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.     

Brain adducin: a protein kinase C substrate that may mediate site-directed assembly at the spectrin-actin junction

Erythrocyte adducin is a membrane skeletal protein that binds to calmodulin, is a major substrate for protein kinase C, and associates preferentially with spectrin-actin complexes. Erythrocyte adducin also promotes association of spectrin with actin, and this activity is inhibited by calmodulin. This study describes the isolation and characterization of a brain peripheral membrane protein closely related to erythrocyte adducin. Brain and erythrocyte adducin have at least 50% antigenic sites in common, each contains a protease-resistant core of Mr = 48,000-48,500, and both proteins are comprised of two partially homologous polypeptides of Mr = 103,000 and 97,000 (erythrocytes) and Mr = 104,000 and 107,000-110,000 (brain). Brain and erythrocyte adducin associate preferentially with spectrin-actin complexes as compared to spectrin or actin alone, and both proteins also promote binding of spectrin to actin. Brain adducin binds calmodulin in a calcium-dependent manner, although the Kd of 1.3 microM is weaker by 5-6-fold than the Kd of erythrocyte adducin for calmodulin. Brain adducin is a substrate for protein kinase C in vitro and can accept up to 2 mol of phosphate/mol of protein. Adducin provides a potential mechanism in cells for mediating site-directed assembly of additional spectrin molecules and possibly other proteins at the spectrin-actin junction. Brain tissue contains 12 pmol of adducin/mg of membrane protein, which is the most of any tissue examined other than erythrocytes, which have 50 pmol/mg. The presence of high amounts of adducin in brain suggests some role for this protein in specialized activities of nerve cells.     

A spectrin-dependent ATPase of the human erythrocyte membrane

Removal of spectrin from erythrocyte membranes results in the simultaneous loss of a calcium-stimulated, magnesium-dependent ATPase with an apparent KD for Ca2+ of 1 microM. This ATPase activity with high Ca2+ affinity is specifically reconstituted by addition of purified spectrin to spectrin-depleted membranes, and the reconstituted activity is directly proportional to the amount of spectrin that is reassociated with the membranes. Spectrin binding and activation of the high Ca2+ affinity Mg2+-ATPase are proportionally inhibited by thermal denaturation, trypsin digestion, or treatment of the membranes with thiol-reactive reagents. Binding of calmodulin to the Ca2+ pump ATPase requires that calmodulin contains bound ca2+. By contrast, spectrin binding to the erythrocyte membrane is Ca2+-independent. Direct assay of calmodulin is purified spectrin and absence of chlorpromazine inhibition of reconstitution demonstrate that activation of the high Ca2+ affinity ATPase resulting from spectrin binding is not a result of contamination of spectrin by calmodulin. Additional evidence that the spectrin-activated ATPase is an entity separate and distinct from the Ca2+ pump is provided by other characteristics of the activation phenomenon. It is suggested that spectrin constitutes part of an ATPase which may function as a component of the "cytoskeleton" controlling erythrocyte shape and membrane flexibility.     

Calmodulin-binding protein of erythrocyte cytoskeleton

Previously we have shown that purified spectrin binds calmodulin in the presence of Ca²⁺ with a Kd value of 3 μM (Sobue, K. et al. (1980) Biochemistry International 1, 561–566). We now provide evidence that the calmodulin-binding activity found in the human erythrocyte cytoskeleton is indeed due to spectrin and no other binding proteins are involved, i.e. the binding activity was purified from the erythrocyte cytoskeleton quantitatively and the purified peak contained spectrin as the only protein constituent. Moreover, Kd value (2.8 μM) and the maximum binding capacity (160,000 – 200,000 calmodulin per cell) obtained from the kinetic analysis of the binding activity in the crude cytoskeleton agreed with the corresponding values reported for purified spectrin. Since the concentration of calmodulin in the erythrocyte cell, which was 2.5 μM or 1.6 × 10⁵ molecules per cell, is close to both the Kd value and the number of the binding sites in the cell, respectively, free calmodulin in the erythrocyte cell may be in a dynamic equilibrium with the spectrin-bound form in vivo depending upon the intracellular concentration of Ca²⁺.     

