Thermal stabilities of brain spectrin and the constituent repeats of subunits

The different genes that encode mammalian spectrins give rise to proteins differing in their apparent stiffness. To explore this, we have compared the thermal stabilities of the structural repeats of brain spectrin subunits (alphaII and betaII) with those of erythrocyte spectrin (alphaI and betaI). The unfolding transition midpoints (T(m)) of the 36 alphaII- and betaII-spectrin repeats extend between 24 and 82 degrees C, with an average higher by some 10 degrees C than that of the alphaI- and betaI-spectrin repeats. This difference is reflected in the T(m) values of the intact brain and erythrocyte spectrins. Two of three tandem-repeat constructs from brain spectrin exhibited strong cooperative coupling, with elevation of the T(m) of the less stable partner corresponding to coupling free energies of approximately -4.4 and -3.5 kcal/mol. The third tandem-repeat construct, by contrast, exhibited negligible cooperativity. Tandem-repeat mutants, in which a part of the "linker" helix that connects the two domains was replaced with a corresponding helical segment from erythroid spectrin, showed only minor perturbation of the thermal melting profiles, without breakdown of cooperativity. Thus, the linker regions, which tolerate few point mutations without loss of cooperative function, have evidently evolved to permit conformational coupling in specified regions. The greater structural stability of the repeats in alphaII- and betaII-spectrin may account, at least in part, for the higher rigidity of brain compared to erythrocyte spectrin.     

c-Src binds alpha II spectrin's Src homology 3 (SH3) domain and blocks calpain susceptibility by phosphorylating Tyr1176

Spectrin is a ubiquitous heterodimeric scaffolding protein that stabilizes membranes and organizes protein and lipid microdomains on both the plasma membrane and intracellular organelles. Phosphorylation of beta-spectrin on Ser/Thr is well recognized. Less clear is whether alpha-spectrin is phosphorylated in vivo and whether spectrin is phosphorylated on tyrosine (pTyr). We affirmatively answer both questions. In cultured Madin-Darby canine kidney cells, alphaII spectrin undergoes in vivo tyrosine phosphorylation. Enhancement of the steady state level of pTyr-modified alphaII spectrin by vanadate, a phosphatase inhibitor, implies a dynamic balance between alphaII spectrin phosphorylation and dephosphorylation. Recombinant peptides containing the Src homology 3 domain of alphaII spectrin (but not the Src homology 3 domain of alphaI spectrin) bind specifically to phosphorylated c-Src in Madin-Darby canine kidney cell lysates, suggesting that this kinase is responsible for its in vivo phosphorylation. pTyr-modified alphaII spectrin is resistant to maitotoxin-induced cleavage by mu-calpain in vivo. In vitro studies of recombinant alphaII spectrin peptides representing repeats 9-12 identify two sites of pTyr modification. The first site is at Tyr(1073), a residue immediately adjacent to a region encoded by alternative exon usage (insert 1). The second site is at Tyr(1176). This residue flanks the major site of cleavage by the calcium-dependent protease calpain, and phosphorylation of Tyr(1176) by c-Src reduces the susceptibility of alphaII spectrin to cleavage by mu-calpain. Calpain cleavage of spectrin, activated by Ca(2+) and calmodulin, contributes to diverse cellular processes including synaptic remodeling, receptor-mediated endocytosis, apoptosis, and the response of the renal epithelial cell to ischemic injury. Tyrosine phosphorylation of alphaII spectrin now would appear to also mediate these events. The spectrin skeleton thus forms a point of convergence between kinase/phosphatase and Ca(2+)-mediated signaling cascades.     

