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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2005-0895
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 11 6131-6140
Copyright © 2005 by The Endocrine Society

Cell Surface Targeting Accounts for the Difference in Iodide Uptake Activity between Human Na+/I– Symporter and Rat Na+/I– Symporter

Zhaoxia Zhang, Yu-Yu Liu and Sissy M. Jhiang

Ohio State Biochemistry Program, Departments of Physiology and Cell Biology and Internal Medicine, Ohio State University College of Medicine, Columbus, Ohio 43210

Address all correspondence and requests for reprints to: Dr. Sissy M. Jhiang, 304 Hamilton Hall, 1645 Neil Avenue, Ohio State University, Columbus, Ohio 43210. E-mail: jhiang.1{at}osu.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Context: The Na+/I– symporter (NIS) has been proposed to serve as an imaging reporter gene to optimize vector delivery, monitor therapeutic gene expression, and map the tissue/organ sites of repopulated progenitor cells in vivo. In addition, NIS can serve as a therapeutic gene to facilitate targeted radionuclide therapy for various cancers.

Objective: It was reported that rat NIS (rNIS) confers higher radioactive iodide uptake (RAIU) activity than human NIS (hNIS). We aim to investigate the mechanism underlying this difference.

Results: We showed that the open reading frames (ORF) of hNIS and rNIS, although encoding for proteins with 83% amino acid identity, exhibit a significant difference in RAIU activity in transfected cells. The ORF rNIS confers four to five times higher RAIU activity as well as cell surface NIS accumulation than ORF hNIS despite similar total NIS protein levels. Multiple regions appear to play roles in the difference in NIS cell surface levels between ORF hNIS and ORF rNIS, indicating that proper folding of NIS in tertiary structure is critical for NIS cell surface targeting. We also showed that the kinetics of Na+ binding are different between ORF hNIS and ORF rNIS, and that site-directed mutation changing Ser200 to other uncharged amino acid significantly increased RAIU activity in ORF hNIS.

Conclusions: NIS transgene could be optimized for cell surface trafficking and RAIU activity to improve its clinical applications.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE Na+/I– SYMPORTER (NIS) is a transmembrane glycoprotein that mediates active iodide uptake into thyroid follicular cells. As a member of the sodium/solute symporter family (SSSF) (1), NIS effectively transports iodide against its concentration gradient from the bloodstream into thyroid follicular cells. Iodide uptake is coupled with Na+ transport down its electrochemical gradient, which is maintained by the activity of Na+,K+-adenosine triphosphatase. Electrophysiological analysis of NIS revealed that two sodium ions are cotransported with one iodide ion, suggesting a 2:1 Na+/I– stoichiometry (2). The predicted membrane topology suggests that NIS contains 13 putative transmembrane domains with an extracellular NH2 terminus and a cytoplasmic COOH terminus (3, 4).

Currently, NIS cDNA sequence has been reported from four species: human (5), rat (6), mouse (7), and porcine (8). Among them, rat NIS (rNIS) and mouse NIS have the highest homology, sharing 95.5% amino acid identity. In comparison, rNIS and human NIS (hNIS), which encode proteins containing 618 and 643 amino acids, respectively, share 83% amino acid identity (Fig. 1Go). Interestingly, it was reported that rNIS confers higher radioactive iodide uptake (RAIU) activity than hNIS (9). In this study, we aim to investigate the mechanism underlying this difference. We showed that cDNA coding for the open reading frame (ORF) of rNIS confers 4- to 5-fold higher RAIU activity than ORF hNIS. Despite similar total NIS protein levels, NIS cell surface level in cells expressing ORF rNIS is 4- to 5-fold higher than ORF hNIS. This finding suggests that the difference in RAIU activity between ORF rNIS and ORF hNIS is mainly due to the difference in NIS cell surface targeting. Multiple regions appear to play roles in the difference in NIS cell surface levels between ORF hNIS and ORF rNIS, indicating that proper folding of NIS in tertiary structure is critical for NIS cell surface accumulation. Finally, we showed that rNIS and hNIS exert a difference in Na+ binding kinetics, and that site-directed mutation changing Ser200 to other uncharged amino acid significantly increased radioiodide uptake activity in ORF hNIS.



