Abstract
Background
The thyroid’s ability to accumulate iodide via the sodium-iodide symporter
(NIS) is utilised to successfully treat most thyroid cancers with
radioiodide. However, approximately 25% of thyroid cancers lose
functional NIS expression and become unresponsive to radioiodide
therapy, resulting in a poorer prognosis. Our knowledge of NIS regulation
is limited, but as dimerisation of NIS has been proposed, we
sought to investigate NIS dimerisation and function.
Methods
A homology model of NIS structure was built based on the bacterial
protein vSGLT using the modeling platform Phyre2, to identify residues
potentially involved in dimerisation, which were mutated via
site-directed mutagenesis. Proximity ligation assays (PLA) were used
to assess dimerisation of wild-type and mutant NIS. Novel constructs
conjugated to fluorescent proteins (cerulean or citrine) were created
to further assess dimerisation using Fӧrster resonance energy transfer
(FRET).
Results
Dimerisation of wild-type NIS was confirmed via PLA in both a
thyroid (SW1736) and non-thyroid (HeLa) cell line. Furthermore, FRET
increased in the presence of both fluorophore-conjugated NIS
constructs compared to single expression (1.52 ± 0.10 vs 1.08 ± 0.18,
P < 0.0001), validating dimerisation. We mutated five residues identified
from our homology model (D237A, Y242A, T243A, Q471A and
A525F), and two putative dimerisation motifs identified in the literature
(glycine- and leucine-zipper motifs). PLA suggested that all mutants
retained the ability to dimerise, indicating that dimerisation
involves multiple or as yet undiscovered residues.
Conclusion
NIS dimerisation has been conclusively demonstrated using two
discrete methodologies. Further work is ongoing to determine the
critical residues, cellular localisation and regulation of NIS dimerisation
and its impact on function.
The thyroid’s ability to accumulate iodide via the sodium-iodide symporter
(NIS) is utilised to successfully treat most thyroid cancers with
radioiodide. However, approximately 25% of thyroid cancers lose
functional NIS expression and become unresponsive to radioiodide
therapy, resulting in a poorer prognosis. Our knowledge of NIS regulation
is limited, but as dimerisation of NIS has been proposed, we
sought to investigate NIS dimerisation and function.
Methods
A homology model of NIS structure was built based on the bacterial
protein vSGLT using the modeling platform Phyre2, to identify residues
potentially involved in dimerisation, which were mutated via
site-directed mutagenesis. Proximity ligation assays (PLA) were used
to assess dimerisation of wild-type and mutant NIS. Novel constructs
conjugated to fluorescent proteins (cerulean or citrine) were created
to further assess dimerisation using Fӧrster resonance energy transfer
(FRET).
Results
Dimerisation of wild-type NIS was confirmed via PLA in both a
thyroid (SW1736) and non-thyroid (HeLa) cell line. Furthermore, FRET
increased in the presence of both fluorophore-conjugated NIS
constructs compared to single expression (1.52 ± 0.10 vs 1.08 ± 0.18,
P < 0.0001), validating dimerisation. We mutated five residues identified
from our homology model (D237A, Y242A, T243A, Q471A and
A525F), and two putative dimerisation motifs identified in the literature
(glycine- and leucine-zipper motifs). PLA suggested that all mutants
retained the ability to dimerise, indicating that dimerisation
involves multiple or as yet undiscovered residues.
Conclusion
NIS dimerisation has been conclusively demonstrated using two
discrete methodologies. Further work is ongoing to determine the
critical residues, cellular localisation and regulation of NIS dimerisation
and its impact on function.
| Original language | English |
|---|---|
| Article number | P2 |
| Pages (from-to) | 5 |
| Number of pages | 1 |
| Journal | Thyroid Research |
| Volume | 10(Suppl 2) |
| Issue number | 5 |
| Publication status | Published - 19 Sept 2017 |
| Event | 65th British Thyroid Association Annual Meeting - London, United Kingdom Duration: 16 May 2017 → 16 May 2017 |
