Beta spectrin bestows protein 4.1 sensitivity on spectrin-actin interactions

The ability of protein 4.1 to stimulate the binding of spectrin to F-actin has been compared by cosedimentation analysis for three avian (erythrocyte, brain, and brush border) and two mammalian (erythrocyte and brain) spectrin isoforms. Human erythroid protein 4.1 stimulated actin binding of all spectrins except the brush border isoform (TW 260/240). These results suggested that the beta subunit determined the protein 4.1 sensitivity of the heterodimer, since all avian alpha subunits are encoded by a single gene. Tissue-specific posttranslational modification of the alpha subunit was excluded by examining the properties of hybrid spectrins composed of the purified alpha subunit from avian erythrocyte or brush border spectrin and the beta subunit of human erythrocyte spectrin. A hybrid composed of avian brush border alpha and human erythroid beta spectrin ran on nondenaturing gels as a discrete band, migrating near human erythroid spectrin tetramers. The actin-binding activity of this hybrid was stimulated by protein 4.1, while either chain alone was devoid of activity. Therefore, although both subunits were required for actin binding, the sensitivity of the spectrin-actin interaction to protein 4.1 is a property uniquely bestowed on the heterodimer by the beta subunit. The singular insensitivity of brush border spectrin to stimulation by erythroid protein 4.1 was also consistent with the absence of proteins in avian intestinal epithelial cells which were immunoreactive with polyclonal antisera sensitive to all of the known avian and human erythroid 4.1 isoforms.     

Caenorhabditis elegans beta-G spectrin is dispensable for establishment of epithelial polarity, but essential for muscular and neuronal function

The Caenorhabditis elegans genome encodes one alpha spectrin subunit, a beta spectrin subunit (beta-G), and a beta-H spectrin subunit. Our experiments show that the phenotype resulting from the loss of the C. elegans alpha spectrin is reproduced by tandem depletion of both beta-G and beta-H spectrins. We propose that alpha spectrin combines with the beta-G and beta-H subunits to form alpha/beta-G and alpha/beta-H heteromers that perform the entire repertoire of spectrin function in the nematode. The expression patterns of nematode beta-G spectrin and vertebrate beta spectrins exhibit three striking parallels including: (1) beta spectrins are associated with the sites of cell-cell contact in epithelial tissues; (2) the highest levels of beta-G spectrin occur in the nervous system; and (3) beta spectrin-G in striated muscle is associated with points of attachment of the myofilament apparatus to adjacent cells. Nematode beta-G spectrin associates with plasma membranes at sites of cell-cell contact, beginning at the two-cell stage, and with a dramatic increase in intensity after gastrulation when most cell proliferation has been completed. Strikingly, depletion of nematode beta-G spectrin by RNA-mediated interference to undetectable levels does not affect the establishment of structural and functional polarity in epidermis and intestine. Contrary to recent speculation, beta-G spectrin is not associated with internal membranes and depletion of beta-G spectrin was not associated with any detectable defects in secretion. Instead beta-G spectrin-deficient nematodes arrest as early larvae with progressive defects in the musculature and nervous system. Therefore, C. elegans beta-G spectrin is required for normal muscle and neuron function, but is dispensable for embryonic elongation and establishment of early epithelial polarity. We hypothesize that heteromeric spectrin evolved in metazoans in response to the needs of cells in the context of mechanically integrated tissues that can withstand the rigors imposed by an active organism.     

Mechanisms of cytoskeletal regulation: functional and antigenic diversity in human erythrocyte and brain beta spectrin

A study of human erythrocyte and brain spectrin with particular emphasis on the beta subunits revealed a structural homology but functional dissimilarity between these two molecules. Six monoclonal antibodies raised to human erythrocyte beta spectrin identify three of the four proteolytically defined domains of erythrocyte beta spectrin. Five of these monoclonal antibodies cross-react with human brain spectrin. None of a previously identified set of alpha erythrocyte spectrin monoclonal antibodies [Yurchenco et al: J Biol Chem 257:9102, 1982] reacted with brain spectrin. A domain map generated by limited tryptic digestion shows that brain spectrin is composed of proteolytically resistant domains analogous to erythrocyte spectrin, but the brain protein is more basic. The binding of brain spectrin to erythrocyte ankyrin, both in solution and on erythrocyte IOVs, yielded an association constant approximately 100 time weaker than for erythrocyte spectrin. The binding of azido-calmodulin under native conditions was specific for the erythrocyte beta subunit but was not calcium dependent. In contrast, azido-calmodulin bound only to the alpha subunit of brain spectrin in a calcium-dependent manner. The similarity of structure but modified functional characteristics of the brain and erythrocyte beta spectrins suggest that these proteins serve different cellular roles.     