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  		FIG. 1. Alignment of amino acid sequences of hNIS and rNIS. The bold areas indicate putative transmembrane domains. Identical amino acids are marked with an asterisk. Gaps, indicated by a hyphen, were created to maximize the alignment.

 
NIS-mediated iodide uptake in thyroid cells is the basis for targeted radionuclide imaging and treatment of differentiated thyroid carcinomas and their metastases (10, 11). Furthermore, NIS expression is increased in the majority of human breast tumors, suggesting that an additional increase in NIS expression/function in breast tumors may enable targeted radionuclide imaging and therapy for patients with breast cancer (12, 13). Finally, we and others have shown that forced expression of exogenous NIS induces radioiodide accumulation in various malignant nonthyroidal cells, suggesting that NIS may serve as an imaging reporter gene to optimize vector delivery, monitor therapeutic gene expression, and map the tissue/organ sites of repopulated progenitor cells in clinical trials (14, 15, 16, 17). In addition, NIS can serve as a therapeutic gene to facilitate targeted radionuclide therapy for various cancers (17, 18, 19, 20). Our study indicates that NIS transgene could be optimized for cell surface trafficking and radioiodide uptake activity to improve its clinical applications.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
NIS cDNA constructs

Table 1Go summarized all primers used to generate various NIS cDNA constructs. ORF hNIS cDNA was PCR amplified using pcDNA3-FLhNIS (5) as a template. ORF rNIS cDNA was amplified by RT-PCR using mRNA isolated from rat thyroid PC CL3 cells. N-Flag was added on ORF hNIS and ORF rNIS to form Flag-hNIS and Flag-rNIS, respectively. All PCR products were cloned into the TA cloning vector pCR2.1 (Invitrogen Life Technologies, Inc., Carlsbad, CA) and then subcloned into pcDNA3 vector. The nucleotide sequences of all NIS cDNA constructs were verified by automated sequencing.


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  		TABLE 1. Primers used for PCR and site-directed mutagenesis

 
All chimeras were generated by fragment exchange between Flag-hNIS and Flag-rNIS using introduced restriction sites: HpaI [situated at 783 nucleotides (nt) of ORF hNIS and ORF rNIS], SunI (situated at 384 nt of ORF hNIS and ORF rNIS), and XhoI (situated at 621 nt of ORF hNIS and at 600 nt of ORF rNIS). ORF hNIS mutants containing mutation F176L, A180T, S200V, S200A, S200G, S200T, S200D, or S200R were generated by site-directed mutagenesis (Table 1Go).

RAIU assay

Monkey kidney COS-7 cells were maintained in DMEM with 10% fetal bovine serum and 1% penicillin-streptomycin. COS-7 cells were seeded in 24-well plates for approximately 24 h. Transfection was performed using FuGene 6 with 0.5 µg of various NIS constructs. Cell number counting and RAIU assay (5) were performed at 48 h after transfection. Briefly, cells were incubated with 2.0 µCi Na125I in 5 µM nonradioactive NaI for 30 min at 37 C with 5% CO2. Cells were then washed twice with cold Hanks’ balanced salt solution and lysed with cold 95% ethanol for 20 min. The cell lysate was collected, and radioactivity was counted by a {gamma}-counter (Packard Instruments, Downers Grove, IL). For I–-dependent kinetic analysis (21), cells were incubated for 2 min with various concentration of NaI (0–600 µM) containing Na125I with a specific activity of 80 mCi/mmol, and the amount of accumulated iodide was measured as described above. The Km and maximum velocity (Vmax) values for I– were derived from a fitted Michaelis-Menten equation according to the Eadie-Hofstee plot. For Na+-dependent kinetic analysis (21), cells were incubated for 2 min with various concentration of NaCl (0–150 mM), and isotonicity was maintained constant with choline chloride. The Km and Vmax values were derived using the equation v = Vmax x [Na+]2/(Km + [Na+]2).