Fodrin CaM-binding domain cleavage by Pet from enteroaggregative Escherichia coli leads to actin cytoskeletal disruption

We have previously shown that the plasmid-encoded toxin (Pet) of enteroaggregative Escherichia coli produces cytotoxic and enterotoxic effects. Pet-intoxicated epithelial cells reveal contraction of the cytoskeleton and loss of actin stress fibres. Pet effects require its internalization into epithelial cells. We have also shown that Pet degrades erythroid spectrin. Pet delivery within the intestine suggests that Pet may degrade epithelial fodrin (non-erythroid spectrin). Here we demonstrate that Pet has affinity for alpha-fodrin (formally named alphaII spectrin) in vitro and in vivo and cleaves epithelial fodrin, causing its redistribution within the cells. When Pet has produced its cytoskeletal effects, fodrin is found in intracellular aggregates as membrane blebs. Pet cleaves recombinant GST-fodrin, generating two breakdown products of 37 and 72 kDa. Sequencing of the 37 kDa fragment demonstrated that the cleavage site occurred within fodrin's 11th repetitive unit between M1198 and V1199, in the calmodulin binding domain. Site-directed mutagenesis of these amino acids prevented fodrin degradation by Pet. Pet also cleaves epithelial fodrin from cultured Pet-treated cells. A mutant in the Pet serine protease motif was unable to cause fodrin redistribution or to cleave GST-fodrin. This is the first report showing cleavage of alpha-fodrin by a bacterial protease. Cleavage occurs in the middle of the calmodulin binding domain, which leads to cytoskeleton disruption.     

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.     

Structural analysis of the alpha N-terminal region of erythroid and nonerythroid spectrins by small-angle X-ray scattering

We used SpalphaI-1-156 peptide, a well-characterized model peptide of the alphaN-terminal region of erythrocyte spectrin, and SpalphaII-1-149, an alphaII brain spectrin model peptide similar in sequence to SpalphaI-1-156, to study their association affinities with a betaI-spectrin peptide, SpbetaI-1898-2083, by isothermal titration calorimetry. We also determined their conformational flexibilities in solution by small-angle X-ray scattering (SAXS) methods. These two peptides exhibit sequence homology and could be expected to exhibit similar association affinities with beta-spectrin. However, our studies show that the affinity of SpalphaII-1-149 with SpbetaI-1898-2083 is much higher than that of SpalphaI-1-156. Our SAXS findings also indicate a significantly more extended conformation for SpalphaII-1-149 than for SpalphaI-1-156. The radius of gyration values obtained by two different analyses of SAXS data and by molecular modeling all show a value of about 25 A for SpalphaI-1-156 and of about 30 A for SpalphaII-1-149, despite the fact that SpalphaI-1-156 has seven amino acid residues more than SpalphaII-1-149. For SpalphaI-1-156, the SAXS results are consistent with a flexible junction between helix C' and the triple helical bundle that allows multiple orientations between these two structural elements, in good agreement with our published NMR analysis. The SAXS findings for SpalphaII-1-149 support the hypothesis that this junction region is rigid (and probably helical) for alphaII brain spectrin. The nature of the junction region, from one extreme as a random coil (conformationally mobile) segment in alphaI to another extreme as a rigid segment in alphaII, determines the orientation of helix C' relative to the first structural domain. We suggest that this particular junction region in alpha-spectrin plays a major role in modulating its association affinity with beta-spectrins, and thus regulates spectrin tetramer levels. We also note that these are the first conformational studies of brain spectrin.     

The spectrin super-family

The review is focused on recent data on the primary sequences of erythroid and non-erythroid spectrins. As in other fields, the techniques of molecular genetics have allowed great advances in our knowledge of the structure and the genetic story of these molecules. Comparison of alpha-chains sequences of the non-erythroid (fodrin) and erythroid spectrin demonstrated that the fodrin alpha-genes are strictly conserved across species, while the mammalian spectrin genes have diverged rapidly. Spectrin and fodrin alpha-chains are largely composed of homologous 106-amino-acid repeat units. Spectrin alpha-chain is lacking a 37 amino-acid sequence which bears the calmodulin-binding site of the fodrin alpha-chain. The highest degree of homology between the spectrin alpha-chain and the fodrin alpha-chain lies in a central atypical segment unrelated to the canonical repeat sequence. This region is closely related to the N-terminal segment of several src-tyrosine kinases and to a domain of phospholipase C. Like the spectrin alpha-chain, the major central part of the spectrin beta-chain is made up of repeat units of 106 amino-acids. The N-terminal domain of the beta-chain, and especially the actin binding site, is the region of greatest homology among members of the spectrin super-family, including Drosophila spectrin beta-chain, dystrophin and alpha-actinin. The C-terminal extremity of the erythroid beta-chain is also of great interest, since tissue-specific differential processing of 3'beta-spectrin gene pre-mRNA generates a beta spectrin-isoform with a unique C-terminus in human skeletal muscle.     

