Activation of NMDA receptors reverses desensitization of mGluR5 in native and recombinant systems

Alagarsamy, Shanmugarathinam; Marino, Michael J.; Rouse, Susan T.; Gereau, Robert W.; Heinemann, Stephen F.; Conn, P. Jeffrey

doi:10.1038/6338

Cited by 184 publications

(143 citation statements)

References 39 publications

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“…Previous studies have shown that NMDA potentiates mGluR5 responses (Challiss et al, 1994;Luthi et al, 1994). We have previously shown that a likely mechanism for this effect is via reversal of PKC-mediated desensitization of mGluR5 (Alagarsamy et al, 1999a). Our previous report and current data are consistent with the hypothesis that NMDA activates PP2B/calcineurin to mediate reversal of mGluR5 desensitization and that calcineurin acts by interaction with, and dephosphorylation of, the C-terminus of mGluR5.…”

Section: Discussionsupporting

confidence: 90%

“…Xenopus oocytes were injected and recorded from as previously described (Alagarsamy et al, 1999a). Briefly, oocytes were injected with in vitro transcribed mRNA encoding mGluR5a alone or in the presence of mRNA for a constitutively active mutant form of calcineurin (CaNa) (Friday et al, 2000).…”

Section: Xenopus Oocyte Recordingsmentioning

confidence: 99%

“…Immunoprecipitation experiments and Western blots were performed as previously described (Alagarsamy et al, 1999a) with some modifications. Hippocampal slices were used instead of cortical cultures, radioactive labeling was omitted and supernatants of homogenized samples were immunoprecipitated with anti-mGluR5 (4 g; Upstate) were probed with either anti-mGluR5 or anti-calcineurin (4 g; Upstate) for Western blots.…”

Section: Coimmunoprecipitationmentioning

confidence: 99%

“…For instance, low concentrations of NMDA can enhance mGluR-mediated increases in phosphoinositide hydrolysis in rat cortex (Challiss et al, 1994;Alagarsamy et al, 1999b). Furthermore, NMDA receptor activation potentiates an inward current induced by 1S,3R-ACPD in hippocampal pyramidal cells (Luthi et al, 1994;Alagarsamy et al, 1999a). We recently reported that NMDA-induced potentiation of group I mGluR responses in recombinant systems and in the hippocampus is mediated by reversal of PKC-induced mGluR desensitization.…”

Section: Introductionmentioning

confidence: 99%

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NMDA-induced potentiation of mGluR5 is mediated by activation of protein phosphatase 2B/calcineurin

Alagarsamy¹,

Saugstad²,

Warren

et al. 2005

Neuropharmacology

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Previous reports have shown that activation of N-methyl-D-aspartate (NMDA) receptors potentiates responses to activation of the group I metabotropic glutamate receptor mGluR5 by reversing PKC-mediated desensitization of this receptor. NMDA-induced reversal of mGluR5 desensitization is dependent on activation of protein phosphatases. However, the specific protein phosphatase involved and the precise mechanism by which NMDA receptor activation reduces mGluR desensitization are not known. We have performed a series of molecular, biochemical, and genetic studies to show that NMDA-induced regulation of mGluR5 is dependent on activation of calcium-dependent protein phosphatase 2B/calcineurin (PP2B/CaN). Furthermore, we report that purified calcineurin directly dephosphorylates the C-terminal tail of mGluR5 at sites that are phosphorylated by PKC. Finally, immunoprecipitation and GST fusion protein pull-down experiments reveal that calcineurin interacts with mGluR5, suggesting that these proteins could be colocalized in a signaling complex. Taken together with previous studies, these data suggest that activation of NMDA receptors leads to activation of calcineurin and that calcineurin modulates mGluR5 function by directly dephosphorylating mGluR5 at PKC sites that are involved in desensitization of this receptor.

