Anionic phospholipids, interfacial binding and the regulation of cell functions

Biochimica et Biophysica Acta 1483 (2000) 199^216 www.elsevier.com/locate/bba

Review

Anionic phospholipids, interfacial binding and the regulation of cell functions Andrew G. Buckland, David C. Wilton * Division of Biochemistry and Molecular Biology, School of Biological Sciences, University of Southampton, Bassett Crescent East, Southampton SO16 7PX, UK Received 5 August 1999; received in revised form 5 October 1999; accepted 25 October 1999

Contents 1.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Interfacial binding and anionic phospholipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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General properties of anionic phospholipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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4.

Anionic phospholipids, interfacial binding and the regulation of enzyme activity 4.1. Phospholipases A2 (PLA2 s) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. sPLA2 s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. PA exposure and the regulation of human sPLA2 activity . . . . . . . . . . . . 4.4. cPLA2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5. PKC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.6. The role of the C2 domain in the modulation of other protein functions . 4.7. CTP:phosphocholine cytidylyl transferase (CT) . . . . . . . . . . . . . . . . . . . . 4.8. Activation of DnaA protein by anionic phospholipids . . . . . . . . . . . . . . .

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Speci¢c anionic phospholipids and their role in the regulation of cell functions . . . 5.1. Phosphatidyl serine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. PS exposure to the extracellular environment . . . . . . . . . . . . . . . . . . . . . . . . 5.3. The antiphospholipid syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4. Annexin V binds to PS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5. PI and phosphoinositides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.6. PH domains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.7. Phospholipase C-N1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.8. Phosphoinositide-3-kinase, FYVE domains and other domains that recognise phosphoinositides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.9. Other structural roles for phosphoinositides . . . . . . . . . . . . . . . . . . . . . . . . . 5.10. PG and CL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.11. PA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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* Corresponding author. 1388-1981 / 00 / $ ^ see front matter ß 2000 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 8 - 1 9 8 1 ( 9 9 ) 0 0 1 8 8 - 2

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Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction A considerable phospholipid asymmetry exists across the bilayer of the biological membrane, particularly the plasma membrane, and this asymmetry plays a major role in cell function [1^4]. The asymmetry is best illustrated by the fact that the external monolayer of the mammalian cell is made up almost exclusively of neutral zwitterionic phospholipids such as phosphatidylcholine (PC) and sphingomyelin (SM) together with some phosphatidylethanolamine (PE). In contrast the monolayer facing the cytoplasm of the cell contains anionic phospholipids as a major component together with the majority of the PE. These anionic phospholipids, which account for about 30% of cell phospholipids, include phosphatidylserine (PS), phosphatidylinositol (PI) and its phosphorylated derivatives, phosphatidylglycerol (PG) and cardiolipin (CL). A number of physiological roles have emerged for these anionic phospholipids that a¡ect intracellular functions and, in the case of PS, the interaction of the cell with extracellular proteins. These biological functions are normally initiated by the binding of speci¢c proteins to the phospholipid interface resulting in a conformational change linked to protein activation and the biological response. The overall phenomenon can be represented as follows: P ‡ phospholipid…n† 3P ‡ L3P L Dbiological response where P is a protein molecule; phospholipid…n† is the phospholipid interface; P* is the activated protein bound to the interface; P*L represents speci¢c binding of a ligand linked to the biological response such as catalysis. This review will concentrate on the role of anionic phospholipids in a non-speci¢c interfacial binding step (P+phospholipid…n† 3P*). Such non-speci¢c interactions are often identi¢ed as involving interac-

tions with PS, this being normally the most abundant anionic phospholipid in mammalian systems. The review will also include examples where binding involves presumptive speci¢c interaction with individual anionic phospholipid molecules at de¢ned sites on the interfacial surface of the protein as part of the overall interfacial binding phenomenon. At this time speci¢c interfacial binding is exempli¢ed by the speci¢c binding to phosphoinositides by way of pleckstrin homology (PH) domains as part of the interaction with the membrane surface. Clearly, in the absence of de¢nitive crystal structures it will often be di¤cult to discriminate what is non-speci¢c interfacial binding and a speci¢c interaction with de¢ned residues on the interfacial surface of the protein. The role of anionic phospholipids as substrates for speci¢c enzymes or ligands for phospholipid transfer proteins will not be considered. As this article covers a wide research area, it will mostly cite references from 1997 and recent reviews. A review on the electrostatics of lipid surfaces by Langner and Kubica [5] was published after submission of this manuscript and is of particular relevance. 2. Interfacial binding and anionic phospholipids As de¢ned, interfacial binding (P+phospholipid…n† 3P*) is relatively non-speci¢c and the nature of the interactions will be dictated both by the phospholipid composition and structure of the interface as well as the nature of the interfacial binding surface of the protein. The presence of the anionic phospholipid will facilitate the binding of the protein by initial electrostatic interactions and a common characteristic of this initial non-speci¢c event is the requirement for a minimum interfacial concentration of anionic phospholipid (anionic charge density) before substantive protein binding or biological activity is observed. In addition to surface interactions, interfacial events can involve partial membrane penetration by

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the protein. The presence of anionic phospholipids usually facilitates this process as a result of e¡ects on phospholipid packing density and perturbation of phospholipid bilayer structure. This membrane penetration by the protein may involve amphipathic helices or amino acids modi¢ed with acyl or prenyl groups [6]. The special role of aromatic residues such as tryptophan in membrane penetration has been highlighted [7^9]. Conformational changes at the interface as a result of high local concentrations of hydrogen ions associated with negatively charged phospholipid head groups have also been discussed [10]. As an alternative mechanism to membrane penetration by the protein, Kinnunen [11] has argued the case for a phospholipid bridging mechanism in which a single acyl chain of the phospholipid is reorientated out of the surface monolayer in an extended conformation and binds to a non-polar cavity of the protein. Such a phenomenon has also been proposed during membrane fusion [12]. The phenomenon of protein translocation to the interface can play a major regulatory role in protein function and anionic phospholipids are an integral part of this phenomenon. Important enzymes that are regulated by translocation to the phospholipid surface and have been subjected to detailed investigations include cytosolic phospholipase A2 (cPLA2 ) [13,14], protein kinase C (PKC) [15,16] and CTP phosphocholine cytidylyl transferase (CT) [17,18]. The distinction between interfacial binding and classical substrate (ligand) binding at an active site may be di¤cult to resolve as both events will participate in the binding of protein to a phospholipid interface. In the case of secretory PLA2 s (sPLA2 ), these enzymes can act in a highly processive scooting mode on anionic vesicles where the enzyme remains bound to the phospholipid surface during many catalytic cycles and proves that the interfacial binding of these enzymes to the membrane (P3P*) and classical formation of the Michaelis complex during catalysis (P*+L3P*L) are distinct steps [19]. 3. General properties of anionic phospholipids Anionic phospholipids will produce a negatively charged interface since, with the exception of sphingosine [20], there are no natural positively charged

