Abstract
Heterotetrameric N-methyl-d-aspartate type glutamate receptors (NMDAR) are cationic channels primarily permeable for Ca2+. NR1 and NR3 subunits bind glycine, while NR2 subunits bind glutamate for full activation. As NR1 may contain a nuclear localization signal (NLS) that is recognized by importin-α, our aim was to investigate if NMDARs are expressed in the nuclei of melanocytes and melanoma cells. A detailed NMDAR subunit expression pattern was examined by RT-PCRs (reverse transcription followed by polymerase chain reaction), fractionated western blots and immunocytochemistry in human epidermal melanocytes and in human melanoma cell lines A2058, HT199, HT168M1, MEL35/0 and WM35. All kind of NMDAR subunits are expressed as mRNAs in melanocytes, as well as in melanoma cells, while NR2B protein remained undetectable in any cell type. Western blots proved the exclusive presence of NR1 and NR3B in nuclear fractions and immunocytochemistry confirmed NR1-NR3B colocalization inside the nuclei of all melanoma cells. The same phenomenon was not observed in melanocytes. Moreover, protein database analysis revealed a putative NLS in NR3B subunit. Our results support that unusual, NR1-NR3B composed NMDAR complexes are present in the nuclei of melanoma cells. This may indicate a new malignancy-related histopathological feature of melanoma cells and raises the possibility of a glycine-driven, NMDA-related nuclear Ca2+-signalling in these cells.
Highlights
Glutamate is the main excitatory neurotransmitter of the mammalian central nervous system (CNS) acting on a broad range of ionotropic and metabotropic glutamate receptors
We aimed to examine the detailed subcellular expression pattern of N-methyl-D-aspartate type glutamate receptors (NMDAR) subunits in melanoma cells and melanocytes
As the most striking novel observation, we found that cells all of the investigated melanoma cell lines possessed full size nuclear NR1 and NR3B, which phenomenon was not observed in normal human epidermal melanocytes (NHEM)
Summary
Glutamate is the main excitatory neurotransmitter of the mammalian central nervous system (CNS) acting on a broad range of ionotropic (iGluR) and metabotropic glutamate receptors (mGluR). Roles of mGluRs in physiological functions [1] or tumorigenesis [2] of various peripheral tissues, including melanocytes and melanoma have been recognized, but less is known about iGluRs in these contexts. According to their pharmacological properties (ligand binding) and sequence analogy iGluRs are divided into four subtypes: N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), kainate and δ type glutamate receptors [3]. Subsequent functional studies concluded that the rare variant of the NMDARs consisting of NR1-NR3 subunits are glycine gated, excitatory ion channels, that are unresponsive to glutamate or NMDA and insensitive to membrane depolarization as they do not have Mg2+-blockade [9]. Ca2+-influx generated by channels consisting of NR1-NR3 subunits has a smaller amplitude in comparison to conventional NMDARs and probably leads to intracellular signals others than those evoked by activation of NR1-NR2 channels [10]
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