Clarifying the role of the TSH receptor protein in the autoimmune process may be the key to understanding the development of Graves' disease. In the present study we used a 16 amino acid peptide of the human TSH receptor (hTSHR) to immunize rabbits. A comparable, but theoretically less immunogenic, peptide was injected into other rabbits. The antibody response against these and other peptides, as well as against solubilized human thyroid membrane (TM) and guinea pig fat cell membrane (GPF) proteins, was tested using ELISA and Western blots. The GPF and TM binding pattern of rabbits' sera was compared to that of Graves' patients' sera. We have identified an area of antigenic cross-reactivity between GPF and TM; a 63 kD protein was present in both GPF and TM, and this protein uniformly bound IgG-s of the rabbits' postimmunization sera and one of eight Graves' patient's serum. We have shown that i) a theoretically immunogenic 16 amino acid peptide was indeed highly immunogenic in rabbits, ii) antibodies binding to GPF and TM were detected after immunization, and iii) the peak of thyroid stimulating immunoglobulin activity of sera was followed by a transient elevation of serum triiodothyronine levels. Further studies investigating the immunogenic epitopes of the hTSHR as well as characterizing the 63 kD protein are indicated.

Amino Acid Sequence↗

PubMedDate not supplied

Graves' IgG stimulation of continuously cultured rat thyroid cells: a sensitive and potentially useful clinical assay.

A continuously cultured line of normal rat thyroid (FRTL) cells can be stimulated by immunoglobulin preparations from patients with Graves' disease as measured by increases in intracellular cAMP levels. Responsiveness is concentration-dependent but is delayed in time relative to thyrotropin. Additionally, the cells respond to Graves' immunoglobulins which have no long-acting thyroid stimulator (LATS) activity and are negative when adenylate cyclase stimulation in human thyroid membrane preparations is assayed. No correlation exists between the stimulation activity and the ability of a Graves' immunoglobulin preparation to inhibit thyrotropin binding; cells are responsive even in the presence of such inhibitor activity.

Adenylyl Cyclases↗

PubMedDate not supplied

Monoclonal antibodies to the thyrotropin receptor: the identification of blocking and stimulating antibodies.

Monoclonal antibodies to the thyrotropin (TSH) receptor have been obtained from fusions of mouse myeloma cells with spleen cells immunized with solubilized thyroid membrane preparations. Two monoclonal antibodies which inhibit 125I-TSH binding and are reactive with the glycoprotein component of the bovine TSH receptor (11E8 and 13D11), are shown to inhibit basal and TSH stimulated adenylate cyclase activity in bovine thyroid membranes and human thyroid cells. Both antibodies also inhibit 125I-TSH binding in vitro, whether binding is measured at pH 6.0 in low salts and at 0-4 C or at pH 7.4 in 50 mM NaCl and at 37 C. The glycoprotein component is thus a portion of the physiologic TSH receptor in vivo and 125I-TSH binding studies apparently measure the high affinity glycoprotein component under nonphysiologic conditions and conditions more representative of the physiologic milieu. A third monoclonal antibody whose interaction with thyroid membranes is prevented by TSH is shown to stimulate adenylate cyclase activity in bovine thyroid membranes and human thyroid cells. This stimulating antibody only weakly inhibits 125I-TSH binding to thyroid membranes or to the glycoprotein component of the TSH receptor. The 22A6 antibody does, however, immunoprecipitate mixed brain gangliosides, in distinct contrast to the monoclonal antibodies to the glycoprotein receptor component, i.e., 11E8 and 13D11. The results support the speculation that autoimmune antibodies which inhibit TSH binding to thyroid membranes are not necessarily identical to antibodies which stimulate function; that antibodies directed at the high affinity initial site of TSH interaction with a cell can behave as blocking rather than stimulating antibodies and that a possible relationship exists between stimulating antibodies and the low affinity TSH binding sites (gangliosides) on thyroid membranes.

Animals↗

PubMedDate not supplied

Multicomponent structure of the thyrotropin receptor: relationship to Graves' disease.

The thyrotropin receptor is proposed to contain both a glycoprotein and a ganglioside component. Monoclonal antibodies have been developed against soluble thyroid TSH receptor preparations and using Graves' lymphocytes. These show that initial recognition of thyrotropin requires the glycoprotein component, but that monoclonal antibodies to this component block thyrotropin function (blocking antibodies) rather than mimic thyrotropin. Monoclonal antibodies which stimulate thyroid activity in cultured cell systems (cAMP increase) or mouse bioassays, all interact with gangliosides. Using monoclonal antibodies to the glycoprotein component of the thyrotropin receptor, we show that two protein bands, molecular weights 18,000-23,000 and 50,000-55,000, are precipitated from detergent-solubilized preparations. Using a crosslinking procedure with 125I-labeled thyrotropin, we show that thyrotropin binding is related to the disappearance of the 18,000-23,000 molecular weight band on sodium dodecylsulfate gels and the appearance of a 30,000-33,000 molecular weight thyrotropin-membrane component complex. Higher molecular weight thyrotropin-membrane complexes of 75,000-80,000 and 250,000 are visualized when binding studies are performed at pH 7.4 in physiologic medium; larger amounts of the 30,000-33,000 complex are evident at pH 6.0 in a low salt medium. It is thus proposed that the glycoprotein component of the thyrotropin receptor is composed of two subunits with apparent molecular weights of 18,000-23,000 and 50,000-55,000; that the 18,000-23,000 subunit interacts with thyrotropin; and that different receptor subunits can exist depending on in vitro binding conditions. Using monoclonal-stimulating antibodies or natural autoimmune IgG preparations from patients' sera, we show that stimulating antibodies exhibit species-specific binding to human thyroid ganglioside preparations. Individual components or determinants of the thyrotropin receptor structure with specific autoimmune immunoglobulins.

Animals↗

PubMedDate not supplied

Effects of thyroglobulin and pendrin on iodide flux through the thyrocyte.

Iodide transport by thyrocytes involves porters on the apical and basal surfaces of the cell facing the follicular lumen and bloodstream, respectively. Recent work identifies pendrin as an apical porter and shows that follicular thyroglobulin is a transcriptional regulator of the gene encoding pendrin and other thyroid-restricted genes. For example, whereas follicular thyroglobulin suppresses the gene expression and activity of the sodium iodide symporter (NIS), it increases pendrin gene expression. A potential new dynamic for iodide flux and thyroid hormone formation in thyrocytes has thus emerged and is supported by in vivo data.

Animals↗