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Spectrin–phospholipid interactions
Chemistry and Physics of Lipids 141 (2006) 133–141
Review
Spectrin–phospholipid interactions Existence of multiple kinds of binding sites? Michał Grzybek a , Anna Chorzalska a , Ewa Bok a , Anita Hryniewicz-Jankowska a , Aleksander Czogalla a , Witold Diakowski a , Aleksander F. Sikorski a,b,∗ a
University of Wrocław, Institute of Biochemistry and Molecular Biology, Przybyszewskiego 63/77, 51 148 Wrocław, Poland b Academic Centre for Biotechnology of Lipid Aggregates, Przybyszewskiego 63/77, 51 148 Wrocław, Poland Received 21 November 2005; accepted 20 February 2006 Available online 13 March 2006
Abstract The object of this paper is to review briefly the studies on the interactions of erythroid and non-erythroid spectrins with lipids in model and natural membranes. An important progress on the identification of lipid-binding sites has recently been made although many questions remain still unanswered. In particular, our understanding of the physiological role of such interactions is still limited. Another important issue is the occurrence of spectrins in membrane rafts, how they are attached to the raft and what is their function in rafts. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Spectrin–lipid interactions; Spectrin; Fodrin; Membrane skeleton; Membrane rafts
Contents 1. 2.
3.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Spectrin-based membrane skeleton . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lipid-binding by spectrins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Erythrocyte spectrin and its nonperythroid analogue bind hydrophobic ligands and phospholipid mono- and bilayers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Lipid-binding sites in spectrins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Possible role of spectrin–phospholipid interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Spectrin–raft interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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1. Introduction 1.1. Spectrin-based membrane skeleton ∗
Corresponding author. Tel.: +4871 3756 233; fax: +4871 3756 208. E-mail address: [email protected] (A.F. Sikorski).
The remarkable mechanical properties of red cell membrane stem from the presence on the cytoplasmic
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surface of a dense, well organised protein network called the membrane skeleton. Its major component is spectrin, a high molecular weight flexible rod-like protein formed by head-to-head association of two heterodimers composed of ␣- (280 kDa) and - (247 kDa) subunits (Sahr et al., 1990; Winkelmann et al., 1990). Spectrin heterodimers are formed by antiparallel double-helical association of ␣- and -subunits, each of which form predominantly a segmental triple-helical molecule. The detailed structure of the triple-helical segment, which is 106 amino acid residues long, was first solved by X-ray crystallography and also by NMR studies on an expressed fragment of Drosophila spectrin (Yan et al., 1993). Five to six spectrin tetramers interact with a short (37 nm) actin protofilament to form a structure known as the junctional complex (Byers and Branton, 1985). Several other proteins: protein 4.1, adducin, dematin, p55, tropomyosin and tropomodulin are also involved in forming the junction. Both protein 4.1 and adducin are known to promote high affinity binding of spectrin to protofilaments (F-actin in the test tube) which otherwise would bind very weakly. Thus the formation of spectrin tetramers (i.e. dimmer–dimer interaction) and the junctional complex are responsible for the planar integrity of the membrane skeleton, and these cytoskeletons can be isolated from either intact cells, or from membranes, by extraction with non-ionic detergent solution (Yu et al., 1973). The membrane skeleton of the red blood cell is attached to the lipid bilayer embedded with integral membrane proteins through two pathways of interaction with transmembrane proteins, i.e. spectrin-ankyrinAE1(band 3) protein (e.g. Bennett and Branton, 1977; Yu and Goodman, 1979; Bennett and Stenbuck, 1979, 1980) and protein 4.1-glycophorin C and D (Pinder et al., 1993) and the ternary interaction protein 4.1-p55-glycophorin C (Hemming et al., 1995), or a recently discovered connection involving Rh, RhAG, CD47, protein 4.2 and/or ankyrin (Mauro-Chanteloup et al., 2003; Nicolas et al., 2003). The structure of the membrane skeleton, the reciprocal interactions of its components and its interactions with membrane proteins in non-erythroid cells are known to a much lesser extent, largely because of their much higher stuctural complexity. However, since many animal cell membranes contain spectrin and spectrinbinding proteins (analogues of erythrocyte membrane proteins and novel spectrin-binding proteins) the existence of a similar protein network, which is tightly associated with membrane proteins, is anticipated (Bennett and Baines, 2001). The above mentioned high affinity protein–protein interactions responsible for membrane skeleton attachment to the membrane have been the subject of many excellent reviews (e.g. Bennett and Baines,
2001). There are however, many indications coming from various studies on cells, isolated membranes, and model systems that direct protein–lipid interactions contribute to the attachment of the membrane skeleton to the membrane hydrophobic domain. 2. Lipid-binding by spectrins 2.1. Erythrocyte spectrin and its nonperythroid analogue bind hydrophobic ligands and phospholipid mono- and bilayers The earliest published studies (Sweet and Zull, 1970; Juliano et al., 1971) on the interaction of spectrin with phospholipids predate the discovery of ankyrin and other proteinaceous receptors in erythrocytes. Further studies showed that purified spectrin had the ability to bind hydrophobic and amphipathic ligands (Isenberg et al., 1981; Sikorski et al., 1987a; Sikorski, 1988) supporting the view that spectrin contained a number of hydrophobic sites. This has been confirmed by analyses of binding isotherms using brominated fatty acids and phospholipid vesicles (Kahana et al., 1992; Bitbol et al., 1989; Sikorski et al., 2000a). Numerous studies on the interaction of erythrocyte spectrin with membrane bilayer phospholipids, from natural (erythrocyte) membranes, liposomes, or monolayer lipid films have been carried out by a variety of techniques. For a review of the older literature see Sikorski et al. (2000b). Mombers et al. (1980) observed a decrease in the enthalpy change associated with the phase transition of anionic phospholipids such as PS, PG or CL in the presence of spectrin. This interaction was pH dependent, being optimal at pH 5.5. Further specificity studies on the interaction of anionic phospholipids monolayers with separated ␣- or -spectrin subunits indicated a preferential binding by the -subunit (Schubert et al., 1981). A similar conclusion was reached when PE/PC monolayers were used (Białkowska et al., 1999). It has been suggested by several groups that spectrin binds preferentially to membrane monolayers or bilayers that contain PS. Indeed some experimental data seem to confirm this hypothesis (Cohen et al., 1986; DeWolf et al., 1996; Maksymiw et al., 1987, pp. 27–29), while our data (Białkowska et al., 1994; Sikorski et al., 1987) and those of others (Bitbol et al., 1989; O’Toole et al., 2000), showed that the affinity of purified spectrin for PS-containing vesicles was not significantly different to that for PC vesicles. Ray and Chakrabarti (2004) studying an interactions of DMPE containing DMPC vesicles with red blood cell spectrin dimer found that KD increased with an
