Association of brain ankyrin with brain membranes and isolation of active proteolytic fragments of membrane-associated ankyrin-binding protein(s)

An assay has been developed to measure association of brain ankyrin with protein site(s) in brain membranes that are independent of spectrin and tubulin, behave as integral membrane proteins, and appear to be similar in several respects to the erythrocyte anion channel. Brain membranes were depleted of ankyrin, spectrin, and other peripheral membrane proteins by a brief incubation in 0.1 M sodium hydroxide. Binding of ankyrin to these membranes fulfilled experimentally testable criteria for a specific protein-protein association. Binding was optimal at physiological values for ionic strength and pH, was of high affinity (Kd = 20-60 nM), and the capacity of 25 pmol/mg of brain membrane protein is in the same range as the number of spectrin tetramers (30 pmol/mg). The membrane-binding site(s) for brain ankyrin are likely to be related in some way to the cytoplasmic domain of the erythrocyte anion channel since binding was inhibited by the anion channel domain and by erythrocyte ankyrin. The binding site(s) for brain ankyrin were released from the membrane by limited proteolysis as active water-soluble fragments capable of inhibiting binding of ankyrin to membranes. Ankyrin-binding fragments of Mr = 40,000 and 68,000 were selectively bound to an erythrocyte ankyrin affinity column. The fragment of Mr = 40,000 is close to the size of the cytoplasmic domain of the erythrocyte anion channel. It is likely based on these results that membrane attachment proteins for ankyrin are present in brain and other tissues and that these membrane proteins have domains homologous at least in conformation to the ankyrin-binding site of the erythrocyte anion channel.     

Ankyrin-independent membrane protein-binding sites for brain and erythrocyte spectrin

Brain spectrin reassociates in in vitro binding assays with protein(s) in highly extracted brain membranes quantitatively depleted of ankyrin and spectrin. These newly described membrane sites for spectrin are biologically significant and involve a protein since (a) binding occurs optimally at physiological pH (6.7-6.9) and salt concentrations (50 mM), (b) binding is abolished by digestion of membranes with alpha-chymotrypsin, (c) Scatchard analysis is consistent with a binding capacity of at least 50 pmol/mg total membrane protein, and highest affinity of 3 nM. The major ankyrin-independent binding activity of brain spectrin is localized to the beta subunit of spectrin. Brain membranes also contain high affinity binding sites for erythrocyte spectrin, but a 3-4 fold lower capacity than for brain spectrin. Some spectrin-binding sites associate preferentially with brain spectrin, some with erythrocyte spectrin, and some associate with both types of spectrin. Erythrocyte spectrin contains distinct binding domains for ankyrin and brain membrane protein sites, since the Mr = 72,000 spectrin-binding fragment of ankyrin does not compete for binding of spectrin to brain membranes. Spectrin binds to a small number of ankyrin-independent sites in erythrocyte membranes present in about 10,000-15,000 copies/cell or 10% of the number of sites for ankyrin. Brain spectrin binds to these sites better than erythrocyte spectrin suggesting that erythrocytes have residual binding sites for nonerythroid spectrin. Ankyrin-independent-binding proteins that selectively bind to certain isoforms of spectrin provide a potentially important flexibility in cellular localization and time of synthesis of proteins involved in spectrin-membrane interactions. This flexibility has implications for assembly of the membrane skeleton and targeting of spectrin isoforms to specialized regions of cells.     

Plasmodium falciparum histidine-rich protein 1 associates with the band 3 binding domain of ankyrin in the infected red cell membrane

Infection of erythrocytes by the malaria parasite Plasmodium falciparum results in the export of several parasite proteins into the erythrocyte cytoplasm. Changes occur in the infected erythrocyte due to altered phosphorylation of proteins and to novel interactions between host and parasite proteins, particularly at the membrane skeleton. In erythrocytes, the spectrin based red cell membrane skeleton is linked to the erythrocyte plasma membrane through interactions of ankyrin with spectrin and band 3. Here we report an association between the P. falciparum histidine-rich protein (PfHRP1) and phosphorylated proteolytic fragments of red cell ankyrin. Immunochemical, biochemical and biophysical studies indicate that the 89 kDa band 3 binding domain and the 62 kDa spectrin-binding domain of ankyrin are co-precipitated by mAb 89 against PfHRP1, and that native and recombinant ankyrin fragments bind to the 5' repeat region of PfHRP1. PfHRP1 is responsible for anchoring the parasite cytoadherence ligand to the erythrocyte membrane skeleton, and this additional interaction with ankyrin would strengthen the ability of PfEMP1 to resist shear stress.     

