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N-methyl-D-aspartate receptors mediate the phosphorylation and desensitization of muscarinic receptors in cerebellar granule neurons.

Butcher AJ, Torrecilla I, Young KW, Kong KC, Mistry SC, Bottrill AR, Tobin AB - J. Biol. Chem. (2009)

Bottom Line: NMDA receptors themselves are subject to regulation through signaling pathways that are activated by G-protein-coupled receptors (GPCRs).We show that NMDA receptor activation results in the phosphorylation and desensitization of M(3)-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 (M(3)-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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Physiology and Pharmacology, University of Leicesterm, Hodgkin Building, Lancaster Road, Leicester LE1 9HN, United Kingdom.

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 G(q/11)-coupled M(3)-muscarinic receptor expressed endogenously in mouse cerebellar granule neurons. We show that NMDA receptor activation results in the phosphorylation and desensitization of M(3)-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 (M(3)-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.

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Activation of NMDA receptors induces phosphorylation of M3-muscarinic receptor. A, representative autoradiograph of M3-muscarinic receptors immunoprecipitated from [32P]orthophosphate labeled CG neurons prepared from wild type (WT) mice or M3-muscarinic receptor knock-out (KO) mice. The CG neurons were stimulated for 5 min with either; vehicle, methacholine (100 μm), or NMDA (100 μm). In these experiments immunoprecipitated proteins resolved by SDS-PAGE were transferred to nitrocellulose, and after exposure for phosphorylation the nitrocellulose was probed using a receptor-specific monoclonal antibody. This served as a loading control for the M3-muscarinic receptor. B, phosphorylation of the M3-muscarinic receptor in response to methacholine (100 μm), with or without atropine (10 μm), and to glutamate (100 μm) or NMDA (100 μm). C, phosphorylation of the M3-muscarinic receptor in CG neurons stimulated with the ionotropic glutamate receptor agonists NMDA, AMPA, and kainic acid (all at 100 μm). D, effect of the NMDA receptor antagonists MK-801 (5 μm, 10 min) and AP-5 (20 μm, 10 min). E, time course of M3-muscarinic receptor phosphorylation following stimulation with NMDA (100 μm). Graphs represent quantification of phosphorylation (means ± S.D.) from at least three replicates, normalized to the basal phosphorylation of the receptor in control cells with no stimulation. *, significant difference between basal and stimulated samples (p < 0.01).
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Figure 1: Activation of NMDA receptors induces phosphorylation of M3-muscarinic receptor. A, representative autoradiograph of M3-muscarinic receptors immunoprecipitated from [32P]orthophosphate labeled CG neurons prepared from wild type (WT) mice or M3-muscarinic receptor knock-out (KO) mice. The CG neurons were stimulated for 5 min with either; vehicle, methacholine (100 μm), or NMDA (100 μm). In these experiments immunoprecipitated proteins resolved by SDS-PAGE were transferred to nitrocellulose, and after exposure for phosphorylation the nitrocellulose was probed using a receptor-specific monoclonal antibody. This served as a loading control for the M3-muscarinic receptor. B, phosphorylation of the M3-muscarinic receptor in response to methacholine (100 μm), with or without atropine (10 μm), and to glutamate (100 μm) or NMDA (100 μm). C, phosphorylation of the M3-muscarinic receptor in CG neurons stimulated with the ionotropic glutamate receptor agonists NMDA, AMPA, and kainic acid (all at 100 μm). D, effect of the NMDA receptor antagonists MK-801 (5 μm, 10 min) and AP-5 (20 μm, 10 min). E, time course of M3-muscarinic receptor phosphorylation following stimulation with NMDA (100 μm). Graphs represent quantification of phosphorylation (means ± S.D.) from at least three replicates, normalized to the basal phosphorylation of the receptor in control cells with no stimulation. *, significant difference between basal and stimulated samples (p < 0.01).

Mentions: In vivo [32P]orthophosphate labeling, receptor solubilization, and immunoprecipitation were conducted as described previously (29). In brief, CG neurons in 6-well plates were washed and incubated for 2 h in CSS-25 buffer (1 ml) containing 100 μCi/ml [32P]orthophosphate (GE Healthcare). The cells were then stimulated with methacholine, glutamate, NMDA, AMPA, or kainic acid at a final concentration of 100 μm for 5 min unless otherwise stated. Where appropriate, antagonists or other compounds were added before the addition of agonists. The cells were then lysed in lysis buffer (10 mm EDTA, 500 mm NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 10 mm Tris, pH 7.4), and the M3-muscarinic receptor was immunoprecipitated using an in house anti-mouse M3 receptor polyclonal antibody (29). Immunoprecipitated proteins were resolved by 8% SDS-PAGE and visualized by autoradiography or using a STORM phosphor-imager (GE Healthcare). To control for equal receptor loading, immunoprecipitated proteins resolved by SDS-PAGE were transferred to nitrocellulose. The membranes were then exposed before being used in Western blots with M3-muscarinic receptor specific monoclonal antibodies (see Figs. 1A and 3A). Quantification of the phosphorylation status of the receptor was determined by analysis using the ImageQuant and AlphaEase softwares.


