Phosphatidylinositol and Related Phosphoinositides

Although it had long been recognized that phosphatidylinositol or 1,2-diacyl-sn-glycero-3-phospho-(1'-myo-inositol) was a key membrane constituent, it was initially something of a surprise when the manifold biological activities of this lipid, and then of the derived phosphatidylinositol phosphates and their hydrolysis products, were discovered in animals, plants and microorganisms. Many years after the initial discoveries in the 1950s, these lipids continue to be a major focus for research efforts around the world with considerable relevance to human health. Phosphatidylinositol and its various metabolites and relevant enzymes can be located and function within different membrane regions in cells, and they form part of what have been termed phosphoinositide and phosphatidylinositol cycles, their versatility stemming from the inositol head group, a six-carbon hexahydroxy-ring, which can be reversibly phosphorylated on the 3, 4 and 5 positions. In addition to their structural role in membranes, these lipids are intimately involved in innumerable aspects of membrane trafficking and signalling in eukaryotic cells, functions that are essential to cell growth and metabolism. Only a brief overview of such a highly complex topic is possible here.

Glycosyl-phosphatidylinositol (GPI) is a related lipid that serves as an anchor for proteins; it is a sufficiently distinctive topic for its own web page (together with phosphatidylinositol mannosides).

1. Phosphatidylinositol

Structure and Occurrence: Phosphatidylinositol is an important lipid, both as a membrane constituent and as a participant in essential metabolic processes in all plants and animals, both directly and via its metabolites. It is an acidic (anionic) phospholipid that in essence consists of a phosphatidic acid backbone linked via the phosphate group to inositol (hexahydroxycyclohexane). In most organisms, the stereochemical form of the last is myo-D-inositol (with one axial hydroxyl in position 2 with the remainder equatorial, i.e., a chair-like structure), although other forms (scyllo- and chiro-) have been found on occasion in plants. The sn‑1‑stearoyl-2-arachidonoyl molecular species, which is of considerable biological importance in animals, is illustrated.

Formula of phosphatidylinositol

Phosphatidylinositol is especially abundant in brain tissue where it can amount to 10% of the phospholipids, but it is present in all tissues, cell types, and membranes at relatively low levels in comparison to many other phospholipids. In rat liver, it amounts to 1.7 micromoles/g., i.e., less than phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine. Under normal conditions, it is present entirely in the inner leaflet of the erythrocyte membrane and of the plasma membrane in nucleated cells. Phosphatidylinositol per se is rarely found in prokaryotes other than the Actinomycetales, although the thermophilic α-proteobacterium Rhodothermus marinus contains dialkylether glycerophosphoinositides.

The fatty acid composition of phosphatidylinositol is rather distinctive as shown in Table 1. Thus, in almost all animal tissues, the characteristic feature is a high content of stearic and arachidonic acids. All the stearic acid is linked to position sn-1 and all the arachidonic acid to position sn-2, and as much as 78% of the total lipid may consist of the single molecular species sn-1-stearoyl-sn-2-arachidonoyl-glycerophosphorylinositol (see Table 2 below). Although 1-alkyl- and alkenyl- forms of phosphatidylinositol are known, they tend to be much less abundant than the diacyl form. In plant phosphatidylinositol, e.g., Arabidopsis thaliana as listed, palmitic acid is the main saturated fatty acid in position sn-1, while linoleic and linolenic acids are the main unsaturated components in position sn-2. Similarly in yeast, palmitic acid is in position sn-1 with oleic and palmitoleic acids in position sn-2 predominantly; the Amoebozoa have a C₁₆ alkyl group in position sn-1 and cis-vaccenic acid in position sn-2.

Tissue	Fatty acids
Table 1. Fatty acid composition of phosphatidylinositol (wt % of the total) in animal and plant tissues.
	16:0	18:0	18:1	18:2	18:3	20:3	20:4	22:3	22:5	22:6

Bovine brain [1]	8	38	10	1	-	5	34	2	tr.	1
Bovine liver [2]	5	32	12	6	1	7	23	4	3	5
Rat liver [3]	5	49	2	2		4	35			1

*A. thaliana* [4]	48	3	2	24	24
[1] = Holub, B.J. et al.. J. Lipid Res.., 11, 558-564 (1970); DOI. [2] = Thompson, W. and MacDonald, G., J. Biol. Chem., 250, 6779-6785 (1975); DOI. [3] = Wood, R. and Harlow, R.D. Arch. Biochem. Biophys., 135, 272-281 (1969); DOI. [4] = Browse, J. et al. Biochem. J., 235, 25-31 (1986); DOI.

Biosynthesis: The basic mechanism for biosynthesis of phosphatidylinositol and phosphatidylglycerol is sometimes termed a branch point in phospholipid synthesis, as phosphatidylcholine and phosphatidylethanolamine are produced by a somewhat different route.