Flow cytometric analysis for cell surface NIS and total NIS protein levels

COS-7 cells were seeded in 100-mm dishes for 24 h, and transfection was performed using FuGene 6 with 5 µg of various NIS constructs. Cells were harvested by 0.05% trypsin at 48 h after transfection. To detect cell surface NIS protein levels, flow cytometric analysis (22, 23) was performed using nonpermeabilized cells with 1:50 diluted mouse anti-FLAG monoclonal antibody M2 (Sigma-Aldrich Corp., St. Louis, MO) that recognizes extracellular FLAG peptide, followed by 1:100 diluted fluorescein isothiocyanate-conjugated goat antimouse IgG (Sigma-Aldrich Corp.). Cells were then washed and resuspended in fixative buffer (1% paraformaldehyde in PBS) for analysis by a FACSCalibur flow cytofluorometer (BD Biosciences, Eerenbodegem, Belgium). Secondary antibody alone was used as a negative control for nonspecific fluorescence. To detect total NIS protein levels, cells were fixed with 1% paraformaldehyde for 10 min on ice and permeabilized with 0.2% saponin for 30 min. All subsequent steps were performed as described above, except including 0.2% saponin in all solutions. For quantification, a positive events region was gated at a fluorescence density that had less than 1% of cells with nonspecific fluorescence. Total NIS protein levels and NIS cell surface levels were quantified by multiplying the percentage of cells by the mean fluorescence density in the gated region (Fig. 2Go).



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  		FIG. 2. ORF rNIS confers higher RAIU activity and cell surface NIS accumulation than ORF hNIS. A, RAIU assay of COS-7 cells transfected with pcDNA3 vector, ORF hNIS, Flag-hNIS, ORF rNIS, or Flag-rNIS. The results are representative of three independent experiments. Each data point was determined in triplicate, and the mean ± SD are shown. *, Statistically significant difference (P < 0.05). B, Flow cytometric analysis of NIS cell surface levels and total NIS protein levels. The NIS cell surface level was detected in nonpermeabilized cells (upper panel), and the total NIS protein level was measured in permeabilized cells (lower panel). Cells were incubated with fluorescein isothiocyanate-labeled goat antimouse antibody alone for nonspecific fluorescence (pink dashed line) or with mouse M2 anti-FLAG monoclonal antibody, followed by fluorescein isothiocyanate-labeled goat antimouse antibody (green solid line). For quantification, the positive events region was gated at a fluorescence density that had less than 1% of cells with nonspecific fluorescence (shown as a horizontal line). C, Total NIS protein detected by Western blot analysis. Membrane fractions (5 µg/lane) of cells transfected with vector pcDNA3, Flag-hNIS, or Flag-rNIS were subjected to SDS-PAGE, and Flag-hNIS/Flag-rNIS was detected using mouse M2 anti-FLAG monoclonal antibody. D, Subcellular localization of NIS protein by indirect immunofluorescence. Permeabilized cells expressing Flag-hNIS or Flag-rNIS were incubated with mouse M2 anti-FLAG monoclonal antibody, followed by CyTM3-labeled antimouse antibody. Representative immunofluorescence (IF) images are shown in the upper panel, and the corresponding differential interference contrast (DIC) images are shown in the lower panel.

 
Western blot analysis for total NIS protein levels

COS-7 cells were seeded and transfected as described above. Membrane fraction was isolated at 48 h after transfection and subjected to 7.5% SDS-PAGE (5 µg/lane). The proteins were transferred to nitrocellulose membrane and then incubated with mouse anti-FLAG monoclonal antibody M2 (1:2000) for 1 h at room temperature, followed by incubation with horseradish peroxidase-conjugated antimouse IgG (1:3000) for 1 h at room temperature. The membrane was incubated with the enhanced chemiluminescence detection reagents for 1 min and exposed to x-ray film. Coomassie staining was performed to confirm equal loading.

