Identification of a binding motif for ankyrin on the alpha-subunit of Na+,K(+)-ATPase.
Cytoskeleton membrane associations are important for a variety of cellular functions. The anion exchanger of erythrocytes (AE1) and Na+,K(+)-ATPase of polarized epithelial cells provide well studied examples of how integral membrane proteins are anchored via the linker molecule ankyrin to the spectrin-based membrane cytoskeleton. In the present study we have generated several recombinant fragments of the large (third) cytoplasmic domain (CD3) of Na+,K(+)-ATPase to define binding sites of ankyrin on CD3 at a molecular level. We provide evidence that a cluster of four amino acids, ALLK, is essential for binding of ankyrin to both recombinant CD3 and to native Na+,K(+)-ATPase. Once bound, conformational changes might uncover further binding sites for ankyrin on Na+,K(+)-ATPase. A motif related to the ALLK cluster is also present in the cytoplasmic domain of AE1 where this sequence (ALLLK) turned out to be also important for ankyrin binding. These motifs are highly conserved during evolution of both Na+,K(+)-ATPase and AE1, further underlining their potential role in cytoskeleton to membrane linkage.
Institute of Anatomy, University of Würzburg, Germany.
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Jordan
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Püschel
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Koob
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Drenckhahn
D000595:Amino Acid Sequence; D000818:Animals; D017487:Ankyrins / Q000378:metabolism*; D001665:Binding Sites; D001710:Biotin; D003599:Cytoskeleton / Q000378:metabolism; D004848:Epithelium / Q000201:enzymology; D004910:Erythrocyte Membrane / Q000378:metabolism; D046911:Macromolecular Substances; D008961:Models, Structural; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000378:metabolism*
D017487:Ankyrins; D046911:Macromolecular Substances; D010446:Peptide Fragments; D011994:Recombinant Proteins; D001710:Biotin; D000254:Sodium-Potassium-Exchanging ATPase