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.
Highlights
At least a quarter of patients with differentiated thyroid cancer (DTC) do not concentrate radioiodide (131I) sufficiently for effective ablation therapy [1,2], which remains a particular problem in metastatic disease
For use in the Forster resonance energy transfer (FRET) experiments, NIS cDNA was inserted into the HindIII and BamHI restriction sites of the cerulean-N1 and citrine-N1 vectors to create NIS constructs conjugated at the C-terminus to a cerulean or citrine fluorophore, respectively
The functional consequences of abrogating putative residues implicated in dimerization Based on the modeling of Figure 3, we considered the region containing D237, Y242, T243, F244, Q471, and A525 to be a ‘‘hot-spot’’ of potential dimerization, and to identify whether these residues might be involved in dimerization, we made the following point mutations: D237A, Y242A, T243A, Q471A, and A525F
Summary
At least a quarter of patients with differentiated thyroid cancer (DTC) do not concentrate radioiodide (131I) sufficiently for effective ablation therapy [1,2], which remains a particular problem in metastatic disease. There are essentially two groups of thyroid cancer patients: those who respond to radioiodide treatment and have an excellent prognosis, and those who do not respond and for whom outcome is dire [3]. Mechanisms that influence treatment success in those thyroid cancers that are radioiodide resistant have been described in numerous investigations. 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 hypothesize that dimerization of NIS is critical to its trafficking to the PM and may represent a new mechanism that would need to be considered in overcoming therapeutic failure in patients with thyroid cancer
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