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increase of DMPE content (from 57 nM for pure DMPC vesicles up to 720 nM for 80% DMPE in these vesicles). However, when vesicles were prepared from pure DMPE the KD values dropped to 0.7 nM in the fluid phase and 2.6 nM in the gel phase. These and other results led authors to the suggestion that binding site for pure DMPE is located only at one end of the spectrin dimer. Non-erythroid spectrins bear a high sequence homology to erythroid spectrins and have ␣- and -subunits of 283 and 275 kDa, respectively (Goodman et al., 1981; Winkelmann and Forget, 1993). As well as being a component of the membrane skeleton non-erythroid spectrin (in particular ␣II1/II1 isoform) is thought to be involved in the regulation of exocytosis, in particular in the regulation of neurotransmitter release, in which its interaction with small synaptic vesicles via synapsin was shown to play a crucial role (Sikorski et al., 1991, 2000b). Numerous interactions of spectrin with other proteins have been inferred from using the yeast two hybrid system (review: De Matteis and Morrow, 2000). Non-erythroid (brain) spectrin also binds to membrane phospholipids (Diakowski and Sikorski, 1995). Saturable binding isotherms were observed for FATliposomes with KD ’s in the nanomolar range (i.e. from 16 nM at pH 7.5 for liposomes prepared from total lipid mixture extracted from synaptic plasma membrane to ∼500 nM for PC liposomes at pH 6.0). Purified brain spectrin induced an increase in surface pressure in lipid monolayers composed of PE/PC, PS/PC (3:2) and PC. The maximal effect (π) was observed when monolayers contained PE in the mixture, in particular when PE was 50–60% of the monolayer forming lipid. This interaction occurred optimally at pH 7.5, both in the pelleting assays and in monolayer experiments. There was also an ionic strength optimum, corresponding to 0.15 M NaCl. Monolayer experiments revealed similarly, as in the case of red blood cell spectrin, that the major lipidbinding site is located in the -subunit of the brain protein (Diakowski et al., 1999). Our monolayer experiments on anionic phospholipids including PI and PIP2 binding to purified brain spectrin showed that a monolayer formed of PI or PI/PC bound brain spectrin efficiently while red blood cell spectrin exerted much smaller effect on these monolayers. However, monolayers of PIP2 or PIP2/PC did not bind purified brain spectrin at all (Diakowski and Sikorski, 2002), suggesting a large affinity of brain spectrin for anionic phospholipids. When natural membranes were treated in such a way as to remove protein receptors, i.e. NaOH extraction and treatment with proteases still bound spectrin, thus indi-
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cating the presence of a protein-independent receptor. The affinities of these stripped membrane preparations for spectrin are similar to those found in other model systems. Moreover, this binding is competitively inhibited by lipid vesicles (Diakowski et al., 2003). Interaction of spectrins with monolayers prepared from membrane phospholipids of various fluidity seem to be dependent on the ways this parameter is regulated. Our data (Diakowski et al., 2006) indicates that the presence of up to 10−20% cholesterol in the PE/PC monolayer facilitates the penetration of the monolayer by both types of spectrin. For monolayers constructed from mixtures of PI/PC and cholesterol, the effect of spectrins was characterised by the presence of two maxima (at 5 and 30% cholesterol) of surface pressure for erythroid spectrin, and a single maximum (at 20% cholesterol) for brain spectrin. The binding assay results indicated a small but easily detectable decrease in the affinity of erythrocyte spectrin for FAT-liposomes prepared from a PE/PC mixture containing cholesterol, and a two- to five-fold increase in maximal binding capacity (Bmax ) depending on the cholesterol content. On the other hand, the results from experiments with a monolayer constructed from homogenous synthetic phospholipids indicated an increase in π change with the decrease in the fluidity of the phospholipids used to prepare the monolayer. This result was further confirmed by a pelleting experiment. Adding spectrins into the subphase of raft-like monolayers constructed from DOPC, SM and cholesterol (1/1/1) induced an increase in surface pressure. The π change values were, however, much smaller than those observed in the case of a natural PE/PC (6/4) monolayer. An increased binding capacity of liposomes prepared from a “raft-like” mixture of lipids for spectrins could also be concluded from the pelleting assay. 2.2. Lipid-binding sites in spectrins Spectrins are multidomain proteins which show specific structural features: one is that it forms an elongated segmental structure built of a series of repeating units, each of about 106 amino acid residues forming a triplehelical motif. The other feature is the fact that outside typical segments and within them there are domains which are responsible for the functions of spectrin. The most important functional domains outside typical spectrin repeats are: actin-binding domain (two CH motifs located at amino terminal region of -subunit of spectrin) (Karinch et al., 1990) and SH3 domain present in atypical, 10th segment of ␣-spectrin. Examples of other domains are: ankyrin-binding domain located in part in
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14th repeat and in 15th unit of -spectrin (Kennedy et al., 1991; Baines, 2003) and Ca2+ -binding EF-hand motifs located in carboxyl terminal region of ␣-subunit (Buevich et al., 2004). The consequence of this multifunctionality is the existence of several lipid-binding activities connected with the occurrence of different binding sites: (1) a “general” phospholipids-binding activity characteristic for the spectrin repeat motif (DeWolf et al., 1997; LeRumeur et al., 2003), (2) specific for aminophospholipids (PE and/or PS-binding sites located in the -subunits (e.g. An et al., 2004a,b, 2005; HryniewiczJankowska et al., 2004) and (3) specific for phosphatidylinositol 4,5-bisphosphate, characteristic only for longer -spectrin isoforms which poses Pleckstrin Homology (PH) domain (Saraste and Hyvonen, 1995; Wang et al., 1996). The first kind of the lipid-binding activity that could be attributed to the spectrin repeat motif was actually studied by using a series of constructs containing the second repeat of the human dystrophin rod domain (Kahana and Gratzer, 1995). DeWolf et al. (1997) observed that properly folded fragment corresponding to the second repeat unit of the rod domain is able to modify the properties of lipid membranes (inducing of a large increase in surface shear viscosity of the monolayer) containing anionic phosphatidylserine. Moreover, this property is dependent on the native structure as the unfolded expressed fragment had no effect. Further studies of LeRumeur et al. (2003) showed the engagement of tryptophan residues in this binding, as in the presence of small, unilammellar vesicles containing PS the fluorescence characteristics changed dramatically indicating the tryptophan residues changed their environment into more hydrophobic. The accessibility of these residues from the hydrophilic solvent for quenchers was also limited. These authors conclude that dystrophin rod lies along the membrane surface. Similar experimental approach should be undertaken to determine whether or not the same conclusion could be drawn with respect of spectrin–phospholipids interactions. The second class of lipid-binding sites seems to be confined to certain regions of spectrin molecule which show some aminophospholipid and anionic phospholipids specificity. One of the approaches to identify this class of sites was to clone and express systematically all the fragments of both subunits of erythrocyte spectrin and test their binding to liposomes prepared from PS and its mixture with PC. Studies of An et al. (2004a,b) indicate that binding sites for PS containing vesicles are located in repeats 8–10 of the ␣-subunit and in repeats 2, 3, 4, 12, 13, 14 and in the non-homologous amino termi-
