Analysis of the ternary interaction of the red cell membrane skeletal proteins spectrin, actin, and 4.1

Spectrin dimers interact weakly with F-actin under physiological solvent conditions (with an association constant of about 5 X 10(3) M-1 at 20 degrees C). In the presence of the membrane skeletal constituent, protein 4.1, strong binding is observed; an analysis of the profiles for formation of a ternary complex leads to an association constant of about 1 X 10(12) M-2. This association becomes weaker at low ionic strength, whereas the opposite applies to the spectrin-actin interaction. The stability of the ternary complex is maximal at physiological ionic strength and somewhat above. The effect of temperature in the range 0-20 degrees C on the formation of the ternary complex is small, whereas the spectrin-actin interaction almost vanishes at low temperature. There is no detectable calcium sensitivity in either the binary or the ternary system within the limits of precision of our assay. The ternary complex resembles the natural system in the membrane in that the actin is resistant to dissociation and unavailable in the deoxyribonuclease assay; after selective proteolytic destruction of spectrin and 4.1, all the actin becomes available. In the absence of 4.1, spectrin dimers do not measurably protect the actin against dissociation.     

Associations of erythrocyte membrane proteins. Binding of purified bands 2.1 and 4.1 to spectrin

Specific associations of spectrin with Bands 2.1 and 4.1 have been examined by measuring the binding of purified 125I-Band 2.1 and 125I-Band 4.1 to [32P]spectrin in solution. Binding of Bands 2.1 and 4.1 to spectrin was measured as 125I radioactivity precipitated by an anti-spectrin.Staphylococcus aureus complex. The association between spectrin and Band 2.1 is characterized by relatively high affinity (Kd congruent to 10(-7) M at pH 7.6) and saturation of available binding sites at a molar ratio of 1:1 (Band 2.1/spectrin heterodimer). Band 4.1 binding to spectrin is characterized by a similar affinity (Kd congruent to 10(-7) M at pH 7.6) with saturation of available sites occurring at a stoichiometric ration of 2:1 (Band 4.1/spectrin heterodimer). Scatchard plots of Band 4.1 binding to spectrin are curvilinear and consistent with a positively cooperative interation. Bands 2.1 and 4.1 bind to different sites on the spectrin molecule: unlabeled Band 4.1 does not competitively displace 125 I-Band 2.1 from spectrin in solution, and low angle rotary-shadowed platinum-carbon replicas of these polypeptides reveal two discrete binding sites.     

Modulation of red cell glycolysis: interactions between vertebrate hemoglobins and cytoplasmic domains of band 3 red cell membrane proteins

Several vital functions/physical characteristics of erythrocytes (including glycolysis, the pentose phosphate pathway, ion fluxes, and cellular deformability) display dependence on the state of hemoglobin oxygenation. The molecular mechanism proposed involves an interaction between deoxyhemoglobin and the cytoplasmic domain of the anion-exchange protein, band 3 (cdB3). Given that band 3 also binds to membrane proteins 4.1 and 4.2, several kinases, hemichromes, and integral membrane proteins, and at least three glycolytic enzymes, it has been suggested that the cdB3-deoxyhemoglobin interaction might modulate the pathways mediated by these associated proteins in an O(2)-dependent manner. We have investigated this mechanism by synthesizing 10-mer peptides corresponding to the NH(2)-terminal fragments of various vertebrate cdB3s, determining their effects on the oxygenation reactions of hemoglobins from the same and different species and examining binding of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase to the erythrocytic membrane of mouse erythrocytes. The cdB3 interaction is strongly dependent on pH and the number of negative and positive charges of the peptide and at the effector binding site, respectively. It lowers the O(2) association equilibrium constant of the deoxygenated (Tense) state of the hemoglobin and is inhibited by magnesium ions, which neutralize cdB3's charge and by 2,3-diphosphoglycerate, which competes for the cdB3-binding site. The interaction is stronger in humans (whose erythrocytes derive energy predominantly from glycolysis and exhibit higher buffering capacity) than in birds and ectothermic vertebrates (whose erythrocytes metabolize aerobically and are poorly buffered) and is insignificant in fish, suggesting that its role in the regulation of red cell glycolysis increased with phylogenetic development in vertebrates.     