Influence of calmodulin on the human red cell membrane skeleton

The calcium receptor calmodulin interacts with components of the human red cell membrane skeleton as well as with the membrane. Under physiological salt conditions, calmodulin has a calcium-dependent affinity for spectrin, one of the major components of the membrane skeleton. It is apparent from our results that calmodulin inhibits the ability of erythrocyte spectrin (when preincubated with filamentous actin) to create nucleation centers and thereby to seed actin polymerization. The gelation of filamentous actin induced by spectrin tetramers is also inhibited by calmodulin. The inhibition is calcium dependent and decreases with increasing pH, similar to the binding of calmodulin to spectrin. Direct binding studies using aqueous two-phase partition indicate that calmodulin interferes with the binding of actin to spectrin. Even in the presence of protein 4.1, which is believed to stabilize the ternary complex, calmodulin has an inhibitory effect. Since calmodulin also inhibits the corresponding activities of brain spectrin (fodrin), it appears likely that calmodulin may modulate the organization of cytoskeletons containing actin and spectrin or spectrin analogues.     

Characterization of calcium binding to spectrins.

Calcium binding to brain and erythrocyte spectrins was studied at physiological ionic strength by a calcium overlay assay and aqueous two-phase partitioning. When the spectrins were immobilized on nylon membranes by slot blotting, the overlay assay showed that even though both spectrins bound 45Ca2+, the brain protein displayed much greater affinity for calcium ions than erythrocyte spectrin did. Since the observed binding was weaker than that displayed by calmodulin under similar conditions, the overlay assay results indicated that the binding must be weaker than 1 microM. The phase partition experiments showed that there are at least two sites for calcium on brain spectrin and that calcium binding to one of these sites is reduced significantly by magnesium ions. From the partition isotherm, the dissociation constants were estimated as 50 microM for the Mg(2+)-independent site and 150 microM for the Mg(2+)-dependent site. The phase partition results also showed that erythrocyte spectrin bound calcium ions at least 1 order of magnitude weaker. By examining calcium binding to slot-blotted synthetic peptides, we identified two binding sites in brain spectrin. One mapped to the second putative calcium binding site (EF-hand) in alpha-spectrin and the other to the 36 amino acid residue long insert in domain 11. In addition, a tryptic fragment derived from the C-terminal of erythrocyte alpha-spectrin, which contained the two postulated EF-hands, also bound calcium. These findings suggest that the calcium signal system may also involve direct binding of calcium to spectrin beside known calcium modulators such as calmodulin and calpain.(ABSTRACT TRUNCATED AT 250 WORDS)     

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)     

Thermal denaturation of the erythrocyte cytoskeleton alters the morphological changes associated with osmotic swelling

1. 1. The shape changes during osmotic swelling of human erythrocytes in a hypotonic medium at room temperature, at 45°C and at the denaturation temperature (49.5°C) of the cytoskeletal protein, spectrin, have been monitored by video microscopy.

2. 2. At room temperature the great majority of cells (which were discoid prior to injection of hypotonic medium) swelled to a spherical shape through an intermediate ellipsoidal form.

3. 3.At 49.5°C (where cells had cupped shapes prior to injection) the transition to the spherical form often involved a stomatocytic rather than ellipsoidal intermediate shape.

4. 4. The cupped form of the cells prior to injection did not account for the high incidence of cells swelling through a stomatocytic intermediate shape at 49.5°C.

5. 5. A theoretical treatment by Svetina and Zeks (1983) attributes the nature of the osmotic swelling transition shape to the difference in area between the outer and inner faces of the membrane. Our results would be consistent with the theoretical predictions if it is assumed that an increase in the area of the inner face of the membrane follows thermal denaturation of the cytoskeleton of cells in hypotonic medium.

The 240-kDa subunit of human erythrocyte spectrin binds calmodulin at micromolar calcium concentrations

The binding of the isolated alpha-subunit of human erythrocyte spectrin to calmodulin is demonstrated by partitioning in aqueous two-phase systems. The affinity of the alpha-subunit for calmodulin is slightly higher than that of the spectrin dimer, whereas the beta-subunit interacts only very weakly. The binding is in all cases calcium-dependent and is abolished on addition of chlorpromazine. At an ionic strength close to physiological conditions, about 1 microM free calcium is required to induce maximum binding of calmodulin to spectrin dimer.     

