Limits...
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.

Show MeSH

Related in: MedlinePlus

Extracellular calcium and CamKII activity is required for NMDA-mediated phosphorylation of M3-muscarinic receptors. CG neurons were [32P]orthophosphate labeled in calcium containing medium. A, cells were subsequently washed with calcium-containing (left panel) or nominally calcium-free (right panel) medium, and stimulated for 5 min with NMDA (100 μm) or methacholine (100 μm). B, phosphorylation of the M3-muscarinic receptor in response to NMDA (100 μm, 5 min) in the presence or absence of inhibitors of CamKII, KN-93 and KN-62, and the inactive analogue, KN-92 (10 μm). C and D, time course of the free intracellular calcium concentration in response to transient stimulation by methacholine or NMDA in the absence (C) or presence (D) of KN-62. The phosphorylation of M3-muscarinic receptors was quantified and normalized to the basal phosphorylation and presented as the mean ± S.D. *, significant difference between basal and stimulated samples (p < 0.01). **, significant difference between basal and stimulated samples (p < 0.05). Calcium traces correspond to the changes in free intracellular calcium in single CG neurons and represent the response of at least 20 cells in three different assays.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2719353&req=5

Figure 2: Extracellular calcium and CamKII activity is required for NMDA-mediated phosphorylation of M3-muscarinic receptors. CG neurons were [32P]orthophosphate labeled in calcium containing medium. A, cells were subsequently washed with calcium-containing (left panel) or nominally calcium-free (right panel) medium, and stimulated for 5 min with NMDA (100 μm) or methacholine (100 μm). B, phosphorylation of the M3-muscarinic receptor in response to NMDA (100 μm, 5 min) in the presence or absence of inhibitors of CamKII, KN-93 and KN-62, and the inactive analogue, KN-92 (10 μm). C and D, time course of the free intracellular calcium concentration in response to transient stimulation by methacholine or NMDA in the absence (C) or presence (D) of KN-62. The phosphorylation of M3-muscarinic receptors was quantified and normalized to the basal phosphorylation and presented as the mean ± S.D. *, significant difference between basal and stimulated samples (p < 0.01). **, significant difference between basal and stimulated samples (p < 0.05). Calcium traces correspond to the changes in free intracellular calcium in single CG neurons and represent the response of at least 20 cells in three different assays.

Mentions: NMDA receptor activation will result in an influx of extracellular calcium and the subsequent activation of a number of calcium-dependent signaling molecules in particular CamKII (3, 4). We show here that the ability of NMDA receptors to stimulate the phosphorylation of M3-muscarinic receptors is dependent on the presence of extracellular calcium in the medium (Fig. 2). Following [32P]orthophosphate labeling, the cells were washed with medium (CSS-25) containing calcium or with nominally calcium-free medium. A subsequent stimulation with NMDA resulted in a robust phosphorylation of the M3-muscarinic receptor only in cells that were incubated in calcium containing medium (Fig. 2A). In contrast, phosphorylation of M3-muscarinic receptors in response to the muscarinic receptor agonist methacholine was not affected by the absence of calcium in the medium (Fig. 2A).


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)

Extracellular calcium and CamKII activity is required for NMDA-mediated phosphorylation of M3-muscarinic receptors. CG neurons were [32P]orthophosphate labeled in calcium containing medium. A, cells were subsequently washed with calcium-containing (left panel) or nominally calcium-free (right panel) medium, and stimulated for 5 min with NMDA (100 μm) or methacholine (100 μm). B, phosphorylation of the M3-muscarinic receptor in response to NMDA (100 μm, 5 min) in the presence or absence of inhibitors of CamKII, KN-93 and KN-62, and the inactive analogue, KN-92 (10 μm). C and D, time course of the free intracellular calcium concentration in response to transient stimulation by methacholine or NMDA in the absence (C) or presence (D) of KN-62. The phosphorylation of M3-muscarinic receptors was quantified and normalized to the basal phosphorylation and presented as the mean ± S.D. *, significant difference between basal and stimulated samples (p < 0.01). **, significant difference between basal and stimulated samples (p < 0.05). Calcium traces correspond to the changes in free intracellular calcium in single CG neurons and represent the response of at least 20 cells in three different assays.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2719353&req=5

Figure 2: Extracellular calcium and CamKII activity is required for NMDA-mediated phosphorylation of M3-muscarinic receptors. CG neurons were [32P]orthophosphate labeled in calcium containing medium. A, cells were subsequently washed with calcium-containing (left panel) or nominally calcium-free (right panel) medium, and stimulated for 5 min with NMDA (100 μm) or methacholine (100 μm). B, phosphorylation of the M3-muscarinic receptor in response to NMDA (100 μm, 5 min) in the presence or absence of inhibitors of CamKII, KN-93 and KN-62, and the inactive analogue, KN-92 (10 μm). C and D, time course of the free intracellular calcium concentration in response to transient stimulation by methacholine or NMDA in the absence (C) or presence (D) of KN-62. The phosphorylation of M3-muscarinic receptors was quantified and normalized to the basal phosphorylation and presented as the mean ± S.D. *, significant difference between basal and stimulated samples (p < 0.01). **, significant difference between basal and stimulated samples (p < 0.05). Calcium traces correspond to the changes in free intracellular calcium in single CG neurons and represent the response of at least 20 cells in three different assays.
Mentions: NMDA receptor activation will result in an influx of extracellular calcium and the subsequent activation of a number of calcium-dependent signaling molecules in particular CamKII (3, 4). We show here that the ability of NMDA receptors to stimulate the phosphorylation of M3-muscarinic receptors is dependent on the presence of extracellular calcium in the medium (Fig. 2). Following [32P]orthophosphate labeling, the cells were washed with medium (CSS-25) containing calcium or with nominally calcium-free medium. A subsequent stimulation with NMDA resulted in a robust phosphorylation of the M3-muscarinic receptor only in cells that were incubated in calcium containing medium (Fig. 2A). In contrast, phosphorylation of M3-muscarinic receptors in response to the muscarinic receptor agonist methacholine was not affected by the absence of calcium in the medium (Fig. 2A).

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