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Failure of Membrane Targeting Causes the Functional Defect of Two Mutant Sodium Iodide Symporters¹

Departments of Medicine (J.P., R.E.W., S.R.) and Pediatrics (J.P., S.R.) and the J. P. Kennedy, Jr., Mental Retardation Research Center (S.R.), University of Chicago, Chicago, Illinois 60637-1470; Department of Medical Genetics (G.V., L.D.) and Institute of Interdisciplinary Research (G.V., L.D., S.C.), Free University of Brussels, Campus Erasme, 1070 Brussels, Belgium

Address all correspondence and requests for reprints to: Dr. Samuel Refetoff, University of Chicago, 5841 South Maryland Avenue, Chicago, Illinois 60637. E-mail: refetoff{at}medicine.bsd.uchicago.edu

	Abstract

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Molecular cloning of the sodium/iodide symporter (NIS) allowed identification of NIS gene mutations in patients with iodide trapping defect. Whereas various mutant human (h) NIS molecules display loss of function when expressed by transfection in mammalian cells, the precise mechanism(s) responsible for the functional abnormality of these proteins remains unknown. With the aim to explore these mechanisms in three natural hNIS mutants identified previously in patients with iodide trapping defect (Q267E, S515X, and C272X), we have prepared tools allowing direct measurement of the protein at its normal location in the plasma membrane.

A COS-7 cell line was made by transfection that stably expressed high levels of wild-type hNIS. It was used to screen by flow cytometry monoclonal antibodies (mAbs) prepared from mice immunized against hNIS. Genetic immunization was performed by im injection of a wild-type hNIS complementary DNA construct, because this procedure has demonstrated the ability to produce antibodies recognizing native membrane proteins. One mAb that recognized an epitope of hNIS exposed on the extracellular side of the plasma membrane was selected for further studies. The epitope was localized on the sixth putative extracellular loop of the protein on the basis that the mAb did not recognize rat NIS, which exhibits major sequence differences in this segment.

When this mAb was used to test by flow cytometry the expression of the three mutant hNIS proteins in transfected COS-7 cells, it detected similar amounts of wild-type, Q267E, and the S515X hNIS molecules in permeabilized cells. In contrast, only the wild-type hNIS was detected at the surface of nonpermeabilized cells. The C272X hNIS truncation mutant was not detected in intact or permeabilized cells. This is consistent with the absence of the mAb epitope from this mutant, which is expected to lack the sixth extracellular loop. Our data demonstrate that faulty membrane targeting is implicated in the mechanisms causing iodide trapping defect in the Q267E and S515X natural hNIS mutants.

	Introduction

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MOLECULAR CLONING of the rat sodium/iodide symporter (NIS) in 1996 (1) followed by the human NIS (2, 3) allowed identification of NIS gene mutations in patients expressing the phenotype of iodide trapping defect (4)_. This disorder is characterized by a reduced ability of the thyroid gland to concentrate iodide, which is an integral element of the thyroid hormone molecule. Although each of the seven mutant human (h) NIS molecules reported to date show loss of function when expressed in heterologous mammalian cells (5, 6, 7, 8, 9, 10, 11), the precise mechanisms responsible for the functional defect have not been fully characterized.

We produced a monoclonal antibody to determine the expression and targeting of three mutant hNIS molecules that we have identified earlier in patients with the iodide trapping detect. One is a missense mutation (Q267E) in the fourth intracellular loop (9), and the other two are nonsense mutations (C272X and S515X) producing truncated molecules (7, 9). All three were shown to be devoid of biological activity when transiently expressed in COS-7 cells.

We now show that the wild-type (WT) as well as Q267E and S515X NIS molecules are expressed in transiently transfected COS-7 cells. However, only the WT hNIS was found on the surface of nonpermeabilized cells. C272X hNIS was not detected, because it lacks the sixth extracellular domain of the molecule that is recognized by the monoclonal antibody.

	Materials and Methods

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Derivation of a stable COS cell line expressing hNIS

hNIS complementary DNA (cDNA) sequence amplified from a normal thyroid gland cDNA (9) was introduced in the pEFIN vector (12) and transfected in COS-7 cells, as previously described (13). Stable cell lines were selected by resistance to geneticin, and one clone (COS NIS-6) with a large capacity to accumulate iodide was selected (see Results). For measurement of iodide uptake, COS NIS-6 cells were seeded in 96-well plates at a density of 30,000 cells/well and cultured in DMEM supplemented with 10% FCS. After 24 h, the medium was replaced with Hank’s medium containing 0.5 µCi carrier-free Na¹²⁵I and 0.1 µmol/L KI in the presence or absence of 10 µmol/L sodium perchlorate. After 1-h incubation at 37 C, the medium was removed, and cells were quickly washed twice with ice-cold medium. The cells were dissolved with 1 N NaOH, and radioactivity was counted in a -scintillation counter. All determinations were performed in triplicate.

