicating loss of the c-myc epitope at residues 338-349. Further, even though the A subunit was not detected in TSHRmyc cells with anti-myc mAb, 125I-TSH cross-linking to the cell surface showed similar A subunit expression in TSHRmyc and wild-type TSHR expressing cells. In summary, our study provides a surprising and novel finding for G protein-coupled receptors. Contrary to the prevailing concept of one cleavage site in the TSHR, we present evidence that there are, in fact, two such sites. The TSHR, like insulin, may release a C peptide during intramolecular cleavage into two subunits.

Wedescribe a familial occurrence of primary hyperparathyroidism. The proband is a 60-yearold womanwho had a parathyroid adenoma. Her older sister had a parathyroid adenoma with cementifying jaw fibroma and her younger sister died of parathyroid adenocarcinoma with pulmonarymetastasis at the age of 38.Wehave not yet obtained evidence for other endocrine disorders suggesting multiple endocrine neoplasia (MEN)in this pedigree. The proband is complicated with Wilms' tumor. It is now widely accepted that respective predisposed genes of MENtype 1 and Wilms' tumor, and PTHgene are located on chromosome ll. The manifestation observed in this case maybe related to mutational abnormalities on chromosomell. (Internal Medicine 33: 123-126, 1994)

The thyrotropin receptor (TSHR) exists in two forms (single polypeptide and two subunits), whereas the lutropin/chorionic gonadotropin receptor (LH/CGR) is a single chain. Recent data suggest that the TSHR cleaves at two sites. We mutagenized selected chimeric TSH-LH/ CGR to localize the cleavage sites in the TSHR. All 23 receptors mutated in the estimated vicinity of the upstream site cleaved into two subunits as determined by 125 I-TSH cross-linking to intact cells. In contrast, in a series of mutations homologous to the noncleaving LH/ CGR, the downstream TSHR cleavage site localized to three amino acids (GQE 367-369 ). Remarkably, group substitution of these residues, but not substitution of individual residues, abolished cleavage. Moreover, the mutation that prevented cleavage (GQE 367-369 NET) transposed a motif (NET [291][292][293] ) that is glycosylated in the LH/CGR. TSHR cleavage or noncleavage after substitution of GQE 367-369 with other triplets (AAA, NQE, and NQT) was consistent with a role for N-linked glycosylation at this site.In summary, our data (i) support the concept that the TSHR cleaves at two sites, (ii) relate TSHR residues GQE 367-369 to cleavage at the second, downstream site, and (iii) suggest that cleavage or noncleavage at site two is related to N-linked glycosylation. These findings provide new insight into the evolutionary divergence of two closely related receptors.The thyrotropin receptor (TSHR) 1 and lutropin/chorionic gonadotropin (LH/CG) receptor are closely related members of the G protein-coupled receptor family with glycoprotein hormone ligands. Both the TSHR and LH/CG receptor have large, heavily glycosylated ectodomains with leucine-rich repeats and are encoded by numerous exons (1-3). Despite their common evolutionary background, these receptors have several remarkable differences aside from the specificity of their ligands. For example, disease-causing autoantibodies to the TSHR are common, whereas autoantibodies to the LH/CG receptor are extremely rare. Thus, engagement of the TSHR by autoantibodies is the direct cause of thyrotoxicosis in Graves' disease, an organ-specific autoimmune disorder affecting only humans. Another striking difference exists at the structural level. As detected by TSH cross-linking to the surface of intact cells, the functional TSHR exists in two forms; a single chain receptor and a receptor with two subunits (4, 5). In contrast, the LH/CG receptor exists only in a single chain form (reviewed in Segaloff and Ascoli (6)). Variable proportions of single chain and twosubunit forms of the TSHR are also observed on immunoprecipitation or immunoblotting of mammalian cell extracts (7-11). The two-subunit TSHR involves a ligand-binding, glycosylated A subunit and a membrane-associated B subunit linked by disulfide bonds (12). Because the TSHR is encoded by a single mRNA species (13-16), the A and B subunits must be formed by intramolecular cleavage, a process believed to involve a matrix metalloprotease (17).Recent evidence raised the surprising pos...

Generation of large amounts of recombinant TSH receptor (TSHR) protein capable of recognition by TSHR autoantibodies is a goal of clinical importance. We expressed in Chinese hamster ovary cells the human TSHR ectodomain (ECD) with a carboxyl-terminus six-histidine tag. After transgene amplification, expressing clones were selected by nickel chelate chromatography in combination with [35S] methionine precursor labeling. An approximately 74-kDa protein was detected in the culture medium, and larger quantities of an approximately 68-kDa protein were found in the cell soluble fraction. Immunoblot analysis with a rabbit antiserum revealed that most of the TSHR-ECD was not secreted, but was retained within the soluble fraction of the cell. Nickel chelate chromatography was not effective in purifying significant quantities of this material. In contrast, with Concanavalin A, but not with wheat germ agglutinin, an approximately 50-fold purification of TSHR-ECD was achieved from the cell soluble fraction. Surprisingly, this affinity-enriched TSHR, containing high mannose carbohydrate, was not recognized by human TSHR autoantibodies in sera from six individuals. By ion exchange chromatography, the autoantibody-neutralizing TSHR in the cell supernatant fraction was found to be nonidentical with TSHR-ECD protein recognized by antisera from immunized animals. The present data indicate the critical relationship between autoantibody recognition and TSHR maturation as reflected in the acquisition of complex carbohydrate. Nonsecretion of the TSHR-ECD appears to be related to the specific protein rather than to the glycosylation apparatus of the host cell. Antibodies from immunized animals may be ineffective in monitoring purification of autoantigen-competent TSHR. Finally, the data explain why soluble recombinant TSHR generated in many expression systems is not recognized satisfactorily by human autoantibodies.