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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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In vitro phosphorylation the M3-muscarinic receptor within the third intracellular loop by CamKII. A, bacterially expressed GST or GST-3iloop (containing the third intracellular loop of the mouse M3-muscarinic receptor) were used in an in vitro phosphorylation using purified CamKII (200 ng). Shown is a representative experiment where the gel is first stained with Coomassie Blue and then an autoradiograph obtained. B, diagram representation of the CamKII phospho-acceptor sites Ser322 and Ser350 as determined by LC-MS/MS (in red) and a peptide that is singularly phosphorylated at one of three sites; serines 384/385 and threonine 397 (indicated in blue). (See supplemental Figs. S2–S4 for detailed traces.)
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Figure 4: In vitro phosphorylation the M3-muscarinic receptor within the third intracellular loop by CamKII. A, bacterially expressed GST or GST-3iloop (containing the third intracellular loop of the mouse M3-muscarinic receptor) were used in an in vitro phosphorylation using purified CamKII (200 ng). Shown is a representative experiment where the gel is first stained with Coomassie Blue and then an autoradiograph obtained. B, diagram representation of the CamKII phospho-acceptor sites Ser322 and Ser350 as determined by LC-MS/MS (in red) and a peptide that is singularly phosphorylated at one of three sites; serines 384/385 and threonine 397 (indicated in blue). (See supplemental Figs. S2–S4 for detailed traces.)

Mentions: The mouse M3-muscarinic receptor contains a large third intracellular loop with 33 serine and 20 threonine residues. This compares with only three threonine and one serine in the short C-terminal tail. For this reason, and for that fact that the sites for agonist-regulated phosphorylation have previously been shown to be in the third intracellular loop (29, 34, 35), it was decided to test whether CamKII could phosphorylate the third intracellular loop in vitro. These experiments demonstrated that a bacterially expressed glutathione S-transferase fusion protein containing the third intracellular loop of the mouse M3-muscarinic receptor (Arg252–Thr491) was phosphorylated specifically in the receptor portion of the fusion protein by CamKII (Fig. 4A).


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)

In vitro phosphorylation the M3-muscarinic receptor within the third intracellular loop by CamKII. A, bacterially expressed GST or GST-3iloop (containing the third intracellular loop of the mouse M3-muscarinic receptor) were used in an in vitro phosphorylation using purified CamKII (200 ng). Shown is a representative experiment where the gel is first stained with Coomassie Blue and then an autoradiograph obtained. B, diagram representation of the CamKII phospho-acceptor sites Ser322 and Ser350 as determined by LC-MS/MS (in red) and a peptide that is singularly phosphorylated at one of three sites; serines 384/385 and threonine 397 (indicated in blue). (See supplemental Figs. S2–S4 for detailed traces.)
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: In vitro phosphorylation the M3-muscarinic receptor within the third intracellular loop by CamKII. A, bacterially expressed GST or GST-3iloop (containing the third intracellular loop of the mouse M3-muscarinic receptor) were used in an in vitro phosphorylation using purified CamKII (200 ng). Shown is a representative experiment where the gel is first stained with Coomassie Blue and then an autoradiograph obtained. B, diagram representation of the CamKII phospho-acceptor sites Ser322 and Ser350 as determined by LC-MS/MS (in red) and a peptide that is singularly phosphorylated at one of three sites; serines 384/385 and threonine 397 (indicated in blue). (See supplemental Figs. S2–S4 for detailed traces.)
Mentions: The mouse M3-muscarinic receptor contains a large third intracellular loop with 33 serine and 20 threonine residues. This compares with only three threonine and one serine in the short C-terminal tail. For this reason, and for that fact that the sites for agonist-regulated phosphorylation have previously been shown to be in the third intracellular loop (29, 34, 35), it was decided to test whether CamKII could phosphorylate the third intracellular loop in vitro. These experiments demonstrated that a bacterially expressed glutathione S-transferase fusion protein containing the third intracellular loop of the mouse M3-muscarinic receptor (Arg252–Thr491) was phosphorylated specifically in the receptor portion of the fusion protein by CamKII (Fig. 4A).

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