Generation of NIS monoclonal antibodies

Ten female 6-week-old BALB/c mice were used for immunization, as previously described (14), by injecting in the anterior tibialis muscle 100 µg of an expression construct made of hNIS cDNA inserted in pcDNA3/Amp (15). Injections were repeated 4 and 8 weeks thereafter. Blood samples were obtained from retroocular capillaries 12 weeks after the initial immunization, and the presence of antibodies against hNIS was determined by flow cytometry on the COS NIS-6 cell line as previously described (14) (data not shown).

One mouse that scored strongly positive in this assay was selected for the generation of monoclonal antibody (mAb). It was boosted 16 weeks after the initial immunization by iv injection of 3 x 10⁶ COS NIS-6 cells and was killed 3 days later. Splenocytes were fused with SP2/0 myeloma cells at a 5:1 ratio using 50% polyethylene glycol (16), and selection of hybridomas was performed as previously described (17). Ten days after the fusion, isolated colonies were removed from the plates and transferred into individual wells of a 96-well tissue culture plate containing 200 µl ClonalCell-HY growth medium (StemCell Technologies, Inc., Vancouver, Canada). When the cells were growing actively, culture media from individual wells were tested for anti-hNIS activity by flow cytometry on COS NIS-6 cells. Selected hybridomas were cloned by limiting dilution. The Ig class of mAb was determined with a mouse mAb isotyping kit (IsoStrip, Roche Molecular Biochemicals, Brussels, Belgium).

Flow cytometry

COS-7 cells expressing WT or mutant hNIS or control COS-7 cells were detached from the plates with phosphate-buffered saline containing ethylenediamine tetraacetate and ethyleneglycol-bis-(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid (5 mmol/L each) and transferred into Falcon 2052 tubes (200,000 cells/tube). The immunoreactivity of intact cells was determined exactly as previously described (14). Propidium iodide (10 µg/ml) was used for detection of damaged cells, which were excluded from the analysis. The immunoreactivity of permeabilized cells was determined after fixing them for 10 min on ice with 1% paraformaldehyde and treating them for 30 min at room temperature with 0.2% saponin. All subsequent steps with antibodies were performed in 0.2% saponin. The fluorescence of 5,000 cells/tube was assayed with a FACScan flow cytofluorometer (Becton Dickinson and Co., Eerenbodegem, Belgium).

Transient transfection of hNIS mutants

pcDNA3/Amp constructs harboring WT or mutant hNIS cDNAs (9) were transfected in COS-7 cells by the diethylaminoethyl-dextran method followed by a dimethylsulfoxide shock (18). Two days after transfection, cells were used for flow immunocytofluorometry. Duplicate dishes were used for each analysis. Each experiment was repeated twice. Cells transfected with pcDNA3/Amp alone were used as controls.

	Results

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Generation and characterization of a stable COS cell line expressing human NIS

A stable cell line expressing high level of hNIS was generated as a screening tool to isolate mAbs recognizing the native hNIS protein inserted in the plasma membrane. After several unsuccessful attempts to generate Chinese hamster ovary cell lines expressing hNIS, using protocols that have demonstrated their efficiency for a series of G protein-coupled receptors (12, 13), we turned to COS-7 cells. These had demonstrated the capability of expressing high levels of functional NIS in transient transfections (1). A stable COS-7 cell line was isolated (COS NIS-6) with a strong capacity to accumulate iodide in a perchlorate-sensitive way (Fig. 1A; 30,000 cells; 801,270 ± 6,600 cpm in COS NIS-6 vs. 2,400 ± 223 cpm in nontransfected COS cells). The high level of functional hNIS protein in COS NIS-6 cells was used to screen for mAbs recognizing the native protein that is inserted into the plasma membrane (see below).

View larger version (13K): [in this window] [in a new window]   Figure 1. Characterization of the COS NIS-6 cell line. A, Iodide uptake of the COS NIS-6 cell line in comparison with WT nontransfected COS cells (COS NT) in the absence (-) or presence (+) of sodium perchlorate. Results are expressed as counts per min of 125I uptake. B, Analysis by flow cytometry of the COS NIS-6 cell line with the VJ2 mAb () compared with WT COS NT cells ().

The complexity of hNIS protein structure, with 12 or 13 transmembrane helexes (19), makes it difficult to predict which epitopes would be exposed at the cell surface. The successful production of mAbs against native G protein-coupled receptors by genetic immunization (14) prompted us to apply this methodology to the generation of anti-hNIS mAbs (see Materials and Methods). Three mAbs (1C6, VJ1, and VJ2) recognizing hNIS at the surface of COS NIS-6 cells were isolated (Fig. 1B; FACS with VJ2 antibody). All three were of the IgG1 isotype and exhibited the same properties. They did not recognize hNIS protein in Western blots after denaturation with SDS and reduction with dithiothreitol (data not shown). In addition, these antibodies did not recognize rat NIS (data not shown) after its transient expression in COS-7 cells (rat NIS cDNA was a gift from Dr. N. Carrasco). Finally, the hNIS mAbs had no effect on the uptake of iodide by COS NIS-6 cells (not shown).

