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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 and calpain activity in SKNBE cells.(A) SKNBE cells were lysed to perform immunoprecipitation with 1 μg of anti-NR1 antibody. The immunoprecipitated material (IP NR-1) was analyzed by immunoblotting (WB) to detect the indicated proteins. Each immunoblot is representative of four different experiments. (B) SKNBE cells (2 × 105) were incubated in 100 μL of isotonic HEPES buffer containing 50 μM t-Boc-Leu-Met-CMAC fluorogenic calpain substrate (see Methods). Cells were then washed and suspended in 100 μL of HEPES buffer containing 10 μM glycine and 1 mM CaCl2. Calpain activity was measured in the absence (Control) or presence (NMDA) of 100 μM NMDA. Calpain activity was also measured in cells pre-incubated for 30 minutes with 1 μM Calpain inhibitor 2 (CI-2). The values reported are the arithmetical mean ± SEM of four different experiments, and p values were calculated according to t-test. (C) Calcium Green™-loaded cells were exposed to the indicated stimuli. Data are means ± SEM from two independent experiments in duplicate.
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pone.0139750.g001: NMDAR cluster and calpain activity in SKNBE cells.(A) SKNBE cells were lysed to perform immunoprecipitation with 1 μg of anti-NR1 antibody. The immunoprecipitated material (IP NR-1) was analyzed by immunoblotting (WB) to detect the indicated proteins. Each immunoblot is representative of four different experiments. (B) SKNBE cells (2 × 105) were incubated in 100 μL of isotonic HEPES buffer containing 50 μM t-Boc-Leu-Met-CMAC fluorogenic calpain substrate (see Methods). Cells were then washed and suspended in 100 μL of HEPES buffer containing 10 μM glycine and 1 mM CaCl2. Calpain activity was measured in the absence (Control) or presence (NMDA) of 100 μM NMDA. Calpain activity was also measured in cells pre-incubated for 30 minutes with 1 μM Calpain inhibitor 2 (CI-2). The values reported are the arithmetical mean ± SEM of four different experiments, and p values were calculated according to t-test. (C) Calcium Green™-loaded cells were exposed to the indicated stimuli. Data are means ± SEM from two independent experiments in duplicate.

Mentions: To define the role of this resident calpain, we have first explored if the protease, in its associated form, expresses the catalytic activity in unstimulated SKNBE cells. As shown in Fig 1A, the immunoprecipitation with an anti-NR1 antibody revealed that, in addition to the native 180 kD NR2B subunit, a second 60 kD NR2B band was present in NMDAR cluster. This low molecular weight NR2B was similar to the fragment generated by calpain-mediated selective cleavage of NR2B at the C-terminal region [10]. The presence of this fragment suggests not only that calpain 1 is specifically and constitutively associated to NMDAR receptor, but also that the protease is partially active, even in the absence of specific cell stimulation. As we have reported previously [34], HSP90 is also present in this cluster, whereas both calpain 2 and calpastatin are not detectable. Thus, the association of calpain 1 to NMDAR is highly specific.


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 and calpain activity in SKNBE cells.(A) SKNBE cells were lysed to perform immunoprecipitation with 1 μg of anti-NR1 antibody. The immunoprecipitated material (IP NR-1) was analyzed by immunoblotting (WB) to detect the indicated proteins. Each immunoblot is representative of four different experiments. (B) SKNBE cells (2 × 105) were incubated in 100 μL of isotonic HEPES buffer containing 50 μM t-Boc-Leu-Met-CMAC fluorogenic calpain substrate (see Methods). Cells were then washed and suspended in 100 μL of HEPES buffer containing 10 μM glycine and 1 mM CaCl2. Calpain activity was measured in the absence (Control) or presence (NMDA) of 100 μM NMDA. Calpain activity was also measured in cells pre-incubated for 30 minutes with 1 μM Calpain inhibitor 2 (CI-2). The values reported are the arithmetical mean ± SEM of four different experiments, and p values were calculated according to t-test. (C) Calcium Green™-loaded cells were exposed to the indicated stimuli. Data are means ± SEM from two independent experiments in duplicate.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0139750.g001: NMDAR cluster and calpain activity in SKNBE cells.(A) SKNBE cells were lysed to perform immunoprecipitation with 1 μg of anti-NR1 antibody. The immunoprecipitated material (IP NR-1) was analyzed by immunoblotting (WB) to detect the indicated proteins. Each immunoblot is representative of four different experiments. (B) SKNBE cells (2 × 105) were incubated in 100 μL of isotonic HEPES buffer containing 50 μM t-Boc-Leu-Met-CMAC fluorogenic calpain substrate (see Methods). Cells were then washed and suspended in 100 μL of HEPES buffer containing 10 μM glycine and 1 mM CaCl2. Calpain activity was measured in the absence (Control) or presence (NMDA) of 100 μM NMDA. Calpain activity was also measured in cells pre-incubated for 30 minutes with 1 μM Calpain inhibitor 2 (CI-2). The values reported are the arithmetical mean ± SEM of four different experiments, and p values were calculated according to t-test. (C) Calcium Green™-loaded cells were exposed to the indicated stimuli. Data are means ± SEM from two independent experiments in duplicate.
Mentions: To define the role of this resident calpain, we have first explored if the protease, in its associated form, expresses the catalytic activity in unstimulated SKNBE cells. As shown in Fig 1A, the immunoprecipitation with an anti-NR1 antibody revealed that, in addition to the native 180 kD NR2B subunit, a second 60 kD NR2B band was present in NMDAR cluster. This low molecular weight NR2B was similar to the fragment generated by calpain-mediated selective cleavage of NR2B at the C-terminal region [10]. The presence of this fragment suggests not only that calpain 1 is specifically and constitutively associated to NMDAR receptor, but also that the protease is partially active, even in the absence of specific cell stimulation. As we have reported previously [34], HSP90 is also present in this cluster, whereas both calpain 2 and calpastatin are not detectable. Thus, the association of calpain 1 to NMDAR is highly specific.

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