NMDA-induced potentiation of mGluR5 is mediated by activation of protein phosphatase 2B/calcineurin

https://doi.org/10.1016/j.neuropharm.2005.05.005Get rights and content

Abstract

Previous reports have shown that activation of N-methyl-d-aspartate (NMDA) receptors potentiates responses to activation of the group I metabotropic glutamate receptor mGluR5 by reversing PKC-mediated desensitization of this receptor. NMDA-induced reversal of mGluR5 desensitization is dependent on activation of protein phosphatases. However, the specific protein phosphatase involved and the precise mechanism by which NMDA receptor activation reduces mGluR desensitization are not known. We have performed a series of molecular, biochemical, and genetic studies to show that NMDA-induced regulation of mGluR5 is dependent on activation of calcium-dependent protein phosphatase 2B/calcineurin (PP2B/CaN). Furthermore, we report that purified calcineurin directly dephosphorylates the C-terminal tail of mGluR5 at sites that are phosphorylated by PKC. Finally, immunoprecipitation and GST fusion protein pull-down experiments reveal that calcineurin interacts with mGluR5, suggesting that these proteins could be colocalized in a signaling complex. Taken together with previous studies, these data suggest that activation of NMDA receptors leads to activation of calcineurin and that calcineurin modulates mGluR5 function by directly dephosphorylating mGluR5 at PKC sites that are involved in desensitization of this receptor.

Introduction

Glutamate elicits synaptic responses by activation of both ionotropic receptors (iGluRs) and G protein-coupled metabotropic glutamate receptors (mGluRs). The iGluRs are ligand-gated cation channels that are classified into the N-methyl-d-aspartate (NMDA), kainate, and [RS]-α-amino-3-hydroxy-5-methyl-isoxazolepropionic acid (AMPA) receptor subtypes (Dingledine et al., 1999). The mGluRs are members of family III G protein-coupled receptors (GPCRs). To date, eight mGluR subtypes (mGluR1-mGluR8) have been cloned from mammalian brain (see Conn and Pin, 1997 for review). These mGluR subtypes can be further classified into three groups. Group I mGluRs consist of mGluRs 1 and 5 and are coupled to Gq and activation of phospholipase C. Group II (mGluRs 2 and 3) and group III (mGluRs 4, 6–8) mGluRs couple to Gi/Go and associated effectors.

One prominent effect of mGluR activation in many neuronal populations is an enhancement of agonist-evoked currents through NMDA receptor channels (Aniksztejn et al., 1991, Jones and Headley, 1995, Bleakman et al., 1992, Harvey and Collingridge, 1993, Fitzjohn et al., 1996, Pisani et al., 1997, Awad et al., 2000). In each of these cases, mGluR-induced potentiation of NMDA receptor currents is mediated by a group I mGluR. However, the specific group I mGluR subtype involved in eliciting this effect can vary in different neuronal populations. For instance, recent pharmacological studies reveal that potentiation of NMDA-evoked currents is mediated by mGluR5 in hippocampal pyramidal cells (Doherty et al., 1997) (Mannaioni et al., 2001) and in neurons in the subthalamic nucleus (Awad et al., 2000), whereas this response is mediated by mGluR1 in cortical cells (Heidinger et al., 2002).

Interestingly, the interaction between NMDA receptors and group I mGluRs is bi-directional. For instance, low concentrations of NMDA can enhance mGluR-mediated increases in phosphoinositide hydrolysis in rat cortex (Challiss et al., 1994, Alagarsamy et al., 1999b). Furthermore, NMDA receptor activation potentiates an inward current induced by 1S,3R-ACPD in hippocampal pyramidal cells (Luthi et al., 1994, Alagarsamy et al., 1999a). We recently reported that NMDA-induced potentiation of group I mGluR responses in recombinant systems and in the hippocampus is mediated by reversal of PKC-induced mGluR desensitization. Furthermore, we found that this response is dependent on activation of a protein phosphatase. Previous reports suggest that PKC desensitizes mGluR5 by phosphorylation of Ser and Thr residues on the C-terminal tail and first and second intracellular loops of the receptor (Gereau and Heinemann, 1998). Taken together, these findings raise the possibility that activation of NMDA receptors induces activation of a protein phosphatase that directly dephosphorylates mGluR5 at sites responsible for desensitization. The studies reported here suggest that NMDA-induced modulation of mGluR5 is dependent on activation of the calcium-dependent protein phosphatase 2B (PP2B), calcineurin. Furthermore, we provide evidence that calcineurin may exist in a signaling complex with mGluR5 in several brain areas and that this phosphatase can directly dephosphorylate mGluR5 at sites that are phosphorylated by PKC on the C-terminal tail of the receptor.

Section snippets

Cell culture and transfection

Chinese hamster ovary (CHO) cells were cultured in DMEM supplemented with 10% fetal bovine serum. The cells were grown to ∼80% confluency in 24-well plates. For expression of the receptors and a constitutively active mutant of calcineurin (CaNa) (Friday et al., 2000), the plasmids were transfected into the CHO cell line, using a calcium phosphate technique. Each plasmid (1 μg) was added to 50 μl CaCl (0.25 mM) and 50 μl BES (5 mM, pH 7.4) into wells containing cells and 1 ml media. After an overnight

Materials

The constitutively active calcineurin construct was a kind gift from Dr. Grace Pavlath of Emory University, antibodies were obtained from Upstate Biotechnology, Lake Placid, NY, and cell culture reagents were obtained from Gibco, Invitrogen, Carlsbad, CA. All other reagents, unless otherwise noted, were obtained from Sigma, St. Louis, MO.

Results

We previously reported that N-methyl-d-aspartate (NMDA) potentiates mGluR5 function by reversing PKC-mediated desensitization of the receptor. Furthermore, studies with protein phosphatase inhibitors suggested that this effect is dependent on activation of a phosphatase. In order to perform more detailed pharmacological studies to gain insight into the phosphatase responsible for this effect of NMDA receptor activation, we determined the effect of NMDA on DHPG-induced phosphoinositide

Discussion

In total, the data presented here suggest that calcineurin and mGluR5 may exist in a signaling complex and that calcineurin is capable of potentiating mGluR5 function by direct dephosphorylation of the receptor at PKC phosphorylation sites. Our biochemical studies provide clear support for the hypothesis that mGluR5 desensitization is reversed by NMDA receptor-mediated activation of calcineurin. Functional studies, coupled with the finding that purified calcineurin dephosphorylates PKC sites on

Acknowledgements

The authors would like to thank Dr. Grace Pavlath for her kind contribution of the constitutively active calcineurin construct and FK506. This work was supported by NIH NINDS grants (S.A. and P.J.C.) and NIMH grants (R.W.G).

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