Brain ankyrin. A membrane-associated protein with binding sites for spectrin, tubulin, and the cytoplasmic domain of the erythrocyte anion channel

Brain ankyrin was purified from pig brain membranes in milligram quantities by a procedure involving affinity chromatography on erythrocyte spectrinagarose. Brain ankyrin included two polypeptides of Mr = 210,000 and 220,000 that were nearly identical by peptide mapping and were monomers in solution. Brain ankyrin and erythrocyte ankyrin are closely related proteins with the following properties in common: 1) shared antigenic sites, 2) high-affinity binding to the spectrin beta subunit at the midregion of spectrin tetramers, 3) a binding site for the cytoplasmic domain of the erythrocyte anion channel, 4) a binding site for tubulin, 5) a similar domain structure with a protease-resistant domain of Mr = 72,000 that contains the spectrin-binding activity and domains of Mr = 95,000 (brain ankyrin) or 90,000 (erythrocyte ankyrin) that contain binding sites for both tubulin and the anion channel. Brain ankyrin is present at about 100 pmol/mg of membrane protein in demyelinated membranes based on radioimmunoassay with antibody raised against brain ankyrin and affinity purified on brain ankyrin-agarose. Brain spectrin tetramers are present at 30 pmol/mg of membrane protein. Brain ankyrin thus is present in sufficient amounts to attach spectrin to membranes. Brain ankyrin also may attach microtubules to membranes independently of spectrin and has the potential to interconnect microtubules and spectrin-associated actin filaments.     

Ankyrin and synapsin: spectrin-binding proteins associated with brain membranes

Brain membranes contain an actin-binding protein closely related in structure and function to erythrocyte spectrin. The proteins that attach brain spectrin to membranes are not established, but, by analogy with the erythrocyte membrane, may include ankyrin and protein 4.1. In support of this idea, proteins closely related to ankyrin and 4.1 have been purified from brain and have been demonstrated to associate with brain spectrin. Brain ankyrin binds with high affinity to the spectrin beta subunit at the midregion of spectrin tetramers. Brain ankyrin also has binding sites for the cytoplasmic domain of the erythrocyte anion channel (band 3), as well as for tubulin. Ankyrins from brain and erythrocytes have a similar domain structure with protease-resistant domains of Mr = 72,000 that contain spectrin-binding activity, and domains of Mr = 95,000 (brain ankyrin) or 90,000 (erythrocyte ankyrin) that contain binding sites for both tubulin and the anion channel. Brain ankyrin is present at about 100 pmol/mg membrane protein, or about twice the number of copies of spectrum beta chains. Brain ankyrin thus is present in sufficient amounts to attach spectrin to membranes, and it has the potential to attach microtubules to membranes as well as to interconnect microtubules with spectrin-associated actin filaments. Another spectrin-binding protein has been purified from brain membranes, and this protein cross-reacts with erythrocyte 4.1. Brain 4.1 is identical to the membrane protein synapsin, which is one of the brain's major substrates for cAMP-dependent and Ca/calmodulin-dependent protein kinases with equivalent physical properties, immunological cross-reaction, and peptide maps. Synapsin (4.1) is present at about 60 pmol/mg membrane protein, and thus is a logical candidate to regulate certain protein linkages involving spectrin.     

Spectrin promotes the association of F-actin with the cytoplasmic surface of the human erythrocyte membrane