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membrane lipids that might be introduced to neutralise this negative charge. Thus attention has focussed on the role of electrostatics in interfacial binding to cationic regions on the protein or peptide. However, the normal anionic phospholipids such as PS, PG, CL and phosphatidic acid (PA) will tend to perturb the planar condensed PC-based bilayer into which they are incorporated. These perturbing e¡ects will be dictated by the size of the head group relative to the acyl chains together with head group repulsion and how this will be neutralised by the presence of counterions such as calcium ions. The net result is a lower packing density and the presence of packing density perturbations or boundary defects that will facilitate non-polar bilayer penetration by the protein. The participation of anionic phospholipids in membrane-protein interactions has been reviewed in detail [1^4,10,18,21^29] with a particular focus on the ability of certain lipids to form non-lamellar phases. Although such non-lamellar phases have yet to be observed in biological as opposed to arti¢cial membranes it may be the propensity of such lipids to form such structures resulting in curvature defects that may have critical e¡ects on the quality of the interface and be the basis of membrane perturbation. The ability of anionic phospholipids to perturb the interface means that they can promote both polar (electrostatic) and non-polar interactions. It is not unreasonable to assume that an initial electrostatic interaction is followed by events involving membrane penetration and non-polar interactions. Non-speci¢c multiple electrostatic interactions normally demonstrate a threshold of anionic charge density that is required to promote the physiological response. This threshold of anionic phospholipid concentration when expressed as a mol% of the total phospholipid is normally between 10 and 20 mol% and there are numerous examples in the literature where the biological response to anionic phospholipid concentration demonstrates this type of sigmoidal relationship. The phenomenon normally shows a lack of head group speci¢city with PS behaving similarly to PG and PI, although PA, which is dianionic at higher pH values in vitro, can be anomalous. It should be noted that interfacial binding is usually more sensitive to the presence of CL (diphosphatidylglycerol) with the threshold for this phospholipid being normally 6 10 mol% re£ecting the presence of two phosphate

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groups per mole of this unusual phospholipid. The polyanionic phosphoinositides can demonstrate similar properties in terms of non-speci¢c binding. These overall binding characteristics re£ect the surface charge density as is characteristic of multiple electrostatic interaction between the protein and phospholipid interface. The theoretical basis of the electrostatic binding of basic peptides and proteins to membranes containing anionic phospholipids has been considered and these models highlight the e¡ect of mol% of acidic lipid, the ionic strength of the solution and the positive charge of the peptide on the strength of these interactions [30^32]. As might be anticipated, binding energies determined from model studies are greater than that predicted from purely electrostatic interaction implying contributions from other types of interactions. The role of non-polar interactions and the relative contribution of polar and non-polar interactions to interfacial binding is an area of considerable interest and, for example, detailed charge reversal mutagenesis studies on bee venom sPLA2 have directly highlighted the role of non-electrostatic interactions in interfacial binding [33]. The role of hydrophobic interaction in interfacial binding and the particular contribution of aromatic residues such as tryptophan have been investigated in detail [7^9]. Recently, model studies with peptides indicate if the hydrophobicity is below a threshold that promotes spontaneous membrane insertion, then primary electrostatic attractions provided by anionic phospholipids become essential for peptide binding and insertion into membranes [34]. In addition to interfacial e¡ects, the role of anionic phospholipids in promoting peptide and protein insertion into biological membranes has been reviewed [10,35]. Similarly, anionic phospholipids have a role in determining the topology of intrinsic membrane proteins. Thus, the orientation of many membrane proteins during assembly may be dictated by the positive-inside rule for the protein re£ecting the asymmetric distribution of anionic phospholipids within cellular membranes [36].

4. Anionic phospholipids, interfacial binding and the regulation of enzyme activity In this section those enzyme will be considered that have been investigated in detail with respect to non-speci¢c interfacial binding, sometimes referred to as membrane translocation. In many cases crystal structures are now available that allow a detailed description of the molecular events involved in binding. 4.1. Phospholipases A2 (PLA2 s) An expanding number of PLA2 s have been identi¢ed [37,38]; however, detailed kinetic and structural information is only available for the groups I, II and II secreted and the group IV cytosolic PLA2 s. A detailed understanding of the group V sPLA2 is emerging [39] but in the absence of a crystal structure is not discussed. 4.2. sPLA2 s The low molecular mass sPLA2 s have a high a¤nity for anionic interfaces and such interfaces have been instrumental in de¢ning the interfacial binding step as a discrete step in the overall mechanism of such enzymes. Indeed, it was the very high a¤nity that allowed the development of the concept of scooting kinetics whereby the enzyme remains bound to the interface during many catalytic cycles and in the case of small unilamellar vesicles (SUVs) will allow the complete hydrolysis of the outer monolayer of the vesicle without release of the enzyme [19,40]. Although it had been presumed that electrostatics would play a very major role in this interfacial binding, the fact that a wide variety of sPLA2 s with a very considerable variation in the cationic character of the interfacial binding surface [41] all showed high a¤nity for anionic interfaces suggested that other factors might be contributing to interfacial binding. The presence of anionic phospholipid will reduce packing density and facilitate non-polar interactions as a result of membrane perturbations in addition to providing the basis for electrostatic interactions. In the case of the bee venom enzyme, extensive charge reversal mutagenesis studies were performed in which up to ¢ve of the six cationic residues on the interfa-