Structural analysis of homologous repeated domains in alpha-actinin and spectrin

The amino acid sequences of chick and slime mould alpha-actinin each contain four repeats of approximately 122 residues. These repeats are homologous to the 18-22 repeats, each of approximately 106 residues, found in the alpha and beta subunits of spectrin and fodrin, and to the multiple repeats of approximately 110 residues found in the Duchenne muscular dystrophy protein (dystrophin). The repeats correspond to the elongated rod-like portion of these molecules. We present a multiple sequence alignment of 21 repeats from this superfamily (8 alpha-actinin and 13 spectrin/fodrin), based on optimal pairwise alignments, from which a characteristic consensus pattern of amino acid types is deduced. Trp 46 is invariant in all but one repeat, and physicochemical classes of amino acids are conserved at 25 other positions. Secondary structure prediction on both the alpha-actinin and spectrin repeats taken together with the distribution of proline residues in the sequences, strongly suggest that each repeated domain consists of a four-helix structure. Our predictions differ significantly from previous three-helix models based on analyses of fewer sequences. To determine possible interdomain regions, sites of limited proteolysis of the native chick alpha-actinin dimer were determined and located in the amino acid sequence. The majority of these sites were in corresponding positions in different repeats within a segment predicted as a long helix. We propose a model, consistent with the overall dimensions of the rod-like portions of the molecules, in which these long, probably interrupted helices, link adjacent domains.     

Phosphatidylserine binding sites in erythroid spectrin: location and implications for membrane stability

The erythrocyte membrane is a composite structure consisting of a lipid bilayer tethered to the spectrin-based membrane skeleton. Two complexes of spectrin with other proteins are known to participate in the attachment. Spectrin has also been shown to interact with phosphatidylserine (PS), a component of the lipid bilayer, which is confined to its inner leaflet. That there may be multiple sites of interaction with PS in the spectrin sequence has been inferred, but they have not hitherto been identified. Here we have explored the interaction of PS-containing liposomes with native alpha- and beta-spectrin chains and with recombinant spectrin fragments encompassing the entire sequences of both chains. We show that both alpha-spectrin and beta-spectrin bind PS and that sites of high affinity are located within 8 of the 38 triple-helical structural repeats which make up the bulk of both chains; these are alpha8, alpha9-10, beta2, beta3, beta4, beta12, beta13, and beta14, and PS affinity was also found in the nonhomologous N-terminal domain of the beta-chain. No other fragments of either chain showed appreciable binding. Binding of spectrin and its constituent chains to mixed liposomes of PS and phosphatidylcholine (PC) depended on the proportion of PS. Binding of spectrin dimers to PS liposomes was inhibited by single repeats containing PS binding sites. It is noteworthy that the PS binding sites in beta-spectrin are grouped in close proximity to the sites of attachment both of ankyrin and of 4.1R, the proteins engaged in attachment of spectrin to the membrane. We conjecture that direct interaction of spectrin with PS in the membrane may modulate its interactions with the proteins and that (considering also the known affinity of 4.1R for PS) the formation of PS-rich lipid domains, which have been observed in the red cell membrane, may be a result.     