We studied the binding of actin to the erythrocyte membrane by a novel application of falling ball viscometry. Our approach is based on the notion that if membranes have multiple binding sites for F-actin they will be able to cross-link and increase the viscosity of actin. Spectrin- and actin-depleted inside-out vesicles reconstituted with purified spectrin dimer or tetramer induce large increases in the viscosity of actin. Comparable concentrations of spectrin alone, inside-out vesicles alone, inside-out vesicles plus heat-denatured spectrin dimmer or tetramer induce large increases in the viscosity of actin. Comparable concentrations of spectrin alone, inside-out vesicles alone, inside-out plus heat denatured spectrin, ghosts, or ghosts plus spectrin have no effect on the viscosity of actin. Centrifugation experiments show that the amount of actin bound to the inside-out vesicles is enhanced in the presence of spectrin. The interactions detected by low-shear viscometry reflect actin interaction with membrane- bound spectrin because (a) prior removal of band 4.1 and ankyrin (band 2.1, the high- affinity membrane attachment site for spectrin) reduces both spectrin binding to the inside-out vesicles and their capacity to stimulate increase in viscosity of actin in the presence of spectrin + actin are inhibited by the addition of the water-soluble 72,000- dalton fragment of ankyrin, which is known to inhibit spectrin reassociation to the membrane. The increases in viscosity of actin induced by inside-out vesicles reconstituted with purified spectrin dimer or tetramer are not observed when samples are incubated at 0 degrees C. This temperature dependence may be related to the temperature-dependent associations we observe in solution studies with purified proteins: addition of ankyrin inhibits actin cross-linking by spectrin tetramer plus band 4.1 at 0 degrees C, and enhances it at 32 degrees C. We conclude (a) that falling ball viscometry can be used to assay actin binding to membranes and (b) that spectrin is involved in attaching actin filaments or oligomers to the cytoplasmic surface of the erythrocyte membrane.     

Ankyrin binds to the 15th repetitive unit of erythroid and nonerythroid beta-spectrin

Ankyrin mediates the attachment of spectrin to transmembrane integral proteins in both erythroid and nonerythroid cells by binding to the beta-subunit of spectrin. Previous studies using enzymatic digestion, 2-nitro-5-thiocyanobenzoic acid cleavage, and rotary shadowing techniques have placed the spectrin-ankyrin binding site in the COOH-terminal third of beta-spectrin, but the precise site is not known. We have used a glutathione S-transferase prokaryotic expression system to prepare recombinant erythroid and nonerythroid beta-spectrin from cDNA encoding approximately the carboxy-terminal half of these proteins. Recombinant spectrin competed on an equimolar basis with 125I-labeled native spectrin for binding to erythrocyte membrane vesicles (IOVs), and also bound ankyrin in vitro as measured by sedimentation velocity experiments. Although full length beta-spectrin could inhibit all spectrin binding to IOVs, recombinant beta-spectrin encompassing the complete ankyrin binding domain but lacking the amino-terminal half of the molecule failed to inhibit about 25% of the binding capacity of the IOVs, suggesting that the ankyrin-independent spectrin membrane binding site must lie in the amino-terminal half of beta-spectrin. A nested set of shortened recombinants was generated by nuclease digestion of beta-spectrin cDNAs from ankyrin binding constructs. These defined the ankyrin binding domain as encompassing the 15th repeat unit in both erythroid and nonerythroid beta-spectrin, amino acid residues 1,768-1,898 in erythroid beta-spectrin. The ankyrin binding repeat unit is atypical in that it lacks the conserved tryptophan at position 45 (1,811) within the repeat and contains a nonhomologous 43 residue segment in the terminal third of the repeat. It also appears that the first 30 residues of this repeat, which are highly conserved between the erythroid and nonerythroid beta-spectrins, are critical for ankyrin binding activity. We hypothesize that ankyrin binds directly to the nonhomologous segment in the 15th repeat unit of both erythroid and nonerythroid beta-spectrin, but that this sequence must be presented in the context of a properly folded spectrin "repeat unit" structure. Future studies will identify which residues within the repeat unit are essential for activity, and which residues determine the specificity of various spectrins for different forms of ankyrin.     

A dynamical study on the interactions between the cytoskeleton components in the human erythrocyte as detected by saturation transfer electron paramagnetic resonance of spin-labeled spectrin, ankyrin, and protein 4.1

Isolated human erythrocyte spectrin, ankyrin, and protein 4.1 have been labeled with the maleimide spin label, 3-maleimido-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl, and studied by saturation transfer electron paramagnetic resonance spectroscopy. The presence of the labels does not affect the reassociation of these proteins with erythrocyte membranes selectively depleted of either spectrin-actin or of all the extrinsic proteins. When maleimide spin-labeled spectrin is reassociated with the erythrocyte membrane in presence of all the cytoskeleton components, including endogeneous or purified muscle actin, spectrin still preserves its flexible character. The rotational mobilities of maleimide spin-labeled ankyrin and maleimide spin-labeled protein 4.1 are of the same order of magnitude (tau c (L"/L) approximately 5 X 10(-5) and 8 X 10(-5) s, respectively, at 2 degrees C), while protein 4.1 is almost three times smaller in size than ankyrin. This result indicates that the movements of membrane-bound maleimide spin-labeled protein 4.1 are more restricted than those of ankyrin. This suggests that their respective binding sites have different structural properties. The rotational movements of both proteins are slowed down on the addition of spectrin indicating that protein 4.1 as well as ankyrin also represents one of the links of the cytoskeleton to the membrane.     

