Calmodulin binding to human spectrin

Berglund, Åsa M. M.; Backman, Lars; Shanbhag, Vithaldas P.

doi:10.1016/0014-5793(84)80884-4

Cited by 23 publications

(6 citation statements)

References 30 publications

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“…To assess whether calmodulin interferes with the binding of filamentous actin to spectrin, we did direct binding studies using the aqueous two-phase partition technique (Backman, 1985). This technique has proven to be well-suited for studying weak interactions, and we have previously used it to characterize the relatively weak association between calmodulin and spectrin (Berglund et al, 1984(Berglund et al, , 1986. It is clearly seen in Figure 5 that increasing amounts of filamentous actin reduced the fraction of calmodulin bound to spectrin; at 10 µ actin in the lower phase, 60-70% of bound calmodulin was released.…”

Section: Resultsmentioning

confidence: 99%

“…Calmodulin, which is known to bind to erythrocyte spectrin (Berglund et al, 1984(Berglund et al, , 1986Husain et al, 1984) and to other spectrins (Glenney et al, 1982), inhibits the fragmenting activity of spectrin on actin. The inhibition is calcium dependent since no reduction of fragmentation was observed when calcium was replaced by magnesium.…”

Section: Discussionmentioning

confidence: 99%

“…The calcium binding protein calmodulin interacts with components of the membrane cytoskeleton as well as with the membrane itself. It has been shown that, under physiological salt conditions, calmodulin has a calcium-dependent affinity for purified spectrin (Berglund et al, 1984;Husain et al, 1984) and isolated membrane skeletons (Burns & Gratzer, 1985). Since the affinity of calmodulin for spectrin and the cytoskeleton is similar (about 10 µ ) and the number of sites for calmodulin in the cytoskeleton corresponds to the concentration of spectrin, it appears likely that spectrin constitutes the major binding site for calmodulin in intact cytoskeletons.…”

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confidence: 99%

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Influence of calmodulin on the human red cell membrane skeleton

Strömqvist¹,

Berglund²,

Vp³

et al. 1988

Biochemistry

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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.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Influence of calmodulin on the human red cell membrane skeleton

Strömqvist¹,

Berglund²,

Vp³

et al. 1988

Biochemistry

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“…Although interaction between CaM and spectrin is weak, with the reported Kd ranging from 2-22 ,uM (22,23,25), FIGURE 5 Effect of CON-A on MAL-6-labeled CaM in solution and bound to ghosts. Tau is the apparent rotational correlation time.…”

Section: Discussionmentioning

confidence: 99%

Spin-labeling studies of the conformation of the Ca(2+)-regulatory protein calmodulin in solution and bound to the membrane skeleton in erythrocyte ghosts: implications to transmembrane signaling

Yacko

Butterfield

1992

Biophysical Journal

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Electron paramagnetic resonance (EPR) studies of the Ca(2+)-regulatory protein calmodulin (CaM) have been performed. The conformation of CaM in solution changes upon binding of Ca2+ allowing the protein to bind to target proteins existing in the red blood cell membrane. In this study a maleimide spin label, covalently attached to the single cysteine residue of CaM located in the first Ca(2+)-binding domain, was used to monitor allosteric conformational changes induced by interaction of CaM with Ca2+ and subsequently with the red blood cell membrane. The results show, relative to apo-CaM, a significant increase in the apparent rotational correlation time, tau, of the spin label when Ca2+ was present in solution (P less than 0.001). When apo-CaM exposed to red blood cell membrane ghosts in the absence of Ca2+, no significant difference in spin label motion was seen relative to solution, consistent with the idea that Ca2+ is required for CaM to bind to skeletal proteins. When Ca2+ was added to CaM which was then exposed to ghosts, a highly significant increase in tau (decrease in motion) (P less than 0.000001) relative to apo-CaM exposed to ghosts was found. This latter increase in tau is significantly greater than that produced by the addition of Ca2+ to CaM in solution (P less than 0.001). The major interaction sites of CaM were found by photoaffinity labeling and autoradiography on SDS-PAGE to be on the principal skeletal protein, spectrin. EPR was also used to investigate the biophysical correlates of transmembrane signaling. Spin-labeled CaM was bound to the membrane skeleton in the presence of Ca2+. On the opposite side of the erythrocyte membrane a lectin was bound to the external glycoconjugate of Band 3, the major transmembrane protein of the erythrocyte. A highly significant increase in T of the maleimide spin probe was found relative to the control system in which the lectin was absent. (P < 0.00001). These results suggest that electron paramagnetic resonance spectra of spin-labeled CaM can provide useful information about protein structure and function when in solution and when bound to membranes.

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“…Surprisingly, the viscosity decreased with further increases in Ca2" concentration. A possible role for CaM in regulating skeletal protein interactions was also suggested by studies that showed that this protein can bind to the erythrocyte membrane (13,14) as well as to various skeletal proteins (spectrin monomer [5,15,16], af3 spectrin dimer [14,17], and protein 4.1 [18]). Gardner and Bennett (19) The property of membrane deformability that determines the extent of membrane deformation that can be induced by a defined level of applied force was also measured by the ektacytometer.…”

Section: Introductionmentioning

confidence: 99%

Modulation of erythrocyte membrane material properties by Ca2+ and calmodulin. Implications for their role in regulation of skeletal protein interactions.

Takakuwa¹,

Mohandas²

1988

J. Clin. Invest.

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Skeletal proteins of the red blood cell apparently play an important role in regulating membrane material properties of deformability and stability. However, the role of various intracellular constituents in regulating membrane properties has not been clearly defined. To determine whether Ca2' and calmodulin might play a role in this regulation, we measured the membrane stability and deformability of resealed ghosts prepared in the presence of varying concentrations of Ca2l and calmodulin (CaM). For membranes resealed in the presence of Ca2' and physiologic concentrations of CaM (2-8, M), membrane stability decreased with increasing Ca2+ concentrations (> 1.0 MM). Moreover, Ca2' and CaM-induced alterations in membrane stability were completely reversible. In the absence of CaM, an equivalent decrease in membrane stability was seen only when Ca2+ concentration was two orders of magnitude higher (> 100 MM). Calmodulin did not alter membrane stability in the absence of Ca2 . Compared with these changes in membrane stability, membrane deformability decreased only at Ca2+ concentrations > 100 MM, and calmodulin had no effect on Ca2+-induced decrease in membrane deformability. Examination of the effects of Ca2+ and CaM on various membrane interactions have enabled us to suggest that spectrin-protein 4.1-actin interaction may be one of the targets for the effect of Ca2' and CaM. These results imply that Ca21 and calmodulin can regulate membrane stability through modulation of skeletal protein interactions, and that these protein interactions are of a dynamic nature on intact membranes.

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Calmodulin binding to human spectrin

Cited by 23 publications

References 30 publications

Influence of calmodulin on the human red cell membrane skeleton

Influence of calmodulin on the human red cell membrane skeleton

Spin-labeling studies of the conformation of the Ca(2+)-regulatory protein calmodulin in solution and bound to the membrane skeleton in erythrocyte ghosts: implications to transmembrane signaling

Modulation of erythrocyte membrane material properties by Ca2+ and calmodulin. Implications for their role in regulation of skeletal protein interactions.

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