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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.

Among the glycoprotein hormone receptors, only the thyrotropin receptor (TSHR) cleaves (at two sites) into disulfide-linked A and B subunits. A 50-amino acid insertion unique to the TSHR ectodomain (residues 317-366) plays no role in ligand binding or signal transduction, but its deletion abrogates cleavage at Site 1, closely upstream of the insertion. We sought to define the region within the 50-amino acid tract involved in TSHR cleavage at Site 1. Mutation of small segments within this region previously failed to prevent cleavage at Site 1. We, therefore, divided the 50-amino acid insertion into quartiles and deleted each one individually (TSHR residues 317-327, 328-338, 339-350, and 351-362). As determined by covalent cross-linking of 125I-TSH to intact cells expressing the mutant receptors, none of these deletions prevented TSHR cleavage at Site 1. Neither did larger deletions of quartiles 1 + 2, 2 + 3, and 3 + 4. However, qualitative differences in the extent of receptor cleavage suggested that quartiles 1 and 4 were playing a greater role in cleavage at Site 1 than were the middle two quartiles. In support of this hypothesis, deletion of these two discontinuous segments almost completely eliminated TSHR cleavage at Site 1. In conclusion, intramolecular cleavage at Site 1 requires the presence of the N-terminal and C-terminal quartiles of the 50-amino acid insertion unique to the TSHR. Taken together with previous observations, our data suggest that this tract may provide a discontinuous binding site for a protease that clips the TSHR at Site 1.

The three-dimensional structure of the Fab of TR1.9, a high-affinity IgG1, kappa human autoantibody to thyroid peroxidase, was determined crystallographically to a resolution of 2.0 A. The combining site was found to be relatively flat, like other antibodies to large proteins. Sequence differences from the most closely related germline genes mainly occur at positions occupied by residues with outward-pointing side chains. An increased deformability of the second and third complementarity-determining regions of the heavy chain may result from the replacement of two germline asparagines and the presence of several glycines, and may allow "induced fit" in the binding to antigen. Four exposed charged residues, resulting from the use of a particular D (diversity) and J (joining) segments in the assembly of the heavy chain, may contribute to the high affinity of antigen binding. The crystal structure of TR1.9 Fab is the first for a human IgG high-affinity autoantibody.

Metmyoglobin (metMb) reduction by metMb reductase from heart muscle requires cytochrome b5 as electron-transfer mediator. The existence of a metMb-ferrous cytochrome b5 complex is demonstrated by mutual perturbation of the proteins' respective electrophoretic titration curves between pH 4 and 7. The same technique shows a preferential binding of cytochrome b5 over metMb by the enzyme. The paramagnetic hyperfine shifts in the cytochrome b5 1H NMR spectrum are perturbed by metMb, indicating the formation of a specific bimolecular complex with a 1:1 stoichiometry and a binding constant estimated to be less than 10 microM. The resonances assigned to the cytochrome b5 heme 6-propionate methylene group exhibit the largest complexation shifts. Computer modeling implicates lysines 47, 50, and 98 of metMb as contact points with cytochrome b5 carboxylate residues 43, 44, 60, and heme 6-propionate. The mechanism of the enzymatic reduction establishes metMb reductase as an NADH-cytochrome b5 oxidoreductase. Cytochrome b5 is reduced at near diffusion-controlled rates by the enzyme with a turnover number of 1000 min-1 X Km for the cytochrome is 0.9 microM versus 100 microM reported for the erythrocyte enzyme. Ferrous cytochrome b5 then reduces metMb nonenzymatically with an apparent rate constant of 4.9 X 10(4) M-1 min-1 X Acetylation of metMb, which does not affect its oxygen affinity or chemical reduction, renders it a poor substrate for enzymatic reduction. This study suggests a function for the three exterior lysine residues conserved in all mammalian myoglobin sequences: they are contact points for complexation with cytochrome b5.

Thyrotropin (TSH) receptor (TSHR) A and B subunits are formed by intramolecular cleavage of the single chain receptor at two separate sites. The region involved in cleavage at Site 2 has been identified, but previous mutagenesis studies failed to identify Site 1. We now report fortuitous observations on the effect of trypsin on the TSHR that localizes a small region harboring Site 1. Thus, as detected by immunoblotting and by 125I-TSH cross-linking to TSHR expressed on the surface of intact CHO cells, trypsin clipped a small polypeptide fragment bearing a glycan moiety from the C terminus of the A subunit. Based on the TSHR primary structure, this small fragment (1-2 kDa) contains Asn-302. This information, together with estimation of the size of the deglycosylated A subunit relative to a series of C-terminal truncated TSHR ectodomain variants, places cleavage Site 1 in the vicinity of, or closely upstream to, residue 317. Remarkably, mutagenesis of every amino acid residue between residues 298-316 (present study) and 317-362 (previous data) did not prevent cleavage at Site 1. However, cleavage at this site was abrogated by deletion of a 50-amino acid segment (residues 317-366) unique to the TSHR in the glycoprotein hormone receptor family. In summary, these data provide novel insight into TSHR intramolecular cleavage. Cleavage at Site 1 does not depend on a specific amino acid motif and differs from cleavage at Site 2 by involvement of a mechanism requiring the presence of the enigmatic TSHR 50-amino acid "insertion."

Previous attempts to generate autoantibody-reactive, secreted thyrotropin receptor (TSHR) ectodomain in mammalian cells have failed because of retention within the cell of material with immature carbohydrate. We have overcome this difficulty by performing progressive carboxyl-terminal truncations of the human TSHR ectodomain (418 amino acid residues including signal peptide). Three ectodomain variants (TSHR-261, TSHR-289, and TSHR-309) were truncated at residues 261, 289, and 309, respectively. Unlike the full ectodomain, ectodomain variants were secreted with an efficiency inversely proportional to their size. Secreted ectodomain variants contained approximately 20 kDa of complex carbohydrate. TSHR-261 was chosen for further study because it was secreted very efficiently and neutralized autoantibodies in Graves' patients' sera. This ectodomain variant was partially purified using sequential lectin and nickel-chelate chromatography, permitting the first direct visualization and quantitation of the mammalian TSHR. Most important, very small (nanogram) quantities of this material neutralized 70-100% of TSHR autoantibody activity in all 18 Graves' sera studied. In summary, carboxyl-terminal truncation of the human TSHR ectodomain generates a secreted protein with complex carbohydrate that neutralizes autoantibodies in Graves' patients' sera. Antigenically active TSHR will be valuable for future studies on the diagnosis, pathogenesis, and immunotherapy of Graves' disease.