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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

Effect of calpain inhibition on NR2B protein levels in SKNBE cells.(A) SKNBE cells were incubated for 24 hours in the absence or presence of 1 μM Calpain inhibitor 2 (CI-2), and lysed to perform immunoprecipitation with 1 μg of anti-NR1 antibody. The immunoprecipitated material (IP NR-1) was analyzed by immunoblotting for NR2B (WB NR2B). (B and C) The protein bands detected in (A) were quantified as described in Methods. Each value represents the arithmetical mean ± SEM of four different experiments. * p < 0.01 vs control (- CI-2), according to t-test.
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pone.0139750.g003: Effect of calpain inhibition on NR2B protein levels in SKNBE cells.(A) SKNBE cells were incubated for 24 hours in the absence or presence of 1 μM Calpain inhibitor 2 (CI-2), and lysed to perform immunoprecipitation with 1 μg of anti-NR1 antibody. The immunoprecipitated material (IP NR-1) was analyzed by immunoblotting for NR2B (WB NR2B). (B and C) The protein bands detected in (A) were quantified as described in Methods. Each value represents the arithmetical mean ± SEM of four different experiments. * p < 0.01 vs control (- CI-2), according to t-test.

Mentions: To characterize the role of calpain in the constitutive digestion of NR2B, SKNBE cells were cultured for 24 hours in the presence of the synthetic calpain inhibitor CI-2 and the levels of NR2B species were measured. As shown in Fig 3A, 3B and 3C untreated SKNBE cells contain, in addition to the native 180 kD NR2B, the 60 kD fragment in amounts ranging from 10 to 15% of total. Instead, cell exposure to CI-2 for 24 hours induced a 2–3-fold increase in the level of native 180 kD NR2B and the complete disappearance of NR2B 60 kD fragment. Our observations demonstrate the direct involvement of the resident calpain 1 in the selective cleavage of NR2B subunit in basal conditions.


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)

Effect of calpain inhibition on NR2B protein levels in SKNBE cells.(A) SKNBE cells were incubated for 24 hours in the absence or presence of 1 μM Calpain inhibitor 2 (CI-2), and lysed to perform immunoprecipitation with 1 μg of anti-NR1 antibody. The immunoprecipitated material (IP NR-1) was analyzed by immunoblotting for NR2B (WB NR2B). (B and C) The protein bands detected in (A) were quantified as described in Methods. Each value represents the arithmetical mean ± SEM of four different experiments. * p < 0.01 vs control (- CI-2), according to t-test.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0139750.g003: Effect of calpain inhibition on NR2B protein levels in SKNBE cells.(A) SKNBE cells were incubated for 24 hours in the absence or presence of 1 μM Calpain inhibitor 2 (CI-2), and lysed to perform immunoprecipitation with 1 μg of anti-NR1 antibody. The immunoprecipitated material (IP NR-1) was analyzed by immunoblotting for NR2B (WB NR2B). (B and C) The protein bands detected in (A) were quantified as described in Methods. Each value represents the arithmetical mean ± SEM of four different experiments. * p < 0.01 vs control (- CI-2), according to t-test.
Mentions: To characterize the role of calpain in the constitutive digestion of NR2B, SKNBE cells were cultured for 24 hours in the presence of the synthetic calpain inhibitor CI-2 and the levels of NR2B species were measured. As shown in Fig 3A, 3B and 3C untreated SKNBE cells contain, in addition to the native 180 kD NR2B, the 60 kD fragment in amounts ranging from 10 to 15% of total. Instead, cell exposure to CI-2 for 24 hours induced a 2–3-fold increase in the level of native 180 kD NR2B and the complete disappearance of NR2B 60 kD fragment. Our observations demonstrate the direct involvement of the resident calpain 1 in the selective cleavage of NR2B subunit in basal conditions.

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