Characterization of the Rat Thyroid Iodide Transporter Using Anti-peptide Antibodies

Abstract

Anti-peptide antibodies directed against the C-terminal portion (amino acids 603-618) of the rat thyroid iodide transporter (rTIT) have been produced to characterize the molecular forms of rTIT in the rat thyroid and in the functional rat thyroid cell line, FRTL-5. rTIT is located on the basolateral membrane of rat thyroid follicular cells and randomly distributed on the plasma membrane of FRTL-5 cells that do not exhibit cell polarity. The major rTIT component corresponds to an 80 -90-kDa glycosylated protein. After treatment of cell membrane fractions with N-glycosidase F or incubation of FRTL-5 cells with tunicamycin, rTIT has an apparent molecular mass of about 55 kDa. FRTL-5 cells cultured in the presence of TSH exhibit a high rTIT content and a high iodide uptake activity (IUA). Upon either removal of TSH or addition of cycloheximide, IUA declines more rapidly than rTIT. The half-life of rTIT was about 4 days. Re-exposure of 7-day TSH-deprived FRTL-5 cells to TSH causes a rapid synthesis of the glycosylated rTIT but a delayed re-induction of IUA. Tunicamycin totally prevents the TSH-dependent re-expression and activity of rTIT. Our data bring basic information on the location, structure, and turnover of rTIT and suggest that its activity is subjected to diverse control mechanisms including regulatory proteins.Iodide trapping by epithelial thyroid cells is the first step of thyroid hormone synthesis within thyroid follicles. Thyroid iodide uptake brings into play a membrane transporter (1) defined as a Na ϩ /iodide symporter (2). After the pioneering studies of Wolff (1), a definite step in the knowledge of the thyroid iodide transporter (TIT), 1 has been made by Dai et al. (3) who cloned the cDNA of the rat TIT (rTIT) allowing the design of tools for further molecular analyses. Secondary structure prediction and hydropathic profile analyses indicated that rTIT is a membrane protein with 12 membrane-spanning domains thus resembling the other Na ϩ -dependent cotransporters. The availability of the rTIT primary sequence prompted us to produce antibodies to characterize the TIT protein of the rat and other animal species. We choose to generate polyclonal antibodies against a peptide corresponding to the last 16 amino acids (amino acids 603-618) of the rTIT. Antibodies that were raised exhibited a very high titer and a high specificity for a rat thyroid membrane glycoprotein fulfilling the criteria for being the rTIT. These antibodies have been used (a) to visualize the rTIT on rat thyroid tissue sections, (b) to characterize the molecular form(s) of rTIT in the rat thyroid and FRTL-5 cells, and (c) to determine the turnover of rTIT in FRTL-5 cells in relation with changes in their capacity to concentrate iodide. EXPERIMENTAL PROCEDURESMaterials-Coon's modified Ham's F-12 medium was obtained from Seromed (Biochrom KG, Berlin). Bovine TSH (2 units/mg), insulin, cortisol, transferrin, glycyl-L-histidyl-L-lysine acetate, tunicamycin, cycloheximide, and anti-rabbit IgG antibody conjugated to alkaline phosphatase ...

Immunoanalysis indicates that the sodium iodide symporter is not overexpressed in intracellular compartments in thyroid and breast cancers

“…3). The 50 kDa band is believed to correspond to partially glycosylated NIS protein as already demonstrated (30,31). Further, an additional, strong 15 kDa band was present in thyroid samples corresponding to Graves' disease.…”

Evidence for Transcriptional and Posttranscriptional Alterations of the Sodium/Iodide Symporter Expression in Hypofunctioning Benign and Malignant Thyroid Tumors

“…Treatment of thyroid membrane fractions by N-glycosidase F in the presence of SDS led to the transformation of the 75-to 80-kd hNIS protein into a main component of ϳ50 kd, likely representing the nonglycosylated (nG) hNIS polypeptide chain as found for rat NIS. 18,19 In the absence of SDS, the enzyme had a lower efficiency and generated two main intermediate forms and a low amount of the 50-kd species. The hNIS deglycosylation pattern ( Figure 6) composed of two partially glycosylated forms named pG1, pG2, and nG was reproducibly obtained.…”

The Biology of the Sodium Iodide Symporter and its Potential for Targeted Gene Delivery

“…Additional use of targeted radiosensitising drugs can lead to further synergistic interactions between radioisotopic irradiation and EBRT. [148] in vitro and in vivo pancreatic cancer functional and therapeutic Adenoviral vector expressing hNIS regulated by MUC1 promoter (Ad-MUC1-NIS) 43-fold increase in iodide uptake in vivo scintigraphy − 13% of injected iodide retained by tumours -optimal uptake at 5 hrs in vivo therapeutic effect with 131 I (>50% reduction in tumour volume) (p<0.0001) Dwyer (2007) [149] in vitro and in vivo ovarian cancer functional and therapeutic [151] in vivo medullary thyroid cancer functional and therapeutic AV vector expressing hNIS regulated by CEA promoter (Ad-CEA-NIS) IT injection -5 × 10 8 PFU iodide retention of 7.5 +/− 1.2% ID/g biological half-life − 6.1 ±0.8hrs significant reduction in tumour growth with 131 I with reduction in calcitonin levels Willhauck (2007) [152] in vitro and in vivo hepatocellular carcinoma functional and therapeutic stably NIS-tranfected murine hepa-1-6 and human hepG2 cell lines > 75% cell-death following exposure to 131 I hepG2 xenografts accumulated 15% of 123 I biological half-life of 8.4 hours tumour-growth inhibition after 55MBq of 131 I Dwyer (2005) [153] in vivo adult male beagle dogs safety and feasibility study Adenoviral vector expressing hNIS regulated by CMV promoter (Ad-CMV-NIS) 123 I scintigraphy using SPECT/CT average-absorbed dose of 23 ±42 cGy after 1 mCi 131 I average-absorbed dose of 1245.1 ±280.9 cGy…”

“…After TSH withdrawal, a reversible reduction of both intracellular cAMP levels and iodide uptake activity are observed in thyroid cell lines [24]. However, NIS protein has an unusually long half-life and remains detectable 10 days after TSH deprivation [41][42][43]. Riedel et al (2001) have reported the half-life of NIS protein to be approximately 5 days in the presence and 3 days in the absence of TSH stimulation [41].…”