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cial binding surface of the protein were changed to glutamates. Such mutations had only a modest e¡ect and have highlighted the role of non-electrostatic interactions in interfacial binding of this enzyme [33]. It is probable that electrostatic interaction plays a major role in the functioning of the highly cationic (pI s 10.5) human group IIa sPLA2 (human sPLA2 ). The physiological function of this enzyme remains to be clearly de¢ned but the enzyme is noted for its poor penetrating properties and its almost zero activity on condensed zwitterionic interfaces and mammalian cell membranes [42^45]. In contrast, the enzyme expresses high activity on anionic interfaces such as that presented by PG. This property is certainly consistent with a proposed major physiological role of the enzyme, namely that of an antibacterial protein capable of hydrolysing the phospholipids in the membrane of Gram-positive bacteria [46^49]. Such bacteria do not normally contain PC but are rich in anionic phospholipids, particularly PG. Therefore the outer monolayer of the cell membrane is anionic and a potential substrate for the enzyme. However, the enzyme must ¢rst penetrate the highly anionic peptidoglycan bacterial cell wall, a process that is probably facilitated by the very cationic nature of this enzyme (Buckland and Wilton, submitted for publication). The potent ability of this human enzyme to hydrolyse the bacterial membrane, once cell wall penetration has been achieved, contrasts with the inability of the enzyme to hydrolyse the host plasma membrane. A major factor in this minimal activity against selfmembranes is a lack of an interfacial tryptophan, a residue now recognised for its ability to penetrate the condensed zwitterionic interface where it achieves maximum stability by residing in the interfacial region of the phospholipid monolayer [7^9]. Mutagenesis of the human enzyme to insert a tryptophan in the interfacial region (a V3W mutant) dramatically enhances the ability of this enzyme to hydrolyse PC vesicles and must be an important contact between protein and membrane [44]. The precise role of electrostatics in interfacial binding of the human enzyme has been investigated by producing a large number of charge reversal mutants and these results [50,51] highlight the non-speci¢c nature of the interaction, the major feature that a¡ects binding is loss of total positive charge. How-

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ever, such comprehensive mutagenesis data provide little information on how the enzyme sits on the interface and, in the absence of a crystal structure for an interfacially bound enzyme, more novel approaches are required. In the case of the bee venom, an elegant electron spin resonance approach has demonstrated that cationic residues on the interfacial binding surface appear not to make direct contact with the interface and that such direct interactions involve hydrophobic residues [52,53]. In this method [52], electron spin resonance, together with membrane impermeant and permeant spin-relaxing agents, are used to determine the distance between nitroxide spin labels, placed at speci¢c points on the protein surface, and the membrane surface. 4.3. PA exposure and the regulation of human sPLA2 activity Although antibacterial activity must be a major role for the human sPLA2 , there is considerable evidence implicating this enzyme in the in£ammatory response of the host cells [38,54]. An important question is how might the human sPLA2 be involved in the in£ammatory response if this extracellular enzyme is unable to hydrolyse the normal cell membrane? The possibility that the enzyme might be capable of attacking the perturbed membrane of normal cells thus becoming pro-in£ammatory has been considered [55]. An attractive hypothesis is the generation of anionic phospholipid in the external monolayer of the plasma membrane and one such anionic phospholipid of particular interest is PA. The role of PA in cell function is an area of active research and speculation. This phospholipid is normally present at very low concentrations in mammalian cell membranes being less than 5 mol% that of the PC concentration. In addition to being an important biosynthetic intermediate this phospholipid is unique in that it can be formed in situ from other phospholipids by the action of phospholipase D (PLD). The phospholipid is also partially dianionic with a pKa2 of 7.6 when present in predominantly PC bilayers [56]. This dianionic character makes PA vesicles particularly sensitive to calcium ions and vesicle aggregation and fusion are promoted both by the calcium ion concentration and the mol% of

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Fig. 1. Stimulation of the ability of human sPLA2 to hydrolyse PC vesicles as a result of the addition of anionic phospholipids. The ability of human sPLA2 to hydrolyse SUVs was assayed using a continuous £uorescence assay that monitors total fatty acid release [159]. The assay was performed in 0.1 M Tris-HCl pH 8.0 containing 0.1 M NaCl and 1 mM CaCl2 . The speci¢c activity was determined using dioleoyl-PC (DOPC) at 63 WM or with increasing amounts of anionic phospholipid, dioleoyl-PA (DOPA) or dioleoyl-PS (DOPS), keeping the total glycerophospholipid concentration constant. For experiments involving DOPS the assays were also performed in the presence of dioleoyl-PE (DOPE; 20 mol%) or cholesterol and SM each added at 15 mol%. An experiment containing DOPE, cholesterol and SM gave a curve virtually identical to that without these additions (DOPC/DOPS only) and has been omitted for clarity. b, DOPC/DOPA ; F, DOPC/DOPS; R, DOPC/cholesterol/SM/ DOPS; S, DOPC/DOPE/DOPS; Values are means þ S.D. ; n = 3.

PA in the vesicle. A further and potentially physiologically important feature is the possible ability of calcium ions to promote PA clustering [57], resulting in phospholipid domain formation. Domain formation remains an elusive biological phenomenon in the absence of proteins binding to the membrane interface. However, it has been argued that domains of anionic phospholipids may be induced or stabilised when basic proteins bind to the interface [58] and such domain formation is supported by in vitro data, e.g. [59,60]. The ability of PA to stimulate human sPLA2 to hydrolyse PC-based interfaces has been investigated [43]. Not only does the addition of low concentrations of PA to vesicles prepared from PC enhance hydrolysis by human sPLA2 (Fig. 1) but also pretreatment of pure PC-containing vesicles with bacterial PLD produce both a time- and dose-dependent

increase in hydrolysis [43]. Of particular interest is the fact that pretreatment of whole mammalian cells in suspension with bacterial PLD dramatically enhances their susceptibility to human sPLA2 . Pretreatment produces a time- and dose-dependent response in that the cell membrane becomes sensitive to hydrolysis by added human sPLA2 . Thus, hydrolysis of RAW 264.7 macrophages is observed at a concentration as low as 100 ng/ml of human sPLA2 in the medium whereas, prior to PLD treatment, in excess of 10 Wg/ml of enzyme is required to detect signi¢cant cell hydrolysis under these assay conditions. Blood levels of human sPLA2 in septic shock can rise over 100-fold to the order of 1 Wg/ml, a concentration that would normally produce minimal hydrolysis of cell membranes. It remains to be established if production of PA in the surface monolayer of intact cells in vivo can provide the necessary activation to initiate an in£ammatory response in the presence of in£ammatory levels of the human sPLA2 . Although there is no indication that cellular PLD is observed extracellularly, an equivalent glycosylphosphatidylinositol-speci¢c PLD is found in serum [61] and could generate PA on the cell surface. In addition an ecto-phosphatidic acid phosphohydrolase has been reported [62] which would suggest external exposure of PA. Human sPLA2 is able to generate lysophosphatidic acid (LPA) from shed microvesicles [63], indicating the presence of PA in these vesicles while LPA is present in arthritic synovial £uid [63] where human sPLA2 is also present at high concentrations. PA, along with PG, is reported to be a preferred substrate for the human enzyme [64]. Overall, it would appear that the human sPLA2 has evolved to hydrolyse only those aggregated phospholipid interfaces that contain a signi¢cant proportion of anionic charge which will facilitate interfacial binding and catalysis. Enzyme speci¢city would suggest that PG and PA are preferred anionic phospholipids. To date the highly anionic bacterial cell membrane appears to best o¡er these characteristics. Although PS represents a classic physiological example of exposure of an anionic phospholipid on the cell surface (see below), we have been unable to demonstrate signi¢cant activation of PC vesicle hydrolysis using concentrations of PS that would be anticipated if this phospholipid reached equilibration

