Red Cell Membranes of Ankyrin-Deficient <i>nb/nb</i> Mice Lack Band 3 Tetramers but Contain Normal Membrane Skeletons

Yi, Su‐Jin; Liu, S C; Derick, Laura H.; Murray, J. G.; Barker, JE; Cho, Michael; Palek, J; Golan, David E.

doi:10.1021/bi9704966

Cited by 62 publications

(62 citation statements)

References 36 publications

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“…To validate these results, two controls were performed. i) Cell surface expression of murine band 3 was found to be reduced by 50% in nb/nb mice, which is in agreement with previous chromatographic analysis of nb/nb RBC membrane proteins (33). ii) The fluorescence given by the binding of phalloidin to actin was analyzed on intact and permeabilized/fixed RBCs; as expected, no signal was obtained in non-treated RBCs (not shown), whereas similar mean fluorescence intensity (MFI) values were obtained with all permeabilized RBCs (Fig.…”

Section: Ankyrin-r-deficient Mouse Rbcssupporting

confidence: 91%

Rh-RhAG/Ankyrin-R, a New Interaction Site between the Membrane Bilayer and the Red Cell Skeleton, Is Impaired by Rhnull-associated Mutation

Nicolas

Kim

Gane

et al. 2003

Journal of Biological Chemistry

118

109

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Several studies suggest that the Rh complex represents a major interaction site between the membrane lipid bilayer and the red cell skeleton, but little is known about the molecular basis of this interaction. We report here that ankyrin-R is capable of interacting directly with the C-terminal cytoplasmic domain of Rh and RhAG polypeptides. We first show that the primary defect of ankyrin-R in normoblastosis (nb/nb) spherocytosis mutant mice is associated with a sharp reduction of RhAG and Rh polypeptides. Secondly, our flow cytometric analysis of the Triton X-100 extractability of recombinant fusion proteins expressed in erythroleukemic cell lines suggests that the C-terminal cytoplasmic domains of Rh and RhAG are sufficient to mediate interaction with the erythroid membrane skeleton. Using the yeast two-hybrid system, we demonstrate a direct interaction between the cytoplasmic tails of Rh and RhAG and the second repeat domain (D2) of ankyrin-R. This finding is supported by the demonstration that the substitution of Asp-399 in the cytoplasmic tail of RhAG, a mutation associated with the deficiency of the Rh complex in one Rh null patient, totally impaired interaction with domain D2 of ankyrin-R. These results identify the Rh/RhAG-ankyrin complex as a new interaction site between the red cell membrane and the spectrin-based skeleton, the disruption of which might result in the stomato-spherocytosis typical of Rh null red cells.

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Section: Ankyrin-r-deficient Mouse Rbcssupporting

confidence: 91%

Rh-RhAG/Ankyrin-R, a New Interaction Site between the Membrane Bilayer and the Red Cell Skeleton, Is Impaired by Rhnull-associated Mutation

Nicolas

Kim

Gane

et al. 2003

Journal of Biological Chemistry

118

109

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“…Such destabilization probably derives from rupture of ϳ50% of the membrane-to-skeleton tethers, as proposed by the "skeleton bridge" hypothesis and supported by observations on nb/nb mice. Thus, erythrocytes from these anemic mice lack ankyrin; and even though their band 3 elutes almost exclusively as a dimer during size-exclusion chromatography, their membranes are extremely unstable (20). Consistent with this interpretation, we have noted that most of the membrane mechanical deficiencies in our studies were manifested at ankyrin fragment concentrations above ϳ9 M (Figs.…”

Section: Concentration Dependence Of 456-kda Ankyrin Fragment Incorpsupporting

confidence: 87%

Analysis of Integral Membrane Protein Contributions to the Deformability and Stability of the Human Erythrocyte Membrane

Dort¹,

Knowles²,

Chasis³

et al. 2001

Journal of Biological Chemistry

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Three major hypotheses have been proposed to explain the role of membrane-spanning proteins in establishing/maintaining membrane stability. These hypotheses ascribe the essential contribution of integral membrane proteins to (i) their ability to anchor the membrane skeleton to the lipid bilayer, (ii) their capacity to bind and stabilize membrane lipids, and (iii) their ability to influence and regulate local membrane curvature. In an effort to test these hypotheses in greater detail, we have modified both the membrane skeletal and lipid binding interactions of band 3 (the major membrane-spanning and skeletal binding protein of the human erythrocyte membrane) and have examined the impact of these modifications on erythrocyte membrane morphology, deformability, and stability. The desired changes in membrane skeletal and protein-lipid interactions were induced by 1) reaction of the cells with 4,4-diisothiocyanostilbene-2,2-disulfonate (DIDS), an inhibitor of band 3-mediated anion transport that dissociates band 3 into dimers (increasing its surface area in contact with lipid) and severs band 3 linkages to the membrane skeleton; 2) a fragment of ankyrin that ruptures the same ankyrin-band 3 bridge to the membrane skeleton, but drives the band 3 subunit equilibrium toward the tetramer (i.e. decreasing the band 3 surface area in contact with lipid); and 3) an antibody to the ankyrin-binding site on band 3 that promotes the same changes in band 3 skeletal and lipid interactions as the ankyrin fragment. We observed that although DIDS induced echinocytic morphological changes in the treated erythrocytes, it had little impact on either membrane deformability or stability. In contrast, resealing of either the ankyrin fragment or anti-band 3 IgG into erythrocytes caused spontaneous membrane fragmentation and loss of deformability/stability. Because these and other new observations cannot all be reconciled with any single hypothesis on membrane stability, we suggest that more than one hypothesis may be operative and provide an explanation of how each might individually contribute to net membrane stability.In mechanistic models, the erythrocyte membrane is often depicted as a composite material with two coupled components surrounding a protein-rich cytoplasm. In this representation, the outer component is composed of a lipid bilayer traversed by multiple transport and structural proteins, predominantly the glycophorins and band 3. In contrast, the inner component consists of a two-dimensional scaffold of interconnecting skeletal proteins, primarily spectrin, protein 4.1R, and actin. Connections between the two components occur at regular intervals and are mainly established by bridging proteins. Prominent among the bridging proteins are ankyrin, which links the ␤-subunit of spectrin to the cytoplasmic domain of band 3, and protein 4.1R, which connects a spectrin-protein 4.1R-actin junctional complex to the cytoplasmic tail of glycophorin C (1). Although other proteins such as protein 4.2, dematin, p55, and adducin undoubtedly...

