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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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Effect of NMDA on the M3-muscarinic receptor phosphoinositide response is dependent on calcium entry and CamKII activity. CG neurons were transfected with eGFP-PHPLCδ1 and grown for 7–9 days before being imaged on an inverted epifluorescence microscope. A, continuously perfused cells were subjected to two stimulations with methacholine (100 μm). Shown is the time course of eGFP-PHPLCδ translocation from the plasma membrane to the cytoplasm in a representative single cell expressed as changes in the cytoplasmic F/F0 self-ratio (see “Experimental Procedures”). Shown also are representative fluorescent images of CG neurons stimulated with methacholine (100 μm) for 0, 3, 15, 24, and 29 min. B, same as A but including a 5-min stimulation with NMDA (100 μm) prior to the second methacholine stimulation. Shown are images of a cell at times 0, 3, 15, 18, 24, and 29 min. C, the effects of the CamKII inhibitor KN-62 (10 μm) added 10 min before NMDA or the effects of removal of extracellular calcium on the NMDA-mediated desensitization of the M3-muscarinic receptor phosphoinositide response. The data are representative traces of 12–18 cells taken from 8–10 independent experiments. The insets represent the means ± S.D. of the S1 and S2 phosphoinositide responses. *, significant difference between S1 and S2 (p < 0.01). **, significant difference between S1 and S2 (p < 0.05).
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Figure 6: Effect of NMDA on the M3-muscarinic receptor phosphoinositide response is dependent on calcium entry and CamKII activity. CG neurons were transfected with eGFP-PHPLCδ1 and grown for 7–9 days before being imaged on an inverted epifluorescence microscope. A, continuously perfused cells were subjected to two stimulations with methacholine (100 μm). Shown is the time course of eGFP-PHPLCδ translocation from the plasma membrane to the cytoplasm in a representative single cell expressed as changes in the cytoplasmic F/F0 self-ratio (see “Experimental Procedures”). Shown also are representative fluorescent images of CG neurons stimulated with methacholine (100 μm) for 0, 3, 15, 24, and 29 min. B, same as A but including a 5-min stimulation with NMDA (100 μm) prior to the second methacholine stimulation. Shown are images of a cell at times 0, 3, 15, 18, 24, and 29 min. C, the effects of the CamKII inhibitor KN-62 (10 μm) added 10 min before NMDA or the effects of removal of extracellular calcium on the NMDA-mediated desensitization of the M3-muscarinic receptor phosphoinositide response. The data are representative traces of 12–18 cells taken from 8–10 independent experiments. The insets represent the means ± S.D. of the S1 and S2 phosphoinositide responses. *, significant difference between S1 and S2 (p < 0.01). **, significant difference between S1 and S2 (p < 0.05).

Mentions: In this study, stimulation of the CG neurons with methacholine evoked a rapid and substantial translocation of eGFP-PHPLCδ1 to the cytosol. The level of the phosphoinositide response (named S1) remained elevated for the length of the stimulation (F/F0 = 1.81 ± 0.12; n = 20; Fig. 6A). The value of S1 served as an internal standard. Following withdrawal of methacholine, the eGFP-PHPLCδ1 probe returned to the plasma membrane within 2 min (Fig. 6A). The cells subjected to a second stimulation with 100 μm methacholine gave rise to a phosphoinositide response (named S2) of very similar shape and amplitude (F/F0 1.73 ± 0.10; n = 12; Student's t test, p > 0.05) to the first (S1). However, when cells were treated with 100 μm NMDA for 5 min before the second stimulation with methacholine, the amplitude of S2 was significantly reduced (F/F0 1.43 ± 0.08 [n = 18]; Fig. 6B). These data demonstrate that NMDA receptor stimulation was able to desensitize the M3-muscarinic receptor phosphoinositide response.


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)

Effect of NMDA on the M3-muscarinic receptor phosphoinositide response is dependent on calcium entry and CamKII activity. CG neurons were transfected with eGFP-PHPLCδ1 and grown for 7–9 days before being imaged on an inverted epifluorescence microscope. A, continuously perfused cells were subjected to two stimulations with methacholine (100 μm). Shown is the time course of eGFP-PHPLCδ translocation from the plasma membrane to the cytoplasm in a representative single cell expressed as changes in the cytoplasmic F/F0 self-ratio (see “Experimental Procedures”). Shown also are representative fluorescent images of CG neurons stimulated with methacholine (100 μm) for 0, 3, 15, 24, and 29 min. B, same as A but including a 5-min stimulation with NMDA (100 μm) prior to the second methacholine stimulation. Shown are images of a cell at times 0, 3, 15, 18, 24, and 29 min. C, the effects of the CamKII inhibitor KN-62 (10 μm) added 10 min before NMDA or the effects of removal of extracellular calcium on the NMDA-mediated desensitization of the M3-muscarinic receptor phosphoinositide response. The data are representative traces of 12–18 cells taken from 8–10 independent experiments. The insets represent the means ± S.D. of the S1 and S2 phosphoinositide responses. *, significant difference between S1 and S2 (p < 0.01). **, significant difference between S1 and S2 (p < 0.05).
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Related In: Results  -  Collection

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Show All Figures
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Figure 6: Effect of NMDA on the M3-muscarinic receptor phosphoinositide response is dependent on calcium entry and CamKII activity. CG neurons were transfected with eGFP-PHPLCδ1 and grown for 7–9 days before being imaged on an inverted epifluorescence microscope. A, continuously perfused cells were subjected to two stimulations with methacholine (100 μm). Shown is the time course of eGFP-PHPLCδ translocation from the plasma membrane to the cytoplasm in a representative single cell expressed as changes in the cytoplasmic F/F0 self-ratio (see “Experimental Procedures”). Shown also are representative fluorescent images of CG neurons stimulated with methacholine (100 μm) for 0, 3, 15, 24, and 29 min. B, same as A but including a 5-min stimulation with NMDA (100 μm) prior to the second methacholine stimulation. Shown are images of a cell at times 0, 3, 15, 18, 24, and 29 min. C, the effects of the CamKII inhibitor KN-62 (10 μm) added 10 min before NMDA or the effects of removal of extracellular calcium on the NMDA-mediated desensitization of the M3-muscarinic receptor phosphoinositide response. The data are representative traces of 12–18 cells taken from 8–10 independent experiments. The insets represent the means ± S.D. of the S1 and S2 phosphoinositide responses. *, significant difference between S1 and S2 (p < 0.01). **, significant difference between S1 and S2 (p < 0.05).
Mentions: In this study, stimulation of the CG neurons with methacholine evoked a rapid and substantial translocation of eGFP-PHPLCδ1 to the cytosol. The level of the phosphoinositide response (named S1) remained elevated for the length of the stimulation (F/F0 = 1.81 ± 0.12; n = 20; Fig. 6A). The value of S1 served as an internal standard. Following withdrawal of methacholine, the eGFP-PHPLCδ1 probe returned to the plasma membrane within 2 min (Fig. 6A). The cells subjected to a second stimulation with 100 μm methacholine gave rise to a phosphoinositide response (named S2) of very similar shape and amplitude (F/F0 1.73 ± 0.10; n = 12; Student's t test, p > 0.05) to the first (S1). However, when cells were treated with 100 μm NMDA for 5 min before the second stimulation with methacholine, the amplitude of S2 was significantly reduced (F/F0 1.43 ± 0.08 [n = 18]; Fig. 6B). These data demonstrate that NMDA receptor stimulation was able to desensitize the M3-muscarinic receptor phosphoinositide response.

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