Protein 4.2 binds to the carboxyl-terminal EF-hands of erythroid alpha-spectrin in a calcium- and calmodulin-dependent manner.
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.
Korsgren
Catherine
C
Division of Hematology/Oncology, Children's Hospital Boston and Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115, USA.
Peters
Luanne L
LL
Lux
Samuel E
SE
D000818:Animals; D001457:Anion Exchange Protein 1, Erythrocyte / Q000235:genetics / Q000378:metabolism; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology*; D002147:Calmodulin / Q000378:metabolism*; D002417:Cattle; D003598:Cytoskeletal Proteins / Q000235:genetics / Q000378:metabolism*; D020832:EF Hand Motifs / Q000235:genetics / Q000502:physiology; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D011485:Protein Binding / Q000187:drug effects; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013049:Spectrin / Q000235:genetics / Q000378:metabolism*; D013552:Swine
D001457:Anion Exchange Protein 1, Erythrocyte; D002147:Calmodulin; D003598:Cytoskeletal Proteins; D008565:Membrane Proteins; D011994:Recombinant Proteins; C028841:erythrocyte membrane band 4.1 protein; C044791:erythrocyte membrane band 4.2 protein; D013049:Spectrin; D002118:Calcium