Protein 4.2 binds to the carboxyl-terminal EF-hands of erythroid alpha-spectrin in a calcium- and calmodulin-dependent manner

J Biol Chem. 2010 Feb 12;285(7):4757-70. doi: 10.1074/jbc.M109.056200. Epub 2009 Dec 11.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Anion Exchange Protein 1, Erythrocyte / genetics
  • Anion Exchange Protein 1, Erythrocyte / metabolism
  • Calcium / metabolism
  • Calcium / pharmacology*
  • Calmodulin / metabolism*
  • Cattle
  • Cytoskeletal Proteins / genetics
  • Cytoskeletal Proteins / metabolism*
  • EF Hand Motifs / genetics
  • EF Hand Motifs / physiology
  • Electrophoresis, Polyacrylamide Gel
  • Humans
  • Mass Spectrometry
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism*
  • Mice
  • Protein Binding / drug effects
  • Protein Multimerization
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Spectrin / genetics
  • Spectrin / metabolism*
  • Swine

Substances

  • Anion Exchange Protein 1, Erythrocyte
  • Calmodulin
  • Cytoskeletal Proteins
  • Membrane Proteins
  • Recombinant Proteins
  • erythrocyte membrane band 4.1 protein
  • erythrocyte membrane band 4.2 protein
  • Spectrin
  • Calcium