Identification of an atypical calcium-dependent calmodulin binding site on the C-terminal domain of GluN2A.
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 Ca(2+)/calmodulin binds GluN2A with high affinity (5.2±2.4 nM) in vitro. Direct interaction of Ca(2+)/calmodulin with GluN2A was not affected by disruption of classic sequence motifs associated with Ca(2+)/calmodulin target recognition, but was critically dependent upon Trp-1014. These findings provide new insight into the potential of Ca(2+)/calmodulin, previously considered a GluN1-binding partner, to influence NMDA receptors by direct association.
Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State University, Corvallis, Oregon 97331, United States.
Gaurav
G
Bajaj
Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State University, Corvallis, Oregon 97331, United States.
Andrew M
AM
Hau
Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State University, Corvallis, Oregon 97331, United States.
Peter
P
Hsu
Proteomics Facility, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, United States.
Philip R
PR
Gafken
Department of Biochemistry & Biophysics, Oregon State University, Corvallis, Oregon 97331, United States.
Michael I
MI
Schimerlik
Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State University, Corvallis, Oregon 97331, United States. Electronic address: jane.ishmael@oregonstate.edu.
Jane E
JE
Ishmael
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D016194:Receptors, N-Methyl-D-Aspartate / Q000737:chemistry* / Q000378:metabolism*
D002118:Calcium; D002147:Calmodulin; D018698:Glutamic Acid; D016194:Receptors, N-Methyl-D-Aspartate
Calcium; Calmodulin; Glutamate; NMDA