N-Methyl-d-aspartate Receptors Mediate the Phosphorylation and Desensitization of Muscarinic Receptors in Cerebellar Granule Neurons
2009; Elsevier BV; Volume: 284; Issue: 25 Linguagem: Inglês
10.1074/jbc.m901031200
ISSN1083-351X
AutoresAdrian J. Butcher, Ignacio Torrecilla, Kenneth W. Young, Kok Choi Kong, Sharad Mistry, Andrew R. Bottrill, Andrew B. Tobin,
Tópico(s)Neuropeptides and Animal Physiology
ResumoChanges 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. 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. Glutamate neurotransmission mediated through the ionotropic N-methyl-d-asparate (NMDA), 3The abbreviations used are: NMDAN-methyl-d-asparateAMPAα-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acidCamKIIcalcium/calmodulin-dependent protein kinase IICGcerebellar granuleeGFPenhanced green fluorescent proteinGPCRG-protein-coupled receptorGRKGPCR kinaseIP3inositol 1,4,5-trisphosphateLTDlong term depressionLTPlong term potentiationPHpleckstrin homologyPIP2phosphoinositide 4,5-bisphosphatePLCphospholipase CsiRNAsmall interfering RNAGSTglutathione S-transferaseMALDI-TOFmatrix-assisted laser desorption ionization time-of-flightLC-MS/MSliquid chromatography-tandem mass spectrometry. 3The abbreviations used are: NMDAN-methyl-d-asparateAMPAα-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acidCamKIIcalcium/calmodulin-dependent protein kinase IICGcerebellar granuleeGFPenhanced green fluorescent proteinGPCRG-protein-coupled receptorGRKGPCR kinaseIP3inositol 1,4,5-trisphosphateLTDlong term depressionLTPlong term potentiationPHpleckstrin homologyPIP2phosphoinositide 4,5-bisphosphatePLCphospholipase CsiRNAsmall interfering RNAGSTglutathione S-transferaseMALDI-TOFmatrix-assisted laser desorption ionization time-of-flightLC-MS/MSliquid chromatography-tandem mass spectrometry. α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and kainate receptors is the primary excitatory stimulus in the central nervous system (1Watkins J.C. Jane D.E. Br. J. Pharmacol. 2006; 147: S100-S108Crossref PubMed Scopus (194) Google Scholar). NMDA and AMPA receptors are found co-expressed at the post-synaptic membrane of glutaminergic synapses. AMPA receptors, permeable to sodium and potassium ions, open in response to glutamate generating rapid excitatory post-synaptic potentials. NMDA receptors allow for the influx of extracellular calcium into the post-synaptic cell in response to glutamate and a concomitant depolarization that relieves the magnesium block of channel conductance. Through the temporal and spatial summation of excitatory post-synaptic potentials and glutamate release, NMDA receptors can contribute significantly to post-synaptic membrane depolarization. N-methyl-d-asparate α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid calcium/calmodulin-dependent protein kinase II cerebellar granule enhanced green fluorescent protein G-protein-coupled receptor GPCR kinase inositol 1,4,5-trisphosphate long term depression long term potentiation pleckstrin homology phosphoinositide 4,5-bisphosphate phospholipase C small interfering RNA glutathione S-transferase matrix-assisted laser desorption ionization time-of-flight liquid chromatography-tandem mass spectrometry. N-methyl-d-asparate α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid calcium/calmodulin-dependent protein kinase II cerebellar granule enhanced green fluorescent protein G-protein-coupled receptor GPCR kinase inositol 1,4,5-trisphosphate long term depression long term potentiation pleckstrin homology phosphoinositide 4,5-bisphosphate phospholipase C small interfering RNA glutathione S-transferase matrix-assisted laser desorption ionization time-of-flight liquid chromatography-tandem mass spectrometry. In addition, NMDA receptors can influence post-synaptic activity in a more subtle manor by allowing for changes in intracellular calcium concentrations that initiate calcium-dependent signaling cascades (2Malenka R.C. Nicoll R.A. Science. 1999; 285: 1870-1874Crossref PubMed Scopus (2206) Google Scholar, 3Sheng M. Kim M.J. Science. 