Prickett Todd D, Gartner Jared J, Samuels Yardena
National Cancer Institute, Surgery Branch, US National Institutes of Health, Bethesda, MD, 20892, USA.
Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, 76100, Israel.
Methods Mol Biol. 2017;1677:93-116. doi: 10.1007/978-1-4939-7321-7_3.
Ionotropic glutamate receptors (iGluRs) are large integral membrane multi-protein complexes that create ion channels in plasma membranes. Upon binding of receptor specific ligands (e.g., glutamate), increased efflux or influx of mono- or divalent cations (e.g., Ca) promotes synaptic transmission, cellular migration, and survival. Three classes of iGluRs were originally defined after their respective agonists: AMPA, kainate, and NMDA receptors (NMDARs). Recently, we examined iGluR families at the genetic level using Next-Generation Sequencing (NGS) (whole-exome sequencing (WES)) and discovered a high prevalence of somatic mutations within the gene for one of the NMDAR subunits, GRIN2A, specifically in malignant melanoma. Following confirmation of the somatic mutations, we focused on functional characterization of a subset of the GRIN2A mutants that demonstrated a loss of NMDAR functionality. We used gene expression and protein biochemistry to examine complex formation between GluN1 subunit (encoded by GRIN1) and GluN2A subunit (encoded by GRIN2A), anchorage-independent growth in soft agar and cellular migration. Furthermore, we used shRNA depletion of endogenous GRIN2A in melanoma cells expressing either wild-type GRIN2A or mutant GRIN2A and measured cellular proliferation compared to negative controls. Our data show that somatic mutation of certain residues in GluN2A results in increased survival and is the first such report to demonstrate the functional importance of GRIN2A mutations in melanoma and the significance ionotropic glutamate receptor signaling plays in malignant melanoma.
离子型谷氨酸受体（iGluRs）是大型整合膜多蛋白复合物，可在质膜中形成离子通道。当受体特异性配体（如谷氨酸）结合后，单价或二价阳离子（如钙离子）外流或内流增加，从而促进突触传递、细胞迁移和存活。最初根据各自的激动剂定义了三类离子型谷氨酸受体：AMPA、海人酸和NMDA受体（NMDARs）。最近，我们使用下一代测序（NGS）（全外显子组测序（WES））在基因水平上研究了离子型谷氨酸受体家族，发现NMDAR亚基之一GRIN2A的基因中存在高频率的体细胞突变，特别是在恶性黑色素瘤中。在确认体细胞突变后，我们重点研究了一组显示NMDAR功能丧失的GRIN2A突变体的功能特征。我们使用基因表达和蛋白质生物化学方法来检测GluN1亚基（由GRIN1编码）和GluN2A亚基（由GRIN2A编码）之间的复合物形成、软琼脂中的非锚定生长和细胞迁移。此外，我们在表达野生型GRIN2A或突变型GRIN2A的黑色素瘤细胞中使用shRNA耗尽内源性GRIN2A，并与阴性对照相比测量细胞增殖。我们的数据表明，GluN2A中某些残基的体细胞突变会导致存活率增加，这是首次有此类报告证明GRIN2A突变在黑色素瘤中的功能重要性以及离子型谷氨酸受体信号传导在恶性黑色素瘤中的意义。