Contributions of the beta-subunit to spectrin structure and function

The three avian spectrins that have been characterized consist of a common alpha-subunit (240 kD) paired with an isoform-specific beta-subunit from either erythrocyte (220 or 230 kD), brain (235 kD), or intestinal brush border (260 kD). Analysis of avian spectrins, with their naturally occurring "subunit replacement" has proved useful in assessing the relative contribution of each subunit to spectrin function. In this study we have completed a survey of avian spectrin binding properties and present morphometric analysis of the relative flexibility and linearity of various avian and human spectrin isoforms. Evidence is presented that, like its mammalian counterpart, avian brain spectrin binds human erythroid ankyrin with low affinity. Cosedimentation analysis demonstrates that 1) avian erythroid protein 4.1 stimulates spectrin-actin binding of both mammalian and avian erythrocyte and brain spectrins, but not the TW 260/240 isoform, 2) calpactin I does not potentiate actin binding of either TW 260/240 or brain spectrin, and 3) erythrocyte adducin does not stimulate the interaction of TW 260/240 with actin. In addition, a morphometric analysis of rotary-shadow images of spectrin isoforms, individual subunits, and reconstituted complexes from isolated subunits was performed. This analysis revealed that the overall flexibility and linearity of a given spectrin heterodimer and tetramer is largely determined by the intrinsic rigidity and linearity of its beta-spectrin subunit. No additional rigidity appears to be imparted by noncovalent associations between the subunits. The scaled flexural rigidity of the most rigid spectrin analyzed (human brain) is similar to that reported for F-actin.     

Mechanisms of cytoskeletal regulation: modulation of membrane affinity in avian brush border and erythrocyte spectrins

The spectrins isolated from chicken erythrocytes and chicken intestinal brush border, TW260/240, share a common alpha subunit and a tissue-specific beta subunit. The ability of these related proteins to bind human erythrocyte inside out vesicles (IOVs) and human erythrocyte ankyrin in vitro have been quantitatively compared with human erythrocyte spectrin. Chicken erythrocyte spectrin binds human IOVs and human ankyrin with affinities nearly identical to that for human erythrocyte spectrin. TW260/240 does not significantly bind to either IOVs or ankyrin. These results demonstrate a remarkable tissue preservation of ankyrin-binding capacity, even between diverse species, and confirm the role of the avian beta-spectrins in modulating this functionality. Avian brush border spectrin may represent a unique spectrin which serves primarily as a filament cross-linker and which does not interact strongly with membrane-associated proteins.     

Beta spectrin in human skeletal muscle. Tissue-specific differential processing of 3' beta spectrin pre-mRNA generates a beta spectrin isoform with a unique carboxyl terminus

Spectrin, an important component of the mammalian erythrocyte membrane skeleton, is a heterodimeric protein with alpha and beta subunits of 280 and 246 kDa, respectively. Spectrin-like proteins have also been demonstrated in a wide variety of nonerythroid cells. To examine the hypothesis that nonerythroid beta spectrins may be encoded by the "erythroid" beta spectrin gene, we have isolated cDNA clones from a human fetal skeletal muscle library by hybridization to a previously described red cell beta spectrin cDNA. Detailed comparison of muscle and erythroid beta spectrin cDNAs has revealed sequence identity over the majority of their lengths, confirming that they are the product of the same gene. However, there is a sharp divergence in sequence at their 3' ends. A consequence of this divergence is the replacement of the carboxyl terminus of erythroid beta spectrin with a different, longer carboxyl-terminal domain in skeletal muscle. We hypothesize that tissue-specific differential polyadenylation leads to the selective activation of a donor splice site within the beta spectrin coding sequence, splicing downstream nonerythroid exons into the mature muscle beta spectrin mRNA. We predict that replacement, in nonerythroid cells, of the beta spectrin carboxyl terminus, known to participate in spectrin self-association and phosphorylation, has significant functional consequences. These data may explain previously reported nonerythroid beta spectrin isoforms that resemble red cell beta spectrin by immunochemical analysis.     