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across the plasma membrane bilayer. In Fig. 1, the e¡ect of an increasing mol% PS in PC vesicles on enzyme activity is plotted in the presence or absence of other lipids (PE, sphingomyelin and cholesterol) also present in the outer monolayer of the plasma membrane. The curve is typical of numerous published examples where a biological activity is plotted against mol% anionic phospholipid and demonstrates the classical response threshold above about 10 mol% anion, characteristic of multiple non-specific electrostatic interactions (see above). The presence of the neutral lipids, PE, sphingomyelin and cholesterol (Fig. 1), does not signi¢cantly a¡ect the threshold of anionic phospholipid density required to enhance human sPLA2 activity, consistent with a dominant role for electrostatics in this process. 4.4. cPLA2 Cytosolic PLA2 is a highly regulated enzyme that speci¢cally releases arachidonic acid from membrane phospholipid and plays an important role in cell regulation and the in£ammatory response [13,14]. The availability of a crystal structure for the C2 domain of cPLA2 [65,66] and the more recent publication of the complete crystal structure [67] provide an opportunity to brie£y highlight a few features of this important interfacial enzyme. A possible role for anionic phospholipids, particularly phosphoinositides, in stimulating enzyme activity had been recognised from kinetic studies [68]. Recently a potent increase in a¤nity for the binding of cPLA2 to PC vesicles has been shown, speci¢c for phosphatidylinositol-4,5-bisphosphate (PI-4,5-P2 ), coupled with an increase in enzyme activity [69]. The authors proposed the presence of a possible PH domain to mediate this speci¢c e¡ect of PI-4,5P2 , through sequence similarity to phospholipase C-Q (PLC-Q) and 2³ structure prediction. While the reported a¤nity of cPLA2 for PI-4,5-P2 appears to be equal to or greater than other PH domains preferentially binding PI-4,5-P2 , dissociation constants are not comparable between studies, as observed by these authors. The activity of cPLA2 is enhanced to varying extents by PI-3,4-P2 , PI-3,4,5-P3 and PI-4-P; however, the e¡ect of these phospholipids on cPLA2 binding a¤nity was not reported. Our own data ([70]; Buck-

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land and Wilton, unpublished work) showed that PI4,5-P2 enhanced hydrolysis of PC substrate but that PI-4-P and PA were stimulatory to a similar extent. Moreover, no inhibition of the stimulatory a¡ect was achieved by addition of the water soluble head group of PI-4,5-P2 , inositol trisphosphate (IP3 ), suggesting that this enzyme does not possess a true PH domain. Certainly, the recently solved crystal structure of cPLA2 reveals no region with structural homology to other PH domains [67]. The enzyme would appear to bind to the membrane surface utilising only two domains, namely the C2 domain and the catalytic domain. This contrasting with PLC-Q where a third, PH, domain may tether the enzyme to speci¢c phospholipid regions. The C2 domain contains clusters of hydrophobic residues that are believed to penetrate the membrane interface [65], a model that is supported by cysteine-substitution mutagenesis studies [71]. Such hydrophobic interactions may be facilitated by the neutralisation of adjacent anionic residues by calcium while NMR studies in the presence or absence of membrane demonstrated that calcium binding does not cause major conformational changes in the protein [72]. This neutralisation by calcium may be an example of a hydrophobic-electrostatic switch mechanism for interfacial binding exempli¢ed in the case of several myristoylated proteins [6] where protein phosphorylation reduces interfacial binding by adversely a¡ecting electrostatic interactions of cationic residues on the protein with anionic phospholipids. Alternatively, calcium binding to the C2 domain may stabilise the membrane binding hydrophobic loops [71] as has also been proposed for the C2 domain of PLC-N1 [73]. Interfacial penetration by the C2 domain to achieve hydrophobic interaction will be facilitated by reduced membrane phospholipid packing density. Such factors as increased membrane curvature, and the presence of membrane perturbing lipids such as diacylglycerol (DAG) and the PLA2 hydrolysis products lysophosphatidylcholine and arachidonic acid have been implicated in this process. Although a patch of basic residues has been noted that could contribute an electrostatic component to interfacial binding, a speci¢c electrostatic role involving anionic phospholipids remains to be established and mutagenesis studies argue against these cationic residues

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playing a signi¢cant role [74]. Recently, the use of an electron paramagnetic resonance approach [75] has provided strong support for models to describe the binding of the C2 domain to the phospholipid interface based on X-ray [65] and NMR [72] studies. A clear picture of the interaction of the catalytic domain of cPLA2 with the phospholipid interface is not available. The crystal structure [67] highlights the potential involvement of a classical `lipase lid' in this interaction and would be consistent with interfacial activation of this enzyme. Whether anionic phospholipids play an important role in the interfacial binding of the catalytic domain remains to be established. The presence of a basic patch on the protein proximal to the membrane surface would be consistent with anionic lipids enhancing membrane binding as well as providing interfacial perturbation to facilitate membrane penetration. A comparison of the properties of cPLA2 and the isolated C2 domain has highlighted that calcium is no longer required when substrate is incorporated into anionic phosphatidyl methanol vesicles [76]. This phenomenon must re£ect interfacial binding of the catalytic domain because binding of the isolated C2 domain to these vesicles has an absolute requirement for calcium ions [76]. Thus anionic phospholipids can play a major role in the binding of the catalytic domain under appropriate conditions. It is not unreasonable to assume that the high anionic charge density of phosphoinositides (see above) could facilitate calcium-independent membrane binding and catalysis via the catalytic domain by promoting non-speci¢c electrostatic interactions as well as producing interfacial perturbations. Overall, interfacial binding will re£ect the combined properties of the C2 and catalytic domains and be a variable balance between electrostatic and non-polar interactions depending on the nature of the phospholipid interface. The binding of this C2 domain has a major non-polar component re£ecting partial membrane penetration and is normally calcium-dependent. A characteristic of cPLA2 is that the calcium requirement for binding and catalysis can be removed by the use of high salt concentration [77], highlighting a potential major role for hydrophobic interactions in membrane binding. The crystal structure does not provide an obvious explanation, in terms of the recognition of a unique phospholipid bilayer structure e.g. phosphoinositide