Indirect immunofluorescence for NIS subcellular localization

COS-7 cells were seeded on 12-mm coverslips for 24 h and transfected as described above. Cells were fixed in 4% paraformaldehyde for 30 min at 48 h after transfection and then permeabilized with PBS containing 0.1% saponin, 0.1% BSA, and 0.02% NaN3. Cells were incubated with mouse anti-FLAG monoclonal antibody M2 (1:500) in permeabilization buffer overnight at 4 C and stained with CyTM3-conjugated donkey antimouse IgG antibody (1:700; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) in PBS for 30 min. Fluorescence and differential interference contrast images were obtained with a Nikon (Melville, NY) Optiphot microscope equipped with a Photometrics Cool Snap fx CCD camera (Roper Scientific, Tucson, AZ).

Statistical analysis

Statistical comparison of radioactive iodide uptake was performed using paired t test. Differences were considered significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
ORF rNIS confers higher RAIU activity and cell surface NIS accumulation than ORF hNIS

ORF hNIS and ORF rNIS were transiently transfected into COS-7 cells, and NIS functional activity was measured by RAIU assay. We found that ORF rNIS confers 4- to 5-fold higher RAIU activity than ORF hNIS (Fig. 2AGo). To determine whether the difference in RAIU activity is due to differences in total NIS protein and/or cell surface NIS protein levels, FLAG was tagged at the N terminus of ORF hNIS and ORF rNIS (Flag-hNIS and Flag-rNIS). Consistent with the finding of the difference in RAIU between ORF hNIS and ORF rNIS, Flag-rNIS confers 4- to 5-fold higher RAIU activity than Flag-hNIS. Using the monoclonal anti-FLAG antibody M2, cell surface NIS and total NIS protein levels were evaluated by flow cytometric analysis in nonpermeabilized and permeabilized transfected COS-7 cells, respectively. Interestingly, cell surface NIS was about 4- to 5-fold higher in cells expressing Flag-rNIS than in cells expressing Flag-hNIS, yet there was not much difference in their total NIS protein levels (Fig. 2BGo). Indeed, Western blot analysis confirmed that the total NIS protein levels were similar in cells expressing Flag-hNIS and Flag-rNIS (Fig. 2CGo), and immunofluorescence staining showed that Flag-rNIS, but not Flag-hNIS, was readily detectable at the cell periphery (Fig. 2DGo). The difference in RAIU activity and cell surface NIS accumulation between Flag-hNIS and Flag-rNIS appeared to be cell type independent, because the difference was also found in transfected HEK 293 and HeLa cells (data not shown).

Differences in RAIU activity and cell surface NIS accumulation between rNIS and hNIS are mostly contributed by the region between the N terminus and putative transmembrane domain 7 (TM7)

To identify the regions responsible for the differences in RAIU and cell surface NIS accumulation between hNIS and rNIS, we first engineered two chimeras, h(N-TM7)-r(TM8-C)NIS and r(N-TM7)-h(TM8-C)NIS. The h(N-TM7)-r(TM8-C)NIS chimera is composed of the N-terminal half of hNIS and the C-terminal half of rNIS, and vice versa for the chimera r(N-TM7)-h(TM8-C)NIS. As shown in Fig 3BGo, although there was no change in total NIS protein level, both RAIU and cell surface NIS levels were significantly increased in the chimera r(N-TM7)-h(TM8-C)NIS compared with hNIS. These results suggest that the differences in RAIU and cell surface NIS accumulation between rNIS and hNIS are mostly contributed by the region between the N terminus and putative TM7.