Sequential degradation of alphaII and betaII spectrin by calpain in glutamate or maitotoxin-stimulated cells

Calpain-catalyzed proteolysis of II-spectrin is a regulated event associated with neuronal long-term potentiation, platelet and leukocyte activation, and other processes. Calpain proteolysis is also linked to apoptotic and nonapoptotic cell death following excessive glutamate exposure, hypoxia, HIV-gp120/160 exposure, or toxic injury. The molecular basis for these divergent consequences of calpain action, and their relationship to spectrin proteolysis, is unclear. Calpain preferentially cleaves II spectrin in vitro in repeat 11 between residues Y1176 and G1177. Unless stimulated by Ca++ and calmodulin (CaM), betaII spectrin proteolysis in vitro is much slower. We identify additional unrecognized sites in spectrin targeted by calpain in vitro and in vivo. Bound CaM induces a second II spectrin cleavage at G1230*S1231. BetaII spectrin is cleaved at four sites. One cleavage only occurs in the absence of CaM at high enzyme-to-substrate ratios near the betaII spectrin COOH-terminus. CaM promotes II spectrin cleavages at Q1440*S1441, S1447*Q1448, and L1482*A1483. These sites are also cleaved in the absence of CaM in recombinant II spectrin fusion peptides, indicating that they are probably shielded in the spectrin heterotetramer and become exposed only after CaM binds alphaII spectrin. Using epitope-specific antibodies prepared to the calpain cleavage sites in both alphaII and betaII spectrin, we find in cultured rat cortical neurons that brief glutamate exposure (a physiologic ligand) rapidly stimulates alphaII spectrin cleavage only at Y1176*G1177, while II spectrin remains intact. In cultured SH-SY5Y cells that lack an NMDA receptor, glutamate is without effect. Conversely, when stimulated by calcium influx (via maitotoxin), there is rapid and sequential cleavage of alphaII and then betaII spectrin, coinciding with the onset of nonapoptotic cell death. These results identify (i) novel calpain target sites in both alphaII and betaII spectrin; (ii) trans-regulation of proteolytic susceptibility between the spectrin subunits in vivo; and (iii) the preferential cleavage of alphaII spectrin vs betaII spectrin when responsive cells are stimulated by engagement of the NMDA receptor. We postulate that calpain proteolysis of spectrin can activate two physiologically distinct responses: one that enhances skeletal plasticity without destroying the spectrin-actin skeleton, characterized by preservation of betaII spectrin; or an alternative response closely correlated with nonapoptotic cell death and characterized by proteolysis of betaII spectrin and complete dissolution of the spectrin skeleton.     

Inter-subunit interactions in erythroid and non-erythroid spectrins

Spectrins comprise α- and β-subunits made up predominantly of a series of homologous repeating units of about 106 amino acids; the α- and β-chains form antiparallel dimers by lateral association, and tetramers through head-to-head contacts between the dimers. Here we consider the first of these interactions. (1) We confirm earlier observations, showing that the first two paired repeats (βIR1 with αIR21, and βIR2 with αRI20) at one end of the erythroid spectrin (αIβI) dimer are necessary and sufficient to unite the chains; (2) we resolve a conflict in published reports by showing that the strength of the interaction is considerably increased on adding the adjoining pair of repeats (βIR3-αIR19); (3) in brain (αIIβII) spectrin the first two pairs of repeats are similarly essential and sufficient for heterodimer formation; (4) this interaction is ~60-fold stronger than that in the erythroid counterpart, but no enhancement can be detected on addition of three further pairs of repeats; (5) formation of a tight αIβI dimer probably depends on structural coupling of the first two repeats in each chain; (6) an analysis of the sequences of the strongly interacting repeats, βIR1, βIIR1, αIR21 and αIIR20 and repeats in α-actinin, which also interact very strongly in forming an antiparallel dimer, affords a possible explanation for the different properties of the two spectrin isoforms in respect of the stability of the inter-chain interactions, and also suggests the evolutionary path by which the erythroid and non-erythroid sequences diverged.     

The ratio of the two light reactions and their coupling in chloroplasts.