2002; 298: 776-780Crossref PubMed Scopus (586) Google Scholar). Prominent among these signaling cascades is the regulation of protein phosphorylation via both calcium/calmodulin-dependent kinase II (CamKII) (3Sheng M. Kim M.J. Science. 2002; 298: 776-780Crossref PubMed Scopus (586) Google Scholar, 4Lisman J. Schulman H. Cline H. Nat. Rev. Neurosci. 2002; 3: 175-190Crossref PubMed Scopus (1443) Google Scholar) and the protein phosphatases protein phosphatase 1 and calcineurin (5Mulkey R.M. Endo S. Shenolikar S. Malenka R.C. Nature. 1994; 369: 486-488Crossref PubMed Scopus (897) Google Scholar). These signaling events are among a number of mechanisms that are proposed to regulate NMDA-mediated changes in synaptic strength (6MacDonald J.F. Jackson M.F. Beazely M.A. Biochim. Biophys. Acta. 2007; 1768: 941-951Crossref PubMed Scopus (59) Google Scholar). Synaptic plasticity of this type is frequently measured as long term potentiation (LTP) and depression (LTD) and is widely regarded as a molecular mechanism that contributes to memory and learning (7Bliss T.V. Collingridge G.L. Nature. 1993; 361: 31-39Crossref PubMed Scopus (9396) Google Scholar, 8Kemp N. Bashir Z.I. Prog. Neurobiol. 2001; 65: 339-365Crossref PubMed Scopus (186) Google Scholar). It is now clear that the clustering of signaling molecules around NMDA and AMPA receptors at specialized post-synaptic sites coordinate signaling pathways involved in the adaptation of synaptic activity (3Sheng M. Kim M.J. Science. 2002; 298: 776-780Crossref PubMed Scopus (586) Google Scholar, 9Okabe S. Mol. Cell. Neurosci. 2007; 34: 503-518Crossref PubMed Scopus (176) Google Scholar). Among these signaling molecules are members of the G-protein-coupled receptor (GPCR) family (6MacDonald J.F. Jackson M.F. Beazely M.A. Biochim. Biophys. 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Neurosci. 2005; 25: 11374-11384Crossref PubMed Scopus (97) Google Scholar), and muscarinic (16Shinoe T. Matsui M. Taketo M.M. Manabe T. J. Neurosci. 2005; 25: 11194-11200Crossref PubMed Scopus (191) Google Scholar) receptors are found closely associated with NMDA receptors. These GPCRs regulate NMDA function through a variety of signaling pathways such as the well established second messenger-regulated pathways of calcium mobilization, protein kinase C (17Lu W.Y. Xiong Z.G. Lei S. Orser B.A. Dudek E. Browning M.D. MacDonald J.F. Nat. Neurosci. 1999; 2: 331-338Crossref PubMed Scopus (347) Google Scholar, 18Xiong Z.G. Raouf R. Lu W.Y. Wang L.Y. Orser B.A. Dudek E.M. Browning M.D. MacDonald J.F. Mol. Pharmacol. 1998; 54: 1055-1063Crossref PubMed Scopus (88) Google Scholar) and cAMP-dependent protein kinase (19Abel T. Nguyen P.V. Barad M. Deuel T.A. Kandel E.R. Bourtchouladze R. 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In the current study we investigate further the relationship between NMDA receptors and GPCRs by focusing on the M3-muscarinic receptor endogenously expressed in cerebellar granule (CG) neurons. Previous studies have determined that muscarinic receptors regulate both NMDA receptor function and contribute to LTP and LTD (16Shinoe T. Matsui M. Taketo M.M. Manabe T. J. Neurosci. 2005; 25: 11194-11200Crossref PubMed Scopus (191) Google Scholar, 24Auerbach J.M. Segal M. J. Physiol. 1996; 492: 479-493Crossref PubMed Scopus (147) Google Scholar, 25Grishin A.A. Benquet P. Gerber U. Neuropharmacology. 2005; 49: 328-337Crossref PubMed Scopus (32) Google Scholar, 26McCutchen E. Scheiderer C.L. Dobrunz L.E. McMahon L.L. J. Neurophysiol. 2006; 96: 3114-3121Crossref PubMed Scopus (26) Google Scholar, 27Semba K. White T.D. J. Neurochem. 