Brain ankyrin. Purification of a 72,000 Mr spectrin-binding domain

Polypeptides of Mr = 190,000-220,000 that cross-react with erythrocyte ankyrin were detected in immunoblots of membranes from pig lens, pig brain, and rat liver. The cross-reacting polypeptides from brain were cleaved by chymotrypsin to fragments of Mr = 95,000 and 72,000 which are the same size as fragments obtained with erythrocyte ankyrin. The brain 72,000 Mr fragment associated with erythrocyte spectrin, and the binding occurred at the same site as that of erythrocyte ankyrin 72,000 Mr fragment since (a) brain 72,000 Mr fragment was adsorbed to erythrocyte spectrin-agarose and (b) 125I-labeled erythrocyte spectrin bound to brain 72,000 Mr fragment following transfer of the fragment from a sodium dodecyl sulfate gel to nitrocellulose paper, and this binding was displaced by erythrocyte ankyrin 72,000 Mr fragment. Brain 72,000 Mr fragment was purified about 400-fold by selective extraction and by continuous chromatography on columns attached in series containing DEAE-cellulose followed by erythrocyte spectrin coupled to agarose, and finally hydroxylapatite. The brain 72,000 Mr fragment was not derived from contaminating erythrocytes since peptide maps of pig brain and pig erythrocyte 72,000 Mr fragments were distinct. The amount of brain 72,000 Mr fragment was estimated as 0.28% of membrane protein or 39 pmol/mg based on radioimmunoassay with 125I-labeled brain fragment and antibody against erythrocyte ankyrin. Brain spectrin tetramer was present in about the same number of copies (30 pmol/mg of membrane protein) based on densitometry of Coomassie blue-stained sodium dodecyl sulfate gels. The binding site on brain spectrin for both brain and erythrocyte ankyrin 72,000 Mr fragments was localized by electron microscopy to the midregion of spectrin tetramers about 90 nM from the near end and 110 nM from the far end. These studies demonstrate the presence in brain membranes of a protein closely related to erythrocyte ankyrin, and are consistent with a function of the brain ankyrin as a membrane attachment site for brain spectrin.     

Calcium/calmodulin inhibits direct binding of spectrin to synaptosomal membranes

Brain spectrin, through its beta subunit, binds with high affinity to protein-binding sites on brain membranes quantitatively depleted of ankyrin (Steiner, J., and Bennett, V. (1988) J. Biol. Chem. 263, 14417-14425). In this study, calmodulin is demonstrated to inhibit binding of brain spectrin to synaptosomal membranes. Submicromolar concentrations of calcium are required for inhibition of binding, with half-maximal effects at pCa = 6.5. Calmodulin competitively inhibits binding of spectrin to protein(s) in stripped synaptosomal membranes, with Ki = 1.3 microM in the presence of 10 microM calcium. A reversible receptor-mediated process, and not proteolysis, is responsible for inhibition since the effect of calcium/calmodulin is reversed by the calmodulin antagonist trifluoperazine and by chelation of calcium with sodium [ethylenebis(oxyethylenenitrilo)]tetraacetic acid. The target of calmodulin is most likely the spectrin attachment protein(s) rather than spectrin itself since: (a) membrane binding of the brain spectrin beta subunit, which does not associate with calmodulin, is inhibited by calcium/calmodulin, and (b) red cell spectrin which binds calmodulin very weakly, is inhibited from interacting with membrane receptors in the presence of calcium/calmodulin. Ca2+/calmodulin inhibited association of erythrocyte spectrin with synaptosomal membranes but had no effect on binding of erythrocyte or brain spectrin to ankyrin in erythrocyte membranes. These experiments demonstrate the potential for differential regulation of spectrin-membrane protein interactions, with the consequence that Ca2+/calmodulin can dissociate direct spectrin-membrane interactions locally or regionally without disassembly of the areas of the membrane skeleton stabilized by linkage of spectrin to ankyrin. A membrane protein of Mr = 88,000 has been identified that is dissociated from spectrin affinity columns by calcium/calmodulin and is a candidate for the calmodulin-sensitive spectrin-binding site in brain.     