Eukaryotes are in general able to synthesise inositol de novo via glucose-6-phosphate. As with phosphatidylglycerol (and thence cardiolipin), phosphatidylinositol is formed biosynthetically from phosphatidic acid via the intermediate cytidine diphosphate diacylglycerol, which is produced by the action of a CDP-diacylglycerol synthase, believed to be the rate-limiting enzyme in phosphatidylinositol biosynthesis. Then, the enzyme CDP-diacylglycerol inositol phosphatidyltransferase ('phosphatidylinositol synthase' or 'PIS') catalyses a reaction with myo-inositol to produce phosphatidylinositol.

Biosynthesis of phosphatidylinositol in eukaryotes

Only one isoform of PIS exists in mammals, and it is located in the endoplasmic reticulum (ER), in part in a sub-compartment of this associated with mitochondria (mitochondria-associated membranes - MAM) and in mitochondria per se. Indeed, it is reported that PIS is present in a mobile ER-derived sub-compartment that makes transient contacts with other organelles, including the plasma membrane, and facilitates distribution of phosphatidylinositol to other subcellular compartments. The other product of the reaction is cytidine monophosphate (CMP). As PIS can catalyse the reverse reaction, the rate of phosphatidylinositol synthesis is determined by the relative concentrations of the precursors and product, and for the reaction to continue, the latter must be transported away from the site of synthesis via lipid exchange proteins and vesicular or tubular carriers. Much of the phosphatidylinositol is delivered to other membranes by vesicular transport, but a family of soluble phosphatidylinositol transfer proteins (PITPα, PITPβ and PITPNC1) provides phosphatidylinositol from the ER to kinases for phosphorylation (see below).

Molecular species specificity: The phosphatidylinositol synthase per se does not exhibit the fatty acyl specificity observed in the final product, but earlier in the biosynthetic process 1-stearoyl-2-arachidonoyl species of diacyl-sn-glycerols are converted preferentially into phosphatidic acid by the epsilon isoform of diacylglycerol kinase (DGKε), anchored to the membrane via its N-terminal hydrophobic helix segment; ATP is the phosphate donor. One of the CDP-diacylglycerol synthases (CDS2) has similar specificity in the generation of the immediate precursor CDP-diacylglycerols from phosphatidic acid, while some specificity may be introduced via lysophosphatidylinositol formed as a by-product of eicosanoid formation (see below) or as an intermediate as part of the normal cycle of deacylation-acylation of phosphatidylinositol in tissues in which the fatty acid composition is remodelled to give the final distinctive composition (the Lands’ cycle - see our web page on phosphatidylcholine for a fuller discussion of this process). A membrane-bound O-acyltransferase (MBOAT7 or LPIAT1) specific for position sn-2 of lysophosphatidylinositol with a marked preference for arachidonoyl-CoA is ubiquitously expressed in animal tissues, and this may be one means by which free arachidonic acid and eicosanoid levels are regulated. Deficiencies in this enzyme have been correlated with various disease states in the liver.

In macrophages subjected to inflammatory stimuli, phosphatidylinositol containing two molecules of arachidonate is produced by remodelling reactions, and there is evidence that it is a novel bioactive phospholipid regulating innate immune responses in these cells. Further specificity may be introduced by lysocardiolipin acyltransferase (LYCAT; also known as LCLAT1 or ALCAT1), which exhibits a preference for lysophosphatidylinositol and lysophosphatidylglycerol over other phospholipids in vitro, and incorporates 18:0 rather than shorter chain fatty acids into position sn-1 of phosphatidylinositol and other phosphoinositides, especially phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3-phosphate; this enzyme may be located adjacent to the phosphatidylinositol synthase in the endoplasmic reticulum. Some of the phosphatidylinositol in membranes is derived from re-cycling of polyphosphoinositides via the phosphatidylinositol cycle, and this could influence the molecular species composition (see below). For example, during agonist stimulation, highly unsaturated polyphosphoinositides are replenished much faster than more saturated species.

The highly specific distribution of fatty acids on the glycerol moiety of phosphatidylinositol breaks down in some cancer cells, especially those with a mutation on the transcription factor p53 gene, which is one of the most highly mutated genes in cancers.

Plants and bacteria: In contrast to animals, plants have two phosphatidylinositol synthase isoforms, PIS1 and PIS2, which display specificities for particular species of the CDP-diacylglycerol substrate. PIS1 generates phosphatidylinositol with saturated or monounsaturated fatty acids preferentially, while PIS2 generates polyunsaturated species; the two forms possibly having different functions. In protozoan parasites, such as Trypanosoma brucei, the active site of phosphatidylinositol synthase may be the lumen of the endoplasmic reticulum and Golgi. There is evidence for two distinct pools of product in this organism, the bulk membrane form derived from inositol imported from the environment, and a second for the synthesis of GPI anchors, which uses myo-inositol synthesised de novo. In yeasts, some biosynthesis may occur on the cytosolic side of the plasma membrane.