Spectrin: a structural mediator between diverse plasma membrane proteins and the cytoplasm

The spectrin skeleton of non-erythroid cells is likely to interact with a variety of integral membrane proteins and participate both in stable linkages as well as dynamic structures capable of rapid disassembly and assembly. The basis for diversity of roles for spectrin includes multiple, functionally distinct isoforms of spectrin, ankyrin and other associated proteins, regulation of protein interactions through phosphorylation and calcium/calmodulin, as well as differential expression of accessory proteins that determine the organization and localization of spectrin in cells. Spectrin is highly conserved from Drosophila to man and is likely to be involved in fundamental aspects of membrane structure requiring long range order and organization. Spectrin is a candidate to interact with many integral membrane proteins in roles basic to metazoan cells which must associate into tissues. Organization of cells into tissues requires loss of cell motility, formation of specialized membrane domains and assembly of cell junctions, which are all activities potentially involving spectrin. Future challenges lie in devising direct experiments to evaluate the functions of spectrin in cells and tissues.     

Mechanism of cytoskeletal regulation (I): functional differences correlate with antigenic dissimilarity in human brain and erythrocyte spectrin

Human erythrocyte and brain spectrin (fodrin, calspectin) have been compared quantitatively with respect to the extent and sites of antigenic and functional similarity. Brain spectrin cross-reacts strongly with approx. 1% of the epitopes in erythrocyte spectrin, but weakly with at least 50%. The distribution of shared determinants is not uniform. Brain spectrin is most deficient in epitopes characteristic of the 80 kDa and 52 kDa domains of the alpha-subunit (alpha-I and alpha-III) and of terminal portions of the 28 kDa and 74 kDa domains of the beta-subunit (beta-I and beta-IV). The functions associated with these domains also differ between the two proteins. Brain spectrin does not undergo extensive polymerization and binds calmodulin at a different site. The unique ability of erythrocyte spectrin to oligomerize beyond the tetramer reflects its role in the membrane skeleton. Non-erythroid spectrins probably function as specific linkers between membrane receptors and the filamentous cytoskeleton. In this sense, they may act as regulated transducers of information flow between the membrane and the cytoplasmic matrix.     

A calmodulin and alpha-subunit binding domain in human erythrocyte spectrin

Human erythrocyte spectrin binds calmodulin weakly under native conditions. This binding is enhanced in the presence of urea. The site responsible for this enhanced binding in urea has now been shown to reside in a specific region of the spectrin beta-subunit. Cleavage of spectrin with trypsin, cyanogen bromide or 2-nitro-5-thiocyanobenzoic acid generates fragments of the molecule which retain the ability to bind calmodulin under denaturing conditions. The origin of these fragments, identified by two-dimensional peptide mapping, is the terminal region of the spectrin beta-IV domain. The smallest peptide active in calmodulin binding is a 10 000 Mr fragment generated by cyanogen bromide cleavage. Only the intact 74 000 Mr fragment generated by trypsin (the complete beta-IV domain) retains the capacity to reassociate with the isolated alpha-subunit of spectrin. The position of a putative calmodulin binding site near a site for subunit-subunit association and protein 4.1 and actin binding suggests a possible role in vivo for calmodulin regulation of the spectrin-actin membrane skeleton or for regulation of subunit-subunit associations. This beta-subunit binding site in erythrocyte spectrin is found in a region near the NH2-terminus at a position analogous to the alpha-subunit calmodulin binding site previously identified in a non-erythroid spectrin by ultrastructural studies.     

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.     

Interaction of plasma lipoproteins with erythrocytes. II. Modulation of membrane-associated enzymes.

When incubated with intact erythrocytes, low density lipoproteins (LDL) decrease the phosphate content of erythrocyte spectrin allowing the cells to undergo morphological transformation. The phosphate content of spectrin depends on the balance between the activity of membrane-associated cyclic AMP-independent protein kinases and phosphoprotein phosphates. LDL do not influence the activity of membrane-associated cyclic AMP-independent protein kinases; these lipoproteins activate by 2-fold and greater membrane-associated phosphatases as determined by hydrolysis of p-nitrophenyl phosphate and by phosphate hydrolysis of phosphorylated erythrocyte membrane proteins. We conclude that LDL interact at the exterior surface of the erythrocyte to stimulate dephosphorylation of spectrin. The significance of this conclusion is augmented by the fact that spectrin, the target for LDL-induced dephosphorylation, specifies cell morphology and modulates the distribution of cell-surface receptors. LDL also render erythrocyte acetylcholinesterase less susceptible to inhition by F-. Lipoproteins in the high density class (HDL) do not stimulate dephosphorylation of spectrin, and they are consequently unable to alter erythrocyte morphology. HDL do prevent the LDL-induced activation of membrane phosphatase. The inhibitory capacity of HDL is observed over the range of LDL:HDL (w/w) which exists in the plasma of normolipemic humans.     