Role of spectrin in cross bonding of the red cell membrane

Membrane cross bonding--an adhesion between opposing areas of the cytoplasmic face of the red cell membrane--was achieved by treating red cells with heat, diamide, N-ethymaleimide, urea, or by ATP depletion in conjunction with cell shrinking. Membrane cross bonding could be recognized by the shape of the cells upon swelling. Quantitated by the percentage of cross-bonded red cells the effectivity of the treatments decreased in the order given above. Cross bonding was hardly reversible by reducing the diamide-induced S-S bonds with dithioerythritol. The effect of heat and urea treatment as well as ATP depletion was partly reversible. Transmission electron micrographs of the cross-bonded region showed basically parallel membranes. The distance between the respective phospholipid bilayers varied between 40 and 120 nm from cell to cell. Hb-free ghosts prepared from diamide-treated red cells could also be cross bonded. The following conclusions are drawn: spectrin provides the molecular cross link in membrane cross bonding. Aggregation and enrichment of spectrin in the cross-bonded region are probably involved in membrane cross bonding.     

The 4.1-like proteins of the bovine lens: spectrin-binding proteins closely related in structure to red blood cell protein 4.1

The superficial cortical fiber cells of the bovine lens contain membrane-associated proteins of 150,000, 80,000, and 78,000 D that cross-react with antisera prepared against red blood cell (RBC) protein 4.1 (Aster, J. C., G. J. Brewer, S. M. Hanash, and H. Maisel, 1984, Biochem. J., 224:609-616). To further study their relationship to protein 4.1, these proteins were immunoprecipitated from detergent extracts of crude lens membranes with purified polyclonal and monoclonal anti-4.1 antibodies and resolved by SDS PAGE. The electrophoretic mobilities of the lens proteins of 80,000 and 78,000 D were found to be identical to bovine RBC protein 4.1a and protein 4.1b, respectively. One- and two-dimensional peptide mapping revealed that a high degree of structural homology exists among all three of the lens 4.1-like proteins and RBC protein 4.1a and protein 4.1b. Despite the large difference in apparent molecular mass, the 150,000-D lens protein showed only minor peptide map differences. A nitrocellulose filter overlay assay showed that all three of the lens 4.1-like proteins bind to RBC and lens spectrins. We conclude that the bovine lens contains proteins of 80,000 and 78,000 D that are highly similar to protein 4.1 in structure and functional capacity. Additionally, the lens also contains a 4.1 isomorph of 150 kD. Analogous to RBC protein 4.1, these proteins may function in the lens by promoting association of spectrin with actin and by playing a role in the coupling of lens cytoskeleton to plasma membrane.     

Interactions among red cell membrane proteins

Interactions between human red band 2.1 with spectrin and depleted inside-out vesicles were studied by fluorescence resonance energy transfer and batch microcalorimetry. The band 2.1-spectrin binding isotherm is consistent with a one to one mole ratio. The association constant of 1.4 X 10(8) M-1 corresponds to the association free energy of -11.1 kcal/mol. Under our experimental conditions, the enthalpy of interaction of band 2.1-spectrin was found to be -10.8 kcal/mol and is independent of the protein mole ratio. The calculated entropic factor (-T delta S = 0.3 kcal/mol) strongly suggests a predominantly enthalpic character of the reaction. In addition, we investigated the role of band 2.1 on the binding of band 4.1 to spectrin [Podgorski, A., & Elbaum, D. (1985) Biochemistry 24, 7871-7876] and concluded that only small, if any, alterations of binding of band 4.1 to spectrin have taken place in the presence or absence of band 2.1. This suggests thermodynamic independence of the binding sites. Although the attachment of the cytoskeletal network to the membrane takes place through, at least, two different interactions, band 2.1-band 3 and 4.1-glycophorin, the relative enthalpy values suggest that band 2.1 contributes significantly more than band 4.1 to the energy of the interaction. In addition, we observed that polymerization of actin is modulated by the cytoskeletons as judged by their effect on the rate of actin polymerization.     

