title: 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.
abstract: 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.
authors.affiliation: Department of Biochemistry, School of Medicine, Tokyo Women's Medical University, Shinjuku, Tokyo 162-8666, Japan.
authors.forename: W
authors.initials: W
authors.lastname: Nunomura
authors.forename: Y
authors.initials: Y
authors.lastname: Takakuwa
authors.forename: M
authors.initials: M
authors.lastname: Parra
authors.forename: J G
authors.initials: JG
authors.lastname: Conboy
authors.forename: N
authors.initials: N
authors.lastname: Mohandas
mesh_terms: 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
chemical_list: 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