Comparison of spectrin isolated from erythroid and non-erythroid sources

Spectrin from erythrocytes and two other tissues (brain and intestine) were isolated from two distant species, pig and chicken; some structural and functional properties were compared. A quantitative antibody inhibition assay was used to determine that antibodies to mammalian red cell spectrin cross-react very poorly, if at all, with their non-erythroid (brain) counterpart and similarly antibodies to pig brain spectrin (fodrin) cross-react very weakly with erythroid spectrin. By contrast, antibodies which were directed against the 240000-Mr subunit of avian fodrin were completely inhibited with avian spectrin and vice versa. To analyze the structural relatedness of these molecules further we compared the chymotryptic iodinated peptide maps generated from each individual subunit. Consistent with the antibody results, we find little (less than 10%) homology between peptides derived from mammalian fodrin and spectrin, but complete homology (100%) of the peptides derived from the 240000-Mr subunits of chicken fodrin, spectrin and another related molecule from intestine, TW260/240. Whereas the peptide maps of fodrin (brain spectrin) revealed striking similarity between divergent species, suggesting a high degree of structural conservation, the peptide maps of erythrocyte spectrin was highly variable between species, indicating that it has diverged considerably in mammalian evolution. In addition we have compared a functional activity of mammalian spectrins, the ability to bind calmodulin, using two different assays. Both results show that, whereas fodrin-calmodulin interaction can be readily demonstrated, the binding to mammalian erythroid spectrin is negligible. This suggests that the high-affinity calmodulin site present on fodrin has been lost from spectrin in mammalian evolution.     

Changes in erythroid membrane proteins during erythropoietin-mediated terminal differentiation

Membrane and membrane skeleton proteins were examined in erythroid progenitor cells during terminal differentiation. The employed model system of erythroid differentiation was that in which proerythroblasts from mice infected with the anemia-inducing strain of Friend virus differentiate in vitro in response to erythropoietin (EP). With this system, developmentally homogeneous populations of cells can be examined morphologically and biochemically as they progress from proerythroblasts through enucleated reticulocytes. alpha and beta spectrins, the major proteins of the erythrocyte membrane skeleton, are synthesized in the erythroblasts both before and after EP exposure. At all times large portions of the newly synthesized spectrins exist in and are turned over in the cytoplasm. The remaining newly synthesized spectrin is found in a cellular fraction containing total membranes. Pulse-chase experiments show that little of the cytoplasmic spectrins become membrane associated, but that the proportion of newly synthesized spectrin which is membrane associated increases as maturation proceeds. A membrane fraction enriched in plasma membranes has significant differences in the stoichiometry of spectrin accumulation as compared to total cellular membranes. Synthesis of band 3 protein, the anion transporter, is induced only after EP addition to the erythroblasts. All of the newly synthesized band 3 is membrane associated. A two-dimensional gel survey was conducted of newly synthesized proteins in the plasma membrane enriched fraction of the erythroblasts as differentiation proceeded. A majority of the newly synthesized proteins remain in the same proportion to each other during maturation; however, a few newly synthesized proteins greatly increase following EP induction while others decrease markedly. Of the radiolabeled proteins observed in two dimensional gels, only the spectrins, band 3 and actin become major proteins of the mature erythrocyte membrane. Examination of total proteins of the plasma membrane enriched fractions of EP-treated erythroblasts using silver staining and 32P autoradiography show that many proteins and phosphoproteins are selectively eliminated from this fraction late in the course of differentiation during the reticulocyte stage. The selective removal of many proteins at the reticulocyte stage of development combined with previous selective synthesis and accumulation of some specific proteins such as alpha and beta spectrin and band 3 in the differentiating erythroblasts lead to the final mammalian erythrocyte membrane structure.     

Calmodulin inhibits the phosphorylation of spectrin in vitro

In vitro phosphorylation of purified spectrin dimer was studied in the presence of Ca2+-calmodulin (CaM). CaM inhibited autophosphorylation of the beta subunit of spectrin. The inhibitory effect (65% at a 32-fold molar excess) appeared to be due to a weak interaction of CaM with spectrin. CaM was similarly effective in a phosphatase-stimulated autothiophosphorylation of the beta subunit with [gamma-35S]ATP. Hence, its inhibitory effect was not due to stimulation of a spectrin-associated phosphatase activity. Phosphorylation of spectrin by the catalytic subunit of a cAMP-dependent protein kinase occurred in both subunits (1984, FEBS Lett. 169, 323). CaM selectively inhibited a cAMP-dependent phosphorylation of the alpha subunit of spectrin to 30% at two CaM per spectrin. It was ineffective on the cAMP-dependent phosphorylation of the beta subunit up to a 32-fold molar excess. These results yield functional evidence for a CaM-spectrin interaction. They further suggest that CaM can regulate the extent of a cAMP-dependent phosphorylation of the alpha subunit of spectrin.     

