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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 translocation in SKNBE cells following activation of resident calpain 1.(A and B) SKNBE cells were incubated for 2 hours with 100 μM NMDA and 10 μM glicine in the absence (NMDA) or presence (NMDA+CI-2) of 1 μM CI-2, or left untreated (Control). The cells were then fixed and NR1 subunit localization (green fluorescence) was determined by confocal microscopy. Confocal microscopy micrographs are representative of four different experiments. Alternatively, after the indicated treatments, cells were then lysed to perform immunoprecipitation with 1 μg of anti-NR1 antibody and the immunoprecipitated material (IP NR1) was analyzed by immunoblotting (WB) to detect the indicated proteins. (C) The protein bands detected in (B) were quantified as described in Methods. Each value represents the arithmetical mean ± SEM of three different experiments. * p < 0.01 vs control and NMDA+CI-2; ** p < 0.01 vs control and p < 0.05 vs NMDA+CI-2, according to ANOVA followed by post-hoc Tukey’s test. (D) Cell surface expression of NR1 and NR2B subunits was also evaluated by biotinylation assay (see Methods) after incubation of SKNBE cells in the absence (Control) or presence (NMDA) of 100 μM NMDA for 2 hours. NR1 and NR2B subunits were detected by immunoblotting on a fraction (30 μL) of eluted (Biotinylated) or unbound (Not Biotinylated) proteins. (E) The protein bands detected in (D) were quantified as described in Methods. Each value represents the arithmetical mean ± SEM of three different experiments. * p < 0.001 and ** p < 0.05 vs the relevant protein in biotinylated material following cell stimulation, according to t-test.
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pone.0139750.g005: NMDAR translocation in SKNBE cells following activation of resident calpain 1.(A and B) SKNBE cells were incubated for 2 hours with 100 μM NMDA and 10 μM glicine in the absence (NMDA) or presence (NMDA+CI-2) of 1 μM CI-2, or left untreated (Control). The cells were then fixed and NR1 subunit localization (green fluorescence) was determined by confocal microscopy. Confocal microscopy micrographs are representative of four different experiments. Alternatively, after the indicated treatments, cells were then lysed to perform immunoprecipitation with 1 μg of anti-NR1 antibody and the immunoprecipitated material (IP NR1) was analyzed by immunoblotting (WB) to detect the indicated proteins. (C) The protein bands detected in (B) were quantified as described in Methods. Each value represents the arithmetical mean ± SEM of three different experiments. * p < 0.01 vs control and NMDA+CI-2; ** p < 0.01 vs control and p < 0.05 vs NMDA+CI-2, according to ANOVA followed by post-hoc Tukey’s test. (D) Cell surface expression of NR1 and NR2B subunits was also evaluated by biotinylation assay (see Methods) after incubation of SKNBE cells in the absence (Control) or presence (NMDA) of 100 μM NMDA for 2 hours. NR1 and NR2B subunits were detected by immunoblotting on a fraction (30 μL) of eluted (Biotinylated) or unbound (Not Biotinylated) proteins. (E) The protein bands detected in (D) were quantified as described in Methods. Each value represents the arithmetical mean ± SEM of three different experiments. * p < 0.001 and ** p < 0.05 vs the relevant protein in biotinylated material following cell stimulation, according to t-test.

Mentions: To further explore the effect of resident calpain 1 on the properties of NMDAR complex, SKNBE cells were exposed to 100 μM NMDA to induce a Ca2+ influx across the receptor channel, a condition that, as previously indicated, activates only resident calpain 1 without affecting cell viability. Confocal microscopy images revealed that NMDAR containing native NR1 subunits was internalized into cytoplasmic vesicles (Fig 5A). Since this process was prevented by the addition of CI-2, we can confirm that resident calpain 1 is directly involved in inducing NMDAR internalization. In NMDA-stimulated cells, only NR2B subunits were extensively digested, whereas both NR1 and HSP90 were not affected (Fig 5B and 5C). As expected, CI-2 prevented NR2B proteolysis.