N-methyl-D-aspartate receptors mediate the phosphorylation and desensitization of muscarinic receptors in cerebellar granule neurons.

Butcher AJ, Torrecilla I, Young KW, Kong KC, Mistry SC, Bottrill AR, Tobin AB - J. Biol. Chem. (2009)

Activation of NMDA receptors induces phosphorylation of M3-muscarinic receptor. A, representative autoradiograph of M3-muscarinic receptors immunoprecipitated from [32P]orthophosphate labeled CG neurons prepared from wild type (WT) mice or M3-muscarinic receptor knock-out (KO) mice. The CG neurons were stimulated for 5 min with either; vehicle, methacholine (100 μm), or NMDA (100 μm). In these experiments immunoprecipitated proteins resolved by SDS-PAGE were transferred to nitrocellulose, and after exposure for phosphorylation the nitrocellulose was probed using a receptor-specific monoclonal antibody. This served as a loading control for the M3-muscarinic receptor. B, phosphorylation of the M3-muscarinic receptor in response to methacholine (100 μm), with or without atropine (10 μm), and to glutamate (100 μm) or NMDA (100 μm). C, phosphorylation of the M3-muscarinic receptor in CG neurons stimulated with the ionotropic glutamate receptor agonists NMDA, AMPA, and kainic acid (all at 100 μm). D, effect of the NMDA receptor antagonists MK-801 (5 μm, 10 min) and AP-5 (20 μm, 10 min). E, time course of M3-muscarinic receptor phosphorylation following stimulation with NMDA (100 μm). Graphs represent quantification of phosphorylation (means ± S.D.) from at least three replicates, normalized to the basal phosphorylation of the receptor in control cells with no stimulation. *, significant difference between basal and stimulated samples (p < 0.01).
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Figure 1: Activation of NMDA receptors induces phosphorylation of M3-muscarinic receptor. A, representative autoradiograph of M3-muscarinic receptors immunoprecipitated from [32P]orthophosphate labeled CG neurons prepared from wild type (WT) mice or M3-muscarinic receptor knock-out (KO) mice. The CG neurons were stimulated for 5 min with either; vehicle, methacholine (100 μm), or NMDA (100 μm). In these experiments immunoprecipitated proteins resolved by SDS-PAGE were transferred to nitrocellulose, and after exposure for phosphorylation the nitrocellulose was probed using a receptor-specific monoclonal antibody. This served as a loading control for the M3-muscarinic receptor. B, phosphorylation of the M3-muscarinic receptor in response to methacholine (100 μm), with or without atropine (10 μm), and to glutamate (100 μm) or NMDA (100 μm). C, phosphorylation of the M3-muscarinic receptor in CG neurons stimulated with the ionotropic glutamate receptor agonists NMDA, AMPA, and kainic acid (all at 100 μm). D, effect of the NMDA receptor antagonists MK-801 (5 μm, 10 min) and AP-5 (20 μm, 10 min). E, time course of M3-muscarinic receptor phosphorylation following stimulation with NMDA (100 μm). Graphs represent quantification of phosphorylation (means ± S.D.) from at least three replicates, normalized to the basal phosphorylation of the receptor in control cells with no stimulation. *, significant difference between basal and stimulated samples (p < 0.01).
Mentions: In vivo [32P]orthophosphate labeling, receptor solubilization, and immunoprecipitation were conducted as described previously (29). In brief, CG neurons in 6-well plates were washed and incubated for 2 h in CSS-25 buffer (1 ml) containing 100 μCi/ml [32P]orthophosphate (GE Healthcare). The cells were then stimulated with methacholine, glutamate, NMDA, AMPA, or kainic acid at a final concentration of 100 μm for 5 min unless otherwise stated. Where appropriate, antagonists or other compounds were added before the addition of agonists. The cells were then lysed in lysis buffer (10 mm EDTA, 500 mm NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 10 mm Tris, pH 7.4), and the M3-muscarinic receptor was immunoprecipitated using an in house anti-mouse M3 receptor polyclonal antibody (29). Immunoprecipitated proteins were resolved by 8% SDS-PAGE and visualized by autoradiography or using a STORM phosphor-imager (GE Healthcare). To control for equal receptor loading, immunoprecipitated proteins resolved by SDS-PAGE were transferred to nitrocellulose. The membranes were then exposed before being used in Western blots with M3-muscarinic receptor specific monoclonal antibodies (see Figs. 1A and 3A). Quantification of the phosphorylation status of the receptor was determined by analysis using the ImageQuant and AlphaEase softwares.

Bottom Line: NMDA receptors themselves are subject to regulation through signaling pathways that are activated by G-protein-coupled receptors (GPCRs).We show that NMDA receptor activation results in the phosphorylation and desensitization of M(3)-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 (M(3)-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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Physiology and Pharmacology, University of Leicesterm, Hodgkin Building, Lancaster Road, Leicester LE1 9HN, United Kingdom.

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 G(q/11)-coupled M(3)-muscarinic receptor expressed endogenously in mouse cerebellar granule neurons. We show that NMDA receptor activation results in the phosphorylation and desensitization of M(3)-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 (M(3)-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.

Show MeSH
Related in: MedlinePlus