Shear-response of the spectrin dimer-tetramer equilibrium in the red blood cell membrane

The red cell membrane derives its elasticity and resistance to mechanical stresses from the membrane skeleton, a network composed of spectrin tetramers. These are formed by the head-to-head association of pairs of heterodimers attached at their ends to junctional complexes of several proteins. Here we examine the dynamics of the spectrin dimer-dimer association in the intact membrane. We show that univalent fragments of spectrin, containing the dimer self-association site, will bind to spectrin on the membrane and thereby disrupt the continuity of the protein network. This results in impairment of the mechanical stability of the membrane. When, moreover, the cells are subjected to a continuous low level of shear, even at room temperature, the incorporation of the fragments and the consequent destabilization of the membrane are greatly accentuated. It follows that a modest shearing force, well below that experienced by the red cell in the circulation, is sufficient to sever dimer-dimer links in the network. Our results imply 1) that the membrane accommodates the enormous distortions imposed on it during the passage of the cell through the microvasculature by means of local dissociation of spectrin tetramers to dimers, 2) that the network in situ is in a dynamic state and undergoes a "breathing" action of tetramer dissociation and re-formation.     

The postsynaptic spectrin/4.1 membrane protein "accumulation machine"

An important aspect of the function of the membrane-associated cytoskeleton has been suggested to be to trap and retain selected transmembrane proteins at points on the cell surface specified by cell adhesion molecules. In the process, cell adhesion molecules are cross-linked to each other, and so junctional complexes are strengthened. In this short review, we will discuss recent advances in understanding the role of this "accumulation machine" in postsynaptic structures. Function in the brain depends on correct ordering of synaptic intercellular junctions, and in particular the recruitment of receptors and other apparatus of the signalling system to postsynaptic membranes. Spectrin has long been known to be a component of postsynaptic densities, and recent advances in molecular cloning indicate that beta spectrins at PSDs are all "long" C-terminal isoforms characterised by pleckstrin homology domains. Isoforms of protein 4.1 are also present at synapses. All four 4.1 proteins are represented in PSD preparations, but it is 4.1R that is most enriched in PSDs. 4.1R binds to several proteins enriched in PSDs, including the characteristic PSD intermediate filament, alpha-internexin. Both 4.1 and spectrin interact with ionotropic glutamate receptors (AMPA and NMDA receptors, respectively): 4.1 stabilises AMPA receptors on the cell surface. By linking these receptors to the cytoskeletal and cell adhesion molecules that specify glutamatergic synapses, the membrane protein accumulation machine is suggested to direct the formation of postsynaptic signalling complexes.     

Modulation of red cell band 4.1 function by cAMP-dependent kinase and protein kinase C phosphorylation

Human erythrocyte protein 4.1 is phosphorylated in vivo by several protein kinases including protein kinase C and cAMP-dependent kinase. We have used cAMP-dependent kinase purified from red cells and protein kinase C purified from brain to test the effects of phosphorylation on band 4.1 function. In solution, each kinase catalyzed the incorporation of 1-4 mol of PO4/mol of band 4.1. Phosphorylation of band 4.1 by each kinase resulted in a significant (50-80%) reduction in the ability of band 4.1 to promote spectrin binding to F-actin. Direct measurement of spectrin-band 4.1 binding showed that phosphorylation by each kinase also caused dramatic reduction in this association. Phosphorylation of band 4.1 by each kinase for increasing time periods enabled us to demonstrate an approximately linear inverse relationship between PO4 incorporation into band 4.1 and spectrin binding. These results show that phosphorylation of band 4.1 by cAMP-dependent kinase and protein kinase C may be central to the regulation of red cell cytoskeletal organization and membrane mechanical properties.     

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)     

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.     

