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. 2019 Oct;29(10):1485-1498.
doi: 10.1089/thy.2019.0034. Epub 2019 Aug 30.

Dimerization of the Sodium/Iodide Symporter

Affiliations

Dimerization of the Sodium/Iodide Symporter

Rebecca J Thompson et al. Thyroid. 2019 Oct.

Abstract

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.

Keywords: NIS; dimerization; radioiodide uptake; thyroid.

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Conflict of interest statement

No competing financial interests exist.

Figures

<b>FIG. 1.</b>
FIG. 1.
(A) Cells were transfected with empty pcDNA3.1+ vector (VO) or differentially tagged NIS variants and radioiodide uptake assays performed. Significant radioiodide uptake compared with VO was observed with both MYC- and HA-tagged NIS variants. Pretreatment with sodium perchlorate (NaClO4) to inhibit NIS (gray bars) demonstrates NIS-specific iodide uptake in black bars. Bars show mean radioiodide uptake in pmol 125I/μg protein ± SEM. (B) Representative co-IP assays demonstrating direct interaction between NIS-MYC and NIS-HA in SW1736 and HeLa cells. Cells were transfected with VO (lane 1), VO+NIS-MYC (lane 2), VO+NIS-HA (lane 3), or NIS-MYC+NIS-HA (lanes 4 and 5) and lysed. Co-IP was performed on all lysates (except no antibody control, lane 4) with mouse anti-MYC (i) or rabbit anti-HA (ii). Western blot using antibody to the other tag detected the presence of the other NIS variant, demonstrating a direct interaction between the two NIS variants (co-IP blots, top panel). (C) PLAs with and without DAPI staining for nuclei (blue) demonstrating close proximity (<40 nm) between NIS-MYC and NIS-HA throughout the cell, including the PM. (D) PLAs with Na+K+ATPase immunofluorescent staining. Areas of colocalization (yellow; white arrowheads) between PLA signal generated by close proximity of NIS-MYC and NIS-HA (in red) and the PM marker Na+K+ATPase (in green) in representative SW1736 (left) and HeLa (right) cells were apparent. *p < 0.05. co-IP, coimmunoprecipitation; NIS, sodium/iodide symporter; PLAs, proximity ligation assays; PM, plasma membrane; SEM, standard error of the mean.
<b>FIG. 2.</b>
FIG. 2.
(A) SW1736 (left) and HeLa (right) cells were transfected with VO or one differentially tagged NIS variant and radioiodide uptake assays were performed. Pretreatment with sodium perchlorate (w. NaClO4) was used to specifically inhibit NIS activity (gray bars). (B) Representative images from FRET analysis of SW1736 cells transfected with cerulean+citrine (Cer+Cit, negative control) and NIS-cerulean+NIS-citrine (NIS-Cer+NIS-Cit; left). Rightcoexpression of both NIS-fluorophore constructs gave significantly greater corrected FRET signals than the negative control, demonstrating a close proximity between the two fluorophores that is indicative of dimerization. Bars show mean corrected FRET signal in arbitrary units and error bars show SEM. One-way ANOVA with Tukey's multiple comparison statistical tests was performed: **p < 0.01, ****p < 0.001 (n = 5 with 10 individual cells in each n). (C) SW1736 (left) and HeLa (right) cells were transfected with NIS-cerulean+NIS-citrine and FRET analysis was performed on linear sections across the cell (image). Arrowheads indicate examples of prominent regions of NIS dimerization at the PM. Graphs depict the corrected FRET signal across the lines, suggesting that NIS dimerization occurs throughout the cell (n = 5 cells measured per experiment). *p < 0.05. FRET, Förster resonance energy transfer.
<b>FIG. 3.</b>
FIG. 3.
(A) Eight novel residues were identified as potentially involved in NIS dimerization using the refined NIS homology model, appearing to form two distinct putative dimerization interfaces, a larger (“primary”) region circled in red and smaller (“secondary”) region in blue. Side-chains of residues found to be <3Å from the opposing monomer are shown in “Stick” form, and monomers are colored cyan or magenta. Structures are viewed in the membrane plane oriented so the extracellular milieu is above the structure. Amino and carboxy termini are labeled (N and C, respectively). Insetmagnified regions of closest interaction. (B) Residues known to be involved in sodium or iodide binding and are shown in “Stick” form and colored in magenta and cyan, respectively, while remaining residues are colored in gray. Structures are viewed in the membrane plane oriented so the extracellular milieu is above the dimer. (C) The putative dimeric structure viewed from the extracellular side.
<b>FIG. 4.</b>
FIG. 4.
(A) Mutating residues within the putative dimerization interface alter NIS glycosylation. Western blots were performed on SW1736 and HeLa cell lysates with HA-tagged WT and mutant NIS expression using the mouse anti-NIS antibody. A band at 75–100 kDa represents the fully glycosylated mature form of NIS, while a band at ∼60 kDa represents the nonglycosylated immature form of NIS. (B) NIS localization of HA-tagged NIS mutation of individual residues of the putative dimerization interface. Immunofluorescence analysis was performed using the mouse anti-HA primary antibody to visualize NIS (green), while the rabbit anti-Na+/K+/ATPase primary antibody was used as a PM marker (red). Nuclei are visualized in blue using Hoechst stain, 40 × magnification. (C) Radioiodide uptake is abolished when residues within the putative dimerization interface are mutated, with the exception of Q471A, in SW1736 and HeLa cells. Bars represent mean relative radioiodide uptake relative to WT ± SEM. *p < 0.005, **p < 0.0005, and ***p < 0.0001 compared with WT (n = 3 with 4 replicates in each n). (D) PLA signal is still observed in HeLa cells when single residues in the putative dimerization interface are mutated, 40 × magnification. (E) Corrected FRET values when residues of the putative dimerization interface were mutated. Data presented as box and whisker plots, with the center line indicating the mean corrected FRET value (in arbitrary units), the box representing the 25th and 75th percentiles, and the whiskers denoting the minimum and maximum values. *p < 0.05. n = 2, with a minimum of 20 cells analyzed in each n. WT, wild type.
<b>FIG. 5.</b>
FIG. 5.
(A) Detailed examination of the primary putative dimerization interface. Two distinct clusters of residues are particularly close and are shown in different planes of view. Cluster 1 (circled in brown) comprises Q471 (red) of the cyan monomer and Y242 (yellow) and T243 (orange) of the opposite magenta monomer. Cluster 2 (circled in gray) comprises A525 (green) of the cyan monomer and D237 (blue) and potentially Y242 of the opposite magenta monomer. (B) FRET analysis of combined putative dimerization interface mutants in SW1736 and HeLa cells cotransfected with differentially tagged (either cerulean or citrine) variants of WT NIS or a combination of two NIS mutants (n = 2, with a minimum of 20 cells analyzed in each n). Representative images captured during FRET analysis are shown. Three concurrent versions of each image were taken: total cerulean (CFP) image (cerulean excitation/cerulean emission [CFP/CFP]), total citrine (YFP) image (YFP excitation/YFP emission [YFP/YFP]), and the FRET image (cerulean excitation/citrine emission [CFP/YFP]). (C) Reduced PLA signal was observed with combinations of mutations within the NIS putative dimerization interface in HeLa cells, 40 × magnification. (D) Radioiodine uptake in SW1736 cells in response to various combinations of cotransfected WT and mutant NIS. As indicated, each bar represents the possibility of multiple simultaneous monomeric and dimeric NIS species. **p < 0.01, compared with WT:WT. N = 3 experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

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