Characterization of natural hNIS mutants responsible for iodide trapping defects

Three mutant hNIS molecules that had been identified in patients with defective iodide trapping were analyzed (15). One had a mutation in position 267, substituting glutamic acid for glutamine (Q267E) (9), and two had nonsense mutations in codon 272 (C272X) (7) and codon 531, creating a cryptic 3'-splice acceptor site that produced a six-amino acid frame shift preceding a stop at codon 515 (S515X) (9). The corresponding mutant cDNA constructs were transiently expressed in COS-7 cells. Immunoreactive hNIS expression at the surface of intact cells or within cells permeabilized with saponin was assayed by flow cytometry using the VJ2 mAb. WT hNIS as well as mutants Q267E and S515X were well recognized by the antibody in permeabilized cells (Fig. 2B). In contrast, only the WT hNIS was readily detected at the surface of nonpermeabilized COS-7 cells (Fig. 2A). The mutant S515X was completely absent from the cell surface, and expression of Q267E was barely detectable (Fig. 2A). Mutant C272X was not detected in either intact or permeabilized cells (Fig. 2, A and B). It is concluded from these experiments that Q267E and S515X mutations yield nonfunctional proteins that do not reach the plasma membrane.

View larger version (17K): [in this window] [in a new window]   Figure 2. Analysis of expression pattern of hNIS mutants by flow immunocytometry. COS-7 cells transfected with the empty vector (pcDNA3), the WT hNIS construct (WT), or the various hNIS mutants (Q267E, C272X, and S515X) were analyzed by flow immunocytometry using the VJ2 mAb (see Materials and Methods). Cells were analyzed intact (A) or after permeabilization by 0.2% saponin (B).

	Discussion

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In this communication we report the production of mAbs that react with the extracellular domain of hNIS. Advantage was taken of the genetic immunization technique, which, by allowing synthesis of the foreign protein by cells of the immunized animal, enhances the likelihood of producing antibodies against the properly targeted extracellular domain of the molecule. Indeed, three mAbs were isolated that recognized hNIS at the surface of COS NIS-6 cells that stably express a functional hNIS. All three were specific for human, but not rat, NIS and reacted with the native extracellular portions of hNIS, but not with the denatured and reduced molecule. Binding of these antibodies did not interfere with the iodide transport function of hNIS.

In contrast to hNIS T354P, the two mutant hNIS molecules, Q267E and S515X, were not found in significant amounts on the surface of transfected COS-7 cells despite the fact that they were expressed equally well as the wild-type NIS. These results indicate that a defect of membrane targeting rather than an intrinsic impairment of function is responsible for the failure to observe NIS-mediated active iodide transport in the patients and in transfected cells. More specifically, these results also indicate the implication of Q267 in the correct routing of hNIS to the plasma membrane. Inability to detect the severely truncated hNIS mutant, C272X, may not be due to failure of synthesis or rapid degradation but, rather, to the absence of the epitope reacting with the mAb.

The results obtained with the VJ2 mAb and natural hNIS mutants have implications for our understanding of the insertion of the native hNIS in the plasma membrane. The epitope recognized by the VJ2 mAb at the cell surface must be located between amino acids 272 and 515 (Fig. 3). According to the structure model for NIS with 13 transmembrane helexes proposed by Levy et al. (19), this segment includes the last 3 extracellular loops. The amino acid sequences of human and rat NIS are 97% and 100% identical in the fourth and fifth extracellular loops, respectively (2). Thus, the failure of the VJ2 mAb to recognize rat NIS is probably due to the sequence divergence of the sixth loop between the two species. Indeed, the sixth loop displays only 57% homology with the rat sequence, which also harbors a 5-amino acid deletion (2). It is thus logical to conclude that the epitope recognized by the VJ2 mAb must contain amino acids located within the sixth extracellular loop.

View larger version (70K): [in this window] [in a new window]   Figure 3. Localization of the epitope recognized by the VJ2 mAb. The locations of Q267E, C272X, and S515X residues are indicated on the revised structure of NIS proposed by Levy et al. (19 ). Amino acids differing in hNIS and rat NIS are indicated (•), and additional amino acids not present in rat NIS are boxed. The shaded area encompassing the last three extracellular loops includes the putative zones of interaction of the VJ2 mAb. Extensive sequence differences between hNIS and rat NIS (not recognized by VJ2 antibody; see Discussion), including a gap of five amino acids in rat NIS (boxed), located the epitope on the sixth extracellular loop. Y, Positions of N-linked glycosylation consensus sequences.

	Footnotes

 
¹ This work was supported in part by NIH Grants DK-15070 and RR-00055 (to S.R. and R.E.W.) and in part by the Belgian Program of University Poles of Attraction initiated by the Belgian State, Prime Minister’s Office, Service for Sciences, Technology and Culture.

² Supported in part by a grant from the Deutsche Forschungsgemenschaft (Po 556/1–1).

Received December 6, 1999.

Revised March 17, 2000.

Accepted March 29, 2000.

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