binding, to explain why this enzyme translocates to the nuclear envelope on activation [78,79]. Therefore, a speci¢c membrane associated docking protein would be the most likely explanation for this targeted translocation of cPLA2 within the cell. 4.5. PKC It has been recognised for over two decades that PS (along with DAG) was an activator of PKC and this lipid binding is associated with two of the four protein domains associated with this enzyme, namely C1 and C2 [15,16]. Although the binding of DAG to a pocket in the C1 domain has been established, the structural basis for the interaction of PKC with PS, via the C2 domain, is still under investigation and has only been modelled based on the ¢rst C2 domain of synaptotagmin [15]. Mutagenesis studies of the C2 domain of PKC-K have identi¢ed speci¢c calcium ions and cationic residues that are directly involved in electrostatic interactions with anionic phospholipids such as PS at the interface [80]. These data provide direct evidence for a role for the C2 domain of a conventional PKC in interfacial binding. Further mutagenesis studies highlighted the involvement of tryptophan residues in membrane penetration and the resulting hydrophobic interactions [80]. Such data reinforce the thesis that interfacial binding is a variable balance between electrostatic and hydrophobic interactions depending on the protein. A comparison of membrane binding and activation mechanisms of PKC-K and the novel PKC-O highlights the di¡erent modes of interaction with the membrane interface of these two enzymes [81]. The stoichiometry of the interaction with anionic phospholipid has been investigated in detail and in the case of PKC-LII, eight PS molecules are involved in an interaction consistent with the PKC as a multivalent protein interacting with multiple independent PS molecules without an apparent need for cooperativity [82]. Although there appeared to be evidence for speci¢c PS binding sites on the protein based on apparent stereospeci¢city of binding of L-PS relative to D-PS [83], these two isomers also had di¡erent physical properties [83]. The di¡erence in physical properties may be explained by the fact that these two isomers are not simple mirror images but are in fact diastereomers [84] due to the presence of an-

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other chiral centre at the sn-2 position of the glycerol backbone. When L-PS (1,2-sn-phosphatidyl-L-serine) was compared to its mirror image (2,3-sn-phosphatidyl-D-serine) which has identical physical properties, only the L-PS caused activation of PKC. Although this result demonstrates a major role for lipid structure rather than membrane structure in the activation process, the observations do not preclude the importance of membrane structure as a contributing factor [84]. Indeed there has been much debate about whether other anionic phospholipids can replace PS. Because PS is normally the most abundant anionic phospholipid in vivo and the activation is highly sensitive to the mol% of PS [82], it could be argued that in vivo only PS has the abundance to provide the necessary activation of conventional PKCs. 4.6. The role of the C2 domain in the modulation of other protein functions Originally identi¢ed as a calcium-regulated phospholipid binding domain in protein kinase C and also discussed above with respect to cPLA2 , the C2 domain is present in a number of functionally distinct proteins that interact with cellular membranes (reviewed in [85]). Two other proteins with C2 domains and where crystal structures are available are synaptotagmin [86] for which multiple non-speci¢c

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interactions with anionic phospholipids have been con¢rmed [87], and PLC-N1 [88,89]. 4.7. CTP:phosphocholine cytidylyl transferase (CT) The translocation of CT from a cytoplasmic to a membrane location is a classic example of this type of enzyme modulation and a parameter for promoting such translocation is the presence of anionic lipids within the membrane [90^92]. Detailed molecular studies using the cloned enzyme have identi¢ed an amphipathic helix (reviewed in [23]) that senses membrane phospholipid composition and interfacial binding is promoted by anionic phospholipids such as PS in a manner consistent with non-speci¢c electrostatic interactions. Moreover, there are synergistic interactions with DAG and the process is facilitated by enzyme dephosphorylation. The interaction involves both electrostatic and non-polar interactions and anionic phospholipids will not only promote electrostatic interactions, but as a result of producing a looser packed bilayer will facilitate membrane penetration to allow non-polar interactions [18,23]. The mechanism of activation of CT as a result of membrane translocation remains obscure. The characterization of a catalytic fragment lacking the membrane/lipid binding domain established that enzyme activity was no longer sensitive to lipids and expressed high activity similar to that expressed by

Fig. 2. A model for the regulation of DnaA activity through membrane interaction (taken from [97] with permission).

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the wild-type enzyme in the presence of lipid [93]. The result is consistent with a model whereby the lipid binding domain is inhibitory in the absence of lipids while binding to the membrane/lipid interface removes the inhibition. This is in contrast to a model where the enzyme is activated by the lipid binding domain when that domain becomes membrane bound. The catalytic fragment is still dimeric thus strongly suggesting that the lipid binding domain, or phosphorylation, has no a¡ect on a monomerdimer equilibrium linked to activation [93]. 4.8. Activation of DnaA protein by anionic phospholipids DnaA protein initiates Escherichia coli chromosomal replication by binding at oriC to form a large nucleosome-like complex. In the presence of other proteins and ATP, DNA strand opening is promoted to generate two replication forks. The initiation activity in vitro is in£uenced by ATP and ADP. ATP strongly promotes strand opening while the binding of ADP inactivates the process. The replacement of ADP by ATP reactivates the protein and the binding of the DnaA to anionic phospholipids promotes this process in vitro [94]. Genetic data support the relevance of this phospholipid-requiring process in vivo as disruption of genes for anionic phospholipid biosynthesis in E. coli inhibits DnaA function [95]. Binding of DnaA to an anionic phospholipid interface is consistent with an initial electrostatic complex followed by an event involving membrane insertion and a conformational change in the protein that promotes nucleotide release (Fig. 2) [96^98]. A number of cationic residues from two separate K-helices have been implicated by mutagenesis as being involved in binding to anionic phospholipids such as cardiolipin [99,100]. At least one tryptophan is implicated in the process of membrane insertion [97]. 5. Speci¢c anionic phospholipids and their role in the regulation of cell functions In this section it is convenient to discuss individual anionic phospholipids and a number of in vivo roles for speci¢c anionic phospholipids in particular cell functions can be identi¢ed. However, in the absence