Water-soluble proteins of the human red cell membrane

Summary Procedures were developed for preparation of red cell membranes almost free of hemoglobin but with minimal loss of membrane proteins. Two water-soluble protein fractions are described, each constituting about 25% of the ghost protein. The first is ionically bonded and can be solubilized in water rapidly at pH 7.0 and more slowly at higher ionic strength solutions, with a minimal rate at 20mm. This fraction contains four major components with molecular weights ranging from 30,000 to 48,000. The second fraction can only be solubilized at an appreciable rate if Ca⁺⁺ is absent and at higher pH (9.0). It is predominantly a single molecular weight component (150,000). It tends to aggregate at higher ionic strength and in the presence of Ca⁺⁺. Evidence is presented suggesting that the water-soluble proteins are present at the inner face of the membrane. The lipids remain in a water-insoluble residue that contains four major protein components ranging in molecular weight from 30,000 to 100,000. The latter is the predominant component. Only the residue contains the Na⁺–K⁺-activated ATPase, the cholinesterase, antigenic activity and most of the sialic acid and carbohydrate. The first water-soluble fraction contains a Mg⁺⁺-activated ATPase. The extraction of the water-soluble proteins is accompanied by anatomical changes resulting finally in the formation of small membranous vesicles.     

Interaction between red cell membrane band 3 and cytosolic carbonic anhydrase

We have previously proposed that a membrane transport complex, centered on the human red cell anion transport protein, band 3, links the transport of anions, cations and glucose. Since band 3 is specialized for HCO ₃ ^– /Cl^– exchange, we thought there might also be a linkage with carbonic anhydrase (CA) which hydrates CO₂ to HCO ₃ ^– . CA is a cytosolic enzyme which is not present in the red cell membrane. The rate of reaction of CA with the fluorescent inhibitor, dansylsulfonamide (DNSA) can be measured by stopped-flow spectrofluorimetry and used to characterize the normal CA configuration. If a perturbation applied to a membrane protein alters DNSA/CA binding kinetics, we conclude that the perturbation has changed the CA configuration by either direct or allosteric means. Our experiments show that covalent reaction of the specific stilbene anion exchange inhibitor, DIDS, with the red cell membrane, significantly alters DNSA/CA binding kinetics. Another specific anion exchange inhibitor, benzene sulfonate (BSate), which has been shown to bind to the DIDS site causes a larger change in DNSA/CA binding kinetics; DIDS reverses the BSate effect. These experiments show that there is a linkage between band 3 and CA, consistent with CA interaction with the cytosolic pole of band 3.This work was supported in part by a grant-in-aid from the American Heart Association, by the Squibb Institute for Medical Research and by The Council for Tobacco Research.We should like to express our thanks to Dr. I.M. Wiener for kindly supplying us with the impermeable sulfonamide, ZBI, which we used in preliminary experiments and to Dr. T.H. Maren for analysis of a sample of BCA II.     

Absence of subunits in high-molecular weight proteins (spectrin) of red cell membranes

It is shown that the high-molecular weight proteins (spectrin), which make up a large part of the total protein of the red cell membrane, are single polypeptide chains. They do not generate smaller subunits on treatment with dilute acid: it is shown that recent observations of the appearance of electrophoretic components of low molecular weight, following incubation in acid solution, and interpreted in terms of a subunit structure for the spectrin chains, can be attributed to degradation by endogenous proteases. It is shown that the experimental conditions used favour such degradation.     

