CRMP2 Interacts with NMDA Receptor and Na+/Ca2+ Exchanger and Regulates Their Functional Activity [Molecular Bases of Disease]

January 28th, 2014 by Brustovetsky, T., Pellman, J. J., Yang, X.-F., Khanna, R., Brustovetsky, N.

Collapsin response mediator protein 2 (CRMP2) is traditionally viewed as an axonal growth protein involved in axon/dendrite specification. Here, we describe novel functions of CRMP2. A 15-amino acid peptide from CRMP2, fused to the tat cell penetrating motif of the HIV-1 protein, tat-CBD3, but not CBD3 sans tat, attenuated N-methyl-D-aspartate receptor (NMDAR) activity and protected neurons against glutamate-induced Ca2+ dysregulation, suggesting the key contribution of CRMP2 in these processes. In addition, tat-CBD3, but not CBD3 sans tat or tat-scramble peptide, inhibited increases in cytosolic Ca2+ mediated by the plasmalemmal Na+/Ca2+ exchanger (NCX) operating in the reverse mode. Co-immunoprecipitation experiments revealed an interaction between CRMP2 and NMDAR as well as NCX3 but not NCX1. Tat-CBD3 disrupted CRMP2-NMDAR interaction without change in NMDAR localization. In contrast, tat-CBD3 augmented the CRMP2-NCX3 co-immunoprecipitation, indicating increased interaction or stabilization of a complex between these proteins. Immunostaining with an anti-NCX3 antibody revealed that tat-CBD3 induced NCX3 internalization, suggesting that both reverse and forward modes of NCX might be affected. Indeed, the forward mode of NCX, evaluated in experiments with ionomycin-induced Ca2+ influx into neurons, was strongly suppressed by tat-CBD3. Knockdown of CRMP2 with short interfering RNA (siRNA) prevented NCX3 internalization in response to tat-CBD3 exposure. Moreover CRMP2 downregulation strongly attenuated tat-CBD3-induced inhibition of reverse NCX. Overall, our results demonstrate that CRMP2 interacts with NCX and NMDAR and that tat-CBD3 protects against glutamate-induced Ca2+ dysregulation most likely via suppression of both NMDAR and NCX activities. Our results further clarify the mechanism of action of tat-CBD3 and identify a novel regulatory checkpoint for NMDAR and NCX function based on CRMP2 interaction with these proteins.