of a crystal structure of the protein-phospholipid complex, a speci¢c binding site for that phospholipid on the protein must remain speculative while often in vitro studies demonstrate a broader speci¢city for anionic phospholipids. Speci¢c binding implies a unique binding pocket on the protein for the anionic phospholipid and such speci¢city would normally be characterised as requiring a low ( 6 5) mol% of the anionic phospholipid in the bilayer. At this time, only the phosphoinositides (see below) demonstrate such high speci¢city and where binding to speci¢c domains (e.g. PH domains) has been characterised. 5.1. Phosphatidyl serine PS accounts for most of the anionic phospholipid within the cell being about 10^20 mol% of the total phospholipid in the plasma membrane and endoplasmic reticulum. Therefore it is this anionic phospholipid that would normally make the largest contribution to interfacial e¡ects involving non-speci¢c electrostatic interactions. E¡ects of PS on particular enzyme activities as a result of interfacial binding have already been discussed. In this section the interaction of speci¢c binding proteins to a PS-rich interface and the resulting biological response will be considered. 5.2. PS exposure to the extracellular environment As stated in the Section 1, the outer phospholipid monolayer of the plasma membrane of eukaryotic cells in contact with the extracellular environment is normally neutral and this monolayer only becomes negatively charged under certain conditions. The classic physiological example of the bulk exposure of an anionic phospholipid on the external surface of the cell is that of PS. This phospholipid is normally located exclusively on the inner monolayer surface of the plasma membrane and other internal cell membranes and the PS is actively transported from the outer to the inner lea£et of the plasma membrane by the aminophospholipid translocase [1,2]. The exposure of PS to the external cell surface is a characteristic of platelet activation [4] and of apoptosis [101]. This PS-containing interface is recognised by proteins involved both in blood clotting and macrophage phagocytosis.

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In the case of blood clotting, the lipid-protein interactions have recently been reviewed [4]. The binding of several enzyme-substrate complexes involved in blood coagulation to membranes containing anionic phospholipids results in a dramatic acceleration of the reaction velocity. Part of this activation is due to the system operating on a two-dimensional surface that will greatly facilitate protein-protein interactions required in the blood clotting cascade. PS is the preferred phospholipid and achieves maximum activation at low mol% values but other anionic phospholipids are also e¡ective in vitro, be it at higher concentrations [4]. The presence of PE potentiates the e¡ectiveness of PS as it does in other examples where proteins bind to PS. This e¡ect of PE may be due, in part, to the ability of the head group of the zwitterionic PE to hydrogen bond within the interface thus restricting phospholipid di¡usion in the bilayer [102,103]. In the case of apoptosis, PS exposure is now recognised as an early event in this process proceeding DNA fragmentation while the binding of labelled annexin V (see below) to the exposed PS is now a standard technique for identifying apoptotic cells. Cell surface exposure of PS is linked to recognition by receptors on the surface of macrophages. This recognition is complex and a number of possible receptors have been identi¢ed including speci¢c PS receptors, macrophage scavenger receptors, lipopolysaccharide receptor or thrombospondin-dependent vitronectin receptors (referenced in [104]). In addition, the serum protein L2 -glycoprotein I has been implicated in a bridging mechanism between apoptotic cells and macrophages [104] and has been implicated in the antiphospholipid syndrome (see below). Although the various stages of apoptosis can readily be demonstrated in cell culture, it is probable that the half-life of such cells in vivo is very short due to the considerable ability of macrophages to remove such cells from the system following binding to receptors. The cell surface exposure of PS as a consequence of the ageing process is more controversial. Recent evidence provides strong support for the hypothesis that the removal of aged normal erythrocytes by macrophages is the result of PS exposure [105]; however, the mechanism for the removal of red blood cells in most forms of haemolytic anaemia remains

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to be established [105]. The relationship between lipid peroxidation, PS oxidation and apoptosis is a related problem of obvious interest particularly since, unlike other phospholipids, PS oxidation may not be prevented by vitamin E analogues [106]. 5.3. The antiphospholipid syndrome The pathological consequences of anionic phospholipid exposure are intriguing. The antiphospholipid syndrome (discussed in [4]) is associated with a variety of immune disorders in which autoantibodies are directed against a variety of phospholipids. Although cardiolipin has historically been used as the antigen, such antibodies demonstrate a broad range of speci¢city to anionic phospholipids. The major clinical manifestation of the syndrome has been an interference with the blood coagulation system [4]. An important target of antibody binding appears to be the human serum protein, human L2 glycoprotein I, which in turn binds anionic phospholipids including PS. A mutagenesis study involving the binding of human L2 -glycoprotein I to endothelial cells established that a cluster of lysines are critical for anionic phospholipid binding [107]. Moreover, binding of antibody to the complex resulted in endothelial cell activation in vitro [108]. Interestingly, the ability of human L2 -glycoprotein I to bind to normal human endothelial cells after previous exposure to antiendothelial cell autoantibodies implies that such antibodies cause PS exposure in these cells [109]. This binding of human L2 -glycoprotein I to PS on the cell surface may be a critical event in the removal of PS-expressing cells by macrophages as a result of the protein providing a bridging moiety [104]. 5.4. Annexin V binds to PS The annexins are a group of calcium ion-dependent phospholipid binding proteins [110^112] of which annexin 1 (lipocortin 1) attracted early attention as an inhibitor of PLA2 and hence a potential modulator of the in£ammatory response [113,114]. This inhibition is now recognised to be due to substrate depletion and re£ects the ability of annexins to cluster on the phospholipid surface, thus preventing access of PLA2 to the interface [115^117]. Other phys-

