MOLECULAR CHARACTERIZATION OF THE IODIDE CARRIER

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The overall objective of this project is to characterize the structure and function of the iodide carrier protein of the thyroid gland. This transporter' responsible for the active Na+-dependent accumulation of iodide against its concentration gradient, is a membrane protein located in the baso-lateral end of the epithelial cells of the thyroid follicles. Iodide transport into these cells is the first step in the synthesis of the thyroid hormones (T3 and T4). Thyroid stimulating hormone (TSH) regulates the expression of the iodide carrier by activating the adenylate cyclase-cAMP- dependent protein kinase system. Despite its fundamental importance in mammalian physiology, little is currently known about the structure, biochemistry and molecular properties of the iodide carrier protein. Preliminary studies contained in this proposal show that iodide transport activity is expressed in Xenopus laevis oocytes microinjected with mRNA isolated from FRTL-5 cells (a continuous line of cultured and fully functional rat thyroid cells), and that the mRNA encoding the transporter is 2.8-4.0 kb in length. Hence, the following is proposed: 1) to determine the derived primary sequence of the iodide carrier protein by constructing a cDNA library from the 2.8-4.0 kb fraction of mRNA. screen it b) functional analysis in the Xenopus laevis translation system and sequence cDNA inserts from positive clones; 2) to investigate structure/function relationships of the iodide carrier protein, by performing studies with site-directed polyclonal antibodies, partial proteolysis, group-specific reagents and site- specific mutagenesis: 3) to study the biosynthesis and post- translational modifications of the I- carrier protein by pulse- chase analysis; 4) to establish the electrogenicity of iodide transport and to determine the Na+/I-stoichiometry by direct flux measurements and by applying a thermodynamic approach.