Protein 4.2 binds to the carboxyl-terminal EF-hands of erythroid alpha-spectrin in a calcium- and calmodulin-dependent manner
| Autor: | Luanne L. Peters, Samuel E. Lux, Catherine Korsgren |
|---|---|
| Rok vydání: | 2009 |
| Předmět: |
Vesicle-associated membrane protein 8
Calmodulin Swine Plasma protein binding macromolecular substances Biochemistry Mass Spectrometry Mice Anion Exchange Protein 1 Erythrocyte Membrane Biology Animals Humans Spectrin EF Hand Motifs Molecular Biology Band 3 biology Binding protein Membrane Proteins Cell Biology Recombinant Proteins Cytoskeletal Proteins biology.protein Biophysics Calcium Cattle Electrophoresis Polyacrylamide Gel Protein G Protein Multimerization Binding domain Protein Binding |
| Zdroj: | The Journal of biological chemistry. 285(7) |
| ISSN: | 1083-351X |
| Popis: | Spectrin and protein 4.1 cross-link F-actin protofilaments into a network called the membrane skeleton. Actin and 4.1 bind to one end of beta-spectrin. The adjacent end of alpha-spectrin, called the EF-domain, is calmodulin-like, with calcium-dependent and calcium-independent EF-hands. It has no known function. However, the sph(1J)/sph(1J) mouse has very fragile red cells and lacks the last 13 amino acids in the EF-domain, suggesting the domain is critical for skeletal integrity. Using pulldown binding assays, we find the alpha-spectrin EF-domain either alone or incorporated into a mini-spectrin binds native and recombinant protein 4.2 at a previously identified region of 4.2 (G(3) peptide). Native 4.2 binds with an affinity comparable with other membrane skeletal interactions (K(d) = 0.30 microM). EF-domains bearing the sph(1J) mutation are inactive. Binding of protein 4.2 to band 3 (K(d) = 0.45 microM) does not interfere with the spectrin-4.2 interaction. Spectrin-4.2 binding is amplified by micromolar concentrations of Ca(2+) (but not Mg(2+)) by three to five times. Calmodulin also binds to the EF-domain (K(d) = 17 microM), and Ca(2+)-calmodulin blocks Ca(2+)-dependent binding of protein 4.2 but not Ca(2+)-independent binding. The data suggest that protein 4.2 is located near protein 4.1 at the spectrin-actin junctions. Because proteins 4.1 and 4.2 also bind to band 3, the erythrocyte anion channel, we suggest that one or both of these proteins cause a portion of band 3 to localize near the spectrin-actin junctions and provide another point of attachment between the membrane skeleton and the lipid bilayer. |
| Databáze: | OpenAIRE |
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