Characterization of the thyroid Na ⁺ /I ⁻ symporter with an anti-COOH terminus antibody

Abstract

The Na ؉ ͞I ؊ symporter (NIS) is the plasma membrane protein that catalyzes active I ؊ transport in the thyroid, the first step in thyroid hormone biogenesis. The cDNA encoding NIS was recently cloned in our laboratory and a secondary structure model proposed, suggesting that NIS is an intrinsic membrane protein (618 amino acids; Ϸ65.2 kDa predicted molecular mass) with 12 putative transmembrane domains. Here we report the generation of a site-directed polyclonal anti-COOH terminus NIS antibody (Ab) that immunoreacts with a Ϸ87 kDa-polypeptide present in membrane fractions from a rat thyroid cell line (FRTL-5). The modelpredicted cytosolic-side location of the COOH terminus was confirmed by indirect immunof luorescence experiments using anti-COOH terminus NIS Ab in permeabilized FRTL-5 cells. Immunoreactivity was competitively blocked by the presence of excess synthetic peptide. Treatment of membrane fractions from FRTL-5 cells, Xenopus laevis oocytes, and COS cells expressing NIS with peptidyl N-glycanase F converted the Ϸ87 kDa-polypeptide into a Ϸ50 kDa-species, the same relative molecular weight exhibited by NIS expressed in E. coli. Anti-NIS Ab immunoprecipitated both the NIS precursor molecule (Ϸ56 kDa) and the mature Ϸ87 kDa form. Furthermore, a direct correlation between circulating levels of thyroidstimulating hormone and NIS expression in vivo was demonstrated.

Ras signaling through PI3K confers hormone-independent proliferation that is compatible with differentiation

“…NIS expression was not detected in cells overexpressing RasS35 by 30-fold. The broad band detected with the NIS antibody in WRT cells is virtually identical to that reported in FRTL-5 cells (Levy et al, 1997). Similar results were obtained in four experiments using RasC40 B3 cells, two experiments with RasC40 P6 cells, and in three experiments in RasS35 cells cells, TSH retains the ability to induce the expression of thyroid-speci®c genes in RasC40 cells.…”

Na + coordination at the Na2 site of the Na + /I − symporter

“…Experimental testing has confirmed this topology and revealed that the N terminus faces the extracellular milieu and the long C terminus the cytosol (14). NIS is glycosylated at three positions-N225, N489, and N502 (15) (Fig.…”

“…SDS/PAGE, electrotransference to PVDF membranes, and immunoblotting were carried out as previously described (14). Membranes were incubated with 4 nM of an affinity-purified polyclonal anti-rat NIS Ab directed against 16 residues of the cytosolic NIS carboxyl terminus.…”

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.…”