Ankyrin binds to two distinct cytoplasmic domains of Na,K-ATPase alpha subunit.
Ankyrin has emerged as a ubiquitous protein linking integral membrane transport proteins such as Na,K-ATPase to an underlying spectrin cytoskeleton. This interaction is mediated by the alpha subunit of Na,K-ATPase; however, the nature of the ankyrin binding site in Na,K-ATPase is unknown. As a step to determine the mechanism of this interaction, the ankyrin binding region of human erythrocyte spectrin and each of five putative cytoplasmic domains of the Na,K-ATPase alpha subunit have been prepared as recombinant fusion proteins in bacteria and analyzed for their interaction with erythrocyte and kidney ankyrin (Ank1 and Ank3, respectively) in vitro. Spectrin binds both Ank1 and Ank3 avidly, as expected. Two of the Na,K-ATPase domains, immobilized on a bioaffinity column, also interact specifically with both of these ankyrins. These ATPase domains are encoded by codons 140-290 (domain II) and 345-784 (domain III), with domain II displaying the greatest apparent affinity. Sequences in domain II are highly conserved between species and isoforms of Na,K-ATPase and are homologous to a cytoplasmic domain in H,K-ATPase and to a limited region of sequence in Ca-ATPase. Conversely, domain II shares no significant homology with other ankyrin binding proteins such as band 3 and Na(+)-channel proteins. These results identify a clear function for a conserved but previously not understood region of the alpha subunit of Na,K-ATPase and suggest that the interaction of ankyrin with membrane transport proteins may involve complex tertiary structural determinants not easily deduced from the primary sequence.
Devarajan
P
P
Department of Pediatrics, Yale University School of Medicine, New Haven, CT 06510.
Scaramuzzino
D A
DA
Morrow
J S
JS
D000595:Amino Acid Sequence; D017487:Ankyrins / Q000378:metabolism*; D001665:Binding Sites; D002460:Cell Line; D003593:Cytoplasm; D003598:Cytoskeletal Proteins / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D011485:Protein Binding; D011994:Recombinant Proteins; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*
D017487:Ankyrins; D003598:Cytoskeletal Proteins; D011994:Recombinant Proteins; D000254:Sodium-Potassium-Exchanging ATPase