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Physiological Roles of Calpain 1 Associated to Multiprotein NMDA Receptor Complex.

Averna M, Pellegrini M, Cervetto C, Pedrazzi M, Bavestrello M, De Tullio R, Salamino F, Pontremoli S, Melloni E - PLoS ONE (2015)

Bottom Line: Since the protease resides at the NMDAR in saturating amounts, variations in Ca2+ influx promote an increase in calpain 1 activity without affecting the amount of the protease originally associated to NMDAR.We here propose that resident calpain 1 is involved in NMDAR turnover, and following an increase in Ca2+ influx, the activated protease, by promoting the removal of NMDAR from the plasma membranes, can decrease Ca2+ entrance through this channel.Observations of different HSP90/calpain 1 ratios in different ultrasynaptic compartments support this conclusion.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Medicine (DIMES)-Biochemistry Section, University of Genova, Viale Benedetto XV, 1-16132, Genova, Italy; Center of Excellence for Biomedical Research (CEBR), University of Genova, Viale Benedetto XV, 1-16132, Genova, Italy.

ABSTRACT
We have recently demonstrated that in resting conditions calpain 1, but not calpain 2, is specifically associated to the N-Methyl-D-Aspartate receptor (NMDAR) multiprotein complex. We are here reporting that in SKNBE neuroblastoma cells or in freshly isolated nerve terminals from adult rat hippocampus, the proteolytic activity of calpain 1 resident at the NMDAR is very low under basal conditions and greatly increases following NMDAR stimulation. Since the protease resides at the NMDAR in saturating amounts, variations in Ca2+ influx promote an increase in calpain 1 activity without affecting the amount of the protease originally associated to NMDAR. In all the conditions examined, resident calpain 1 specifically cleaves NR2B at the C-terminal region, leading to its internalization together with NR1 subunit. While in basal conditions intracellular membranes include small amounts of NMDAR containing the calpain-digested NR2B, upon NMDAR stimulation nearly all the receptor molecules are internalized. We here propose that resident calpain 1 is involved in NMDAR turnover, and following an increase in Ca2+ influx, the activated protease, by promoting the removal of NMDAR from the plasma membranes, can decrease Ca2+ entrance through this channel. Due to the absence of calpastatin in such cluster, the activity of resident calpain 1 may be under the control of HSP90, whose levels are directly related to the activation of this protease. Observations of different HSP90/calpain 1 ratios in different ultrasynaptic compartments support this conclusion.

No MeSH data available.


Related in: MedlinePlus

NMDAR cluster modifications in rat hippocampal synaptosomes following calpain activation.(A) Rat hippocampal synaptosomes were incubated at 37°C for 12 min with (NMDA) or without (Control) 100 μM NMDA in HEPES buffer containing 1 μM glycine and 1 mM CaCl2. Synaptosomes were lysed and immunoprecipitation was carried out with 1 μg of anti-NR1 antibody. The immunoprecipitated material (IP NR1) was analyzed by immunoblotting (WB) to detect the indicated proteins. (B) Surface localization of NR1 subunit was also evaluated by biotinylation assay in rat hippocampal synaptosomes treated as in (A). NR1 was detected on a fraction (30 μL) of eluted proteins (biotinylated) by immunoblotting. Each immunoblot is representative of four different experiments.
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pone.0139750.g006: NMDAR cluster modifications in rat hippocampal synaptosomes following calpain activation.(A) Rat hippocampal synaptosomes were incubated at 37°C for 12 min with (NMDA) or without (Control) 100 μM NMDA in HEPES buffer containing 1 μM glycine and 1 mM CaCl2. Synaptosomes were lysed and immunoprecipitation was carried out with 1 μg of anti-NR1 antibody. The immunoprecipitated material (IP NR1) was analyzed by immunoblotting (WB) to detect the indicated proteins. (B) Surface localization of NR1 subunit was also evaluated by biotinylation assay in rat hippocampal synaptosomes treated as in (A). NR1 was detected on a fraction (30 μL) of eluted proteins (biotinylated) by immunoblotting. Each immunoblot is representative of four different experiments.

Mentions: To obtain more information on calpain-mediated proteolysis/internalization of NMDAR, we have investigated on the NR1/NR2B receptor present in hippocampal synaptosomes purified from rat nerve terminals. As shown in Fig 6A, in total synaptosome preparation the NR1-immunoprecipitated multiprotein complex contained calpain 1, and neither calpain 2 nor calpastatin. Moreover, HSP90 was present together with native NR2B, and small amounts of the 60 kD NR2B fragment. Thus, calpain 1 results to be constitutively associated also to the NMDAR localized in synaptosomes. Presumably the presence of the 60 kD NR2B fragment in unstimulated synaptosomes is the result of a calpain-mediated digestion of native NR2B. Indeed, when synaptosomes were exposed to 100 μM NMDA, the concentration inducing the maximal efflux of glutamate [35], a remarkable disappearance of the native NR2B subunit occurred, together with the accumulation of the 60 kD NR2B digested form (Fig 6A). In addition, the amount of resident calpain 1 did not increase following NMDA stimulation, and neither calpain 2 nor calpastatin were recruited in the NMDAR complex. Moreover, in these structures, HSP90 was extensively digested and NR2B degradation corresponded to the removal of NMDAR from the plasma membranes, as indicated by the disappearance of the biotinylated native NR1 subunit (Fig 6B). Thus, the NR1/NR2B-containing NMDAR cluster, present in SKNBE cells and in synaptosomes, contains a resident calpain 1 that is partially active in basal conditions and becomes highly activated following stimulation of NMDAR.


