Characterisation of the purified human sodium/iodide symporter reveals that the protein is mainly present in a dimeric form and permits the detailed study of a native C-terminal fragment.

The sodium/iodide symporter is an intrinsic membrane protein that actively transports iodide into thyroid follicular cells. It is a key element in thyroid hormone biosynthesis and in the radiotherapy of thyroid tumours and their metastases. Sodium/iodide symporter is a very hydrophobic protein that belongs to the family of sodium/solute symporters. As for many other membrane proteins, particularly mammalian ones, little is known about its biochemistry and structure. It is predicted to contain 13 transmembrane helices, with an N-terminus oriented extracellularly. The C-terminal, cytosolic domain contains approximately one hundred amino acid residues and bears most of the transporter's putative regulatory sites (phosphorylation, sumoylation, di-acide, di-leucine or PDZ-binding motifs). In this study, we report the establishment of eukaryotic cell lines stably expressing various human sodium/iodide symporter recombinant proteins, and the development of a purification protocol which allowed us to purify milligram quantities of the human transporter. The quaternary structure of membrane transporters is considered to be essential for their function and regulation. Here, the oligomeric state of human sodium/iodide symporter was analysed for the first time using purified protein, by size exclusion chromatography and light scattering spectroscopy, revealing that the protein exists mainly as a dimer which is stabilised by a disulfide bridge. In addition, the existence of a sodium/iodide symporter C-terminal fragment interacting with the protein was also highlighted. We have shown that this fragment exists in various species and cell types, and demonstrated that it contains the amino-acids [512-643] from the human sodium/iodide symporter protein and, therefore, the last predicted transmembrane helix. Expression of either the [1-512] truncated domain or the [512-643] domain alone, as well as co-expression of the two fragments, was performed, and revealed that co-expression of [1-512] with [512-643] allowed the reconstitution of a functional protein. These findings constitute an important step towards an understanding of some of the post-translational mechanisms that finely tune iodide accumulation through human sodium/iodide symporter regulation.

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

OBJECTIVE: The active transport of iodide into thyroid cells is mediated by the Na(+)/I(-) symporter (NIS) located in the basolateral membrane. Strong intracellular staining with anti-NIS antibodies has been reported in thyroid and breast cancers. Our initial objective was to screen tumour samples for intracellular NIS staining and then to study the mechanisms underlying the altered subcellular localization of the transporters. METHODS: Immunostaining using three different anti-NIS antibodies was performed on paraffin-embedded tissue sections from 93 thyroid or breast cancers. Western blot experiments were carried out to determine the amount of NIS protein in 20 samples. RESULTS: Using three different anti-NIS antibodies, we observed intracellular staining in a majority of thyroid tumour samples. Control immunohistochemistry and western blot experiments indicated that this intracellular staining was due to non-specific binding of the antibodies. In breast tumours, very weak intracellular staining was observed in some samples. Western blot experiments suggest that this labelling is also non-specific. CONCLUSIONS: Our results strongly indicate that the NIS protein level is low in thyroid and breast cancers and that the intracellular staining obtained with anti-NIS antibodies corresponds to a non-specific signal. Accordingly, to increase the efficiency of radiotherapy for thyroid cancers and to enable the use of radioiodine in the diagnosis and therapy of breast tumours, improving NIS targeting to the plasma membrane will not be sufficient. Instead, increasing the expression level of NIS should remain the major goal of this field.

Comparison of expressed human and mouse sodium/iodide symporters reveals differences in transport properties and subcellular localization.

The active transport of iodide from the bloodstream into thyroid follicular cells is mediated by the Na+/I- symporter (NIS). We studied mouse NIS (mNIS) and found that it catalyzes iodide transport into transfected cells more efficiently than human NIS (hNIS). To further characterize this difference, we compared (125)I uptake in the transiently transfected human embryonic kidney (HEK) 293 cells. We found that the V(max) for mNIS was four times higher than that for hNIS, and that the iodide transport constant (K(m)) was 2.5-fold lower for hNIS than mNIS. We also performed immunocytolocalization studies and observed that the subcellular distribution of the two orthologs differed. While the mouse protein was predominantly found at the plasma membrane, its human ortholog was intracellular in approximately 40% of the expressing cells. Using cell surface protein-labeling assays, we found that the plasma membrane localization frequency of the mouse protein was only 2.5-fold higher than that of the human protein, and therefore cannot alone account for the difference in the obtained V(max) values. We reasoned that the observed difference could also be caused by a higher turnover number for iodide transport in the mouse protein. We then expressed and analyzed chimeric proteins. The data obtained with these constructs suggest that the iodide recognition site could be located in the region extending from the N-terminus to transmembrane domain 8, and that the region between transmembrane domain 5 and the C-terminus could play a role in the subcellular localization of the protein.

Internalization-dependent regulation of HT29 cell proliferation by neurotensin.

In this study, we have investigated the involvement of the internalization process induced by neurotensin (NT) on MAP kinases Erk1/2 activation, inositol phosphates (IP) accumulation and cell growth in the human colonic cancer cell line HT29. Reversible blocking of NT/neurotensin receptor (NTR) complex endocytosis by hyperosmolar sucrose totally abolished both the phosphorylation of the MAP kinases Erk1/2 and the [3H]-thymidine incorporation induced by the peptide. By contrast, NT-evoked IP formation was not affected by sucrose treatment. These results therefore indicate that NT/NTR complex endocytosis triggers MAP kinase activation and subsequently the growth of HT29 cells. This property could be useful for the development of novel anticancer treatments.

Shedding of the luminal domain of the neurotensin receptor-3/sortilin in the HT29 cell line.

The neurotensin (NT) receptor-3/sortilin (NTR3) belongs to the new receptor family of VPS10P domain containing receptors. The NTR3 is expressed in all cancer cells on which NT activates cell growth and its cellular location is mainly intracellular within the endoplasmic reticulum and the trans-Golgi network. However, the NTR3 is also present at the cell surface of the HT29 cell line from which it is released by a mechanism activated by phorbol 12-myristate 13-acetate (PMA). The shedding of the NTR3 is sensitive to protein kinase C (PKC) and mitogen-activated protein (MAP) kinase inhibitors and to 1,10-phenanthroline and BB3103, suggesting the activation of zinc-metalloproteases and the ADAM10 (a desintegrin and metalloprotease). The shedding of the membrane NTR3 leads to a soluble protein able to bind exogenous NT, suggesting a role of this process in the biological activity of the peptide.

Neurotensin receptor-1 and -3 complex modulates the cellular signaling of neurotensin in the HT29 cell line.

BACKGROUND & AIMS: The neuropeptide neurotensin (NT) exerts its intracellular effect by interacting with 3 different receptors. Two of these receptors (NTR1 and NTR2) belong to the G protein-coupled receptor family, whereas the third one (NTR3) is a type I receptor with a single transmembrane domain. We recently showed that the 2 structurally different receptors NTR1 and NTR3 were coexpressed in several human cancer cells on which NT exerts proliferative effects. METHODS: Here, by an immunoprecipitation approach, we provide biochemical evidence for an endogenous heterodimerization of the G protein-coupled receptor NTR1 with the NTR3 in the human adenocarcinoma cell line HT29. RESULTS: We show that both receptors are expressed and colocalized within the cell surface of HT29 cells where they already interact to form a heterodimer. The NTR1-NTR3 complex is then internalized on NT stimulation. CONCLUSIONS: The complex formed between these 2 structurally unrelated NT receptors modulates both the NT-induced phosphorylation of mitogen-activated protein kinases and the phosphoinositide (PI) turnover mediated by the NTR1.