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.
Highlights
From the ‡Department of Biochemistry, School of Medicine, Tokyo Women’s Medical University, Shinjuku, Tokyo 1628666, Japan and §Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720
1 The abbreviations used are: CaM, calmodulin; 4.1R, human erythrocyte protein 4.1; Fmoc, N-(9-fluorenyl)methyloxycarbonyl; Recombinant 30-kDa domain of 4.1R (r30kDa), recombinant 30-kDa domain of 4.1R; ⌬Ex.5, ⌬Ex.11, and ⌬Ex.9/11, r30kDa from which sequence encoded by exons 5, 11, and both 9 and 11 was deleted, respectively; K(D)kin, dissociation constant from kinetic analysis; K(D)Scat, dissociation constant from Scatchard plot analysis
Four major structural domains of 4.1R with apparent molecular masses of 30, 16, 10, and 22–24 kDa were identified. 4.1R interacts with spectrin and actin through its 10-kDa domain and with integral membrane proteins glycophorin C and band 3 through its 30-kDa domain
Summary
CaM, calmodulin; 4.1R, human erythrocyte protein 4.1; Fmoc, N-(9-fluorenyl)methyloxycarbonyl; r30kDa, recombinant 30-kDa domain of 4.1R; ⌬Ex.5, ⌬Ex., and ⌬Ex.9/11, r30kDa from which sequence encoded by exons 5, 11, and both 9 and 11 was deleted, respectively; K(D)kin, dissociation constant from kinetic analysis; K(D)Scat, dissociation constant from Scatchard plot analysis. Present study is the role played by CaM in modulating various protein-protein interactions in the erythrocyte membrane involving 4.1R. CaM is present in human erythrocytes at micromolar concentration [3,4,5] In these cells, CaM binds to Ca2ϩ-ATPase [6, 7] with a dissociation constant on the order of 10 nM [8], while it binds to membrane skeletal proteins, 4.1R, and adducin with a dissociation constant on the order of 0.1– 0.2 M (3, 9 –12). By modulating the affinities of these different protein-protein interactions, Ca2ϩ/CaM can play a significant role in regulating the function of the erythrocyte membrane [10, 11, 15, 16]. Ca2ϩ had no effect on the affinity of the band 3 interaction with the 30-kDa domain when either the Ca2ϩ-independent or both the Ca2ϩ-dependent and -independent CaM binding sites were deleted. We propose that two distinct domains in 4.1R are responsible for CaM binding and that one of these domains is responsible for Ca2ϩ-sensitive regulation of 4.1-R interactions with membrane proteins
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