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Section: Discussionsupporting

confidence: 90%

Section: Xenopus Oocyte Recordingsmentioning

confidence: 99%

Section: Coimmunoprecipitationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

NMDA-induced potentiation of mGluR5 is mediated by activation of protein phosphatase 2B/calcineurin

Alagarsamy¹,

Saugstad²,

Warren

et al. 2005

Neuropharmacology

View full text Add to dashboard Cite

show abstract

“…Interestingly, NMDARs have been reported to stimulate mGluRs, and vice versa (Aniksztejn et al, 1992;Harvey and Collingridge, 1993;Challiss et al, 1994;Lüthi et al, 1994;Fitzjohn et al, 1996;Gereau and Heinemann, 1998;Alagarsamy et al, 1999;Collett and Collingridge, 2004). This feedback loop between mGluR and NMDARs may be important in regulating both NMDAR-dependent and mGluR-dependent forms of synaptic plasticity and could contribute to pathological responses induced by NMDA receptor activation.…”

Section: Inhibition Of Glun2b Suppresses Ltdmentioning

confidence: 99%

Dysregulated NMDA-Receptor Signaling Inhibits Long-Term Depression in a Mouse Model of Fragile X Syndrome

2016

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Fragile X syndrome (FXS) is a neurodevelopmental disease. It is one of the leading monogenic causes of intellectual disability among boys with most also displaying autism spectrum disorder traits. Here we investigated the role of NMDA receptors on mGluR-dependent long-term depression (mGluR-LTD), a key biomarker in the disease, at four different developmental stages. First, we applied the mGluR agonist 3,5-dihydroxyphenylglycine in the absence or presence of the NMDAR blocker, APV, hereby unmasking the NMDAR component in this process. As expected, in the presence of APV, we found more LTD in the mouse KO than in WT. This, however, was only observed in the p30 -60 age group. At all other age groups tested, mGluR-LTD was almost identical between KO and WT. Interestingly, at p60, in the absence of APV, no or very little LTD was found in KO that was completely restored by application of APV. This suggests that the underlying cause of the enhanced mGluR-LTD in KO (at p30) is caused by dysregulated NMDAR signaling. To investigate this further, we next used NMDAR-subunit-specific antagonists. Inhibition of GluN2B, but not GluN2A, blocked mGluR-LTD only in WT. This was in contrast in the KO where blocking GluN2B rescued mGluR-LTD, suggesting GluN2B-containing NMDARs in the KO are hyperactive. Thus, these findings suggest strong involvement of GluN2B-containing-NMDARs in the pathophysiology of FXS and highlight a potential path for treatment for the disease.

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Characterization of phosphoproteins from electrophoretic gels by nanoscale Fe(III) affinity chromatography with off-line mass spectrometry analysis

2001

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Detailed characterization of phosphoproteins as well as other post-translationally modified proteins is required to fully understand protein function and regulatory events in cells and organisms. Here we present a mass spectrometry (MS) based experimental strategy for the identification and mapping of phosphorylation site(s) using only low-picomole amounts of phosphoprotein starting material. Miniaturized sample preparation methods for MS facilitated localization of phosphorylation sites in phosphoproteins isolated by polyacrylamide gel electrophoresis. Custom made, nanoscale immobilized Fe(III) affinity chromatography (Fe(III)-IMAC) columns were employed for enrichment of phosphorylated peptides from crude peptide mixtures prior to off-line analysis by matrix-assisted laser desorption/ionization (MALDI) MS or nanoelectrospray tandem mass spectrometry (MS/MS). An optimized and sensitive procedure for alkaline phosphatase treatment of peptide mixtures was implemented, which in combination with nano-scale Fe(III)-IMAC and MALDI-MS allowed unambiguous identification of phosphopeptides by observation of 80 Da mass shifts. Nanoelectrospray MS/MS was used for phosphopeptide sequencing for exact determination of phosphorylation sites. The advantages and limitations of the experimental strategy was demonstrated by enrichment, identification and sequencing of phosphopeptides from the model proteins ovalbumin and bovine beta-casein isolated by gel electrophoresis. Furthermore, an autophosphorylation site at Ser-3 in recombinant human casein kinase-2 beta subunit was determined. The potential of miniaturized Fe(III)-IMAC and MALDI-MS for characterization of in vivo phosphorylated proteins was demonstrated by identification of tryptic phosphopeptides derived from the human p47/phox phosphoprotein isolated by two-dimensional gel electrophoresis.

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Activation of NMDA receptors reverses desensitization of mGluR5 in native and recombinant systems

Cited by 184 publications

References 39 publications

NMDA-induced potentiation of mGluR5 is mediated by activation of protein phosphatase 2B/calcineurin

NMDA-induced potentiation of mGluR5 is mediated by activation of protein phosphatase 2B/calcineurin

Dysregulated NMDA-Receptor Signaling Inhibits Long-Term Depression in a Mouse Model of Fragile X Syndrome

Characterization of phosphoproteins from electrophoretic gels by nanoscale Fe(III) affinity chromatography with off-line mass spectrometry analysis

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