The band 3 protein of the human red cell membrane: A review

Band 3 is the predominant polypetide and the purported mediator of anion transport in the human erythrocyte membrane. Against a background of minor and apparently unrelated polypeptides of similar electrophoretic mobility, and despite apparent heterogeneity in its glycosylation, the bulk of band 3 exhibits uniform and characteristic behavior. This integral glycoprotein appears to exist as a noncovalent dimer of two ～ 93,000-dalton chains which span the membrane asymmetrically. The protein is hydrophobic in its composition and in its behaviour in aqueous solution and is best solubilized and purified in detergent. It can be cleaved while membrane-bound into large, topographically defined segments. An integral, outer-surface, 38,000-dalton fragment bears most of the band 3 carbohydrate. A 17,000-dalton, hydrophobic glycopeptide fragment spans the membrane. A ～ 40,000-dalton hydrophilic segment represents the cytoplasmic domain. In vitro, glyceraldehyde 3-P dehydrogenase and aldolase bind reversibly, in a metabolite-sensitive fashion, to this cytoplasmic segment. The cytoplasmic domain also bears the amino terminus of this polypetide, in contrast to other integral membrane proteins. Recent electron microscopic analysis suggests that the poles of the band 3 molecule can be seen by freezeetching at the two original membrane surfaces, while freeze-fracture reveals the transmembrane disposition of band 3 dimer particles. There is strong evidence that band 3 mediates 1:1 anion exchange across the membrane through a conformational cycle while remaining fixed and asymmetrical. Its cytoplasmic pole can be variously perturbed and even excised without a significant alteration of transport function. However, digestion of the outer-surface region leads to inhibition of transport, so that both this segment and the membrane-spanning piece (which is slectively labeled by covalent inhibitors of transport) may be presumed to be involved in transport. Genetic polymorphism has been observed in the structure and immunogenicity of the band 3 polypeptide but this feature has not been related to variation in anion transport or other band 3 activities.     

The interaction of deoxyhemoglobin with the red cell membrane

The interaction of deoxyhemoglobin with the red cell membrane is characterized by comparing the affinity of deoxyhemoglobin for the membrane with that of oxyhemoglobin. The two techniques used, namely light scattering induced changes and quenching of the fluorescence intensity of a membrane embedded probe, demonstrate that deoxyhemoglobin exhibits a much lower affinity for the membrane than that of oxyhemoglobin. The binding constant of 2×10 M^?1 calculated for deoxyhemoglobin at 5 mM phosphate buffer and pH=6.0 is two orders of magnitude lower than the one calculated for oxyhemoglobin. It is estimated that under physiological conditions the only species capable of interacting with the membrane is the oxyhemoglobin.     

Ubiquitination of spectrin regulates the erythrocyte spectrin-protein-4.1-actin ternary complex dissociation: implications for the sickle cell membrane skeleton.

It has been demonstrated by our laboratory that the irreversibly sickled cell (ISC) spectrin-4.1-actin complex dissociates slowly as compared to ternary complexes formed out of control (AA) and reversibly sickle cell (RSCs) core skeletons. These studies indicated that the molecular basis for the inability of irreversibly sickled cells (ISCs) to change shape is a skeleton that disassembles, and therefore reassembles, very slowly. The present study is based on the following observations: a) alpha-spectrin repeats 20 and 21 contain ubiquitination sites, and b) The spectrin repeats beta-1 and beta-2 are in direct contact with spectrin repeats alpha-20 and alpha-21 during spectrin heterodimer formation, and contain the protein 4.1 binding domain. We demonstrate here that alpha-spectrin ubiquitination at repeats 20 and 21 increases the dissociation of the spectrin-protein-4.1-actin ternary complex thereby regulating protein 4.1's ability to stimulate the spectrin-actin interaction. Performing in vitro ternary complex dissociation assays with AA control and sickle cell SS spectrin (isolated from high-density sickle cells), we further demonstrate that reduced ubiquitination of alpha-spectrin is, in part, responsible for the locked membrane skeleton in sickle cell disease.     

Calspectin (fodrin or nonerythroid spectrin)-actin interaction: a possible involvement of 4.1-related protein

The calspectin/actin complex extracted from the bovine brain membrane crosslinks F-actin, resulting in the increasing viscosity of F-actin determined by low-shear viscometry. We demonstrated the presence of a protein factor in this complex, which regulated the calspectin-F-actin interaction in a Ca2+- and calmodulin-dependent manner. Erythrocyte protein 4.1, but not synapsin I, mimics the function of this brain factor using a reconstitution system including purified calspectin, calmodulin and F-actin. In the brain complex, the Mr 120,000 and the Mr 80,000/77,000 polypeptides were detected to crossreact with anti-protein 4.1 antibody.