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“…These additional protein deficiencies clearly contribute to membrane instability. Similarly, our data predict that protein deficiencies beyond ankyrin occur in nb/nb erythrocytes (20).…”

Section: Discussionsupporting

confidence: 71%

“…Erythrocytes from mice homozygous for the loop deletion (ld/ld) were mildly spherocytic and osmotically fragile, and inside-out vesicles from ld/ld erythrocytes were incapable of binding ankyrin. Despite the lack of ankyrin binding, the erythrocyte membrane proteins were assembled in normal amounts, and ld/ld erythrocytes were not as osmotically fragile and survived longer in the circulation than erythrocytes from nb/nb or Slc4a1 Ϫ/Ϫ mice (20)(21)(22). We conclude from these data that in addition to the ankyrin-cdb3 linkage, additional proteins are involved in stabilizing the linkage of the spectrin-actin complex to the erythrocyte membrane.…”

mentioning

confidence: 79%

“…Defects of ankyrin or band 3 cause Ϸ75% of HS cases (12,13,(17)(18)(19). Erythrocytes from ankyrin-deficient (nb/nb) and band 3-deficient (Slc4a1 Ϫ/Ϫ ) mice exhibit a severe loss of mechanical stability, altered morphology, and dramatically reduced survival (20)(21)(22). We and others have recently solved the crystal structure of cdb3 (23) and the cdb3 binding domain of ankyrin (24), respectively.…”

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confidence: 99%

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An 11-amino acid β-hairpin loop in the cytoplasmic domain of band 3 is responsible for ankyrin binding in mouse erythrocytes

Stefanovic

Markham

Parry

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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The best-studied cytoskeletal system is the inner surface of the erythrocyte membrane, which provides an erythrocyte with the structural support needed to be stable yet flexible as it passes through the circulation. Current structural models predict that the spectrin-actin-based cytoskeletal network is attached to the plasma membrane through interactions of the protein ankyrin, which binds to both spectrin and the cytoplasmic domain of the transmembrane protein band 3. The crystal structure of the cytoplasmic domain of band 3 predicted that the ankyrin binding site was located on a ␤-hairpin loop in the cytoplasmic domain. In vitro, deletion of this loop eliminated ankyrin affinity for band 3 without affecting any other protein-band 3 interaction. To evaluate the importance of the ankyrin-band 3 linkage to membrane properties in vivo, we generated mice with the nucleotides encoding the 11-aa ␤-hairpin loop in the mouse Slc4a1 gene replaced with sequence encoding a diglycine bridge. Mice homozygous for the loop deletion were viable with mildly spherocytic and osmotically fragile erythrocytes. In vitro, homozygous ld/ld erythrocytes were incapable of binding ankyrin, but contrary to all previous predictions, abolishing the ankyrin-band 3 linkage destabilized the erythrocyte membrane to a lesser degree than complete deficiencies of either band 3 or ankyrin. Our data indicate that as yet uncharacterized interactions between other membrane proteins must significantly contribute to linkage of the spectrin-actin-based membrane cytoskeleton to the plasma membrane.cytoskeleton ͉ membrane ͉ transgenic ͉ spherocytosis

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Red Cell Membranes of Ankyrin-Deficient nb/nb Mice Lack Band 3 Tetramers but Contain Normal Membrane Skeletons

Cited by 62 publications

References 36 publications

Rh-RhAG/Ankyrin-R, a New Interaction Site between the Membrane Bilayer and the Red Cell Skeleton, Is Impaired by Rhnull-associated Mutation

Rh-RhAG/Ankyrin-R, a New Interaction Site between the Membrane Bilayer and the Red Cell Skeleton, Is Impaired by Rhnull-associated Mutation

Analysis of Integral Membrane Protein Contributions to the Deformability and Stability of the Human Erythrocyte Membrane

An 11-amino acid β-hairpin loop in the cytoplasmic domain of band 3 is responsible for ankyrin binding in mouse erythrocytes

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