N-Methyl-d-aspartate Receptors Mediate the Phosphorylation and Desensitization of Muscarinic Receptors in Cerebellar Granule Neurons

Adrian J Butcher,Ignacio Torrecilla,Kenneth W Young,Kok Choi Kong,Sharad C Mistry,Andrew R Bottrill,Andrew B Tobin

doi:10.1074/jbc.m901031200

Adrian J Butcher, Ignacio Torrecilla + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m901031200

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Abstract

Changes in synaptic strength mediated by ionotropic glutamate N-methyl-d-asparate (NMDA) receptors is generally considered to be the molecular mechanism underlying memory and learning. NMDA receptors themselves are subject to regulation through signaling pathways that are activated by G-protein-coupled receptors (GPCRs). In this study we investigate the ability of NMDA receptors to regulate the signaling of GPCRs by focusing on the Gq/11-coupled M3-muscarinic receptor expressed endogenously in mouse cerebellar granule neurons. We show that NMDA receptor activation results in the phosphorylation and desensitization of M3-muscarinic receptors through a mechanism dependent on NMDA-mediated calcium influx and the activity of calcium-calmodulin-dependent protein kinase II. Our study reveals a complex pattern of regulation where GPCRs (M3-muscarinic) and NMDA receptors can feedback on each other in a process that is likely to influence the threshold value of signaling networks involved in synaptic plasticity.

Highlights

Glutamate neurotransmission mediated through the ionotropic N-methyl-D-asparate (NMDA),3 ␣-amino-3-hydroxy-5methyl-4-isoxazolepropionic acid (AMPA), and kainate receptors is the primary excitatory stimulus in the central nervous system [1]
In the current study we demonstrate that stimulation of glutamate receptors using NMDA resulted in an increase in the phosphorylation state of the M3 muscarinic receptor (Fig. 1A)
It is clear from a large number of studies that G-protein-coupled receptor (GPCR) can influence the activity of NMDA receptors and in so doing regulate physiological responses such as long term potentiation (LTP) and LTD [6]

Summary

EXPERIMENTAL PROCEDURES

Primary Cell Culture—Mouse CG neurons were cultured as described previously [28]. In brief, cerebella from 7– 8-day-old Balb/c pups were mechanically and enzymatically (trypsin) dissociated. NMDA Desensitization of the M3-muscarinic Receptor Phosphoinositide Response—CG neurons expressing eGFP-PHPLC␦1 were perfused with CSS-25 containing a maximal concentration of methacholine (100 ␮M) for 2 min. This generated a phosphoinositide response that was termed S1 and served as an internal control. In each case it was clear as to the identity of the peptide (and that the peptide was phosphorylated), but for some MS/MS spectra it was not possible to validate the MASCOT assignment of the residue position for phosphorylation, because of low abundance of the required fragment ions In these cases (because the sample amount was not limiting), the samples were repeated using multiple reaction monitoring inclusion lists comprising ion pairs consisting of the precursor ion [Mϩ2H]2ϩ and that of the neutral ion loss [Mϩ2H-H3PO4]2ϩ, such that the MS/MS data were obtained over a longer time period (5.86 s), which resulted in significantly improved data quality.

RESULTS

Does Not Affect Muscarinic Receptor

DISCUSSION