Nonerythrocyte spectrins: actin-membrane attachment proteins occurring in many cell types

The properties of brain fodrin have been analyzed and compared with those of erythrocyte spectrin. Both proteins consist of high molecular weight polypeptide doublets on SDS polyacrylamide gels and in solution behave as very large asymmetric molecules. Both proteins show a characteristic increase in sedimentation coefficient in the presence of 20 mM KCl. Antibodies against the brain protein cross-react with erythrocyte spectrin and cross-react with similar high molecular weight doublet polypeptides in SDS polyacrylamide gels of other cell types and plasma membrane preparations. Both proteins bind actin. The brain protein and erythrocyte spectrin show specific and competitive binding to erythrocyte membranes and this binding is inhibited by antibodies against erythrocyte ankyrin. Several of these properties distinguish these proteins from the class of high molecular weight actin-binding proteins that includes filamin and macrophage actin-binding protein. We conclude that together with erythrocyte spectrin, the brain protein and equivalent, immunologically related proteins in other cell types belong to a single class of proteins with the common function of attachment of actin to plasma membranes. Based on the structural and functional similarities, the name spectrin would seem appropriate for this whole class of proteins.     

Phosphorylation of ankyrin down-regulates its cooperative interaction with spectrin and protein 3

Ankyrin mediates the primary attachment between beta spectrin and protein 3. Ankyrin and spectrin interact in a positively cooperative fashion such that ankyrin binding increases the extent of spectrin tetramer and oligomer formation (Giorgi and Morrow: submitted, 1988). This cooperative interaction is enhanced by the cytoplasmic domain of protein 3, which is prepared as a 45-41-kDa fragment generated by chymotryptic digestion of erythrocyte membranes. Using sensitive isotope-ratio methods and nondenaturing PAGE, we now demonstrate directly (1) the enhanced affinity of ankyrin for spectrin oligomers compared to spectrin dimers; (2) a selective stimulation of the affinity of ankyrin for spectrin oligomer by the 43-kDa cytoplasmic domain of protein 3; and (3) a selective reduction in the affinity of ankyrin for spectrin tetramer and oligomer after its phosphorylation by the erythrocyte cAMP-independent membrane kinase. The phosphorylation of ankyrin does not affect its binding to spectrin dimer. Ankyrin also enhances the rate of interconversion between dimer-tetramer-oligomer by 2-3-fold at 30 degrees C, and in the presence of the 43-kDa fragment, ankyrin stimulates the rate of oligomer interconversions by nearly 40-fold at this temperature. These results demonstrate a long-range cooperative interaction between an integral membrane protein and the peripheral cytoskeleton and indicate that this linkage may be regulated by covalent protein phosphorylation. Such interactions may be of general importance in nonerythroid cells.     

Association of kidney and parotid Na+, K(+)-ATPase microsomes with actin and analogs of spectrin and ankyrin

Kidney Na+,K(+)-ATPase has been recently shown to bind erythroid ankyrin and to colocalize with ankyrin at the basolateral cell surface of kidney epithelial cells. These observations suggest that Na+,K(+)-ATPase is linked via ankyrin to the spectrin/actin-based membrane cytoskeleton. In the present study we show that Na+,K(+)-ATPase and analogs of spectrin, ankyrin and actin copurify from detergent extracts of pig kidney and parotid gland membranes. Actin, spectrin and ankyrin were extracted from purified Na+,K(+)-ATPase microsomes at virtually identical conditions as their counterparts from the erythrocyte membrane, i.e., 1 mM EDTA (spectrin, actin) and 1 M KCl (ankyrin). Visualization of the stripped proteins by rotary shadowing revealed numerous elongated spectrin-like dimers (100 nm) and tetramers (215 nm), a fraction of which (17%) was associated with globular (10 nm) ankyrin-like particles. Like erythrocyte ankyrin, kidney ankyrin was cleaved into a soluble 72 kDa fragment and a membrane-bound 90 kDa fragment. Consistent with our previous immunocytochemical findings on the pig kidney, Na+,K(+)-ATPase and ankyrin were found to be colocalized at the basolateral plasma membrane of striated ducts and acini of the pig parotid gland. The present findings confirm and extend the recently proposed concept that in polarized epithelial cells Na+,K(+)-ATPase may serve as major attachment site for the spectrin-based membrane cytoskeleton to the basolateral cell domain. Connections of integral membrane proteins to the cytoskeleton may help to place these proteins at specialized domains of the cell surface and to prevent them from endocytosis.     