1997; 69: 1066-1072Crossref PubMed Scopus (11) Google Scholar). Here we demonstrate that the NMDA receptor-mediated activation of CamKII results in the phosphorylation of the M3-muscarinic receptor and subsequent uncoupling of the receptor from the Gq/11/phosphoinositide pathway. Our study demonstrates that there is a complex reciprocal relationship between GPCRs and NMDA receptors where each is able to regulate the activity of the other in a process that is likely to be essential in establishing the threshold activities that contribute to synaptic plasticity. Mouse CG neurons were cultured as described previously (28Leist M. Volbracht C. Kuhnle S. Fava E. Ferrando-May E. Nicotera P. Mol. Med. 1997; 3: 750-764Crossref PubMed Google Scholar). In brief, cerebella from 7–8-day-old Balb/c pups were mechanically and enzymatically (trypsin) dissociated. The cells were subsequently plated on poly-l-lysine-coated 6-well plates (Nunclon) or on round glass coverslips (25-mm diameter) at a cell density of 0.25 × 106 cells/cm2, in basal medium Eagle (Invitrogen) supplemented with 20 mm KCl, penicillin/streptomycin, 10% fetal calf serum. Cytosine arabionoside (10 μm) was added 48 h after plating to prevent glial cell proliferation. The cultures were incubated at 37 °C in a humidified atmosphere with 5% CO2 and used after 7–8 days. The experiments were performed in CSS-25 buffer (120 mm NaCl, 1.8 mm CaCl2, 15 mm glucose, 25 mm KCl, 25 mm HEPES, pH 7.4) or in a modified CSS-25 buffer as indicated). For transfection of siRNA duplexes CG neurons cultured for 5 days were transfected with either siRNA duplexes specific for CamKII-β (purchased as verified siRNA from Santa Cruz Biotechnology, Inc.) or scrambled siRNA control. CG neurons were transfected with 80 pmol of scrambled siRNA or 80 pmol of specific siRNA using 5 μl of FuGENE HD transfection reagent (Roche Applied Science) per transfection. The cells were used for phosphorylation experiments 48 h post-transfection. In vivo [32P]orthophosphate labeling, receptor solubilization, and immunoprecipitation were conducted as described previously (29Torrecilla I. Spragg E.J. Poulin B. McWilliams P.J. Mistry S.C. Blaukat A. Tobin A.B. J. Cell Biol. 2007; 177: 127-137Crossref PubMed Scopus (74) Google Scholar). 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 (29Torrecilla I. Spragg E.J. Poulin B. McWilliams P.J. Mistry S.C. Blaukat A. Tobin A.B. J. Cell Biol. 2007; 177: 127-137Crossref PubMed Scopus (74) Google Scholar). 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.FIGURE 3siRNA knock-down of CamKII reduces NMDA-mediated M3-muscarinic receptor phosphorylation. CG neurons on day 5 of culture were sham-transfected (NT) or transfected with scrambled siRNA duplexes or siRNA directed against CamKII-β (80 pmol). A, after a further 2 days the cells were metabolically labeled with [32P]orthophosphate and stimulated with NMDA (100 μm for 5 min). The cells were then solubilized, and M3-muscarinic receptor was immunoprecipitated. The gel was transferred to nitrocellulose and exposed to reveal the phosphorylated receptor, after which the membrane was processed by Western blot to determine equal receptor loading. B, a small sample of the lysate from each condition was retained for Western blotting for CamKII-β, GRK-2, and GRK-6. Shown are the data from a typical experiment carried out at least three times. The graphs represent the cumulative data (mean ± S.E.) of at least three experiments carried out in duplicate.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Experiments examining changes in free intracellular Ca2+ were conducted on CG neurons cultured on coverslips. The cells were loaded with the Ca2+ indicator fura-2 AM (2 μm, 30 min) (Molecular Probes) in CSS-25 at 37 °C and mounted on to a Zeiss Axiovert 200 inverted epifluorescence microscope fitted with a perfusion chamber (flow rate at 3 ml/min). The cells were excited at 340 and 380 nm at a sample rate of 0.67 Hz by means of an excitation filter wheel. Sequential fluorescent image pairs were collected at wavelengths >510 nm via a cooled CCD camera (ORCA-ER, Hamamatsu) and converted to pseudo color images after background subtraction. Free intracellular Ca2+ signal was expressed as 340/380 ratio of fluorescence. Approximately 10–15 cells were analyzed in each experiment, and the mean values from different cultures were averaged. For experiments