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  		FIG. 3. The differences in RAIU activity and cell surface NIS accumulation between rNIS and hNIS are mostly contributed by the region between the N terminus and putative TM7, yet the region of TM4–6 contributes more differences in RAIU activity than cell surface NIS accumulation. A, Schematic diagram indicating interchanged regions. B, Summary of flow cytometric analysis, evaluating total NIS protein levels and NIS cell surface levels as well as RAIU assay in cells expressing various chimeric constructs. Flow cytometric analysis was performed as described in Fig. 2Go. Total NIS protein levels and NIS cell surface levels were quantified by multiplying the percentage of cells with the mean of fluorescence density in the gated region. hNIS was arbitrarily set at 1.0, and various NIS constructs are shown relative to hNIS. Most experiments were performed at least twice, and the average and the range of variation are shown.

 
The region between putative TM4 and TM6 contributes more differences in RAIU activity than cell surface NIS accumulation between hNIS and rNIS

The region between the N terminus and putative TM7 was further divided into three regions (Fig. 3AGo), and six chimeras were engineered. The chimera r(N-TM3)-h(TM4-C) is composed of the region from the N terminus to the putative TM3 of rNIS and the region from the putative TM4 to C-terminus of hNIS, and vice versa for the chimera h(N-TM3)-r(TM4-C). The chimera h(N-TM3)-r(TM4-TM6)-h(TM6-C) is composed mostly of hNIS, except that the region between putative TM4 and TM6 was replaced by the corresponding region of rNIS, and vice versa for the chimera r(N-TM3)-h(TM4-TM6)-r(TM6-C). The chimera h(N-TM6)-r(TM6-TM7)-h(TM8-C) is composed mostly of hNIS, except that the region between putative TM6 and TM7 was replaced by the corresponding region of rNIS, and vice versa for the chimera r(N-TM6)-h(TM6-TM7)-r(TM8-C).

As shown in Fig. 3BGo, both regions N-TM3 and TM4-TM6 contribute to the difference in cell surface NIS levels between hNIS and rNIS. However, the region of TM4-TM6 contributes to the difference in RAIU more than to the difference in cell surface NIS levels between hNIS and rNIS. The region of TM6-TM7 appeared to play no role in the difference in RAIU or cell surface NIS level between hNIS and rNIS.

Site-directed mutation changing Ser200 to other uncharged amino acids in hNIS leads to increased RAIU activity

In the region of TM4-TM6, only five amino acid residues are different between hNIS and rNIS (Fig. 4AGo). The residues I138, F176, A180, S200, and V205 in hNIS are replaced by L138, L176, T180, V200, and I205 in rNIS, respectively. Because there is no difference in polarity and the difference in the sizes of the side chains is minimal between Ile and Leu or between Val and Ile, we choose to focus on the possible effects of site-directed mutations F176L, A180T, and S200V on hNIS. We showed that the S200V mutation in hNIS leads to most significant increase in RAIU activity (Fig. 4BGo). In addition, the RAIU activity of the triple-mutant hNIS (F176L, A180T, and S200V) was not significantly different from that of hNIS (S200V; data not shown). This finding suggests that Ser200 in hNIS is the major determinant for the difference in RAIU activity contributed by the region of TM4–6. However, the reverse mutation V200S in rNIS fails to decrease RAIU activity in rNIS (data not shown), indicating that amino acid residue 200 may interact with other regions/residues to account for the difference in RAIU activity within the region of TM4–6.



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  		FIG. 4. Site-directed mutation changing Ser200 to other uncharged amino acid in hNIS leads to increased RAIU activity. A, Schematic diagram showing the differences in amino acid residues in the region of TM4–6 between hNIS and rNIS. B, Summary of flow cytometric analysis, evaluating total NIS protein levels and NIS cell surface levels as well as RAIU assay in cells expressing hNIS mutant F176L, A180T, S200V, S200A, S200G, S200T, S200D, or S200R. Flow cytometric analysis was performed as described in Fig. 2Go. hNIS was arbitrarily set at 1.0 and various NIS constructs are shown relative to hNIS. Most experiments were performed at least twice, and the average and the range of variation are shown.

