Abstract
Clathrin-dependent endocytosis is mediated by a tightly regulated network of molecular interactions that provides essential protein-protein and protein-lipid binding activities. Here we report the hydrolysis of the alpha- and beta2-subunits of the tetrameric adaptor protein complex 2 by calpain. Calcium-dependent alpha- and beta2-adaptin hydrolysis was observed in several rat tissues, including brain and primary neuronal cultures. Neuronal alpha- and beta2-adaptin cleavage was inducible by glutamate stimulation and was accompanied by the decreased endocytosis of transferrin. Heterologous expression of truncated forms of the beta2-adaptin subunit significantly decreased the membrane recruitment of clathrin and inhibited clathrin-mediated receptor endocytosis. Moreover, the presence of truncated beta2-adaptin sensitized neurons to glutamate receptor-mediated excitotoxicity. Proteolysis of alpha- and beta2-adaptins, as well as the accessory clathrin adaptors epsin 1, adaptor protein 180, and the clathrin assembly lymphoid myeloid leukemia protein, was detected in brain tissues after experimentally induced ischemia and in cases of human Alzheimer disease. The present study further clarifies the central role of calpain in regulating clathrin-dependent endocytosis and provides evidence for a novel mechanism through which calpain activation may promote neurodegeneration: the sensitization of cells to glutamate-mediated excitotoxicity via the decreased internalization of surface receptors.
Highlights
The tetrameric AP-2 complex is composed of ␣, 2, 2, and -subunits [3, 4]
The AP-2 core adaptor complex is important for the overall capacity of clathrin-dependent endocytosis and is crucial for the internalization of particular cargoes such as the transferrin receptor [9]
We report that calpain activation leads to the proteolysis of the ␣- and 2-subunits of the core AP-2 clathrin adaptor complex and that this cleavage contributes significantly to the calpain-mediated inhibition of clathrin-dependent receptor endocytosis
Summary
Clathrin Adaptor Expression Vectors—cDNAs encoding fulllength rat 2-adaptin (with C-terminal FLAG-, 6ϫ-histidine-, or hemagglutinin (HA) tags), N-terminal or C-terminal rat 2-adaptin fragments (amino acids 1–691, with no tag or C-terminal HA tag; amino acids 692–951, with C-terminal 6ϫ-histidine or HA tags), and human AP180 (with an N-terminal HA tag) were created using PCRs with specific primers Protein extracts were cleared of cell debris by centrifugation at 20,000 ϫ g for 15 min at 4 °C, separated by SDS-PAGE on a 12% polyacrylamide gel, and analyzed by immunoblotting. Immunoprecipitation of ␣-Adaptin—Mouse monoclonal antibodies to ␣-adaptin (Novus Biologicals) were conjugated to Protein G-Sepharose 4 Fast Flow beads (Amersham Biosciences) and used as described previously [27] to immunoprecipitate ␣-adaptin-containing protein complexes from HEK 293T cells transfected with SIN-W-TRE plasmid vectors encoding either full-length or N-terminal and C-terminal fragments of rat 2-adaptin, all with C-terminal HA tags. Neuronal Survival Assessed by NeuN-positive Cell Counting— DIV 11 striatal neurons were exposed for 15 min to 50 M glutamate in culture medium supplemented with 50 M glycine and 2.7 mM CaCl2. The one-way analysis of variance was used to compare multiple conditions, and the two-tailed Student’s t test was used for two-group comparisons. p Ͻ 0.05 was set as the threshold for statistical significance
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