Unequal synthesis and differential degradation of alpha and beta spectrin during murine erythroid differentiation

Murine erythroleukemia (MEL) cells represent a valuable system to study the biogenesis of the cytoskeleton during erythroid differentiation. When attached to fibronectin-coated dishes MEL cells induce, upon addition of DMSO, a 7-d differentiation process during which they enucleate and reach the reticulocyte stage (Patel, V. P., and H. F. Lodish. 1987. J. Cell Biol. 105:3105-3118); they accumulate band 3, spectrin, and ankyrin in amounts equivalent to those found in mature red blood cells. To follow the biosynthesis of spectrin during differentiation, membranes and cytoskeletal proteins of cells metabolically labeled with [35S]methionine were solubilized by SDS and alpha and beta spectrins were recovered by specific immunoadsorption. In both uninduced and 3-d induced cells, the relative synthesis of alpha/beta spectrin is approximately 1:3. In uninduced MEL cells newly synthesized alpha and beta spectrins are degraded with a similar half-life of approximately 10 h. In contrast, in 3-d differentiated MEL cells newly made beta spectrin is much more unstable than alpha spectrin; the half-lives of alpha and beta spectrin chains are approximately 22 and 8 h, respectively. Thus, accumulation of equal amounts of alpha and beta spectrin is caused by unequal synthesis and unequal degradation. As judged by Northern blot analyses, the level of actin mRNA is relatively constant throughout the 7-d differentiation period. alpha and beta spectrin mRNAs are barely detectable in uninduced cells, increase during the first 4 d of induction, and remain constant thereafter. In contrast, band 3 mRNA is first detectable on day 4 of differentiation. Thus, most of the spectrin that accumulates in enucleating reticulocytes is synthesized during the last few days of erythropoiesis, concomitant with the onset of band 3 synthesis. To determine whether this was occurring in normal mouse erythropoiesis, we analyzed the rate of appearance of labeled membrane proteins in mature erythrocytes after a single injection of [35S]methionine. Our results show that most of the spectrin and band 3 in mature erythrocytes is synthesized during the last days of bone marrow erythropoiesis, and that, in the marrow, band 3 and protein 4.1 are synthesized at a somewhat later stage of development than are alpha and beta spectrin, ankyrin, and actin.     

Intragenic duplication and divergence in the spectrin superfamily of proteins

Many structural, signaling, and adhesion molecules contain tandemly repeated amino acid motifs. The alpha-actinin/spectrin/dystrophin superfamily of F-actin-crosslinking proteins contains an array of triple alpha-helical motifs (spectrin repeats). We present here the complete sequence of the novel beta-spectrin isoform beta(Heavy)- spectrin (beta H). The sequence of beta H supports the origin of alpha- and beta-spectrins from a common ancestor, and we present a novel model for the origin of the spectrins from a homodimeric actin-crosslinking precursor. The pattern of similarity between the spectrin repeat units indicates that they have evolved by a series of nested, nonuniform duplications. Furthermore, the spectrins and dystrophins clearly have common ancestry, yet the repeat unit is of a different length in each family. Together, these observations suggest a dynamic period of increase in repeat number accompanied by homogenization within each array by concerted evolution. However, today, there is greater similarity of homologous repeats between species than there is across repeats within species, suggesting that concerted evolution ceased some time before the arthropod/vertebrate split. We propose a two-phase model for the evolution of the spectrin repeat arrays in which an initial phase of concerted evolution is subsequently retarded as each new protein becomes constrained to a specific length and the repeats diverge at the DNA level. This evolutionary model has general applicability to the origins of the many other proteins that have tandemly repeated motifs.      

Sequencing of the chicken non-erythroid spectrin cDNA reveals an internal repetitive structure homologous to the human erythrocyte spectrin.

Immunological screening of a chicken gizzard cDNA expression library was used to isolate two clones encoding a part of the non-erythroid spectrin-like protein. Clones were identified by immunoblotting of the polypeptides synthesized in Escherichia coli cells transformed with cDNA cloned in the pUC8 plasmid vector using polyclonal rabbit antibodies raised against bovine non-erythroid spectrin. The sequence of an approximately 1.5-kb cDNA insert of one clone was determined. Analysis of the predicted amino acid sequence reveals that, despite differences in immunological cross-reactivity and peptide maps, the chicken non-erythroid and the human erythrocyte spectrins are highly homologous proteins. Like the human erythrocyte spectrin, the chicken smooth muscle spectrin appears also to be constructed from repeated, homologous structures of 106 amino acid residues. This is probably a universal structure motif of spectrins.     