 
In addition to changing Ser200 to Val (a small nonpolar residue), changing Ser200 to Ala (a nonpolar and {alpha}-helix-stabilizing residue), Gly (a small uncharged polar residue), or Thr (an uncharged polar residue) results in significantly increased RAIU activity in hNIS (Fig. 4BGo). However, changing Ser200 to Asp (a negatively charged residue) or Arg (a positively charged residue) results in loss of RAIU activity in hNIS (Fig. 4BGo). The loss of RAIU activity in these two mutants is due to significantly decreased cell surface NIS levels (Fig. 4BGo). Taken together, amino acid residue 200 appears to play an important role in NIS structural and functional integrity.

There are 25 Ser residues located within the putative TM domains of hNIS, and 20 of them are conserved in rNIS (Fig. 1Go). To investigate whether Ser200 in putative TM6 is uniquely important for NIS structural and functional integrity, we performed site-directed mutagenesis to generate mutants hNIS(S64A), hNIS(S98A), hNIS(S294A), and hNIS(S456A). Interestingly, none of these mutants had altered RAIU activity (data not shown). These results also indicate that Ser200 plays a unique role in NIS structural and functional integrity.

The kinetics of Na+ binding, but not I– binding, are different in hNIS and rNIS

To further investigate the mechanisms underlying the difference in RAIU activity between rNIS and hNIS, the kinetic properties of I– uptake in COS-7 cells expressing ORF hNIS and ORF rNIS were analyzed. As shown in Fig. 5AGo, the Vmax-I– of rNIS is four to five times higher than that of hNIS. This is in agreement with the finding that the NIS cell surface level is about four to five times higher in rNIS than hNIS. The Km-I– for hNIS and rNIS are 132 and 122 µM, respectively, which are not significantly different. This finding suggests that the difference in RAIU between hNIS and rNIS is not mainly due to a difference in I– binding affinity. Indeed, the extent of difference in RAIU activity between hNIS and rNIS is proportional to that in NIS cell surface levels.



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  		FIG. 5. The kinetics of Na+ binding, but not I– binding, are different between hNIS and rNIS. A, Kinetics analysis of iodide uptake on I–, showing that the Vmax of ORF rNIS is four to five times higher than that of ORF hNIS. Initial velocities of iodide uptake were determined at various concentrations of iodide ranging from 0–600 µM. The results are representative of two independent experiments. Each data point was determined in triplicate, and the mean ± SD are shown. The data were plotted according to Eadie-Hofstee. The Vmax-I– of hNIS and rNIS are 29,310 and 122,470 cpm/105 cells·2 min, respectively. The Km-I– for hNIS and rNIS are 132 and 122 µM, respectively. B, Kinetics analysis of iodide uptake on Na+, indicating that Na+ binding kinetics are different between ORF hNIS and ORF rNIS. Initial velocities of iodide uptake were determined at various concentrations of sodium ranging from 0–150 mM. The results are representative of two independent experiments. Each data point was determined in triplicate, and the mean ± SD are shown. The data points acquired from cells expressing ORF rNIS could be well fitted to the Michaelis-Menten equation, and the Vmax-Na+ and Km-Na+ for ORF rNIS were 26,759.93 ± 4,066.88 cpm/105 cells·2 min and 22.06 ± 5.08 mM, respectively. The data points acquired from cells expressing ORF hNIS did not fit into the Michaelis-Menten equation, and initial rate of I– uptake could not reach a plateau. C, Na+ binding kinetics of chimera h(N-TM3)-r(TM4-TM6)-h(TM6-C) and hNIS(S200V) were more similar to ORF rNIS. Kinetic properties of Na+-dependent I– uptake in cells expressing chimera h(N-TM3)-r(TM4-TM6)-h(TM6-C) and hNIS(S200V) were more similar to those of ORF rNIS than those of ORF hNIS. Vmax-Na+ and Km-Na+ for h(N-TM3)-r(TM4-TM6)-h(TM6-C) were 26,528.50 ± 1,447.39 cpm/105 cells·2 min and 36.40 ± 1.05 mM, respectively. Vmax-Na+ and Km-Na+ for hNIS(S200V) were 20,207.26 ± 1,401.22 cpm/105 cells·2 min and 35.20 ± 3.04 mM, respectively.