The kinetics of the absorbance changes of chlorophyll alphaI (P-700) and plastoquinone induced by xenon flashes of saturating intensity were studied in spinach chloroplasts. 1. The total amount of chlorophyll alphaI is compared with that amount being reduced via the rate-limiting step between the light reactions. This is based on the amplitudes of the absorbance changes of chlorophyll alphaI after chemical reduction and after a group of flashes following far-red preillumination. It is concluded that only 75% of chlorophyll alphaI is coupled to chlorophyll alphaII via linear electron transport and that the remaining 25% is functionally isolated. 2. A ratio of 0.85 for coupled chlorophyll alphaI to chlorophyll alphaII is estimated from the time course of the absorbance changes of plastoquinone and chlorophyll alphaI in two independent ways. 3. The oxygen yield per flash is used to calculate the difference extinction coefficient of chlorophyll alphaI at the maximum of the red absorbance band in spinach chloroplasts: delta xi703 = (6.7 +/- 0.7)-10(4) M-1-cm-1. The assumption of a quantitative electron transfer from water via plastoquinone to coupled chlorophyll alphaI is supported by the same extinction coefficient reported by Hiyama and Ke for Photosystem I particles. The location and function of the different chlorophylls alphaI is discussed in detail.     

Conformational stabilities of the structural repeats of erythroid spectrin and their functional implications

The two polypeptide chains of the erythroid spectrin heterodimer contain between them 36 structural repeating modules, which can function as independently folding units. We have expressed all 36 and determined their thermal stabilities. These vary widely, with unfolding transition mid-points (T(m)) ranging from 21 to 72 degrees C. Eight of the isolated repeats are largely unfolded at physiological temperature. Constructs comprising two or more adjacent repeats show inter-repeat coupling with coupling free energies of several kcal mol(-1). Constructs comprising five successive repeats from the beta-chain displayed cooperativity and strong temperature dependence in forced unfolding by atomic force microscopy. Analysis of aligned sequences and molecular modeling suggests that high stability is conferred by large hydrophobic side chains at position e of the heptad hydrophobic repeats in the first helix of the three-helix bundle that makes up each repeat. This inference was borne out by the properties of mutants in which the critical residues have been replaced. The marginal stability of the tertiary structure at several points in the spectrin chains is moderated by energetic coupling with adjoining structural elements but may be expected to permit adaptation of the membrane to the large distortions that the red cell experiences in the circulation.     

Binding of brain spectrin to the 70-kDa neurofilament subunit protein

Brain spectrin, or fodrin, a major protein of the subaxolemmal cytoskeleton, associates specifically in in vitro assays with the 70-kDa neurofilament subunit (NF-L) and with glial filaments from pig spinal cord. As an initial approach to the identification of the fodrin-binding proteins, a crude preparation of neurofilaments was resolved by electrophoresis on SDS/polyacrylamide gels and then transferred to nitrocellulose paper, which was 'blotted' with 125I-fodrin. A significant binding of fodrin was observed on polypeptides of 70 kDa, 52 kDa and 20 kDa. These polypeptides were further purified and identified respectively as the NF-L subunit of neurofilaments, the glial fibrillary acidic protein (GFP) and the myelin basic protein. The binding of fodrin to NF-L was reversible and concentration-dependent. The ability of the pure NF-L and GFP to form filaments was used to quantify their association with fodrin. a) The binding of fodrin to reassembled NF-L was saturable with a stoichiometry of 1 mol fodrin bound/50 +/- 10 mol NF-L and an apparent dissociation constant Kd = 4.3 x 10(-7) M. b) The binding involved the N-terminal domain of the polypeptide chain derived from the [2-(2-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine] cleavage of NF-L. c) Binding occurred optimally at physiological pH (6.8-7.2) and salt concentrations (50 mM). d) Interestingly, calmodulin, a Ca2+-binding protein, which has been shown to bind to fodrin, was found to reinforce the binding of fodrin to the NF-L, at Ca2+ physiological concentrations. The binding of fodrin to pure neurofilaments was not affected by the presence of the 200-kDa (NF-H) and the 160-kDa (NF-M) subunits. The apparent dissociation constant for the binding of fodrin to NF-L in the pure NF was 1.0 x 10(-6) M with 1 mol fodrin bound/80 +/- 10 mol NF-L. Moreover, the binding of fodrin to GFP, demonstrated in blot assays, was confirmed by cosedimentation experiments. The apparent dissociation constant Kd for the fodrin binding was 2.8 x 10(-7) M and the maximum binding was 1 mol fodrin/55 +/- 10 mol GFP.     