High molecular weight microtubule-associated proteins from pig brain are immunologically related to human erythrocyte membrane proteins spectrin, ankyrin, proteins 4.1 and 4.2

The microtubule-associated proteins MAPs 1 and 2 from pig brain have been found to react with antibodies directed against human ankyrin and spectrin, respectively (Bennett and Davis, 1981; Davis and Bennett, 1982). In a complementary approach we have prepared antibodies against MAP1 alpha. MAP1 gamma and MAP2 purified from pig brain and tested their reactivity with human erythrocyte membrane proteins. Anti-MAP1 alpha was shown to react with alpha and beta-spectrin and with protein 4.1; anti-MAP1 gamma reacted with alpha-spectrin and ankyrin and with a 60 K peptide which copurified with human spectrin. Finally anti-MAP2 was specific for beta-spectrin and protein 4.2. The biological function of protein 4.2 is still unknown but details on the interactions between ankyrin, spectrin and protein 4.1 and their role in mediating the linkage of oligomeric actin on the erythrocyte membrane are well documented. The present results, which demonstrate extended immunological analogies between pig brain high molecular weight MAPs and human erythrocyte membrane proteins, may reflect the presence, in the two families of proteins, of similar functionally important epitopes.     

Phosphorylation of ankyrin decreases its affinity for spectrin tetramer

The effects of phosphorylation on the interaction between spectrin and ankyrin were investigated. Spectrin and ankyrin were phosphorylated using purified human erythrocyte membrane and cytosolic (casein kinase A) kinases. These two kinases have similar properties as well as activities toward spectrin and ankyrin. Both kinases catalyzed the incorporation of about 2 mol of phosphate/mol of spectrin and about 7 mol of phosphate/mol of ankyrin. These phosphates were incorporated primarily into seryl and threonyl residues of the proteins. The phosphopeptide maps of ankyrin phosphorylated by the membrane kinase and casein kinase A were identical. Binding studies indicate that ankyrin exhibits different affinities for spectrin dimers (KD = 2.5 +/- 0.9 X 10(-6) M) and tetramers (KD = 2.7 +/- 0.8 X 10(-7) M). These dissociation constants were not appreciably affected by the phosphorylation of spectrin. On the other hand, phosphorylation of ankyrin was found to significantly reduce its affinity for either phosphorylated or unphosphorylated spectrin tetramers (KD = 1.2 +/- 0.1 X 10(-6) M) but not spectrin dimers (KD = 2.5 +/- 0.4 X 10(-6) M). The same results were obtained using either the membrane kinase or casein kinase A as the phosphorylating enzyme. The above observation suggests that ankyrin phosphorylation may provide an important mechanism for the regulation of the erythrocyte membrane cytoskeletal network.     

Colocalization and coprecipitation of ankyrin and Na+,K+-ATPase in kidney epithelial cells

Interactions between integral proteins of the plasma membrane and the cytoskeleton may be important for localizing certain membrane proteins in a nonrandom fashion at specialized domains of the cell surface. Here, we show that ankyrin, the key protein for the linkage of the erythrocyte anion exchanger (band 3) to the spectrin-based membrane cytoskeleton, is also present in kidney distal tubular cells where ankyrin is precisely colocalized with Na+,K+-ATPase. Both proteins are confined to the basolateral plasma membrane and are absent from the apical membrane, the junctional complex and the membrane surface that contacts the basal lamina. Purified Na+,K+-ATPase of sheep and pig kidney contains a binding site for erythrocyte ankyrin as demonstrated by immunoprecipitation experiments. A band 3-like binding site for ankyrin is likely, since binding of ankyrin to Na+,K+-ATPase could be inhibited in a competitive fashion by the isolated cytoplasmic domain of erythrocyte band 3.     

Organizing the fluid membrane bilayer: diseases linked to spectrin and ankyrin

Ankyrin and spectrin were first discovered as binding partners in the membrane skeleton of human erythrocytes. Mutations in genes encoding these proteins cause hereditary spherocytosis. Recent advances reveal that ankyrin and spectrin are required for organization of a surprisingly diverse set of proteins, including ion channels and cell adhesion molecules that are localized in specialized membrane domains in many cell types. New insights into the cell biology of ankyrin and spectrin reveal that these proteins actively participate in assembly of specialized membrane domains in addition to their conventional maintenance role as scaffolding proteins. Recently described inherited human diseases due to defects in spectrin or ankyrin include spinocerebellar ataxia type 5 and a cardiac arrhythmia, termed sick sinus syndrome with bradycardia or ankyrin-B syndrome. Together, these studies identify an emerging paradigm for pathogenesis of human disease where failure in cellular localization of membrane-spanning proteins results in loss of physiological function.     