 
The kinetic properties of Na+-dependent I– uptake in COS-7 cells expressing ORF hNIS and ORF rNIS were also investigated. As shown in Fig. 5BGo, the data points acquired from cells expressing rNIS was fitted nicely to the Michaelis-Menten equation, and the Vmax-Na+ and Km-Na+ for rNIS were determined to be 26,759.93 ± 4,066.88 cpm/105 cells·2 min and 22.06 ± 5.08 mM, respectively. The value of Km-Na+ for rNIS acquired in this study is very similar to other published data (21). Surprisingly, the data points acquired from cells expressing hNIS did not fit the Michaelis-Menten equation, and initial rate of I– uptake could not reach a plateau even at 200 mM Na+ (data not shown). In comparison, the kinetic properties of Na+-dependent I– uptake in cells expressing chimera h(N-TM3)-r(TM4-TM6)-h(TM6-C) and hNIS(S200V) are very similar to those of rNIS than hNIS (Fig. 5CGo). Taken together, our data indicate that the region of putative TM4-TM6, in particular the amino acid residue 200, may be directly or indirectly involved in Na+ binding.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study we showed that the difference in RAIU activity between ORF hNIS and ORF rNIS is mainly due to the difference in NIS cell surface level despite similar total NIS protein levels. Multiple regions appear to play roles in the difference in NIS cell surface levels between ORF hNIS and ORF rNIS, suggesting that proper folding of NIS in tertiary structure is critical for NIS cell surface accumulation. Furthermore, we showed that site-directed mutation changing Ser200 to other uncharged amino acid significantly increased radioiodide uptake activity in ORF hNIS. This finding suggests that the NIS transgene could be optimized for cell surface trafficking and RAIU activity to improve its clinical applications.

It is the NIS protein located at the cell surface, but not the total amount of NIS protein, that determines the degree of NIS-mediated RAIU activity. It seems that NIS cell surface trafficking and accumulation are extremely susceptible to disruption. Clinically, some thyroid tumors had increased NIS expression, but with defects in cell surface accumulation (24, 25); thus, these tumors could not benefit from NIS-mediated radionuclide imaging or therapy. Furthermore, patients with iodide transport defects often carry a mutation in NIS, such that the NIS mutant fails to target to the cell surface (26, 27, 28). Experimentally, we found that NIS cell surface trafficking is extremely susceptible to small substitutions between hNIS and rNIS (as shown in this study) as well as substitutions between hNIS and human sodium-dependent multivitamin transporter (Zhang, Z., and S. M. Jhiang, unpublished observations). Our findings suggest that the tertiary structure formed from discontinuous regions of NIS plays a critical role in cell surface trafficking. In addition, we found that deletion of the 31 amino acids at the C terminus decreases NIS cell surface levels (our unpublished observations) and that the 5'-untranslated region of hNIS plays an important role in NIS cell surface trafficking (Lin, X., Z. Zhang, and S. M. Jhiang, manuscript in preparation). Additional investigation of the mechanism underlying NIS cell surface trafficking is not only physiologically relevant, but also clinically significant.