involving analysis of phosphoinositide signaling, the translocation of eGFP-PHPLCδ1 from the plasma membrane to the cytosol was conducted as described previously (30Nash M.S. Schell M.J. Atkinson P.J. Johnston N.R. Nahorski S.R. Challiss R.A. J. Biol. Chem. 2002; 277: 35947-35960Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 31Young K.W. Nash M.S. Challiss R.A. Nahorski S.R. J. Biol. Chem. 2003; 278: 20753-20760Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Plasmid DNA carrying the eGFP-PHPLCδ1 construct was transfected into CG neurons via the calcium-phosphate precipitation method. Briefly, 1 day after preparation of CG neurons, 3 μg of DNA/well in a volume of 10 μl of H2O was added to 85 μl of HEPES-buffered saline (137 mm NaCl, 5.6 mm glucose, 50 mm KCl, 0.7 mm Na2HPO4, 21 mm HEPES, pH 7.1) and to 5 μl 2.5 m CaCl2. After 20 min, the solution was added to the cells. The following day, the medium was replaced with fresh medium, and the cells were cultured for a further 6–7 days. The coverslips were then mounted on to the microscope, and images of eGFP were captured at a rate of 1 Hz. Phosphoinositide responses were measured as the relative change in fluorescence intensity in the cytosol. The data are expressed as a self-ratio (F/F0) resulting from subtraction of background fluorescence followed by dividing the fluorescence intensity at a given time by the initial fluorescence within each region of interest. Where NMDA was used to stimulate CG neurons, the cells were perfused with Mg2+-free buffer before the addition of NMDA. CG neurons expressing eGFP-PHPLCδ1 were perfused with CSS-25 containing a maximal concentration of methacholine (100 μm) for 2 min. This generated a phosphoinositide response that was termed S1 and served as an internal control. Methacholine was then removed, and a lag time of 12 min was allowed for the cells to recover. NMDA (100 μm) was then applied for 5 min, followed by a wash-out period of 2 min. The cells were then stimulated with a second application of methacholine, and the resulting phosphoinositide response was termed S2. Desensitization of the M3-muscarinic receptor response was determined as the difference in the amplitude between the S1 and S2 responses. CG neurons cultured in 6-well plates were incubated with CSS-25 containing NMDA (100 μm) and/or methacholine (100 μm) for the indicated times. The cells were then washed three times with ice-cold CSS-25 and incubated with CSS-25 containing a saturating concentration of the muscarinic receptor antagonist [3H]N-methylscopolamine (0.5 nm) for 4 h at 4 °C. The cells were then washed three times with ice-cold CSS-25 and solubilized by the addition of lysis buffer (200 μm). Receptor expression was determined by liquid scintillation counting. Nonspecific binding was determined by the inclusion of 10 μm atropine. GST fusion constructs containing the full mouse third intracellular loop R252 to T491 (GST-3iloop) or GST alone were expressed in Escherichia coli BL21 (DE3) IRL bacteria and purified as described previously (32Tobin A.B. Keys B. Nahorski S.R. FEBS Lett. 1993; 335: 353-357Crossref PubMed Scopus (22) Google Scholar). 5 μg of protein was incubated with 200 ng of CaM kinase II (New England Biolabs) in assay buffer (10 mm HEPES, pH 7.4, 2.5 mm β-glycerophosphate, 0.5 mm CaCl2, 5 mm MgCl2, 1 mm dithiothreitol, 0.03 mg/ml calmodulin (Calbiochem)) containing 50 μm ATP and 10 μCi of [γ-32P]ATP. The reactions were incubated for 30 min at 37 °C and stopped by the addition of an equal volume of 2× SDS-PAGE sample buffer. The reactions were separated by SDS-PAGE on 12% gels, dried, and exposed to autoradiography film. For mass spectrometric experiments, the reactions were carried out in assay buffer containing 1 mm ATP. After separation by SDS-PAGE, the proteins were transferred to nitrocellulose, and the protein bands were revealed by staining with Ponceau S (Sigma). The protein bands were excised from the membrane and blocked with 0.5% polyvinylpyrrolidone in 0.6% acetic acid