The successful iodination of thyroglobulin (Tg) leading to coupling of di-iodotyrosyl residues within Tg for the synthesis of thyroxine (the major form of thyroid hormone produced by the thyroid gland), requires that the thyrocyte precisely coordinate a number of distinct cell surface activities. Amongst other proteins, on the basolateral cell surface resides the thyrotropin (TSH) receptor, sodium/potassium ATPase, and sodium-iodide symporter (NIS), while the apical cell surface expresses thyroperoxidase (TPO) and pendrin activities, not to mention the production and release of hydrogen peroxide and the exocytosis of newly-synthesized Tg for hormonogenesis via a """"""""last-come, first-served"""""""" mechanism. This grant, which serves as the sole source of thyroid-related funding for our lab, covers a spectrum of related topics pertinent to thyrocyte cell biology, with a focus on secretory pathway delivery of proteins essential for thyroid function. In earlier cycles of this grant, we defined steps in the normal folding pathway of newly-synthesized Tg; determined that the human illness described as """"""""congenital goiter with deficient thyroglobulin"""""""" is an endoplasmic reticulum storage disease caused by the misfolding and ER retention of mutant Tg (mediated by ER-specific molecular chaperones); and identified the cog/cog mouse as a putative animal model of this illness. In papers published in the most recent five years of this grant (1997-2002), among other findings, we have pinpointed the molecular defect in the cog cog mouse as a single point mutation more than 2,000 amino acids downstream in the Tg coding sequence, and we began to concentrate on the intracellular disposal of the mutant Tg protein by ER-associated degradation (ERAD). We now present compelling evidence to support that the protein domain harboring the cog mutation -- the acetylcholinesterase-(AChE)-Iike domain, plays an essential role in normal Tg folding and homodimerization. By replacing this domain with authentic AChE (which also homodimerizes), we have created a powerful new enzymatically-active native reporter at the C-terminus of Tg, greatly enabling studies of Tg intracellular transport (Aim 1). We also wish to examine quality control signaling proteins and their roles in the ER retention and ERAD of mutant Tg (Aims 2 and 3). Finally we have made steady progress in our studies of the trafficking of TPO (and there are a few surprises!), and we now propose to take these studies in new directions relating to surface expression in both thyrocyte cell lines and the thyroid gland in situ (Aim 4).
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