Physiological Roles of Calpain 1 Associated to Multiprotein NMDA Receptor Complex.

Averna M, Pellegrini M, Cervetto C, Pedrazzi M, Bavestrello M, De Tullio R, Salamino F, Pontremoli S, Melloni E - PLoS ONE (2015)

NMDAR cluster modifications in rat hippocampal synaptosomes following calpain activation.(A) Rat hippocampal synaptosomes were incubated at 37°C for 12 min with (NMDA) or without (Control) 100 μM NMDA in HEPES buffer containing 1 μM glycine and 1 mM CaCl2. Synaptosomes were lysed and immunoprecipitation was carried out with 1 μg of anti-NR1 antibody. The immunoprecipitated material (IP NR1) was analyzed by immunoblotting (WB) to detect the indicated proteins. (B) Surface localization of NR1 subunit was also evaluated by biotinylation assay in rat hippocampal synaptosomes treated as in (A). NR1 was detected on a fraction (30 μL) of eluted proteins (biotinylated) by immunoblotting. Each immunoblot is representative of four different experiments.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4592069&req=5

pone.0139750.g006: NMDAR cluster modifications in rat hippocampal synaptosomes following calpain activation.(A) Rat hippocampal synaptosomes were incubated at 37°C for 12 min with (NMDA) or without (Control) 100 μM NMDA in HEPES buffer containing 1 μM glycine and 1 mM CaCl2. Synaptosomes were lysed and immunoprecipitation was carried out with 1 μg of anti-NR1 antibody. The immunoprecipitated material (IP NR1) was analyzed by immunoblotting (WB) to detect the indicated proteins. (B) Surface localization of NR1 subunit was also evaluated by biotinylation assay in rat hippocampal synaptosomes treated as in (A). NR1 was detected on a fraction (30 μL) of eluted proteins (biotinylated) by immunoblotting. Each immunoblot is representative of four different experiments.
Mentions: To obtain more information on calpain-mediated proteolysis/internalization of NMDAR, we have investigated on the NR1/NR2B receptor present in hippocampal synaptosomes purified from rat nerve terminals. As shown in Fig 6A, in total synaptosome preparation the NR1-immunoprecipitated multiprotein complex contained calpain 1, and neither calpain 2 nor calpastatin. Moreover, HSP90 was present together with native NR2B, and small amounts of the 60 kD NR2B fragment. Thus, calpain 1 results to be constitutively associated also to the NMDAR localized in synaptosomes. Presumably the presence of the 60 kD NR2B fragment in unstimulated synaptosomes is the result of a calpain-mediated digestion of native NR2B. Indeed, when synaptosomes were exposed to 100 μM NMDA, the concentration inducing the maximal efflux of glutamate [35], a remarkable disappearance of the native NR2B subunit occurred, together with the accumulation of the 60 kD NR2B digested form (Fig 6A). In addition, the amount of resident calpain 1 did not increase following NMDA stimulation, and neither calpain 2 nor calpastatin were recruited in the NMDAR complex. Moreover, in these structures, HSP90 was extensively digested and NR2B degradation corresponded to the removal of NMDAR from the plasma membranes, as indicated by the disappearance of the biotinylated native NR1 subunit (Fig 6B). Thus, the NR1/NR2B-containing NMDAR cluster, present in SKNBE cells and in synaptosomes, contains a resident calpain 1 that is partially active in basal conditions and becomes highly activated following stimulation of NMDAR.

Bottom Line: Since the protease resides at the NMDAR in saturating amounts, variations in Ca2+ influx promote an increase in calpain 1 activity without affecting the amount of the protease originally associated to NMDAR.We here propose that resident calpain 1 is involved in NMDAR turnover, and following an increase in Ca2+ influx, the activated protease, by promoting the removal of NMDAR from the plasma membranes, can decrease Ca2+ entrance through this channel.Observations of different HSP90/calpain 1 ratios in different ultrasynaptic compartments support this conclusion.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Medicine (DIMES)-Biochemistry Section, University of Genova, Viale Benedetto XV, 1-16132, Genova, Italy; Center of Excellence for Biomedical Research (CEBR), University of Genova, Viale Benedetto XV, 1-16132, Genova, Italy.

ABSTRACT
We have recently demonstrated that in resting conditions calpain 1, but not calpain 2, is specifically associated to the N-Methyl-D-Aspartate receptor (NMDAR) multiprotein complex. We are here reporting that in SKNBE neuroblastoma cells or in freshly isolated nerve terminals from adult rat hippocampus, the proteolytic activity of calpain 1 resident at the NMDAR is very low under basal conditions and greatly increases following NMDAR stimulation. Since the protease resides at the NMDAR in saturating amounts, variations in Ca2+ influx promote an increase in calpain 1 activity without affecting the amount of the protease originally associated to NMDAR. In all the conditions examined, resident calpain 1 specifically cleaves NR2B at the C-terminal region, leading to its internalization together with NR1 subunit. While in basal conditions intracellular membranes include small amounts of NMDAR containing the calpain-digested NR2B, upon NMDAR stimulation nearly all the receptor molecules are internalized. We here propose that resident calpain 1 is involved in NMDAR turnover, and following an increase in Ca2+ influx, the activated protease, by promoting the removal of NMDAR from the plasma membranes, can decrease Ca2+ entrance through this channel. Due to the absence of calpastatin in such cluster, the activity of resident calpain 1 may be under the control of HSP90, whose levels are directly related to the activation of this protease. Observations of different HSP90/calpain 1 ratios in different ultrasynaptic compartments support this conclusion.

No MeSH data available.


Related in: MedlinePlus