nal region of the -subunit. Their further studies (An et al., 2005) concerning non-erythroid spectrin indicated that PS-binding site is located in repeats 9–11 and in the amino terminus of ␣-subunit and in amino terminal region and repeat units 2 and 3 of the -subunit. Our early attempt to identify the main amphipathic compound-binding site of spectrin revealed its close proximity to the ankyrin-binding domain (Sikorski, 1988) what suggested a functional relationship between ankyrin and lipid binding by erythroid spectrin. Indeed when the effect of purified ankyrin on spectrin binding to phospholipid vesicles was tested, an inhibition of this interaction was observed (Białkowska et al., 1994). The effect was greater for the vesicles containing PE (PE/PC 3:2) for which 60% inhibition was found compared to 10–20% inhibition for PS/PC vesicles. Almost identical results were obtained using a monolayer technique. Dixon-type analysis indicated a competitive mechanism of inhibition of PE/PC vesicles or monolayer binding to spectrin by ankyrin. Tetrameric spectrin bound similarly to PE/PC monolayer but inhibition with ankyrin suggests that only one of the two possible binding sites is engaged in this interaction (Białkowska et al., 1999). Moreover, when interactions of non-erythroid (brain) spectrin with PE/PC monolayers in the presence of ankyrin were analysed, a similar level of inhibition of these interactions by ankyrin was observed (Diakowski et al., 1999). Also, when isolated erythroid spectrin -subunit was introduced into the subphase of PE/PC monolayers in the presence of ankyrin the inhibition was even stronger, i.e. a three-fold lower concentration of ankyrin was needed to induce the same effect. If the ␣-subunit was used instead of  its effect on the monolayer surface pressure was small and entirely insensitive to incubation with ankyrin (Białkowska et al., 1999; Diakowski et al., 1999). It should be noted also that binding of brain spectrin to anionic phospholipid monolayers was also competitively inhibited by purified erythrocyte ankyrin (Diakowski and Sikorski, 2002). As this event could be a subject of the regulation and could be engaged in the possible mechanism of function of the membrane skeleton; we decided to test whether an ankyrin-binding domain which was identified by Kennedy et al. (1991) would show the same lipid-binding properties as spectrin molecule and to identify an ankyrin-sensitive, lipid-binding site in erythroid and non-erythroid spectrins (Hryniewicz-Jankowska et al., 2004; Bok et al., unpublished). The results of the experiments document further the ability of cloned, expressed and purified erythroid -spectrin’s ankyrinbinding domain to bind PE-rich mono- and bilayers. We found that full length ankyrin-binding domain binds
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PE/PC mono- and bilayers with affinity similar to this of native spectrin dimer and that this binding is inhibited by erythrocyte ankyrin. Binding studies were performed by: (i) pelleting assay using PE/PC liposomes and fluorescently labelled expressed polypeptides, (ii) inhibition assay in which unlabelled, expressed polypeptides were used to inhibit binding of purified, labelled red blood cell spectrin to PE/PC liposomes and (iii) in surface plasmon resonance technique experiments where binding of purified, expressed polypeptides to PE/PC monolayer deposited on hydrophobic sensor surface. Results of these experiments indicate that ankyrin-binding domain truncated mutants, that retain amino terminal part bind the PE/PC mono- and bilayers with comparable to full length ankyrin-binding domain affinity and capacity. They also effectively compete for lipids with purified, labelled spectrin. On the other hand, truncated mutants which lack eight or 38 amino acid residues from Nterminal region show, at least an order of magnitude lower affinity and much higher maximal binding capacity. They also compete weakly with spectrin for phospholipid vesicles. The expressed full length domain induced small decrease in order parameter of PE/PC membranes when probed with 5 -doxylstearate similar to the effect of purified spectrin while mutant lacking 38 residues from amino terminus induced small increase in order parameter which was similar to the effect of bovine serum albumin. Similar results were obtained with ankyrin-binding domain of non-erythroid -spectrin. Cloned and expressed fragments of non-erythroid (human brain) -spectrin encompassing a sequence corresponding to an ankyrin-binding domain was found to bind phospholipids mono- and bilayers in a similar way to intact molecule of brain spectrin or erythroid spectrin. Again truncated mutant of this domain which retained amino terminal region bound lipid mono- and bilayers with the affinity similar to full-length domain. Mutants in which this region was deleted bound phospholipids with lower affinities and higher capacities and this binding was insensitive to inhibition with purified ankyrin. Transient expression of HeLa cells with GFPconjugated construct encoding full-length ankyrinbinding domain of either erythroid or non-erythroid -spectrins induced changes in cell morphology and aggregation of membrane skeleton. These changes were observed neither in cells transfected with a construct encoding GFP-conjugated ankyrin-binding domain truncated at amino terminus by 38 amino acid residues nor in cells transfected with a vector encoding only GFP (Bok et al., submitted for publication).
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The above mentioned experimental results indicate that at least in erythroid spectrin the binding site located within or close to ankyrin-binding domain were identified by our and Mohandas groups. It seems that there is a good chance that these sites (segment 12–14 and amino terminal part of the ankyrin-binding domain) are overlapping. The difference concerns nonerythroid -spectrin; our results indicate essentially the same features for both -spectrins while Mohandas group could not identify this site in their constructs. It should be noted that rather high degree of identity between erythroid and non-erythroid spectrins can be found comparing the amino terminal regions ankyrinbinding domains (helix C of repeat 14) of these proteins (Kennedy et al., 1991; Baines, 2003). In our hands binding of PS/PC (3:2) monolayer to bacterially expressed ankyrin-binding domain and its deletion mutants retaining N-terminal region of non-erythroid spectrin occurred with KD ’s in nanomolar range what was measured by SPR technique (Bok, submitted for publication). Pleckstrin homology domain is an example of the third kind of lipid-binding sites specific for phosphoinositides. This kind of PIP2 binding site which is present in non-erythroid spectrins, I2 and II2 and also in many regulatory and cytoskeletal proteins (Wang et al., 1996; Niggli, 2001) is approximately 100 amino acid residues in length. Binding of PIP2 is inhibited competitively by IP3 indicating the specificity for inositol ring (Saraste and Hyvonen, 1995). When PH domain of I2 spectrin was fused with green fluorescent protein and expressed in COS7 cell, it was localized to plasma membrane (Wang et al., 1996). 2.3. Possible role of spectrin–phospholipid interactions It was suggested previously that sites of interactions of spectrin with lipids function as additional support points to the membrane bilayer increasing its mechanical stability. This would be the function of the “general” sites, best characterized in the case of dystrophin repeat segment (see above). It should be noted that almost every fragment of spectrin studied in our laboratory to date, display lipid-binding activity, characterised with a KD in order of 105 –10−6 M. The lipidbinding activities are confined to the specific regions or domains such as PH domain and are highly specific towards PIP2 and are characteristic only for certain isoforms. Lipid-binding sites of limited specificity and moderate-to-high affinity (KD ∼ 10−7 –10−8 M) are