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iological e¡ects of annexins may be due to their ability to prevent the interfacial binding, or restrict the interfacial mobility, of other proteins thus modulating their activity. However, the precise roles of these proteins in cellular function have yet to be elucidated. Annexin V, a calcium and phospholipid binding protein with a high a¤nity for PS, was ¢rst identi¢ed as a vascular anticoagulant protein that bound with high a¤nity to such surfaces and inhibited blood coagulation [118,119]. It is one of the simpler and more abundant members of the annexin family. As a result, this protein provided the ¢rst crystal structure [120] and has become a well-studied model for this family of proteins. In the case of annexin V, translocation to the membrane surface is promoted by a number of anionic phospholipids but a speci¢c interaction with PS has been proposed in a Ca2‡ bridging mechanism based on modelling studies [121]. There is instantaneous release of annexin V from membranes on removal of calcium ions by chelating agents. Concentrations of calcium ions of the order of 100 WM or greater are required for optimal association of annexin V with anionic phospholipid interfaces in vitro when the mol% of anionic phospholipid is ¢xed at the level expected in vivo [122,123]. This calcium requirement highlights a conceptual problem of relating many in vitro binding studies to in vivo function and suggests that other factors may be involved in vivo to promote binding to membranes. Annexin V translocates to nuclear associated membranes on cell activation [124,125]. The driving force for this translocation is not clear because the intracellular calcium ion concentration would not be suf¢cient to promote e¡ective binding to the phospholipid interface under normal conditions. However, binding to biological membranes under conditions of low calcium ion concentrations cannot be released by EGTA and may re£ect binding to an annexin V binding protein [126]. A similar nuclear translocation problem has been highlighted in the case of cPLA2 [78,79], again suggesting the possible involvement of a binding or docking protein. Alternatively, the formation of a highly favourable phospholipid domain in the nuclear membrane or the presence of high local concentrations of calcium ions must be theoretical possibilities that could allow speci¢c transloca-

Fig. 3. A model of the membrane-bound PI-4,5-P2 :spectrin PH domain complex (taken from [131] with permission). The side chains of the positively charged residues surrounding the I1,4,5-P3 binding site are shown by ball and stick. The I-1,4,5-P3 moiety is based on the coordinates of the crystal structure whereas the rest of the phospholipid is a model. Phospholipid head groups are orientated towards the positively charged surface of the PH domain. The ¢gure was prepared with MOLSCRIPT [160].

tion to the nuclear envelope. Annexin V shows relatively weak binding to PI, possibly due to steric reasons [127] which would also argue against any speci¢city towards phosphoinositides. Evidence for an extracellular role for annexin V is lacking although release from the cell would occur as a result of cell damage. The presence of a high concentration of annexin V in vascular endothelial cells (approx. 2.5 mg/ml) would allow release of annexin V after cell injury to interfere with the coagulation

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cascade and limit clot formation by reducing access to exposed anionic interfaces [111]. Fluorescently labelled annexin V, either with £uorescent reagents or as a construct with green £uorescent protein, now provides a standard marker for PS exposure in apoptotic cells [128] where a rapid binding response is observed up to about 6 mol% PS using mM calcium ion concentrations [129]. 5.5. PI and phosphoinositides The inositol phospholipids appear to have crucial roles in protein interfacial binding and regulation of these proteins at the interface as well as being substrates for the generation of important signalling molecules. The phosphoinositides are polyanionic and can be very e¡ective in non-speci¢c electrostatic interactions as low mol% of these phospholipids (like cardiolipin) can generate a high anionic charge density. However, the role of these phospholipids will be discussed at this stage with the emphasis on protein domains identi¢ed in speci¢c phosphoinositide binding. To date, this is best illustrated by the PH domains where crystal structures of some examples have allowed a modelling of the speci¢c interaction with membrane phosphoinositides (Fig. 3) [130,131]. However, the potential for multiple phosphorylated species of PI allows a variety of potential recognition domains for recruitment of speci¢c proteins to cell membranes [132]. 5.6. PH domains PH domains were ¢rst identi¢ed as a novel repeated protein module of approx. 120 amino acids in pleckstrin and have subsequently been found in over 100 di¡erent eukaryotic proteins. Despite minimal sequence homology, PH domains share a common tertiary structure (or predicted structure) and mediate protein-lipid or protein-protein interactions [133^136]. Proposed inositol lipid ligands vary for di¡erent PH domains including I-1,4,5-P3 , PI-4,5-P2 and PI3,4-P2 , along with binding speci¢cities and a¤nities. While the majority of these proteins need to be membrane associated to function, and the PH domain appears to have a role in membrane targeting, the physiological ligands and therefore the importance of

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phosphoinositides for PH domain and thus protein localisation, have been elucidated for only a few proteins [137]. Because of the minimal sequence homology for this domain, the absolute identi¢cation of such a domain from the primary sequence can be di¤cult. Moreover, although the binding to phosphoinositides is a common characteristic of this domain, such binding and biological activation can also be seen with respect to C2 domains and other proteins where interfacial binding is promoted by anionic phospholipids. The high charge density of the phosphoinositides means that interaction with other protein domains may be achieved at a low mol% of the phosphoinositide. The problem of being sure that phosphoinositide activation is due to speci¢c binding is compounded by the di¤culty of performing binding assays using phospholipid aggregates. In addition to binding phosphoinositides, PH domains will also bind water soluble inositol polyphosphates such as IP3 and competition between these two types of ligands provides support for a genuine PH domain. It should be stressed that the binding speci¢city of many PH domains is unresolved and the problems of speci¢city and a¤nity have been reviewed elsewhere [134]. 5.7. Phospholipase C-N1 All of the PLC isoenzymes are multi-domain containing one or more PH domains. The best characterised PH domain in terms of structure and ligand binding properties is that of PLC-N1 . Native PLC-N1 binds with high a¤nity to phospholipid vesicles containing PI-4,5-P2 [138] compared with PS or PI for example, while the isolated recombinant PH domain from the enzyme binds PI-4,5-P2 and I-1,4,5-P3 ^ the soluble head group of PI-4,5-P2 ^ with high a¤nity and speci¢city [139,140]. Other inositol polyphosphates bind considerably weaker (15^62-fold) [140], as do PI-3,4-P2 , PI-4-P and PI, although PI-3,4,5-P2 has been reported to bind with comparable a¤nity to PI-4,5-P2 [139]. Localisation of PLC-N1 PH domain to the plasma membrane has been demonstrated through immuno£uorescence and using green £uorescent protein labelled PH domain [141,142]. PI-4,5-P2 binding to the PH domain speci¢cally enhances the catalytic activity of the enzyme [143]. Upon determination of the crys-