Binding sites of calmodulin and actin on the brain spectrin, calspectin

We used rotary-shadowing electron microscopy to map the calmodulin-and actin-binding sites on the brain spectrin, calspectin (or fodrin). Calspectin dimers appeared as rods 110 nm long and joined in a head-to- head manner to form tetramers 220 nm long. We determined calmodulin- binding sites by a ferritin-labeling method combined with biotin-avidin complex formation. Ferritin particles were found to attach to the head parts of calspectin dimers at a position 10-20 nm from the top of the head. The number of the calmodulin-binding sites seemed to be only one for each dimer and two for each tetramer. In contrast, the actin- binding sites were localized at the tail ends of the calspectin molecules. The tetramers attached to muscle F-actin with their tail ends and often cross-linked adjacent filaments. The results are discussed in view of the analogy to the erythrocyte spectrin.     

Molecular epitopes of the ankyrin-spectrin interaction

Isoforms of ankyrin and its binding partner spectrin are responsible for a number of interactions in a variety of human cells. Conflicting evidence, however, had identified two different, non-overlapping human erythroid ankyrin subdomains, Zu5 and 272, as the minimum binding region for beta-spectrin. Complementary studies on the ankyrin-binding domain of spectrin have been somewhat more conclusive yet have not presented binding in terms of well-phased, integral numbers of spectrin repeats. Thus, the objective of this study was to clearly define and characterize the minimal ankyrin-spectrin binding epitopes. Circular dichroism (CD) wavelength spectra of the aforementioned ankyrin subdomains show that these fragments are 30-60% unstructured. In contrast, human erythroid beta-spectrin repeats 13, 14, 15, and 16 (prepared in all combinations of two adjacent repeats) demonstrated proper folding and stability as determined by CD and tryptophan wavelength and heat denaturation scans. Native polyacrylamide gel electrophoresis (PAGE) gel shifts as well as affinity pull-down assays implicated Zu5 and beta-spectrin repeats 14-15 as the minimum binding epitopes. These results were confirmed by analytical ultracentrifugation to sedimentation equilibrium by which a 1:1 complex was obtained if and only if Zu5 was mixed with beta-spectrin constructs containing repeats 14 and 15 in tandem. Surface plasmon resonance yielded a K D of 15.2 nM for binding of beta-spectrin fragments to the ankyrin subdomain Zu5, accounting for all of the binding observed between the intact molecules. Collectively, these results show the 14th and 15th beta-spectrin repeats comprise the minimal, phased region of beta-spectrin, which binds ankyrin at the Zu5 subdomain with high affinity.     

Interaction of calmodulin with the red cell and its membrane skeleton and with spectrin

The binding of calmodulin to red cell membrane cytoskeletons and to purified spectrin from red cells and bovine brain spectrin (fodrin) has been examined. Under physiological solvent conditions binding can be measured by ultracentrifugal pelleting assays. The membrane cytoskeletons contained a single class of binding sites, with a concentration similar to that of spectrin dimers and an association constant of 1.5 X 10(5) M-1. Binding is calcium dependent and is suppressed by the calmodulin inhibitor trifluoperazine. The binding showed a marked dependence on ionic strength, with a maximum at 0.05 M, and a steep dependence on pH, with a maximum at pH 6.5. It was unaffected by 5 mM magnesium. An azidocalmodulin derivative, under the conditions of our experiments, did not label the spectrin-containing complex, although it could be used to demonstrate binding to fodrin. Binding of calmodulin to spectrin tetramers and fodrin in solution could be demonstrated by a pelleting assay after addition of F-actin. Calculations (which are necessarily rough) suggest that at the free calcium concentration prevailing in a normal red cell about 1 in 20 of the calmodulin binding sites in spectrin will be occupied; this proportion will rise rapidly with increasing intracellular calcium. To determine whether inhibition of calmodulin binding to red cell proteins disturbs the control of cell shape, as has been suggested, calcium ions were removed from the cell by addition of an ionophore and of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid to the external medium. This did not affect the discoid shape. Trifluoperazine still induced stomatocytosis, exactly as in untreated cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Co-expression of spectrin and fodrin in friend erythroleukemic cells treated with DMSO

