THE NA+/I- SYMPORTER: CHARCTERIZATION & TSH REGULATION

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The present proposal continues a project initiated two years ago. Its overall objectives are the molecular characterization of the thyroid Na+/I- symporter and the study of the mechanism by which thyrotropin (TSH) regulates the symporter's activity. Our knowledge of the molecular properties of several thyroid-specific proteins, including thyroid peroxidase, thyroglobulin, and the TSH receptor, has increased substantially in recent years. However, a major gap exists in the understanding of the Na+/i- symporter, a physiologically important plasma membrane protein that mediates I- accumulation in the thyroid. I- uptake is the first step in the biogenesis of the thyroid hormones T3 and T4. To date, the symporter molecule has not been characterized; its size and amino acid sequence are unknown, and anti-symporter Abs are not available. The Na+/I- symporter has been expressed in X. laevis oocytes injected with poly A+ RNA from FRTL-5 cells, a line of highly functional rat thyroid cells. After fractionation by sucrose gradient centrifugation and injection of the resulting fractions, the poly A+ RNA encoding the symporter was found in a fraction containing messages of 2.8-4.O kb in length. A approximate 10 (6) independent-clone cDNA library has been constructed from this fraction. RNA made in vitro from cDNA template of a pool of 3,600 clones has been shown to induce I- uptake activity in oocytes. Harmaline and TRP-P-2, two terpenes, have been identified as novel Na+- site competitive inhibitors of the symporter, using FRTL-5 cells and calf thyroid membrane vesicles as model systems. TRP-P-2 has been employed in affinity chromatography studies aimed at identifying the symporter. A approximate 120 kDA polypeptide was selectively retained by the affinity matrix. This polypeptide might be the symporter, but more definitive experiments are necessary. Observations have been made that suggest the Na+/I- symport process is not-electrogenic and the Na+/I- stoichiometry is 1:1. Unexpectedly, mixed membrane vesicles prepared from FRTL-5 cells maintained in a TSH free medium [TSH] (-)] accumulate I-, even though the corresponding TSH (-) intact cells display no such transport. The Specific Aims of the proposal are: 1. Identification of the Na+/I- symporter and determination of its derived primary sequence through continued functional screening of the cDNA pool of 3,600 clones. 2. Molecular characterization of the symporter, by performance of : a) structure/function studies, employing Abs and modifications of the symporter molecule, and b) biogenesis studies, by pulse-chase analysis. 3. TSH regulation of the symporter, by testing two proposed hypotheses of TSH action via subcellular fractionation.