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confined essentially to two regions of spectrin molecule: one located to the centre of the tetramer and another located near its end and they coincide with, or are close to, the attachment sites for the proteins that mediate linkage with membrane bilayer. An et al. (2004a) suggested that the interactions with PS modulate interactions of spectrin with linking proteins such as ankyrin or protein 4.1. They also suggest that spectrin regulates the state of the bilayer, possibly generating or stabilising PS patches (An et al., 2004b). As was mentioned above, we found that binding of aminophospholipid mono- or bilayers at one of these sites was inhibited by ankyrin (Białkowska et al., 1994, 1999). The same was observed for non-erythroid spectrin (Diakowski et al., 1999) and moreover, for cloned and artificially expressed ankyrinbinding domain (Hryniewicz-Jankowska et al., 2004) and its truncated mutants (for details see above). We have proposed that PE-rich domains (and also to lesser extent PS or anionic phospholipids-rich domains) would serve as membrane attachment site for spectrin in situations in which ankyrin is either deficient or its affinity for spectrin is reduced (Białkowska et al., 1994; Sikorski and Białkowska, 1996). Some examples of evidence of such situations are: (i) erythrocytes of mutant mice, whose erythroblasts fail to synthesise ankyrin, still accumulate about 50% of normal amount of spectrin (Bodine et al., 1984), (ii) erythrocytes of ankyrin-deficient mice which contain normal skeletons but lack AE1 tetramers (Yi et al., 1997), (iii) also the phosphorylation of ankyrin decreases its ability to bind spectrin (Liu et al., 1985). In all these situations, aminophospholipid-rich (primarily PE) domains would serve as anchors substituting ankyrin and ensuring preservation of the mechanical properties in the membrane skeletal lattice. This would explain why mice with a disrupted gene coding for AE1 protein are characterized by severe spherocytosis despite the presence of normal membrane skeletons (Southgate et al., 1996) in contrast to the above-mentioned cases of ankyrin deficiency. Possibly in this case ankyrin inhibits binding of spectrin to the inner layer of membrane lipids. 3. Spectrin–raft interactions Plasma membrane is laterally inhomogeneous. Although the presence of lipid rafts has not been proved to appear in vivo so far (Lichtenberg et al., 2005), they remain an attractive subject. Lipid raft model is build mostly on the data aquired through the isolation of so called DRMs (detergent-resistant membranes). From what we know, DRMs are rich in cholesterol and glycosphingolipids and are resistant to the extraction with cold non-ionic detergent (the most often used is 1%
Triton X-100). They contain a specific subset of proteins such as caveolin, flotillins, stomatin, proteins that anchor to the membrane via glycosylphosphatidylinositol molecule or mirystoyl or palmitoyl residue. Lipid rafts are thought to be engaged not only in signaling pathways, sorting of membrane proteins and lipids but also play role in pathogen entry into the cell or virus budding (reviewed by Grzybek et al., 2005). Although spectrin is observed as a part of DRM proteome in erythroid (Salzer and Prohaska, 2001) and non-erythroid cells (von Haller et al., 2001; Nebl et al., 2002; Leshchyns’ka et al., 2003) its presence in the DRMs is controversial (Samuel et al., 2001; Murphy et al., 2004). Spectrin is co-isolated with the cholesterol enriched fraction depending on the concentration of the detergent, pH and concentration of the protein in the sample (Ciana et al., 2005; Murphy et al., 2004). The concentration of the used detergent, its type and even the presence during the gradient centrifugation may drastically change both the quality and quantity of proteins isolated with the DRMs fraction (Schuck et al., 2003; Murphy et al., 2004; Korzeniowski et al., 2003). Remarkably, the presence of spectrin in the DRMs is not followed by the presence of the usual anchors of the membrane skeleton to the membrane. The normal band 3, ankyrin, spectrin or the junctional complex (actin, protein 4.1, p55, spectrin) are not represented in the DRM proteom (Salzer and Prohaska, 2001). Another group (Samuel et al., 2001; Murphy et al., 2004), however, showed the presence of band 3 in the erythroid rafts but they simultaneously found an absence of spectrin in this fraction. An important question is however, if spectrin is associated with lipid rafts, then how it is connected to them. Some data suggest that these interactions are sensitive to pH or ionic strength (Ciana et al., 2005). The protein candidates for spectrin anchoring to the membrane must then be a part of the usually isolated DRM proteome. Stomatin (1,00,000 copies/cell) which is abundantly represented in the DRMs could be one of such proteins keeping in mind that the stomatinlike protein 2 (4000 copies/cell) was proved to associate with spectrin (Wang and Morrow, 2000). This hypothesis has only one drawback—stomatocytes (erythrocytes that lack stomatin in their membranes) still have spectrin associated with lipid rafts (Salzer and Prohaska, 2001). Alternatively to protein–protein interactions the protein–lipid interactions are possible. If this hypothesis would be correct then the lipids themselves should be responsible for the maintenance of spectrin in rafts. Spectrin’s affinity for special groups of phospholipids such as PE, PS and PI has been well established (see above). In fact recent data suggest that PI and PS occur in DRM in similar proportions as in the whole cell membrane
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(Koumanov et al., 2005). Another possibility of anchoring spectrin to lipid rafts is its palmitoylation. The existence of erythrocyte protein-palmitoyl acyltransferase which could be responsible for such activity supports such a possibility (Das et al., 1997). Altogether, the recruitment of spectrin to lipid rafts via interactions with lipid molecules cannot be excluded. Red blood cells are often used in raft studies because they lack inner cell organelles. However, the advantage of the isolation of DRMs only from the plasma membrane has also some disadvantages, because nobody can answer the question what the rafts are responsible for in erythrocytes. Therefore erythrocyte membrane rafts may be completely inactive and it could be difficult to compare to the other cells. Surprisingly, in more complex cells, such as neurons, the question of spectrin presence in rafts seems to be much easier to explain. The Triton-insoluble I-spectrin is responsible for the NCAM/PKC2 redistribution in neurons and takes part in the neurite growth. In neurons all major NCAMs bind to spectrin and promote spectrin incorporation to DRMs, however not all interactions between various NCAMs and spectrin are maintained after the disruption of rafts with methyl-betacyclodextrin. GPI-anchored NCAM 120, associates with the I-spectrin through lipid rafts (Leshchyns’ka et al., 2003). More complex picture of spectrin association with lipid rafts could be concluded after the proteomical studies on leukocytic DRM (von Haller et al., 2001; Nebl et al., 2002; Li et al., 2004). In contrast to erythrocytes, these cells have a vast number of proteins associated with DRMs, that greatly exceeds the number of proteins in RBCs. The presence of actin, myosin or supervilin together with other cytoskeleton associated proteins allows to create much more sophisticated model of a spectrin–DRM interaction. One of the groups (Nebl et al., 2002) proposed such a possibility. The typical raft integral proteins such as flotillins and stomatin are accompanied by the GPIanchored proteins (extracellular) and by the dually acylated signaling proteins (intracellular). Fodrin and Factin attaches to the DRM via their interactions with supervilin, ␣-actinin, myosin IIa and myosin Ig. To prove this and other models further studies need to be undertaken. The question, whether the isolated fractions correspond to real “rafts” (Heerklotz, 2002) remains unanswered. However, the discovery of spectrin–DRM interaction may solve many intriguing questions concerning one of the roles of spectrin in the cell. It seems that spectrin, originally recognized as structural protein, still has many hidden secrets.
