Abstract
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.
Highlights
Cellular differentiation and several cellular functions depend to a large degree on the compartmentalization of particular membrane proteins such as receptors, adhesion molecules or ion translocating proteins to specialized domains of the cell surface
We confined this study to the large cytoplasmic domain (CD3) because we found in a screening approach that a recombinant protein containing a portion of CD3 blocked binding of erythrocyte ankyrin to native kidney Naϩ,Kϩ-ATPase
To determine whether the ALLLK cluster in the cytoplasmic domain of the erythrocyte AE1 is essential for ankyrin binding, we extended binding studies to the purified cytoplasmic domain of AE1 (CD-AE1)
Summary
Cellular differentiation and several cellular functions depend to a large degree on the compartmentalization of particular membrane proteins such as receptors, adhesion molecules or ion translocating proteins to specialized domains of the cell surface (for review, see Refs. 1–3). We confined this study to the large cytoplasmic domain (CD3) because we found in a screening approach that a recombinant protein containing a portion of CD3 blocked binding of erythrocyte ankyrin to native kidney Naϩ,Kϩ-ATPase.
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