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 translocation in SKNBE cells following activation of resident calpain 1.(A and B) SKNBE cells were incubated for 2 hours with 100 μM NMDA and 10 μM glicine in the absence (NMDA) or presence (NMDA+CI-2) of 1 μM CI-2, or left untreated (Control). The cells were then fixed and NR1 subunit localization (green fluorescence) was determined by confocal microscopy. Confocal microscopy micrographs are representative of four different experiments. Alternatively, after the indicated treatments, cells were then lysed to perform immunoprecipitation with 1 μg of anti-NR1 antibody and the immunoprecipitated material (IP NR1) was analyzed by immunoblotting (WB) to detect the indicated proteins. (C) The protein bands detected in (B) were quantified as described in Methods. Each value represents the arithmetical mean ± SEM of three different experiments. * p < 0.01 vs control and NMDA+CI-2; ** p < 0.01 vs control and p < 0.05 vs NMDA+CI-2, according to ANOVA followed by post-hoc Tukey’s test. (D) Cell surface expression of NR1 and NR2B subunits was also evaluated by biotinylation assay (see Methods) after incubation of SKNBE cells in the absence (Control) or presence (NMDA) of 100 μM NMDA for 2 hours. NR1 and NR2B subunits were detected by immunoblotting on a fraction (30 μL) of eluted (Biotinylated) or unbound (Not Biotinylated) proteins. (E) The protein bands detected in (D) were quantified as described in Methods. Each value represents the arithmetical mean ± SEM of three different experiments. * p < 0.001 and ** p < 0.05 vs the relevant protein in biotinylated material following cell stimulation, according to t-test.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4592069&req=5

pone.0139750.g005: NMDAR translocation in SKNBE cells following activation of resident calpain 1.(A and B) SKNBE cells were incubated for 2 hours with 100 μM NMDA and 10 μM glicine in the absence (NMDA) or presence (NMDA+CI-2) of 1 μM CI-2, or left untreated (Control). The cells were then fixed and NR1 subunit localization (green fluorescence) was determined by confocal microscopy. Confocal microscopy micrographs are representative of four different experiments. Alternatively, after the indicated treatments, cells were then lysed to perform immunoprecipitation with 1 μg of anti-NR1 antibody and the immunoprecipitated material (IP NR1) was analyzed by immunoblotting (WB) to detect the indicated proteins. (C) The protein bands detected in (B) were quantified as described in Methods. Each value represents the arithmetical mean ± SEM of three different experiments. * p < 0.01 vs control and NMDA+CI-2; ** p < 0.01 vs control and p < 0.05 vs NMDA+CI-2, according to ANOVA followed by post-hoc Tukey’s test. (D) Cell surface expression of NR1 and NR2B subunits was also evaluated by biotinylation assay (see Methods) after incubation of SKNBE cells in the absence (Control) or presence (NMDA) of 100 μM NMDA for 2 hours. NR1 and NR2B subunits were detected by immunoblotting on a fraction (30 μL) of eluted (Biotinylated) or unbound (Not Biotinylated) proteins. (E) The protein bands detected in (D) were quantified as described in Methods. Each value represents the arithmetical mean ± SEM of three different experiments. * p < 0.001 and ** p < 0.05 vs the relevant protein in biotinylated material following cell stimulation, according to t-test.
Mentions: To further explore the effect of resident calpain 1 on the properties of NMDAR complex, SKNBE cells were exposed to 100 μM NMDA to induce a Ca2+ influx across the receptor channel, a condition that, as previously indicated, activates only resident calpain 1 without affecting cell viability. Confocal microscopy images revealed that NMDAR containing native NR1 subunits was internalized into cytoplasmic vesicles (Fig 5A). Since this process was prevented by the addition of CI-2, we can confirm that resident calpain 1 is directly involved in inducing NMDAR internalization. In NMDA-stimulated cells, only NR2B subunits were extensively digested, whereas both NR1 and HSP90 were not affected (Fig 5B and 5C). As expected, CI-2 prevented NR2B proteolysis.

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