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Review
Spectrin–phospholipid interactions Existence of multiple kinds of binding sites? Michał Grzybek a , Anna Chorzalska a , Ewa Bok a , Anita Hryniewicz-Jankowska a , Aleksander Czogalla a , Witold Diakowski a , Aleksander F. Sikorski a,b,∗ a
University of Wrocław, Institute of Biochemistry and Molecular Biology, Przybyszewskiego 63/77, 51 148 Wrocław, Poland b Academic Centre for Biotechnology of Lipid Aggregates, Przybyszewskiego 63/77, 51 148 Wrocław, Poland Received 21 November 2005; accepted 20 February 2006 Available online 13 March 2006
Abstract The object of this paper is to review briefly the studies on the interactions of erythroid and non-erythroid spectrins with lipids in model and natural membranes. An important progress on the identification of lipid-binding sites has recently been made although many questions remain still unanswered. In particular, our understanding of the physiological role of such interactions is still limited. Another important issue is the occurrence of spectrins in membrane rafts, how they are attached to the raft and what is their function in rafts. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Spectrin–lipid interactions; Spectrin; Fodrin; Membrane skeleton; Membrane rafts
Contents 1. 2.
3.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Spectrin-based membrane skeleton . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lipid-binding by spectrins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Erythrocyte spectrin and its nonperythroid analogue bind hydrophobic ligands and phospholipid mono- and bilayers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Lipid-binding sites in spectrins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Possible role of spectrin–phospholipid interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Spectrin–raft interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
133 133 134 134 135 137 138 139 139
1. Introduction 1.1. Spectrin-based membrane skeleton ∗
Corresponding author. Tel.: +4871 3756 233; fax: +4871 3756 208. E-mail address: [email protected] (A.F. Sikorski).
The remarkable mechanical properties of red cell membrane stem from the presence on the cytoplasmic
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surface of a dense, well organised protein network called the membrane skeleton. Its major component is spectrin, a high molecular weight flexible rod-like protein formed by head-to-head association of two heterodimers composed of ␣- (280 kDa) and - (247 kDa) subunits (Sahr et al., 1990; Winkelmann et al., 1990). Spectrin heterodimers are formed by antiparallel double-helical association of ␣- and -subunits, each of which form predominantly a segmental triple-helical molecule. The detailed structure of the triple-helical segment, which is 106 amino acid residues long, was first solved by X-ray crystallography and also by NMR studies on an expressed fragment of Drosophila spectrin (Yan et al., 1993). Five to six spectrin tetramers interact with a short (37 nm) actin protofilament to form a structure known as the junctional complex (Byers and Branton, 1985). Several other proteins: protein 4.1, adducin, dematin, p55, tropomyosin and tropomodulin are also involved in forming the junction. Both protein 4.1 and adducin are known to promote high affinity binding of spectrin to protofilaments (F-actin in the test tube) which otherwise would bind very weakly. Thus the formation of spectrin tetramers (i.e. dimmer–dimer interaction) and the junctional complex are responsible for the planar integrity of the membrane skeleton, and these cytoskeletons can be isolated from either intact cells, or from membranes, by extraction with non-ionic detergent solution (Yu et al., 1973). The membrane skeleton of the red blood cell is attached to the lipid bilayer embedded with integral membrane proteins through two pathways of interaction with transmembrane proteins, i.e. spectrin-ankyrinAE1(band 3) protein (e.g. Bennett and Branton, 1977; Yu and Goodman, 1979; Bennett and Stenbuck, 1979, 1980) and protein 4.1-glycophorin C and D (Pinder et al., 1993) and the ternary interaction protein 4.1-p55-glycophorin C (Hemming et al., 1995), or a recently discovered connection involving Rh, RhAG, CD47, protein 4.2 and/or ankyrin (Mauro-Chanteloup et al., 2003; Nicolas et al., 2003). The structure of the membrane skeleton, the reciprocal interactions of its components and its interactions with membrane proteins in non-erythroid cells are known to a much lesser extent, largely because of their much higher stuctural complexity. However, since many animal cell membranes contain spectrin and spectrinbinding proteins (analogues of erythrocyte membrane proteins and novel spectrin-binding proteins) the existence of a similar protein network, which is tightly associated with membrane proteins, is anticipated (Bennett and Baines, 2001). The above mentioned high affinity protein–protein interactions responsible for membrane skeleton attachment to the membrane have been the subject of many excellent reviews (e.g. Bennett and Baines,
2001). There are however, many indications coming from various studies on cells, isolated membranes, and model systems that direct protein–lipid interactions contribute to the attachment of the membrane skeleton to the membrane hydrophobic domain. 2. Lipid-binding by spectrins 2.1. Erythrocyte spectrin and its nonperythroid analogue bind hydrophobic ligands and phospholipid mono- and bilayers The earliest published studies (Sweet and Zull, 1970; Juliano et al., 1971) on the interaction of spectrin with phospholipids predate the discovery of ankyrin and other proteinaceous receptors in erythrocytes. Further studies showed that purified spectrin had the ability to bind hydrophobic and amphipathic ligands (Isenberg et al., 1981; Sikorski et al., 1987a; Sikorski, 1988) supporting the view that spectrin contained a number of hydrophobic sites. This has been confirmed by analyses of binding isotherms using brominated fatty acids and phospholipid vesicles (Kahana et al., 1992; Bitbol et al., 1989; Sikorski et al., 2000a). Numerous studies on the interaction of erythrocyte spectrin with membrane bilayer phospholipids, from natural (erythrocyte) membranes, liposomes, or monolayer lipid films have been carried out by a variety of techniques. For a review of the older literature see Sikorski et al. (2000b). Mombers et al. (1980) observed a decrease in the enthalpy change associated with the phase transition of anionic phospholipids such as PS, PG or CL in the presence of spectrin. This interaction was pH dependent, being optimal at pH 5.5. Further specificity studies on the interaction of anionic phospholipids monolayers with separated ␣- or -spectrin subunits indicated a preferential binding by the -subunit (Schubert et al., 1981). A similar conclusion was reached when PE/PC monolayers were used (Białkowska et al., 1999). It has been suggested by several groups that spectrin binds preferentially to membrane monolayers or bilayers that contain PS. Indeed some experimental data seem to confirm this hypothesis (Cohen et al., 1986; DeWolf et al., 1996; Maksymiw et al., 1987, pp. 27–29), while our data (Białkowska et al., 1994; Sikorski et al., 1987) and those of others (Bitbol et al., 1989; O’Toole et al., 2000), showed that the affinity of purified spectrin for PS-containing vesicles was not significantly different to that for PC vesicles. Ray and Chakrabarti (2004) studying an interactions of DMPE containing DMPC vesicles with red blood cell spectrin dimer found that KD increased with an
