Identification of an atypical calcium-dependent calmodulin binding site on the C-terminal domain of GluN2A

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• 作者：Gaurav Bajaj^a ; ¹ ; Andrew M. Hau^a ; Peter Hsu^a ; Philip R. Gafken^b ; Michael I. Schimerlik^c ; Jane E. Ishmael^a ; ^{jane.ishmael@oregonstate.edu" class="auth_mail}
• 关键词：Calmodulin ; Calcium ; NMDA ; Glutamate
• 刊名：Biochemical and Biophysical Research Communications
• 出版年：21 February, 2014
• 年：2014
• 卷：444
• 期：4
• 页码：588-594
• 全文大小：2127 K

文摘

N-methyl-d-aspartate (NMDA) receptors are calcium-permeable ion channels assembled from four subunits that each have a common membrane topology. The intracellular carboxyl terminal domain (CTD) of each subunit varies in length, is least conserved between subunits, and binds multiple intracellular proteins. We defined a region of interest in the GluN2A CTD, downstream of well-characterized membrane-proximal motifs, that shares only 29% sequence similarity with the equivalent region of GluN2B. GluN2A (amino acids 875-1029) was fused to GST and used as a bait to identify proteins from mouse brain with the potential to bind GluN2A as a function of calcium. Using mass spectrometry we identified calmodulin as a calcium-dependent GluN2A binding partner. Equilibrium fluorescence spectroscopy experiments indicate that Ca²⁺/calmodulin binds GluN2A with high affinity (5.2 卤 2.4 nM) in vitro. Direct interaction of Ca²⁺/calmodulin with GluN2A was not affected by disruption of classic sequence motifs associated with Ca²⁺/calmodulin target recognition, but was critically dependent upon Trp-1014. These findings provide new insight into the potential of Ca²⁺/calmodulin, previously considered a GluN1-binding partner, to influence NMDA receptors by direct association.