Spectrin ubiquitination and oxidative stress: potential roles in blood and neurological disorders

This review covers the observations that erythrocyte spectrin has a E2 ubiquitin conjugating enzymatic activity that allows it to transfer ubiquitin to a target site in the alpha-spectrin repeats 20/21. The position of this ubiquitination site suggests that ubiquitination may regulate alpha beta spectrin heterodimer nucleation, spectrin-4.1-actin ternary complex formation, and adducin stimulated spectrin-actin attachment in the mature erythrocyte. In sickle cells, which contain altered redox status (high GSSG/GSH ratio), ubiquitin attachment to the E2 and target sites in alpha-spectrin is greatly diminished. We propose that this attenuated ubiquitination of spectrin may be due to glutathiolation of the E2 active site cysteine leading to diminished ubiquitin-spectrin adduct and conjugate formation. Furthermore we propose that lack of ubiquitin-spectrin complex formation leads to dysregulation of the membrane skeleton in mature SS erythrocytes and may diminish spectrin turnover in SS erythropoietic cells via the ubiquitin proteasome machinery. In hippocampal neurons, spectrin is the major ubiquitinated protein and a component of the cytoplasmic ubiquitinated inclusions observed in Alzheimer's and Parkinson's diseases. The two primary neuronal spectrin isoforms: alpha SpI Sigma*/beta SpI Sigma 2 and alpha SpII Sigma 1/beta SpII Sigma 1 are both ubiquitinated. Future work will resolve whether neuronal spectrins also contain E2-ubiquitin conjugating activity and the molecular basis for formation of ubiquitinated inclusions in neurological disorders.     

A beta-spectrin isoform from Drosophila (beta H) is similar in size to vertebrate dystrophin

Spectrins are a major component of the membrane skeleton in many cell types where they are thought to contribute to cell form and membrane organization. Diversity among spectrin isoforms, especially their beta subunits, is associated with diversity in cell shape and membrane architecture. Here we describe a spectrin isoform from Drosophila that consists of a conventional alpha spectrin subunit complexed with a novel high molecular weight beta subunit (430 kD) that we term beta H. The native alpha beta H molecule binds actin filaments with high affinity and has a typical spectrin morphology except that it is longer than most other spectrin isoforms and includes two knoblike structures that are attributed to a unique domain of the beta H subunit. Beta H is encoded by a different gene than the previously described Drosophila beta-spectrin subunit but shows sequence similarity to beta-spectrin as well as vertebrate dystrophin, a component of the membrane skeleton in muscle. By size and sequence similarity, dystrophin is more similar to this newly described beta-spectrin isoform (beta H) than to other members of the spectrin gene family such as alpha-spectrin and alpha- actinin.     

In vitro proteolysis of brain spectrin by calpain I inhibits association of spectrin with ankyrin-independent membrane binding site(s).

This report demonstrates that specific proteolysis of brain spectrin by a calcium-dependent protease, calpain I, abolishes association of brain spectrin with the ankyrin-independent binding site(s) in brain membranes. Calpain I cleaves the beta subunit of spectrin at the N-terminal end leaving a 218-kDa fragment and cleaves the alpha subunit in the midregion to produce 150- and 130-kDa fragments. Calpain-proteolyzed spectrin almost completely loses the capacity to displace binding of intact spectrin to membranes. Spectrin digested by calpain I under conditions that almost completely destroyed membrane-binding remained associated as a tetramer and retained about 60% of the ability to associate with actin filaments. Cleavage of spectrin occurred at sites distinct from the membrane-binding site which is located on the beta subunit since the isolated 218-kDa fragment of the beta subunit as well as a reconstituted complex of alpha and 218-kDa beta subunit fragment partially regained binding activity. Moreover, cleavage of the alpha subunit alone reduced the affinity of spectrin for membranes by 2-fold. A consequence of distinct sites for calpain I cleavage and membrane-binding is that calpain I can digest spectrin while spectrin is complexed with other proteins and therefore has the potential to mediate disassembly of a spectrin-actin network from membranes.     