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increase of DMPE content (from 57 nM for pure DMPC vesicles up to 720 nM for 80% DMPE in these vesicles). However, when vesicles were prepared from pure DMPE the KD values dropped to 0.7 nM in the fluid phase and 2.6 nM in the gel phase. These and other results led authors to the suggestion that binding site for pure DMPE is located only at one end of the spectrin dimer. Non-erythroid spectrins bear a high sequence homology to erythroid spectrins and have ␣- and -subunits of 283 and 275 kDa, respectively (Goodman et al., 1981; Winkelmann and Forget, 1993). As well as being a component of the membrane skeleton non-erythroid spectrin (in particular ␣II1/II1 isoform) is thought to be involved in the regulation of exocytosis, in particular in the regulation of neurotransmitter release, in which its interaction with small synaptic vesicles via synapsin was shown to play a crucial role (Sikorski et al., 1991, 2000b). Numerous interactions of spectrin with other proteins have been inferred from using the yeast two hybrid system (review: De Matteis and Morrow, 2000). Non-erythroid (brain) spectrin also binds to membrane phospholipids (Diakowski and Sikorski, 1995). Saturable binding isotherms were observed for FATliposomes with KD ’s in the nanomolar range (i.e. from 16 nM at pH 7.5 for liposomes prepared from total lipid mixture extracted from synaptic plasma membrane to ∼500 nM for PC liposomes at pH 6.0). Purified brain spectrin induced an increase in surface pressure in lipid monolayers composed of PE/PC, PS/PC (3:2) and PC. The maximal effect (π) was observed when monolayers contained PE in the mixture, in particular when PE was 50–60% of the monolayer forming lipid. This interaction occurred optimally at pH 7.5, both in the pelleting assays and in monolayer experiments. There was also an ionic strength optimum, corresponding to 0.15 M NaCl. Monolayer experiments revealed similarly, as in the case of red blood cell spectrin, that the major lipidbinding site is located in the -subunit of the brain protein (Diakowski et al., 1999). Our monolayer experiments on anionic phospholipids including PI and PIP2 binding to purified brain spectrin showed that a monolayer formed of PI or PI/PC bound brain spectrin efficiently while red blood cell spectrin exerted much smaller effect on these monolayers. However, monolayers of PIP2 or PIP2/PC did not bind purified brain spectrin at all (Diakowski and Sikorski, 2002), suggesting a large affinity of brain spectrin for anionic phospholipids. When natural membranes were treated in such a way as to remove protein receptors, i.e. NaOH extraction and treatment with proteases still bound spectrin, thus indi-
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cating the presence of a protein-independent receptor. The affinities of these stripped membrane preparations for spectrin are similar to those found in other model systems. Moreover, this binding is competitively inhibited by lipid vesicles (Diakowski et al., 2003). Interaction of spectrins with monolayers prepared from membrane phospholipids of various fluidity seem to be dependent on the ways this parameter is regulated. Our data (Diakowski et al., 2006) indicates that the presence of up to 10−20% cholesterol in the PE/PC monolayer facilitates the penetration of the monolayer by both types of spectrin. For monolayers constructed from mixtures of PI/PC and cholesterol, the effect of spectrins was characterised by the presence of two maxima (at 5 and 30% cholesterol) of surface pressure for erythroid spectrin, and a single maximum (at 20% cholesterol) for brain spectrin. The binding assay results indicated a small but easily detectable decrease in the affinity of erythrocyte spectrin for FAT-liposomes prepared from a PE/PC mixture containing cholesterol, and a two- to five-fold increase in maximal binding capacity (Bmax ) depending on the cholesterol content. On the other hand, the results from experiments with a monolayer constructed from homogenous synthetic phospholipids indicated an increase in π change with the decrease in the fluidity of the phospholipids used to prepare the monolayer. This result was further confirmed by a pelleting experiment. Adding spectrins into the subphase of raft-like monolayers constructed from DOPC, SM and cholesterol (1/1/1) induced an increase in surface pressure. The π change values were, however, much smaller than those observed in the case of a natural PE/PC (6/4) monolayer. An increased binding capacity of liposomes prepared from a “raft-like” mixture of lipids for spectrins could also be concluded from the pelleting assay. 2.2. Lipid-binding sites in spectrins Spectrins are multidomain proteins which show specific structural features: one is that it forms an elongated segmental structure built of a series of repeating units, each of about 106 amino acid residues forming a triplehelical motif. The other feature is the fact that outside typical segments and within them there are domains which are responsible for the functions of spectrin. The most important functional domains outside typical spectrin repeats are: actin-binding domain (two CH motifs located at amino terminal region of -subunit of spectrin) (Karinch et al., 1990) and SH3 domain present in atypical, 10th segment of ␣-spectrin. Examples of other domains are: ankyrin-binding domain located in part in
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14th repeat and in 15th unit of -spectrin (Kennedy et al., 1991; Baines, 2003) and Ca2+ -binding EF-hand motifs located in carboxyl terminal region of ␣-subunit (Buevich et al., 2004). The consequence of this multifunctionality is the existence of several lipid-binding activities connected with the occurrence of different binding sites: (1) a “general” phospholipids-binding activity characteristic for the spectrin repeat motif (DeWolf et al., 1997; LeRumeur et al., 2003), (2) specific for aminophospholipids (PE and/or PS-binding sites located in the -subunits (e.g. An et al., 2004a,b, 2005; HryniewiczJankowska et al., 2004) and (3) specific for phosphatidylinositol 4,5-bisphosphate, characteristic only for longer -spectrin isoforms which poses Pleckstrin Homology (PH) domain (Saraste and Hyvonen, 1995; Wang et al., 1996). The first kind of the lipid-binding activity that could be attributed to the spectrin repeat motif was actually studied by using a series of constructs containing the second repeat of the human dystrophin rod domain (Kahana and Gratzer, 1995). DeWolf et al. (1997) observed that properly folded fragment corresponding to the second repeat unit of the rod domain is able to modify the properties of lipid membranes (inducing of a large increase in surface shear viscosity of the monolayer) containing anionic phosphatidylserine. Moreover, this property is dependent on the native structure as the unfolded expressed fragment had no effect. Further studies of LeRumeur et al. (2003) showed the engagement of tryptophan residues in this binding, as in the presence of small, unilammellar vesicles containing PS the fluorescence characteristics changed dramatically indicating the tryptophan residues changed their environment into more hydrophobic. The accessibility of these residues from the hydrophilic solvent for quenchers was also limited. These authors conclude that dystrophin rod lies along the membrane surface. Similar experimental approach should be undertaken to determine whether or not the same conclusion could be drawn with respect of spectrin–phospholipids interactions. The second class of lipid-binding sites seems to be confined to certain regions of spectrin molecule which show some aminophospholipid and anionic phospholipids specificity. One of the approaches to identify this class of sites was to clone and express systematically all the fragments of both subunits of erythrocyte spectrin and test their binding to liposomes prepared from PS and its mixture with PC. Studies of An et al. (2004a,b) indicate that binding sites for PS containing vesicles are located in repeats 8–10 of the ␣-subunit and in repeats 2, 3, 4, 12, 13, 14 and in the non-homologous amino termi-
