Collapsin response mediator protein 2 (CRMP2) interacts with N-methyl-D-aspartate (NMDA) receptor and Na+/Ca2+ exchanger and regulates their functional activity

Tatiana Brustovetsky, Jessica J. Pellman, Xiao Fang Yang, Rajesh Khanna, Nickolay Brustovetsky

Research output: Contribution to journalArticle

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Abstract

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 without 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 withoutTATor TAT-scramble peptide, inhibited increases in cytosolic Ca2+ mediated by the plasmalemmal Na+/Ca 2+ exchanger (NCX) operating in the reverse mode. Coimmunoprecipitation 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 theCRMP2-NCX3co-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 down-regulation 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 NCXfunction based on CRMP2 interaction with these proteins.

Original languageEnglish (US)
Pages (from-to)7470-7482
Number of pages13
JournalJournal of Biological Chemistry
Volume289
Issue number11
DOIs
StatePublished - Mar 14 2014

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N-Methyl-D-Aspartate Receptors
Sodium-Calcium Exchanger
Neurons
Glutamic Acid
Proteins
collapsin response mediator protein-2
Human Immunodeficiency Virus Proteins
Peptides
Ionomycin
Dendrites
Immunoprecipitation
Small Interfering RNA
Axons
HIV-1
Anti-Idiotypic Antibodies
Down-Regulation
Stabilization
Experiments
Specifications
Amino Acids

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

Collapsin response mediator protein 2 (CRMP2) interacts with N-methyl-D-aspartate (NMDA) receptor and Na+/Ca2+ exchanger and regulates their functional activity. / Brustovetsky, Tatiana; Pellman, Jessica J.; Yang, Xiao Fang; Khanna, Rajesh; Brustovetsky, Nickolay.

In: Journal of Biological Chemistry, Vol. 289, No. 11, 14.03.2014, p. 7470-7482.

Research output: Contribution to journalArticle

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abstract = "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 without 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 withoutTATor TAT-scramble peptide, inhibited increases in cytosolic Ca2+ mediated by the plasmalemmal Na+/Ca 2+ exchanger (NCX) operating in the reverse mode. Coimmunoprecipitation 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 theCRMP2-NCX3co-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 down-regulation 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 NCXfunction based on CRMP2 interaction with these proteins.",
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AU - Pellman, Jessica J.

AU - Yang, Xiao Fang

AU - Khanna, Rajesh

AU - Brustovetsky, Nickolay

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AB - 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 without 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 withoutTATor TAT-scramble peptide, inhibited increases in cytosolic Ca2+ mediated by the plasmalemmal Na+/Ca 2+ exchanger (NCX) operating in the reverse mode. Coimmunoprecipitation 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 theCRMP2-NCX3co-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 down-regulation 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 NCXfunction based on CRMP2 interaction with these proteins.

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