Mitoxantrone changes spectrin-aminophospholipid interactions

Understanding drug-membrane and drug-membrane protein interactions would be a crucial step towards understanding the action and biological properties of anthracyclines, as the cell membrane with its integral and peripheral proteins is the first barrier encountered by these drugs. In this paper, we briefly describe mitoxantrone-monolayer and mitoxantrone-bilayer interactions, focusing on the effect of mitoxantrone on the interactions between erythroid or nonerythroid spectrin with phosphatidylethanolamine-enriched mono- and bilayers. We found that mitoxantrone markedly modifies the interaction of erythroid and nonerythroid spectrins with phosphatidylethanolamine/phosphatidylcholine (PE/PC) monolayers. The change in delta pi induced by spectrins is several-fold larger in the presence of 72 nM mitoxantrone than in its absence: spectrin/mitoxantrone complexes induced a strong compression of the monolayer. Spin-labelling experiments showed that spectrin/mitoxantrone complexes caused significant changes in the order parameter measured using a 5'-doxyl stearate probe in the bilayer, but they practically did not affect the mobility of 16'-doxyl stearate. These results indicate close-to-surface interactions/penetrations without significant effect on the mid-region of the hydrophobic core of the bilayer. The obtained apparent equilibrium dissociation constants indicated relatively similar mitoxantrone-phospholipid and mitoxantrone-spectrin (erythroid and nonerythroid) binding affinities. These results might in part, explain the effect of mitoxantrone on spectrin distribution in the living cells.     

Immunocytochemical studies of spectrin in hamster cardiac tissue

The spectrins are a family of cytoskeletal-membrane proteins that have a wide tissue distribution. In the present study, we employed polyclonal antibodies made against mammalian and avian erythroid spectrins as well as mammalian brain spectrin to assess their presence and distributions in the mammalian heart. Western blot analyses revealed that all three antibodies were specific for a 240,000 molecular weight alpha-spectrin subunit found in hamster erythrocyte ghost homogenates, whole hamster heart, and isolated hamster cardiac myofibril homogenates. Spectrin staining was absent from the Triton X-100-extracted supernatant fraction of myofibril preparations, suggesting that the protein is linked to the myofibril precipitate after exposure to the detergent. Frozen, unfixed, 2-microns-thick; sections of adult. Syrian golden hamster cardiac tissue exhibited strong immunofluorescent staining of intercalated discs and Z-bands using all three antibodies. In addition, the mammalian erythroid spectrin antibodies showed staining of the sarcolemma, and in cross section, revealed a delicate internal network of staining that appears to surround individual myofibrils. This may be T-tubule-associated staining. Myofibrils isolated from cardiac myocytes using Triton X-100 show positive Z-band staining using all three antibodies. Double staining with Texas Red-labeled monoclonal desmin and FITC-labeled polyclonal spectrin antibodies revealed that both stained the myofibrillar Z-line regions. These results demonstrate that spectrin is closely associated with the membranes, myofibrils, and intermediate filaments in the mammalian heart.     

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.     

Mitoxantrone changes spectrin-aminophospholipid interactions

Understanding drug-membrane and drug-membrane protein interactions would be a crucial step towards understanding the action and biological properties of anthracyclines, as the cell membrane with its integral and peripheral proteins is the first barrier encountered by these drugs. In this paper, we briefly describe mitoxantrone-monolayer and mitoxantrone-bilayer interactions, focusing on the effect of mitoxantrone on the interactions between erythroid or nonerythroid spectrin with phosphatidylethanolamine-enriched mono- and bilayers. We found that mitoxantrone markedly modifies the interaction of erythroid and nonerythroid spectrins with phosphatidylethanolamine/phosphatitydcholine (PE/PC) monolayers. The change in Δπ induced by spectrins is several-fold larger in the presence of 72?nM mitoxantrone than in its absence: spectrin/mitoxantrone complexes induced a strong compression of the monolayer. Spin-labelling experiments showed that spectrin/mitoxantrone complexes caused significant changes in the order parameter measured using a 5′-doxyl stearate probe in the bilayer, but they practically did not affect the mobility of 16′-doxyl stearate. These results indicate close-to-surface interactions/penetrations without significant effect on the mid-region of the hydrophobic core of the bilayer. The obtained apparent equilibrium dissociation constants indicated relatively similar mitoxantrone-phospholipid and mitoxantrone-spectrin (erythroid and nonerythroid) binding affinities. These results might in part, explain the effect of mitoxantrone on spectrin distribution in the living cells.