nal region of the -subunit. Their further studies (An et al., 2005) concerning non-erythroid spectrin indicated that PS-binding site is located in repeats 9–11 and in the amino terminus of ␣-subunit and in amino terminal region and repeat units 2 and 3 of the -subunit. Our early attempt to identify the main amphipathic compound-binding site of spectrin revealed its close proximity to the ankyrin-binding domain (Sikorski, 1988) what suggested a functional relationship between ankyrin and lipid binding by erythroid spectrin. Indeed when the effect of purified ankyrin on spectrin binding to phospholipid vesicles was tested, an inhibition of this interaction was observed (Białkowska et al., 1994). The effect was greater for the vesicles containing PE (PE/PC 3:2) for which 60% inhibition was found compared to 10–20% inhibition for PS/PC vesicles. Almost identical results were obtained using a monolayer technique. Dixon-type analysis indicated a competitive mechanism of inhibition of PE/PC vesicles or monolayer binding to spectrin by ankyrin. Tetrameric spectrin bound similarly to PE/PC monolayer but inhibition with ankyrin suggests that only one of the two possible binding sites is engaged in this interaction (Białkowska et al., 1999). Moreover, when interactions of non-erythroid (brain) spectrin with PE/PC monolayers in the presence of ankyrin were analysed, a similar level of inhibition of these interactions by ankyrin was observed (Diakowski et al., 1999). Also, when isolated erythroid spectrin -subunit was introduced into the subphase of PE/PC monolayers in the presence of ankyrin the inhibition was even stronger, i.e. a three-fold lower concentration of ankyrin was needed to induce the same effect. If the ␣-subunit was used instead of  its effect on the monolayer surface pressure was small and entirely insensitive to incubation with ankyrin (Białkowska et al., 1999; Diakowski et al., 1999). It should be noted also that binding of brain spectrin to anionic phospholipid monolayers was also competitively inhibited by purified erythrocyte ankyrin (Diakowski and Sikorski, 2002). As this event could be a subject of the regulation and could be engaged in the possible mechanism of function of the membrane skeleton; we decided to test whether an ankyrin-binding domain which was identified by Kennedy et al. (1991) would show the same lipid-binding properties as spectrin molecule and to identify an ankyrin-sensitive, lipid-binding site in erythroid and non-erythroid spectrins (Hryniewicz-Jankowska et al., 2004; Bok et al., unpublished). The results of the experiments document further the ability of cloned, expressed and purified erythroid -spectrin’s ankyrinbinding domain to bind PE-rich mono- and bilayers. We found that full length ankyrin-binding domain binds
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PE/PC mono- and bilayers with affinity similar to this of native spectrin dimer and that this binding is inhibited by erythrocyte ankyrin. Binding studies were performed by: (i) pelleting assay using PE/PC liposomes and fluorescently labelled expressed polypeptides, (ii) inhibition assay in which unlabelled, expressed polypeptides were used to inhibit binding of purified, labelled red blood cell spectrin to PE/PC liposomes and (iii) in surface plasmon resonance technique experiments where binding of purified, expressed polypeptides to PE/PC monolayer deposited on hydrophobic sensor surface. Results of these experiments indicate that ankyrin-binding domain truncated mutants, that retain amino terminal part bind the PE/PC mono- and bilayers with comparable to full length ankyrin-binding domain affinity and capacity. They also effectively compete for lipids with purified, labelled spectrin. On the other hand, truncated mutants which lack eight or 38 amino acid residues from Nterminal region show, at least an order of magnitude lower affinity and much higher maximal binding capacity. They also compete weakly with spectrin for phospholipid vesicles. The expressed full length domain induced small decrease in order parameter of PE/PC membranes when probed with 5 -doxylstearate similar to the effect of purified spectrin while mutant lacking 38 residues from amino terminus induced small increase in order parameter which was similar to the effect of bovine serum albumin. Similar results were obtained with ankyrin-binding domain of non-erythroid -spectrin. Cloned and expressed fragments of non-erythroid (human brain) -spectrin encompassing a sequence corresponding to an ankyrin-binding domain was found to bind phospholipids mono- and bilayers in a similar way to intact molecule of brain spectrin or erythroid spectrin. Again truncated mutant of this domain which retained amino terminal region bound lipid mono- and bilayers with the affinity similar to full-length domain. Mutants in which this region was deleted bound phospholipids with lower affinities and higher capacities and this binding was insensitive to inhibition with purified ankyrin. Transient expression of HeLa cells with GFPconjugated construct encoding full-length ankyrinbinding domain of either erythroid or non-erythroid -spectrins induced changes in cell morphology and aggregation of membrane skeleton. These changes were observed neither in cells transfected with a construct encoding GFP-conjugated ankyrin-binding domain truncated at amino terminus by 38 amino acid residues nor in cells transfected with a vector encoding only GFP (Bok et al., submitted for publication).
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The above mentioned experimental results indicate that at least in erythroid spectrin the binding site located within or close to ankyrin-binding domain were identified by our and Mohandas groups. It seems that there is a good chance that these sites (segment 12–14 and amino terminal part of the ankyrin-binding domain) are overlapping. The difference concerns nonerythroid -spectrin; our results indicate essentially the same features for both -spectrins while Mohandas group could not identify this site in their constructs. It should be noted that rather high degree of identity between erythroid and non-erythroid spectrins can be found comparing the amino terminal regions ankyrinbinding domains (helix C of repeat 14) of these proteins (Kennedy et al., 1991; Baines, 2003). In our hands binding of PS/PC (3:2) monolayer to bacterially expressed ankyrin-binding domain and its deletion mutants retaining N-terminal region of non-erythroid spectrin occurred with KD ’s in nanomolar range what was measured by SPR technique (Bok, submitted for publication). Pleckstrin homology domain is an example of the third kind of lipid-binding sites specific for phosphoinositides. This kind of PIP2 binding site which is present in non-erythroid spectrins, I2 and II2 and also in many regulatory and cytoskeletal proteins (Wang et al., 1996; Niggli, 2001) is approximately 100 amino acid residues in length. Binding of PIP2 is inhibited competitively by IP3 indicating the specificity for inositol ring (Saraste and Hyvonen, 1995). When PH domain of I2 spectrin was fused with green fluorescent protein and expressed in COS7 cell, it was localized to plasma membrane (Wang et al., 1996). 2.3. Possible role of spectrin–phospholipid interactions It was suggested previously that sites of interactions of spectrin with lipids function as additional support points to the membrane bilayer increasing its mechanical stability. This would be the function of the “general” sites, best characterized in the case of dystrophin repeat segment (see above). It should be noted that almost every fragment of spectrin studied in our laboratory to date, display lipid-binding activity, characterised with a KD in order of 105 –10−6 M. The lipidbinding activities are confined to the specific regions or domains such as PH domain and are highly specific towards PIP2 and are characteristic only for certain isoforms. Lipid-binding sites of limited specificity and moderate-to-high affinity (KD ∼ 10−7 –10−8 M) are
