Modulation of Graves' disease autoantibody stimulation of recombinant human thyrotrophin receptor.

BACKGROUND: The cAMP response of porcine thyrocytes to the immunoglobulin (Ig) fraction from Graves' sera is increased if these fractions are prepared with higher than usual concentrations of polyethylene glycol (PEG; 22.5% rather than 13.5%), leading to the suggestion that additional factors might exist in serum which influence the ability of autoantibodies to stimulate thyroid cells. METHODS: We characterised the stimulatory activity of fractions prepared by differential PEG precipitation of Graves' sera, using heterologous eukaryotic cells expressing recombinant human thyrotrophin (TSH) receptor. RESULTS: We found no evidence that material soluble in 13.5% PEG but precipitated by 22.5% PEG was stimulatory either on its own or in combination with the immunoglobulin-containing material precipitated by 13.5% PEG. Indeed, the stimulatory effect was reproduced by simply including low concentrations of PEG (1-4%) in the diluted sera from the majority of Graves' patients (12/18 tested) added to the target cells. Intriguingly, PEG had no effect on basal levels, with normal sera or stimulation by thyrotrophin. CONCLUSIONS: The increase in stimulation reported when using immunoglobulins prepared with higher concentrations of PEG is attributable to a direct effect of the extra PEG (which had not been removed from the preparations) during the stimulation of the cells. Because the effect is observed with recombinant heterologous cells, it must be caused by interaction between PEG and the autoantibodies and/or the receptor-it cannot involve any other thyroid-specific molecule, although an involvement of other molecules widely distributed on many cell types is possible.

Bag1-L is a phosphorylation-dependent coactivator of c-Jun during neuronal apoptosis.

In the nervous system, cell death by apoptosis plays a critical role during normal development and pathological neurodegeneration. Jun N-terminal kinases (JNKs) are essential regulators of neuronal apoptosis. The AP-1 transcription factor c-Jun is phosphorylated at multiple sites within its transactivation domain by the JNKs, and c-Jun phosphorylation is required for JNK-induced neurotoxicity. While the importance of c-Jun as a mediator of apoptotic JNK signaling in neurons is firmly established, the molecular mechanism underlying the requirement for c-Jun N-terminal phosphorylation is enigmatic. Here we identify the multifunctional protein Bag1-L as a coactivator of phosphorylated c-Jun. Bag1-L preferentially interacts with N-terminally phosphorylated c-Jun, and Bag1-L greatly augments transcriptional activation by phosphorylated c-Jun. Chromatin immunoprecipitation experiments revealed binding of Bag1-L to the promoters of proapoptotic AP-1 target genes, and overexpression of Bag1-L augmented cell death in primary neurons. Therefore, Bag1-L functions as a coactivator regulating neurotoxicity mediated by phosphorylated c-Jun.

A functional site on the human TSH receptor: a potential therapeutic target in Graves' disease.

OBJECTIVE: Identifying sites on the TSH-receptor that are involved in the pathological stimulation of the thyroid by autoantibodies in Graves' disease would aid the development of new therapies. We tested a series of monoclonal antibodies that recognize the native receptor for their ability to inhibit stimulation of the receptor in vitro. PATIENTS AND METHODS: Heterologous cells expressing the recombinant human TSH-receptor were stimulated with TSH or serum samples from 13 Graves' disease patients or the MRC Long-Acting Thyroid Stimulator standard B (LATS-B) and their cAMP responses measured. The effect on this stimulation of various doses of purified monoclonal antibodies with defined epitopes was determined. RESULTS: Antibodies against one epitope (residues 381-384) inhibited TSH-stimulated cyclic adenosine monophosphate (cAMP) production (1 microg/ml causing 50% inhibition of the response to 100 microU/ml TSH) and also inhibited cAMP production induced by sera from approximately 40% (6/14) of Graves' disease patients, including the MRC LATS-B standard. CONCLUSIONS: Residues 381-384 of the human TSH-receptor are important in the physiological and pathological stimulation of the thyroid. This opens the possibility of more specific therapy of some Graves' disease patients by agents directed against this epitope.