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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

HSP90 levels and 180 kD NR2B degradation in rat hippocampal synaptosomes.(A) The immunoreactive bands (corresponding to HSP90, NR1 subunit, and Calpain 1) obtained by immunoprecipitation from SKNBE cells (see Fig 1A and relevant legend) and rat hippocampal synaptosomes (see Fig 6A and relevant legend) were quantified and the indicated protein ratio were calculated. The values reported are the arithmetical mean ± SEM of four different experiments. * p < 0.05, according to ANOVA followed by post-hoc Tukey’s test. (B) Quantification of the immunoreactive bands corresponding to 180 kD NR2B subunit immunoprecipitated from the indicated samples incubated for 2 hours at different concentrations of NMDA. (C) Quantification of the immunoreactive bands corresponding to 180 kD NR2B subunit immunoprecipitated from the indicated samples incubated with 100 μM NMDA at different times. The values reported are the arithmetical mean ± SEM of three different experiments.
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pone.0139750.g007: HSP90 levels and 180 kD NR2B degradation in rat hippocampal synaptosomes.(A) The immunoreactive bands (corresponding to HSP90, NR1 subunit, and Calpain 1) obtained by immunoprecipitation from SKNBE cells (see Fig 1A and relevant legend) and rat hippocampal synaptosomes (see Fig 6A and relevant legend) were quantified and the indicated protein ratio were calculated. The values reported are the arithmetical mean ± SEM of four different experiments. * p < 0.05, according to ANOVA followed by post-hoc Tukey’s test. (B) Quantification of the immunoreactive bands corresponding to 180 kD NR2B subunit immunoprecipitated from the indicated samples incubated for 2 hours at different concentrations of NMDA. (C) Quantification of the immunoreactive bands corresponding to 180 kD NR2B subunit immunoprecipitated from the indicated samples incubated with 100 μM NMDA at different times. The values reported are the arithmetical mean ± SEM of three different experiments.

Mentions: Taken together these findings indicate that the NMDAR complexes in SKNBE cells and hippocampal synaptosomes contain the same components although in different ratios (Fig 7A). Specifically, in the NMDAR cluster from synaptosomes, the HSP90/calpain ratio is 3−4-fold lower than in unstimulated SKNBE cells. As a consequence, calpain 1 resident in the NMDAR of synaptosomes appears less efficiently controlled. The lack of regulation by HSP90 is further confirmed by the fact that in synaptosomes, unlike in SKNBE cells, NR2B is nearly completely degraded already at 10 μM NMDA (Fig 7B), and the rate of this digestion is about 10–fold faster than that observed in SKNBE cells (Fig 7C).


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)

HSP90 levels and 180 kD NR2B degradation in rat hippocampal synaptosomes.(A) The immunoreactive bands (corresponding to HSP90, NR1 subunit, and Calpain 1) obtained by immunoprecipitation from SKNBE cells (see Fig 1A and relevant legend) and rat hippocampal synaptosomes (see Fig 6A and relevant legend) were quantified and the indicated protein ratio were calculated. The values reported are the arithmetical mean ± SEM of four different experiments. * p < 0.05, according to ANOVA followed by post-hoc Tukey’s test. (B) Quantification of the immunoreactive bands corresponding to 180 kD NR2B subunit immunoprecipitated from the indicated samples incubated for 2 hours at different concentrations of NMDA. (C) Quantification of the immunoreactive bands corresponding to 180 kD NR2B subunit immunoprecipitated from the indicated samples incubated with 100 μM NMDA at different times. The values reported are the arithmetical mean ± SEM of three different experiments.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0139750.g007: HSP90 levels and 180 kD NR2B degradation in rat hippocampal synaptosomes.(A) The immunoreactive bands (corresponding to HSP90, NR1 subunit, and Calpain 1) obtained by immunoprecipitation from SKNBE cells (see Fig 1A and relevant legend) and rat hippocampal synaptosomes (see Fig 6A and relevant legend) were quantified and the indicated protein ratio were calculated. The values reported are the arithmetical mean ± SEM of four different experiments. * p < 0.05, according to ANOVA followed by post-hoc Tukey’s test. (B) Quantification of the immunoreactive bands corresponding to 180 kD NR2B subunit immunoprecipitated from the indicated samples incubated for 2 hours at different concentrations of NMDA. (C) Quantification of the immunoreactive bands corresponding to 180 kD NR2B subunit immunoprecipitated from the indicated samples incubated with 100 μM NMDA at different times. The values reported are the arithmetical mean ± SEM of three different experiments.
Mentions: Taken together these findings indicate that the NMDAR complexes in SKNBE cells and hippocampal synaptosomes contain the same components although in different ratios (Fig 7A). Specifically, in the NMDAR cluster from synaptosomes, the HSP90/calpain ratio is 3−4-fold lower than in unstimulated SKNBE cells. As a consequence, calpain 1 resident in the NMDAR of synaptosomes appears less efficiently controlled. The lack of regulation by HSP90 is further confirmed by the fact that in synaptosomes, unlike in SKNBE cells, NR2B is nearly completely degraded already at 10 μM NMDA (Fig 7B), and the rate of this digestion is about 10–fold faster than that observed in SKNBE cells (Fig 7C).

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