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tal structure of PLC-N1 [89], a tether and ¢x model was proposed for its binding to its target interface, involving the PH domain. Speci¢c high a¤nity binding of the PH domain to PI-4,5-P2 tethers PLC to the membrane, while a second important region, the C2 domain, ¢xes the low a¤nity catalytic domain into a productive orientation on the membrane. C2 domains are known to bind calcium ions, phospholipids including phosphoinositides and inositol polyphosphates, or other proteins (see above); however, the speci¢city of the C2 domain from PLC-N1 is yet to be resolved [85]. While PI-4,5-P2 is thought to allow processive catalysis at the membrane, high levels of IP3 , resulting for example from PLC enzyme activity, may compete with PI-4,5-P2 for PH domain binding and reduce membrane association and catalytic e¤ciency [130,144]. The relative importance of various phosphoinositides to speci¢c PH domains as with other phosphoinositide binding domains will be affected by the relative abundance of such molecules in the cell membranes. PI-4,5-P2 is much more abundant than PI-3,4,5-P2 (see below) even in activated cells [135,137], and as such PLC-N1 is most likely regulated by PI-4,5-P2 . 5.8. Phosphoinositide-3-kinase, FYVE domains and other domains that recognise phosphoinositides The more recent discovery of phosphoinositide-3kinase suggested important roles for phosphoinositides phosphorylated at the 3 position (D3) and it is interesting that subsequent studies with various D3 phosphoinositides, e.g. PI-3,4-P2 , have shown many proteins to exhibit preferential binding to these over D4 phosphoinositides. Protein domains that show speci¢city for D3 phosphoinositides have been described, and one such group (FYVE domains) which show this speci¢city have recently been reviewed [132]. It is clear that multiple types of domains have independently evolved sites for the recognition of speci¢c phosphoinositides linked to a diverse range of cell functions. Other well known domains that have been reported to bind phosphoinositides include phosphotyrosine binding (PTB) domains and Src homology 2 (SH2) domains [134].

5.9. Other structural roles for phosphoinositides A role for phosphoinositides in cytoskeletal protein functioning is emerging and has recently been reviewed [145]. Only in the case of vinculin is structural information available relating to anionic phospholipids [146,147]. 5.10. PG and CL PG is a minor mammalian phospholipid, although relatively abundant in lung surfactant, but is a major constituent of bacterial membranes. The function of PG in lung surfactant function remains unclear [148]; however, a role in binding surfactant proteins in vivo would be anticipated. PI is the major anionic phospholipid of lung surfactant in a number of species, a fact that highlights the importance of the negative charge rather than an absolute requirement for PG. Recently, PG has been identi¢ed as a physiological activator of nuclear PKC, an e¡ect that also shows some speci¢city in terms of the molecular species of the PG [149]. CL is a major component of mitochondrial phospholipid where it is found on the inner mitochondrial membrane [150]. CL is believed to have a speci¢c role in mitochondrial function as a result of interactions with a number of mitochondrial proteins. It may also play an important role in protein import into the mitochondria [35]. However, disruption of CL synthesis in yeast results in a complete absence of CL [151] and a 5-fold increase in PG without any apparent deleterious e¡ects on gross mitochondrial function [152], indicating that there is no absolute requirement for CL. The highly anionic nature of the bacterial cell membrane, which is rich in PG and CL, has already been highlighted in terms of the antimicrobial properties of the highly cationic human sPLA2 (see above). Vertebrates produce a wide variety of antimicrobial peptides [153]. The recurring theme is that these cationic peptides utilise electrostatic interactions for initial membrane binding prior to membrane insertion and the requirement for anionic phospholipid head groups may confer relative selectivity towards bacteria as opposed to host cells [153].

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5.11. PA A possible role of PA in promoting human sPLA2 activity has already been discussed (see above). The role of PA in vesicle formation and transport within the cell has attracted considerable attention linked to the observation that PLD was activated by ADPribosylation factor (ARF), a key protein in coated vesicle formation. However, a direct demonstration of elevated levels of PA within the Golgi membranes during ARF-stimulated vesicle formation is lacking; indeed PA levels within the Golgi membranes appear to decline during vesicle formation [154]. Alternatively, PA generated by PLD may be involved in signal transduction. A direct role for PA as a lipid signalling molecule remains unproven, but is discussed in recent reviews on PLD [155^157]. The more interesting possibility is the role of PA as a precursor of LPA and one route for the generation of this bioactive lipid is as a result of PA hydrolysis by a PLA2 . The opposing e¡ects of PA and LPA on membrane curvature and membrane ¢ssion during vesicle formation have recently been highlighted linked to the lysophosphatidic acyl transferase activity of the presynaptic protein, endophilin 1 [158]. The change of a cone-shaped lipid (PA) to an invertedcone-shaped lipid (LPA) will facilitate the negative membrane curvature required for membrane ¢ssion. 6. Conclusions Anionic phospholipids provide a fertile environment for the interfacial binding of a wide range of proteins. The interaction will normally involve initial electrostatic interactions but the ability of anionic phospholipids to perturb the bilayer would allow subsequent membrane penetration and non-polar interactions. This overall phenomenon tends to be characterised by low phospholipid speci¢city and require a threshold concentration of anionic charge density (normally s 10 mol%) before a biological response is observed consistent with multiple nonspeci¢c electrostatic interactions. The threshold mol% of anionic of anionic phospholipid will appear to be much lower where the phospholipid used is dior polyanionic as is the case with CL, phosphoinositides and possibly PA. In any particular system, the

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balance between electrostatic and non-polar interactions will depend on both the phospholipid composition of the interface and the structure of the interfacial surface of the protein. Because PS is the most abundant anionic phospholipid in mammalian systems, it is this phospholipid that probably makes the major contribution to these non-speci¢c electrostatic interactions. Membrane penetration by the protein provides a mechanism for a more permanent docking to the interface and is facilitated by nonpolar amino acid residues, particularly tryptophan, and also the presence of acylated or prenylated amino acid derivatives. The speci¢c binding of proteins to individual anionic phospholipid molecules is seen in a variety of physiological situations and is characterised by requiring a low mol% of the phospholipid in the interface (usually 6 5 mol%) to obtain a biological response. This characteristic is clearly seen in the case of the phosphoinositides where speci¢c interactions with PH domains are now well recognised within the cell. Other domains that recognise speci¢c phosphoinositides, such as FYVE domains, are extending the complexity of this membrane targeting mechanism. However, the polyanionic nature of the phosphoinositides means that they will also promote non-speci¢c electrostatic interaction at low mol% concentration often making it di¤cult to distinguish speci¢c and non-speci¢c events. An increasing role for the minor polyanionic phospholipids in the regulation of cell functions will emerge in parallel with the discovery of new areas of signal transduction. The external exposure of PS also promotes speci¢c interactions with binding proteins as a trigger for a variety of extracellular events and this phenomenon operates at a low mol% of the PS, although this phenomenon can be mimicked by other anionic phospholipids in vitro. In summary, anionic phospholipids play a major role in the regulation of cell function as a result of promoting the binding of speci¢c proteins to the membrane interface. Acknowledgements Work from the author's laboratory is supported by the Wellcome Trust.

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