for 30 min at 37 °C before digestion with trypsin (1 μg) in 50 mm ammonium bicarbonate, overnight at 37 °C. Tryptic peptides were collected, dried in a rotary evaporator, and resuspended in 50% acetonitrile, 0.1% formic acid. Enrichment of phosphopeptides was carried out using titanium dioxide contained in a MonoTip (GL Sciences Inc) according to the manufacturer's instructions, and the phosphopeptides were eluted in a solution of 5% ammonium hydroxide containing 20% acetonitrile. Where indicated, the enriched phosphopeptides were dried and resuspended in 10 mm Tris, pH 7.4, 10 mm CaCl2 and subjected to further proteolytic digestion by the addition of 1 μg of chymotrypsin (Roche Applied Science) for 2 h at 25 °C. Samples resulting from trypsin or chymotrypsin digestion were acidified with formic acid and mixed 1:1 with a solution containing 10 mg/ml of 2,5-dihydroxybenzoic acid (Sigma) in 50% acetonitrile, 1% phosphoric acid. An aliquot of the resulting sample (0.5 μl) was spotted onto a stainless steel target plate. Analysis of peptide digests was carried out using a Voyager DE-STR MALDI-TOF mass spectrometer (Applied Biosystems, Warrington, UK) in positive ion reflectron mode over the m/z range 800–7000. LC-MS/MS was carried out upon each sample using a 4000 Q-Trap mass spectrometer (Applied Biosystems, Warrington, UK). Peptides resulting from proteolytic digestion were loaded at high flow rate onto a reverse phase trapping column (0.3 mm inner diameter × 1 mm), containing 5 μm C18 300 Å Acclaim PepMap media (Dionex) and eluted through a reverse phase capillary column (75 μm inner diameter × 150 mm) containing Jupiter Proteo 4 μm 90 Å media (Phenomenex, UK) that was self-packed using a high pressure packing device (Proxeon Biosystems, Odense, Denmark). The output from the column was sprayed directly into the nanospray ion source of the 4000 Q-Trap mass spectrometer. The analysis was carried out in positive ion mode using data-dependent switching. Fragment ion spectra generated by LC-MS/MS were searched using the MASCOT search tool (Matrix Science Ltd., London, UK) against an updated copy of the SwissProt protein data base using appropriate parameters. The criteria for protein identification were based on the manufacturer's definitions (Matrix Science Ltd.) (33Perkins D.N. Pappin D.J. Creasy D.M. Cottrell J.S. Electrophoresis. 1999; 20: 3551-3567Crossref PubMed Scopus (6661) Google Scholar) Candidate peptides with probability-based Mowse scores exceeding threshold (p < 0.05), thus indicating a significant or extensive homology, were referred to as "hits." Protein scores were derived from peptide ion scores as a non-probability basis for ranking proteins. For the phosphopeptides elucidated by MASCOT, individual MS/MS spectra were interrogated manually to validate both the peptide identity and position of assignment. In each case it was clear as to the identity of the peptide (and that the peptide was indeed 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 [M+2H]2+ and that of the neutral ion loss [M+2H-H3PO4]2+, such that the MS/MS data were obtained over a longer time period (5.86 s), which resulted in significantly improved data quality. This allowed unambiguous assignment of the position of phosphorylation. The statistic used was S.D. of at least three determinations, and significance was determined using Student's t test or one-way analysis of variance for multiple comparisons. Significance was accepted when p < 0.05. The phosphorylation status of the M3-muscarinic receptor can be monitored by prelabeling cells with [32P]orthophosphate followed by solubilization of the receptor and immunoprecipitation with receptor specific antibodies (34Budd D.C. McDonald J.E. Tobin A.B. J. Biol. Chem. 2000; 275: 19667-19675Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 35Budd D.C. Willars G.B. McDonald J.E. Tobin A.B. J. Biol. Chem. 2001; 276: 4581-4587Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Experiments of this type have previously demonstrated that the M3-muscarinic receptor endogenously expressed in mouse CG neurons is phosphorylated upon stimulation with the muscarinic receptor agonist methacholine (29Torrecilla I. Spragg E.J. Poulin B. McWilliams P.J. Mistry S.C. Blaukat A. Tobin A.B. J. Cell Biol. 2007; 177: 127-137Crossref PubMed Scopus (74) Google Scholar). 