Departments of Physiology and Internal Medicine The Ohio State University Columbus, Ohio 43210
Address correspondence and requests for reprints to: Sissy M. Jhiang, Ph.D., Departments of Physiology and Internal Medicine, The Ohio State University, 302 Hamilton Hall, 1645 Neil Avenue, Columbus, Ohio 43210. E-mail: jhiang.l{at}osu.edu
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Thus far, seven NIS gene mutations have been identified in patients with iodide trapping defectsG93R, Q267E, C272X, T354P, G395R, Y531X with the transcription product of S515X, and G543E (4, 5). The evidence that these NIS mutations directly cause the iodide transport defects in these patients is indicated by the lack of perchlorate-sensitive iodide uptake activity in mammalian cells expressing these human NIS (hNIS) mutants. Although all these hNIS mutants fail to transport iodide, they do not seem to interfere with the iodide transport function of wild-type hNIS. Therefore, patients with iodide transport defects are always either homozygous with one type of hNIS mutation or compound heterozygous with two different types of hNIS mutations. To elucidate hNIS structural-functional relationship, it has been of great interest to determine whether these hNIS mutants are either rapidly degraded, failed to be targeted to the plasma membrane at the cell surface, or had impaired function in iodide transport. The availability of antibodies recognizing the extracellular domain of hNIS will be most useful to identify the hNIS mutants that are not properly targeted to the plasma membrane.
In this issue of the journal, Pohlenz et al. (6) report the development of a monoclonal antibody (mAb) that recognizes an epitope of hNIS exposed on the extracellular side of the plasma membrane. It is believed that the epitope of this mAb is localized to the sixth putative extracellular loop of hNIS, on the basis that the mAb does not recognize hNIS mutant C272X and that the mAb does not recognize rat NIS, which has major sequence differences in this segment. This mAb does not recognize denatured or reduced hNIS proteins by Western blot analysis, and the binding of this mAb does not interfere with the iodide transport function of hNIS. Using this mAb to perform flow immunocytometry on cells expressing wild-type hNIS and hNIS mutants Q267E, C272X, and S515X, the investigators showed that similar amounts of the wild-type, the Q267E, and the S515X hNIS molecules were detected in permeabilized cells. In contrast, only the wild-type hNIS was detected at the surface of nonpermeabilized cells. Therefore, the investigators conclude that the functional defects of hNIS mutants Q267E and S515X are most likely due to the defects of membrane trafficking in which these two hNIS mutants fail to reach the plasma membrane at the cell surface. This is the first report indicating that failure of membrane targeting is one of the underlying mechanisms for the loss-of-function of hNIS mutants found in patients with iodide transport defects.
Among other hNIS mutants identified, it has been shown that the loss-of-function of the T354P hNIS mutant is not due to a structural change of hNIS but due to the absence of a hydroxyl group at the ß-carbon of the amino acid residue at position 354 (7). Now with the availability of mAbs recognizing the extracellular domain of hNIS, it will be of great interest to further confirm that the T354P mutant does not have a defect in membrane trafficking, by using the mAb to perform flow immunocytometry of cells expressing the T354P hNIS mutant. Furthermore, the mechanisms of the loss-of-function for the other hNIS mutants (G93R, G395R, and G543E) have not yet been investigated. In addition to investigating the mutations identified in patients with iodide transport defects, additional loss-of-function hNIS mutations may be found in some thyroid cancers that are known to have decreased iodide uptake activity but have overexpression of hNIS (8). Finally, it may be desirable to investigate additional hNIS mutants generated by random or site-directed mutagenesis. To elucidate the structural-functional relationship of hNIS in detail, it is important to distinguish the mutations that result in defects in membrane trafficking from the mutations that result in impairment of iodide transport function. Once the sites and/or the types of mutations are identified, it is possible to conduct further analysis to uncover the mechanism(s) underlying the defects of membrane targeting as well as the mechanism(s) underlying the impairment of iodide transport function. The information acquired will be invaluable to elucidate the structural-functional relationship of hNIS and may be used to engineer a hNIS molecule with desired features.
The ability of the mAb to detect surface expression of hNIS in mammalian cells will allow the sorting of cell populations based on the expression levels of hNIS on cell surface by fluorescent-activated cell sorting (FACS). Therefore, this mAb may be useful in identifying genes that up-regulate or down-regulate hNIS expression in cells of interest. For example, thyroid cells expressing endogenous hNIS may be treated with a ribozyme library to inactivate various genes. FACS can be used to isolate cells with high hNIS expression from cells with low hNIS expression in the presence or absence of TSH stimulation. The genes responsible for up-regulation or down-regulation of hNIS can then be identified by examining the ribozymes introduced in the cells collected by FACS.
In summary, the mAb recognizing the extracellular domain of hNIS will serve as an invaluable reagent to identify the amino acid residues that are important for hNIS membrane trafficking and the structural conformation that are important for the maintenance of hNIS iodide transport function. This information is not only important to elucidate the pathobiological mechanism(s) underlying the loss-of-function of hNIS in patients with iodide transport defects and in patients with thyroid cancers that fail to concentrate radioiodide, it will also provide vast information to elucidate the structural-functional relationship of hNIS for future clinical applications. Finally, the mAb is also a useful tool to identify genes that up-regulate or down-regulate hNIS expression in cells of interest.
Received May 17, 2000.
Accepted May 17, 2000.
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