To date, the amino acid residues involved in Na+ binding in SSSF members are poorly defined. The consensus is that multiple regions contribute to efficient substrate binding, yet the binding sites for Na+ and solute are proposed to be in close proximity in the tertiary structure to ensure strong cooperativity (29). Jung and colleagues suggested that Asp55 and Met56 in putative TM2 may be located at or close to the Na+ binding site, and that Ser340/Thr341 in putative TM9, Arg40 at the cytoplasmic end of putative TM2 as well as Asp187 in the cytoplasmic loop between putative TM5 and TM6, may directly and indirectly participate in the formation of Na+ binding sites in Na+/proline transporter (PutP) of Escherichia coli (30, 31, 32, 33, 34). Structural and functional studies of another SSSF member, sodium glucose transporter (SGLT1), indicate that the region between putative TM4 and TM5 (amino acid residues 162–173) participates in the formation of an Na+ pore (35). In NIS, although some amino acid residues were found to be pertinent for NIS function (36, 37, 38, 39), none of them has been implicated directly in Na+ binding. In this study we showed that the region of putative TM4-TM6, in particular the amino acid residue 200 in putative TM6, may be directly or indirectly involved in Na+ binding. However, the difference in Na+ binding affinity between hNIS and rNIS does not contribute greatly to the difference in NIS-mediated RAIU activity. This may be due to the fact that Na+ binding affinity in eukaryotic SSSF members is generally low with a Km value in the mM range, vs. a Km value in the µM rangee of E. coli PutP. It is important to note that we did not expect to identify the amino acid residues essential for Na+ binding, because these residues should be conserved between hNIS and rNIS. Most of the amino acid residues identified to be important for Na+ binding in PutP of E. coli, such as Arg40, Met56, Asp187, and Ser340/Thr341, are conserved in NIS. Furthermore, three of 12 amino acid residues in the region between putative TM4 and TM5 of SGLT1 are conserved in NIS. It is most likely that these corresponding amino acid residues in NIS may also play important roles in Na+ binding.

Currently, publications on structural/functional studies of NIS are limited to those amino acid residues involved in mutations found in patients with iodide transport defects (21, 38, 39). It has been shown that a hydroxyl group at Thr354 in putative TM9, corresponding to Thr341 in PutP of E. coli, is essential for NIS function (38). Furthermore, an uncharged amino acid residue with a small side chain at G395 is optimal for NIS function (21). Finally, changing Glu267 to any charged residues significantly decreased NIS activity, yet substitution with neutral residues had a mild effect on NIS activity (39). In this study we showed that changing Ser200 to a charged amino acid residue, such as Asp or Arg, dramatically decreased NIS cell surface accumulation and abolished NIS activity. However, substitution of Ser200 with a nonpolar amino acid residue (Ala or Val) or with other uncharged polar amino acid residue (Gly or Thr) increased NIS activity.

In summary, we showed that the difference in RAIU activity conferred by ORF rNIS and ORF hNIS is mostly due to a difference in cell surface NIS levels, and this difference is mainly contributed by the region between the N terminus and the putative TM7. We also determined that the region of putative TM4–6 contributes more to differences in RAIU activity than cell surface NIS levels, and Ser200 is the major determinant for the difference. We showed that the kinetics of Na+ binding are different between ORF hNIS and ORF rNIS, and that Ser200 of hNIS may be directly or indirectly involved in Na+ binding. Additional investigation of the structural basis underlying NIS cell surface targeting and substrate binding kinetics is highly warranted, so that clinical application of NIS as an imaging reporter gene and a therapeutic gene can be optimized in the clinical setting. Finally, it is also important to examine ways to increase iodide retention if NIS is to be used as a therapeutic gene.


   Footnotes
 
This work was supported by National Institute of Biomedical Imaging and Bioengineering Grant 1-R01-EB-001876-01 and National Cancer Institute Grant 1-R21-CA-108876-01 (to S.M.J.) from the National Institutes of Health.

First Published Online August 16, 2005

Abbreviations: h, Human; NIS, Na+/I– symporter; nt, nucleotide; ORF, open reading frame; r, rat; RAIU, radioactive iodide uptake; SSSF, sodium/solute symporter family; TM, transmembrane domain; Vmax, maximum velocity.

Received April 25, 2005.

Accepted August 10, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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