N-Methyl-D-aspartate Receptor Subunits Are Non-myosin Targets of Myosin Regulatory Light Chain

Abstract

Excitatory synapses contain multiple members of the myosin superfamily of molecular motors for which functions have not been assigned. In this study we characterized the molecular determinants of myosin regulatory light chain (RLC) binding to two major subunits of the N-methyl-D-aspartate receptor (NR). Myosin RLC bound to NR subunits in a manner that could be distinguished from the interaction of RLC with the neck region of non-muscle myosin II-B (NMII-B) heavy chain; NR-RLC interactions did not require the addition of magnesium, were maintained in the absence of the fourth EF-hand domain of the light chain, and were sensitive to RLC phosphorylation. Equilibrium fluorescence spectroscopy experiments indicate that the affinity of myosin RLC for NR1 is high (30 nM) in the context of the isolated light chain. Binding was not favored in the context of a recombinant NMII-B subfragment one, indicating that if the RLC is already bound to NMII-B it is unlikely to form a bridge between two binding partners. We report that sequence similarity in the "GXXXR" portion of the incomplete IQ2 motif found in NMII heavy chain isoforms likely contributes to recognition of NR2A as a non-myosin target of the RLC. Using sitedirected mutagenesis to disrupt NR2A-RLC binding in intact cells, we find that RLC interactions facilitate trafficking of NR1/ NR2A receptors to the cell membrane. We suggest that myosin RLC can adopt target-dependent conformations and that a role for this light chain in protein trafficking may be independent of the myosin II complex.Regulation and maintenance of glutamate receptor numbers at postsynaptic sites are critical for excitatory neurotransmission in the central nervous system. Mechanisms that underlie targeting of glutamate receptors to synapses almost certainly involve a regulated series of protein-protein interactions between the receptor and various binding partners. In trying to understand how these events are temporally and spatially coordinated, the intracellular carboxyl termini of glutamate receptor subunits have attracted much attention as they have been shown to bind directly and indirectly to a number of cytoskeletal and motor proteins, scaffolding proteins, enzymes, and other signaling molecules (1). We have previously demonstrated that three major subunits of the N-methyl-D-aspartate (NMDA) 2 subtype of ionotropic glutamate receptor bind directly to myosin II regulatory light chain (RLC) (2). Myosin RLC is an accessory light chain of the actin-based motor myosin II, which is a hexameric complex composed of two heavy chains, two RLCs, and two essential light chains (ELC) (3). Myosin II light chains are, like calmodulin, members of the EF-hand family of calcium-binding proteins (4). However, the interaction of myosin RLC with NMDA receptor (NR) subunits could be distinguished from the strictly Ca 2ϩ -dependent interaction of calmodulin with the NR1 subunit (2).The light chains of myosin II are integral components of the myosin II complex that bind to tandem IQ motifs located in the neck regio...

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

“…Plasmids containing rat GluN2A, GluN2B and GluN1-1a cDNAs, and the construction of GluN1-C0 (834-864), GluN1 (834-938), GluN2A (838-874), GluN2A (875-1029), GluN2A (1030-1464) and GluN2A (838-1464) in pGEX vectors (GE Healthcare, Piscataway, NJ have been described previously [9,10]. GluN2A (991-1029) was amplified by PCR and inserted into pGEX-6P-3 for fluorescence spectroscopy experiments.…”

“…Postsynaptic density protein of 95 kDa (PSD-95) and related scaffold proteins bind a common distal ESDV motif [6], whereas the membrane-proximal regions of GluN2A and GluN2B share tyrosine-based recognition motifs for the clathrin-dependent, endocytic adapter protein 2 complex (AP-2) [7,8]. We have previously shown that the same membrane-proximal regions of GluN2A and GluN2B also bind a light chain of the actin-based motor myosin II that likely serves an early trafficking function [9]. As the membrane-proximal regions of the GluN2A and GluN2B C-termini appear to have some functional equivalency, the goal of this study was to analyze the region of GluN2A immediately downstream of the paired endocytic motifs and minimal myosin light chain interaction domain.…”

Nonmuscle Myosin II Is Required for Internalization of the Epidermal Growth Factor Receptor and Modulation of Downstream Signaling

“…These receptors including CXCR4, Ins(1,4,5)P3-R, and NMDA-R are not single transmembrane receptors with the exception of the discoidin domain receptor 1 (DDR1) which is a tyrosine kinase receptor activated by type I collagen (23)(24)(25)(26). The EGFR is identified as an NM II binding partner in this study.…”

“…Recent publications have reported that NM II interacts with several kinds of receptors including CXCR4 (chemokine receptor), N-methyl-D-aspartate (NMDA receptor), DDR1 (as a collagen receptor), and the Ins (1,4,5)P 3 receptor (23)(24)(25)(26). However, to date, the potential involvement of NM II in EGFRmediated intracellular signaling has not been studied in depth.…”

Actin in dendritic spines: connecting dynamics to function

“…NMDA receptor reg ulates actin cytoskeleton in at least two ways: (1) by mediating the influx of Ca 2+ ions into postsynaptic neurons, which modulate the activity of many actin binding proteins, e.g., CaMKII (Lisman et al, 2002) and gelsolin (Nag et al, 2009);and (2) by binding directly to actinbinding or regulating proteins, e.g., CaMKII (Raveendran et al, 2009), actinin (Wyszynski et al, 1997), and myosin regulatory light chain (Bajaj et al, 2009). Also, various receptor tyrosine kinases, such as members of the Trk (BDNF receptor; Menna et al, 2009) and Eph/ephrin families (Schubert and Dotti, 2007), as well as synaptic adhesion molecules (Yoshihara et al, 2009), have been shown to be important in regulating actin in spines.…”