Friend erythroleukemic cells can be used as a model of erythroid cell differentiation with the synthesis of the erythrocyte-specific products hemoglobin and spectrin stimulated by agents such as DMSO. In the present study we investigated the expression of both erythroid spectrin and non-erythroid fodrin in uninduced and DMSO-treated Friend cells. We report that both spectrin and fodrin co-exist at low levels in uninduced Friend cells and both are induced by treatment with DMSO. After longer times both spectrin and fodrin appear to undergo rearrangements into submembranous ‘patches’ and ‘caps’. Although both molecules co-localize in most of these cells, they can be independently immunoprecipitated, suggesting that significant amounts of hybrid molecules are not formed.     

Tyrosine phosphatase activity of lymphoma CD45 (GP180) is regulated by a direct interaction with the cytoskeleton.

GP180 is one of the major transmembrane glycoproteins in mouse T-lymphoma cells. This molecule is an isoform of CD45 and is known to contain an intrinsic protein tyrosine phosphatase (PTPase) activity. Using several complementary biochemical techniques, we have found that fodrin (a spectrin-like protein) is preferentially co-isolated with CD45 (GP180), suggesting that a complex between CD45 (GP180) and the cytoskeleton exists in mouse T-lymphoma cells. Furthermore, we have determined that this CD45 (GP180)-fodrin complex is dissociated by high salt treatment. Using in vitro binding assays, we have shown that CD45 (GP180) binds directly and specifically to fodrin (Kd approximately 1.1 nM) or spectrin (Kd approximately 3.2 nM) in a saturable manner. Additional analyses indicate that a 48-kDa phosphopeptide of CD45 (GP180) contains the fodrin/spectrin-binding domain. Most importantly, the direct binding of fodrin/spectrin to CD45 (GP180) is found to significantly stimulate the PTPase activity of CD45. Enzyme kinetic analysis indicates that fodrin and spectrin increase the Vmax of CD45 (GP180)-mediated dephosphorylation by 7.5 and 3.2-fold, respectively, without significantly changing the Km value. These results strongly suggest that the cytoskeletal proteins, fodrin and spectrin, play an important role in the regulation of the CD45 (GP180) PTPase activity during lymphocyte activation.     

Characterization of calcium binding to brain spectrin.

Brain spectrin alpha and beta chains bind 45Ca2+, as shown by the calcium overlay method. Flow dialysis measurements revealed eight high affinity binding sites/tetramer that comprise two binding components (determined by nonlinear regression analysis). The first component has one or two sites (kd = 2-30 x 10(-8) M), depending on the ionic strength of the binding buffer, with the remaining high affinity sites in the second component (kd = 1-3 x 10(-6) M). In addition, there is a variable, low affinity binding component (n = 100-400, kd = 1-2 x 10(-4) M). Magnesium inhibits calcium binding to the low affinity sites with a K1 = 1.21 mM. Proteolytic fragments from trypsin or chymotrypsin digests of brain spectrin bind 45Ca2+ if they include alpha domain IV, alpha domain III, or the amino-terminal half of the beta chain (but more than 25 kDa from the amino-terminal). These data suggest that calcium ions bind with high affinity to the putative EF-hands in alpha domain IV and to one site in the amino-terminal half of the beta chain that is associated with alpha domain IV in the native dimer. The localization is consistent with a direct calcium modulation of the spectrin-actin-protein 4.1 interaction. In addition, there appears to be one high affinity site near the hypersensitive region of alpha brain spectrin. All four proposed binding sites occur near probable calmodulin-binding or calcium-dependent protease cleavage sites.