Ca(2+)-dependent and Ca(2+)-independent calmodulin binding sites in erythrocyte protein 4.1. Implications for regulation of protein 4.1 interactions with transmembrane proteins.
In vitro protein binding assays identified two distinct calmodulin (CaM) binding sites within the NH(2)-terminal 30-kDa domain of erythrocyte protein 4.1 (4.1R): a Ca(2+)-independent binding site (A(264)KKLWKVCVEHHTFFRL) and a Ca(2+)-dependent binding site (A(181)KKLSMYGVDLHKAKDL). Synthetic peptides corresponding to these sequences bound CaM in vitro; conversely, deletion of these peptides from a 30-kDa construct reduced binding to CaM. Thus, 4.1R is a unique CaM-binding protein in that it has distinct Ca(2+)-dependent and Ca(2+)-independent high affinity CaM binding sites. CaM bound to 4.1R at a stoichiometry of 1:1 both in the presence and absence of Ca(2+), implying that one CaM molecule binds to two distinct sites in the same molecule of 4.1R. Interactions of 4.1R with membrane proteins such as band 3 is regulated by Ca(2+) and CaM. While the intrinsic affinity of the 30-kDa domain for the cytoplasmic tail of erythrocyte membrane band 3 was not altered by elimination of one or both CaM binding sites, the ability of Ca(2+)/CaM to down-regulate 4. 1R-band 3 interaction was abrogated by such deletions. Thus, regulation of protein 4.1 binding to membrane proteins by Ca(2+) and CaM requires binding of CaM to both Ca(2+)-independent and Ca(2+)-dependent sites in protein 4.1.
Nunomura
W
W
Department of Biochemistry, School of Medicine, Tokyo Women's Medical University, Shinjuku, Tokyo 162-8666, Japan.
Takakuwa
Y
Y
Parra
M
M
Conboy
J G
JG
Mohandas
N
N
D000595:Amino Acid Sequence; D000818:Animals; D001457:Anion Exchange Protein 1, Erythrocyte / Q000378:metabolism; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology*; D002147:Calmodulin / Q000378:metabolism*; D002417:Cattle; D017124:Conserved Sequence; D003598:Cytoskeletal Proteins; D004910:Erythrocyte Membrane / Q000737:chemistry*; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation; D009479:Neuropeptides; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins
D001457:Anion Exchange Protein 1, Erythrocyte; D002147:Calmodulin; D003598:Cytoskeletal Proteins; D008565:Membrane Proteins; D009479:Neuropeptides; D010446:Peptide Fragments; D011994:Recombinant Proteins; C028841:erythrocyte membrane band 4.1 protein; C120557:erythrocyte membrane protein band 4.1-like 1; D002118:Calcium