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J. Biol. Chem., Vol. 284, Issue 2, 1252-1266, January 9, 2009

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

Gaurav Bajaj, Yong Zhang, Michael I. Schimerlik, Andrew M. Hau, Jing Yang, Theresa M. Filtz, Chrissa Kioussi, and Jane E. Ishmael¹

From the Department of Pharmaceutical Sciences, College of Pharmacy, and the Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon 97331

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 site-directed 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.

Received for publication, March 7, 2008 , and in revised form, October 7, 2008.

^* This work was supported, in part, by National Institutes of Health Grants R01AR054406 and P30-ES000210. This work was also supported by American Heart Association Award 0060435Z, P30-ES000210. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed. Tel.: 541-737-5783; Fax: 541-737-3999; E-mail: jane.ishmael{at}oregonstate.edu.

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