Human erythrocyte calmodulin. Further chemical characterization and the site of its interaction with the membrane.

Human erythrocyte and bovine brain calmodulins were indistinguishable by tryptic peptide mapping, indicating that the primary sequence of the two proteins is either very similar or identical. Calcium binding determinations of human erythrocyte calmodulin, by equilibrium dialysis and fluorescence titration, were in close agreement with previous studies on other calmodulins. The calcium-activated adenosine triphosphatase which is stimulated by calmodulin was shown to be firmly associated with smooth erythrocyte plasma membranes devoid of spectrin and actin. Kinetic titration demonstrated that there are 4500 calmodulin binding sites per erythrocyte and that the turnover number of this calcium-activated adenosine triphosphatase is 3000 mumol of Pi . (mumol of site)-1 . min-1 which is similar to the turnover numbers of other transport adenosine triphosphatases. Furthermore, calmodulin stimulates calcium-activated adenosine triphosphatase by a simple enzyme-ligand association.     

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.     

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.     

Erythrocyte adducin: a calmodulin-regulated actin-bundling protein that stimulates spectrin-actin binding

Adducin is an erythrocyte membrane skeletal phosphoprotein comprised of two related subunits of 105,000 and 100,000 Mr. These peptides form a functional heterodimer, and the smaller of the two binds calmodulin in a calcium-dependent fashion. Although this protein has been physicochemically characterized, its function remains unknown. We have examined the interaction of human adducin with actin and with human erythrocyte spectrin using sedimentation, electrophoretic, and morphologic techniques. Purified adducin binds actin at physiologic ionic strength and bundles it into arrays of laterally arranged filaments, the adducin forming cross-bridges between the filaments at 35.2 /- 3.8 (2 SD) nm intervals. The stoichiometry of high affinity adducin binding to actin at saturation is 1:7, corresponding to a dimer of adducin for every actin helical unit. Adducin also promotes the binding of spectrin to actin independently of protein 4.1. At saturation, each adducin promotes the association of one spectrin heterodimer. The formation of this ternary spectrin-actin-adducin complex is independent of the assembly path, and the complex exists in a readily reversible equilibrium with the free components. The binding of adducin to actin and its ability to stimulate spectrin-actin binding is down-regulated by calmodulin in a calcium-dependent fashion. These results thus identify a putative role for adducin, and define a calcium- and calmodulin-dependent mechanism whereby higher states of actin association and its interaction with spectrin in the erythrocyte may be controlled.     

Hemoglobin enhances the self-association of spectrin heterodimers in human erythrocytes

Spectrin in isolated erythrocyte membranes is known to undergo tetramer to dimer transformation upon hypotonic incubation at 37 degrees C. In the present study, we detect no such transformation in intact erythrocytes in which hypotonicity is achieved by valinomycin treatment followed by hypotonic swelling. The inhibition of spectrin tetramer to dimer transformation is attributable to intracellular hemoglobin, since the addition of hemoglobin to isolated membranes or spectrin extracts blocks a similar spectrin transformation. However, the inhibitory effect is not limited to hemoglobin; other proteins including heme-containing proteins and basic proteins such as cytochrome c, ribonuclease, and albumin are also effective. The magnitude of their effect is proportional to the increased pI value of these proteins. We conclude that the stabilizing effect of these proteins on spectrin tetramers under hypotonic conditions is partly due to their non-ideality, which excludes water from spectrin and thus increases the effective concentration of spectrin, and to their electrostatic interactions with spectrin. In addition, promotion of spectrin self-association by hemoglobin under hypotonic conditions increases the stability of membrane skeletons against mechanical shearing. More importantly, the hemoglobin effect on spectrin self-association is demonstrable at physiological hemoglobin concentration, pH, and osmolarity, suggesting that in intact red cells the spectrin dimer-dimer association, as well as the membrane skeletal structure, is strengthened by intracellular hemoglobin.