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). In these experiments the phosphorylated M3-muscarinic receptor appears as a band running at just less than 100 kDa. Western blots of the nitrocellulose membrane containing the radiolabeled phosphorylated receptor demonstrated that equal amounts of receptor are immunoprecipitated under various stimulation conditions (Fig. 1A). Importantly, no M3-muscarinic receptor was detected in immunoprecipitates from CG neurons prepared from M3-muscarinic receptor knock-out mice (Fig. 1A). Glutamate increased muscarinic receptor phosphorylation by 2.21 ± 0.25-fold (n = 5) over basal and NMDA by 2.55 ± 0.33-fold [n = 5] (Fig. 1B). This response appears to be mediated via NMDA receptors because neither AMPA nor kainate had any significant effect on M3-muscarinic receptor phosphorylation (Fig. 1C). Whereas addition of the muscarinic receptor antagonist atropine was able to completely block the phosphorylation mediated by direct activation of the M3-muscarinic receptor using the muscarinic agonist methacholine (Fig. 1B), atropine had no effect on the NMDA- or glutamate-mediated phosphorylation (data not shown). Furthermore, combined stimulation with NMDA and methacholine did not enhance the M3-muscarinic receptor phosphorylation over that induced by the agonists individually (data not shown). A role of NMDA receptors was confirmed by use of the NMDA receptor-specific antagonists MK-801 and AP-5, which were able to block NMDA-mediated phosphorylation of the M3-muscarinic receptor (Fig. 1D). The NMDA showed a transient time course that reached a maximum at 5 min followed by a steady decrease to basal levels by 30 min (Fig. 1E). 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 (3Sheng M. Kim M.J. Science. 2002; 298: 776-780Crossref PubMed Scopus (586) Google Scholar, 4Lisman J. Schulman H. Cline H. Nat. Rev. Neurosci. 2002; 3: 175-190Crossref PubMed Scopus (1443) Google Scholar). 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). To investigate the role of CamKII in the NMDA-mediated response CG neurons were pretreated with the selective CamKII inhibitors KN-93 or KN-62. Both of these pharmacological inhibitors prevented NMDA- mediated muscarinic receptor phosphorylation, whereas the inactive analogue KN-92 had no effect (Fig. 2B). To control for the possibility that the CamKII inhibitors were working in an indirect manner by affecting free intracellular calcium concentrations, we measured the rise in intracellular calcium following NMDA treatment in the absence or presence of KN-62. As illustrated in Fig. 2 (C and D), the CamKII inhibitor did not alter the ability of NMDA to increase intracellular free calcium. These pharmacological studies indicated that CamKII was involved in phosphorylation of M3-muscarinic receptors in response to NMDA-stimulation. To further test this, we used siRNA duplexes against the β-isoform of CamKII, which we found to be highly expressed in mouse CG neurons (supplemental Fig. S1). These siRNA duplexes significantly decreased CamKII-β by more than 70% but had no off target effects on kinases that have previous been shown to phosphorylate the GPCRs, i.e. GRK2, GRK6 (Fig. 3). Under conditions where CamKII-β was knocked down, NMDA-mediated phosphorylation of the M3-muscarinic receptor was decreased by 45.6 + 8.1% (n = 3 + S.D.). These results indicated that CamKII-β, acting downstream of the NMDA receptor, either directly phosphorylates the M3-muscarinic receptor or is on a kinase cascade that results in the phosphorylation of the receptor. The mouse M3-muscarinic receptor contains a large third intracellular loop with 33 serine
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