The objective of this proposal is to examine the role of oxidation- reduction reactions in TSH receptor binding and bioactivity. The interaction of TSH with its receptor underlies important aspects of thyroid physiology and clinical endocrinology. TSH stimulates the synthesis and release of thyroid hormones, and is also a mediator of thyroid gland growth. Inappropriate activation of the TSH receptor is associated with diseases such as thyroid cancer and Graves' disease. Thus, a better understanding of how TSH binds to and activates its receptor would provide new insights into thyroid pathophysiology. Such studies would also represent a first step in the development of clinically useful TSH agonists and antagonists. TSH and the other glycoprotein hormones (LH, FSH, and CG) contain a four amino acid region homologous to the active site of the protein thioredoxin. Thioredoxin is an oxidoreductase that re-activates reduced and denatured proteins by catalyzing the reformation of their disulfide bonds, an activity also present in LH and FSH. Initial studies will determine if TSH shares this thioredoxin-like ability to catalyze protein reactivation, and whether chemical or genetic modification of the TSH thioredoxin-like site alters this activity. It is also proposed that TSH-catalyzed oxidation-reduction reactions represent an initial event in TSH ligand binding and receptor activation. To test this hypothesis, site-directed mutagenesis will be used to produce recombinant human thyrotropins with mutated thioredoxin-like regions. The goal of this work is to demonstrate that mutations which inhibit TSH-catalyzed protein refolding activity also reduce TSH receptor binding and bioactivity. For receptor binding and bioassay studies, the human TSH receptor CDNA will be transfected into a hormonally responsive cell line. Currently, most TSH bioassays use CAMP production as a measure of bioactivity. However, CAMP production is often a less sensitive indicator of hormone action than other biologic responses. To address this limitation of TSH bioassays, the recombinant human TSH receptor will be expressed in a mouse Leydig tumor cell line that normally produces progesterone in response to LH or CG stimulation. Conferring TSH responsiveness on this cell line would couple TSH action to progesterone production, and it is anticipated that this will result in a TSH bioassay of greater sensitivity than CAMP-based assays.
| Hussain, A; Zimmerman, C A; Boose, J A et al. (1996) Large scale synthesis of recombinant human thyrotropin using methotrexate amplification: chromatographic, immunological, and biological characterization. J Clin Endocrinol Metab 81:1184-8 |
