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The iodide-transport-defect-causing mutation R124H: a ?-amino group at position 124 is critical for maturation and trafficking of the Na+/I- symporter.

ABSTRACT: Na(+)/I(-) symporter (NIS)-mediated active accumulation of I(-) in thyrocytes is a key step in the biosynthesis of the iodine-containing thyroid hormones T3 and T4. Several NIS mutants have been identified as a cause of congenital I(-) transport defect (ITD), and their investigation has yielded valuable mechanistic information on NIS. Here we report novel findings derived from the thorough characterization of the ITD-causing mutation R124H, located in the second intracellular loop (IL-2). R124H NIS is incompletely glycosylated and colocalizes with endoplasmic reticulum (ER)-resident protein markers. As a result, R124H NIS is not targeted to the plasma membrane and therefore does not mediate any I(-) transport in transfected COS-7 cells. Strikingly, however, the mutant is intrinsically active, as revealed by its ability to mediate I(-) transport in membrane vesicles. Of all the amino acid substitutions we carried out at position 124 (K, D, E, A, W, N and Q), only Gln restored targeting of NIS to the plasma membrane and NIS activity, suggesting a key structural role for the ?-amino group of R124 in the transporter's maturation and cell surface targeting. Using our NIS homology model based on the structure of the Vibrio parahaemolyticus Na(+)/galactose symporter, we propose an interaction between the ?-amino group of either R or Q124 and the thiol group of C440, located in IL-6. We conclude that the interaction between IL-2 and IL-6 is critical for the local folding required for NIS maturation and plasma membrane trafficking.

SUBMITTER: Paroder V

PROVIDER: S-EPMC3730242 | biostudies-literature | 2013 Aug

REPOSITORIES: biostudies-literature

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The iodide-transport-defect-causing mutation R124H: a δ-amino group at position 124 is critical for maturation and trafficking of the Na+/I- symporter.

Paroder Viktoriya V Nicola Juan P JP Ginter Christopher S CS Carrasco Nancy N

Journal of cell science 20130520 Pt 15

Na(+)/I(-) symporter (NIS)-mediated active accumulation of I(-) in thyrocytes is a key step in the biosynthesis of the iodine-containing thyroid hormones T3 and T4. Several NIS mutants have been identified as a cause of congenital I(-) transport defect (ITD), and their investigation has yielded valuable mechanistic information on NIS. Here we report novel findings derived from the thorough characterization of the ITD-causing mutation R124H, located in the second intracellular loop (IL-2). R124H ...[more]

PMID: 23690546

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Project description:Background: The sodium/iodide (Na+/I-) symporter (NIS) mediates active transport of I- into the thyroid gland. Mutations in the SLC5A5 gene, which encodes NIS, cause I- transport defects (ITDs)-which, if left untreated, lead to congenital hypothyroidism and consequent cognitive and developmental deficiencies. The ITD-causing NIS mutation Y348D, located in transmembrane segment (TMS) 9, was reported in three Sudanese patients. Methods: We generated cDNAs coding for Y348D NIS and mutants with other hydrophilic and hydrophobic amino acid substitutions at position 348 and transfected them into cells. The activity of the resulting mutants was quantitated by radioiodide transport assays. NIS glycosylation was investigated by Western blotting after endoglycosidase H (Endo H) and PNGase-F glycosidase treatment. Subcellular localization of the mutant proteins was ascertained by flow cytometry analysis, cell surface biotinylation, and immunofluorescence. The intrinsic activity of Y348D was studied by measuring radioiodide transport in membrane vesicles prepared from Y348D-NIS-expressing cells. Our NIS homology models and molecular dynamics simulations were used to identify residues that interact with Y348 and investigate possible interactions between Y348 and the membrane. The sequences of several Slc5 family transporters were aligned, and a phylogenetic tree was generated in ClustalX. Results: Cells expressing Y348D NIS transport no I-. Furthermore, Y348D NIS is only partially glycosylated, is retained intracellularly, and is intrinsically inactive. Hydrophilic residues other than Asp at position 348 also yield NIS proteins that fail to be targeted to the plasma membrane (PM), whereas hydrophobic residues at this position, which we show do not interact with the membrane, rescue PM targeting and function. Conclusions: Y348D NIS does not reach the PM and is intrinsically inactive. Hydrophobic amino acid substitutions at position 348, however, preserve NIS activity. Our findings are consistent with our homology model's prediction that Y348 should face the side opposite the TMS9 residues that coordinate Na+ and participate in Na+ transport, and with the notion that Y348 interacts only with hydrophobic residues. Hydrophilic or charged residues at position 348 have deleterious effects on NIS PM targeting and activity, whereas a hydrophobic residue at this position rescues NIS activity.

Project description:Background: The ability of thyroid follicular epithelial cells to accumulate iodide via the sodium/iodide symporter (NIS) is exploited to successfully treat most thyroid cancers, although a subset of patients lose functional NIS activity and become unresponsive to radioiodide therapy, with poor clinical outcome. Our knowledge of NIS regulation remains limited, however. While numerous membrane proteins are functionally regulated via dimerization, there is little definitive evidence of NIS dimerization, and whether this might impact upon radioiodide uptake and treatment success is entirely unknown. We hypothesized that NIS dimerizes and that dimerization is a prerequisite for iodide uptake. Methods: Coimmunoprecipitation, proximity ligation, and Förster resonance energy transfer (FRET) assays were used to assess NIS:NIS interaction. To identify residues involved in dimerization, a homology model of NIS structure was built based on the crystal structure of the dimeric bacterial protein vSGLT. Results: Abundant cellular NIS dimerization was confirmed in vitro via three discrete methodologies. FRET and proximity ligation assays demonstrated that while NIS can exist as a dimer at the plasma membrane (PM), it is also apparent in other cellular compartments. Homology modeling revealed one key potential site of dimeric interaction, with six residues <3Å apart. In particular, NIS residues Y242, T243, and Q471 were identified as critical to dimerization. Individual mutation of residues Y242 and T243 rendered NIS nonfunctional, while abrogation of Q471 did not impact radioiodide uptake. FRET data show that the putative dimerization interface can tolerate the loss of one, but not two, of these three clustered residues. Conclusions: We show for the first time that NIS dimerizes in vitro, and we identify the key residues via which this happens. We hypothesize that dimerization of NIS is critical to its trafficking to the PM and may therefore represent a new mechanism that would need to be considered in overcoming therapeutic failure in patients with thyroid cancer.

Dataset Information

The iodide-transport-defect-causing mutation R124H: a ?-amino group at position 124 is critical for maturation and trafficking of the Na+/I- symporter.

Publications

The iodide-transport-defect-causing mutation R124H: a δ-amino group at position 124 is critical for maturation and trafficking of the Na+/I- symporter.

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