Characterization of the functional and signaling properties of the Intracellular domain of the NG2 proteoglycan
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Abstract
The NG2 proteoglycan is a molecular marker of oligodendrocyte precursor cells (OPCs) and is abundantly expressed by a variety of tumours. Published results show that NG2 influences proliferation, migration, and has neuromodulatory effects. Like other type-1 transmembrane proteins, NG2 is subjected to regulated intramembrane proteolysis (RIP), generating a large ectodomain, a C-terminal fragment (CTF) and an intracellular domain (ICD) by the sequential action of α- and γ-secretases. The cleavage of NG2 is constitutive and enhanced by neuronal activity. Functional roles of the NG2 protein domains have so far been shown for the full-length protein, the released ectodomain and CTF, but not for the ICD.
This study characterized the role of the NG2 ICD in a murine OPC cell line (Oli-neu), primary OPCs and human embryonic kidney (HEK) cells. It was found that the cleaved NG2 ICD has a predominantly cytosolic expression including the distal processes of OPCs. A small fraction of the protein exhibits a nuclear localization. Immunoprecipitation coupled with Mass Spectrometric analysis indicated that the NG2 ICD modulates mRNA translation and cell-cycle kinetics. Functional studies showed that ICD overexpression results in an upregulated translation of mRNAs as well as a shift of the cell population towards S-phase. NG2 ICD increases levels of the active (phosphorylated) form of mTOR and modulates downstream signaling cascades. These include increased phosphorylation of p70S6K1 and increased expression of eEF2. Strikingly, levels of FMRP, a translation repressor and RNA-binding protein that is regulated by mTOR/S6K1/eEF2 were decreased upon ICD overexpression. De novo proteomic profiling by pulsed-SILAC method after NG2 ICD overexpression revealed that proteins regulating DNA replication, cell differentiation and apoptosis were highly enriched in the NG2 ICD-overexpressing population.
This study establishes that the NG2 ICD, after release by cleavage, acts as a regulator of translation and cell cycle progression in OPCs by modulating the mTOR pathway, a known global translational regulator. It also influences levels of FMRP, a protein responding to network activity and a well-characterized repressor of translation. Findings from this study additionally help to explain why tumours exhibiting enhanced rates of translation and rapid cell cycle kinetics express high levels of NG2.