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confined essentially to two regions of spectrin molecule: one located to the centre of the tetramer and another located near its end and they coincide with, or are close to, the attachment sites for the proteins that mediate linkage with membrane bilayer. An et al. (2004a) suggested that the interactions with PS modulate interactions of spectrin with linking proteins such as ankyrin or protein 4.1. They also suggest that spectrin regulates the state of the bilayer, possibly generating or stabilising PS patches (An et al., 2004b). As was mentioned above, we found that binding of aminophospholipid mono- or bilayers at one of these sites was inhibited by ankyrin (Białkowska et al., 1994, 1999). The same was observed for non-erythroid spectrin (Diakowski et al., 1999) and moreover, for cloned and artificially expressed ankyrinbinding domain (Hryniewicz-Jankowska et al., 2004) and its truncated mutants (for details see above). We have proposed that PE-rich domains (and also to lesser extent PS or anionic phospholipids-rich domains) would serve as membrane attachment site for spectrin in situations in which ankyrin is either deficient or its affinity for spectrin is reduced (Białkowska et al., 1994; Sikorski and Białkowska, 1996). Some examples of evidence of such situations are: (i) erythrocytes of mutant mice, whose erythroblasts fail to synthesise ankyrin, still accumulate about 50% of normal amount of spectrin (Bodine et al., 1984), (ii) erythrocytes of ankyrin-deficient mice which contain normal skeletons but lack AE1 tetramers (Yi et al., 1997), (iii) also the phosphorylation of ankyrin decreases its ability to bind spectrin (Liu et al., 1985). In all these situations, aminophospholipid-rich (primarily PE) domains would serve as anchors substituting ankyrin and ensuring preservation of the mechanical properties in the membrane skeletal lattice. This would explain why mice with a disrupted gene coding for AE1 protein are characterized by severe spherocytosis despite the presence of normal membrane skeletons (Southgate et al., 1996) in contrast to the above-mentioned cases of ankyrin deficiency. Possibly in this case ankyrin inhibits binding of spectrin to the inner layer of membrane lipids. 3. Spectrin–raft interactions Plasma membrane is laterally inhomogeneous. Although the presence of lipid rafts has not been proved to appear in vivo so far (Lichtenberg et al., 2005), they remain an attractive subject. Lipid raft model is build mostly on the data aquired through the isolation of so called DRMs (detergent-resistant membranes). From what we know, DRMs are rich in cholesterol and glycosphingolipids and are resistant to the extraction with cold non-ionic detergent (the most often used is 1%
Triton X-100). They contain a specific subset of proteins such as caveolin, flotillins, stomatin, proteins that anchor to the membrane via glycosylphosphatidylinositol molecule or mirystoyl or palmitoyl residue. Lipid rafts are thought to be engaged not only in signaling pathways, sorting of membrane proteins and lipids but also play role in pathogen entry into the cell or virus budding (reviewed by Grzybek et al., 2005). Although spectrin is observed as a part of DRM proteome in erythroid (Salzer and Prohaska, 2001) and non-erythroid cells (von Haller et al., 2001; Nebl et al., 2002; Leshchyns’ka et al., 2003) its presence in the DRMs is controversial (Samuel et al., 2001; Murphy et al., 2004). Spectrin is co-isolated with the cholesterol enriched fraction depending on the concentration of the detergent, pH and concentration of the protein in the sample (Ciana et al., 2005; Murphy et al., 2004). The concentration of the used detergent, its type and even the presence during the gradient centrifugation may drastically change both the quality and quantity of proteins isolated with the DRMs fraction (Schuck et al., 2003; Murphy et al., 2004; Korzeniowski et al., 2003). Remarkably, the presence of spectrin in the DRMs is not followed by the presence of the usual anchors of the membrane skeleton to the membrane. The normal band 3, ankyrin, spectrin or the junctional complex (actin, protein 4.1, p55, spectrin) are not represented in the DRM proteom (Salzer and Prohaska, 2001). Another group (Samuel et al., 2001; Murphy et al., 2004), however, showed the presence of band 3 in the erythroid rafts but they simultaneously found an absence of spectrin in this fraction. An important question is however, if spectrin is associated with lipid rafts, then how it is connected to them. Some data suggest that these interactions are sensitive to pH or ionic strength (Ciana et al., 2005). The protein candidates for spectrin anchoring to the membrane must then be a part of the usually isolated DRM proteome. Stomatin (1,00,000 copies/cell) which is abundantly represented in the DRMs could be one of such proteins keeping in mind that the stomatinlike protein 2 (4000 copies/cell) was proved to associate with spectrin (Wang and Morrow, 2000). This hypothesis has only one drawback—stomatocytes (erythrocytes that lack stomatin in their membranes) still have spectrin associated with lipid rafts (Salzer and Prohaska, 2001). Alternatively to protein–protein interactions the protein–lipid interactions are possible. If this hypothesis would be correct then the lipids themselves should be responsible for the maintenance of spectrin in rafts. Spectrin’s affinity for special groups of phospholipids such as PE, PS and PI has been well established (see above). In fact recent data suggest that PI and PS occur in DRM in similar proportions as in the whole cell membrane
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(Koumanov et al., 2005). Another possibility of anchoring spectrin to lipid rafts is its palmitoylation. The existence of erythrocyte protein-palmitoyl acyltransferase which could be responsible for such activity supports such a possibility (Das et al., 1997). Altogether, the recruitment of spectrin to lipid rafts via interactions with lipid molecules cannot be excluded. Red blood cells are often used in raft studies because they lack inner cell organelles. However, the advantage of the isolation of DRMs only from the plasma membrane has also some disadvantages, because nobody can answer the question what the rafts are responsible for in erythrocytes. Therefore erythrocyte membrane rafts may be completely inactive and it could be difficult to compare to the other cells. Surprisingly, in more complex cells, such as neurons, the question of spectrin presence in rafts seems to be much easier to explain. The Triton-insoluble I-spectrin is responsible for the NCAM/PKC2 redistribution in neurons and takes part in the neurite growth. In neurons all major NCAMs bind to spectrin and promote spectrin incorporation to DRMs, however not all interactions between various NCAMs and spectrin are maintained after the disruption of rafts with methyl-betacyclodextrin. GPI-anchored NCAM 120, associates with the I-spectrin through lipid rafts (Leshchyns’ka et al., 2003). More complex picture of spectrin association with lipid rafts could be concluded after the proteomical studies on leukocytic DRM (von Haller et al., 2001; Nebl et al., 2002; Li et al., 2004). In contrast to erythrocytes, these cells have a vast number of proteins associated with DRMs, that greatly exceeds the number of proteins in RBCs. The presence of actin, myosin or supervilin together with other cytoskeleton associated proteins allows to create much more sophisticated model of a spectrin–DRM interaction. One of the groups (Nebl et al., 2002) proposed such a possibility. The typical raft integral proteins such as flotillins and stomatin are accompanied by the GPIanchored proteins (extracellular) and by the dually acylated signaling proteins (intracellular). Fodrin and Factin attaches to the DRM via their interactions with supervilin, ␣-actinin, myosin IIa and myosin Ig. To prove this and other models further studies need to be undertaken. The question, whether the isolated fractions correspond to real “rafts” (Heerklotz, 2002) remains unanswered. However, the discovery of spectrin–DRM interaction may solve many intriguing questions concerning one of the roles of spectrin in the cell. It seems that spectrin, originally recognized as structural protein, still has many hidden secrets.
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