Diversity in protein associations of the spectrin-based membrane skeleton of nonerythroid cells

Summary The purpose of this review is to summarize recent progress in understanding interactions of spectrin with membranes from brain and other tissues. Spectrin has at least two choices in linkages with the membrane, one through ankyrin, which in turn is associated with integral membrane proteins, and another linkage directly with integral membrane sites identified recently in brain membranes. Some of the integral membrane protein sites in brain bind preferentially with one spectrin isoform, while some can interact with both erythroid and the general isoform of spectrin. Ankyrin also has different isoforms, and these exhibit specificity in binding to spectrin isoforms and associate with distinct integral membrane proteins. The membrane binding sites for ankyrin include several integral membrane proteins, which are differentially expressed in different cells: the anion exchanger of intercalated cells of mammalian kidney, the sodium/potassium ATPase of kidney, and the voltage-dependent sodium channel of neurons. Ankyrin is present in many other cell types and it is likely that additional ankyrin-binding proteins will be identified. Each of the proteins that now are candidates for ankyrin binding proteins are ion channels or transporters and are localized in specialized cellular domains. The polarized localization of the ankyrin-associated membrane proteins is an essential aspect of their function at a physiological level. Spectrin and ankyrin thus exhibit an unsuspected diversity in protein linkages and have the potential for cell domain-specific interactions with a variety of membrane proteins.     

Self-assembly of spectrin oligomers in vitro: a basis for a dynamic cytoskeleton

Purified human erythrocyte spectrin is able to form large oligomeric species without the collaboration of any other proteins. This reversible self-assembly process is both temperature and concentration dependent and seems to be mediated by the same kinds of low affinity noncovalent associations between spectrin monomers that promote tetramer formation. Low ionic strength extracts of erythrocyte membranes also contain these oligomeric species. These results support the idea that spectrin oligomers and the factors that regulate their formation may be responsible for both the stability and the versatility of the erythrocyte membrane cytoskeleton. It is postulated that the high concentrations of spectrin necessary for oligomerization are maintained in vivo by a high-affinity interaction with ankyrin. Such a coupling of high and low affinity interactions in multifunctional proteins may have significant implications for membrane structure and function.     

Ultrastructural localization of erythrocyte cytoskeletal and integral membrane proteins in Plasmodium falciparum-infected erythrocytes

The distributions of ankyrin, spectrin, band 3, and glycophorin A were examined in Plasmodium falciparum-infected erythrocytes by immunoelectron microscopy to determine whether movement of parasite proteins and membrane vesicles between the parasitophorous vacuole membrane and erythrocyte surface membrane involves internalization of host membrane skeleton proteins. Monospecific rabbit antisera to spectrin, band 3 and ankyrin and a mouse monoclonal antibody to glycophorin A reacted with these erythrocyte proteins in infected and uninfected human erythrocytes by immunoblotting. Cross-reacting malarial proteins were not detected. The rabbit sera also failed to immunoprecipitate [3H]isoleucine labeled malarial proteins from Triton X-100 and sodium dodecyl sulfate (SDS) extracts of infected erythrocytes. These three antibodies as well as the monoclonal antibody to glycophorin A bound to the membrane skeleton of infected and uninfected erythrocytes. The parasitophorous vacuole membrane was devoid of bound antibody, a result indicating that this membrane contains little, if any, of these host membrane proteins. With ring-, trophozoite- and schizont-infected erythrocytes, spectrin, band 3 and glycophorin A were absent from intracellular membranes including Maurer's clefts and other vesicles in the erythrocyte cytoplasm. In contrast, Maurer's clefts were specifically labeled by anti-ankyrin antibody. There was a slight, corresponding decrease in labeling of the membrane skeleton of infected erythrocytes. A second, morphologically distinct population of circular, vesicle-like membranes in the erythrocyte cytoplasm was not labeled with anti-ankyrin antibody. We conclude that membrane movement between the host erythrocyte surface membrane and parasitophorous vacuole membrane involves preferential sorting of ankyrin into a subpopulation of cytoplasmic membranes.