Few species of bacteria appear to contain phosphatidylinositol, although it is essential for Mycobacteria, and it is present in some species of the Bacteroidetes and other proteobacteria. Phosphatidylinositol-containing lipids are found in the Actinomycetes (lipophosphoglycans). In these and many other bacterial species, CDP-diacylglycerols and inositol phosphate are combined by a phosphatidylinositol-phosphate synthase to generate phosphatidylinositol phosphate, which is subsequently dephosphorylated to generate phosphatidylinositol. Archaeal ether lipids include analogues of phosphatidylinositol, and these are synthesised by a comparable mechanism, i.e., by reaction of inositol 1-phosphate with CDP-archaeol to form archaetidylinositol 3-phosphate and thence archaetidylinositol (see our web page on Archaeal lipids for a more detailed discussion). In contrast, in the thermophilic bacterium Rhodothermus marinum, L-myo-inositol-1-phosphate cytidylyltransferase catalyses the formation of CDP-inositol from inositol-1-phosphate and CTP, before a synthase catalyses the transfer of the inositol-1-phosphate group from CDP-inositol to dialkylether glycerols to produce phosphoinositol ether lipids; this differs from all other pathways, which involve activated forms of the lipid moiety as intermediates.

Biosynthesis of phosphatidylinositol in Mycobacteria and Rhodothermus marinum

Function: In addition to functioning as negatively charged building blocks of membranes, the inositol phospholipids (including the phosphatidylinositol phosphates or 'polyphosphoinositides' discussed below) have crucial roles in interfacial binding of proteins and in the regulation of protein activity at the cell interface. As phosphoinositides are polyanionic, they can be very effective in non-specific electrostatic interactions with proteins. However, they are especially efficient in specific binding to so-called ‘PH’ domains of cellular proteins. At least three phosphatidylinositol molecules are present in the crystal structure of human erythrocyte glycophorin, for example, and they are believed to influence binding to other proteins via their head groups. The lipid is a structural component of yeast cytochrome bc₁.

In animal tissues, phosphatidylinositol is the primary source of the arachidonic acid required for biosynthesis of eicosanoids, including prostaglandins, via the action of the enzyme phospholipase A₂, which releases the fatty acids from position sn-2.

Release of arachidonic acid from phosphatidylinositol

Similarly, phosphatidylinositol and the phosphatidylinositol phosphates are the main source of diacylglycerols that serve as signalling molecules in animal and plant cells via the action of a family of enzymes collectively known as phospholipase C (see below and our web pages on diacylglycerols for further discussion). In brief, diacylglycerols regulate the activity of a group of at least a dozen related enzymes known as protein kinase C, which in turn control many key cellular functions, including differentiation, proliferation, metabolism and apoptosis. Indeed, the biological actions of the various components released have been the subject of intensive study over many years. 2‑Arachidonoylglycerol, an endogenous cannabinoid receptor ligand, may be a further product of phosphatidylinositol metabolism. 1,2-Dioleoyl-phosphatidylinositol is signalling lipid, derived from the action of the stearoyl-CoA desaturase (SCD1), and acts as a lipokine to respond to stresses that activate protein degradation, apoptosis and autophagy.

2. Phosphatidylinositol Phosphates (Polyphosphoinositides) in Animals

Structure and Occurrence: The pioneering work of Mable and Lowell Hokin in the 1950s led to the discovery that phosphatidylinositol was converted to polyphosphoinositides with important signalling and other functional activities, including cell communication via signal transduction, cell survival and proliferation, membrane trafficking and modulation of gene expression. Phosphatidylinositol is now known to be phosphorylated by a number of substrate-selective kinases that place the phosphate moiety on positions 3, 4 and/or 5 of inositol with the balance among them maintained by distinct phosphatases and phospholipases. Seven different isomers are known (mono-, bis- and tris-phosphorylated), which are produced in a tightly coordinated manner, and all of these have characteristic biological activities. They each turn over much more rapidly than the parent phosphatidylinositol molecule. Further, there can be an array of molecular species of each of these isomers that differ in the nature of the fatty acyl groups. Although the most significant in quantitative and possibly biological terms were long thought to be phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5‑bisphosphate, it is now recognized that phosphatidylinositol 3-phosphate and its metabolites are as important biologically at least.

Formulae of phosphatidylinositol phosphates

These lipids are usually present at low levels only in tissues, typically at about 0.5 to 1% of the total lipids of the inner leaflet of the plasma membrane, so they are unlikely to have an appreciable structural role. On the other hand, static measurements of lipids that turn over very rapidly do not provide a meaningful assessment of their cellular functions. The positional distributions of fatty acids in the phosphatidylinositol, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate of ox brain are listed in Table 2. In each the saturated fatty acids are concentrated in position sn-1 and polyunsaturated, especially arachidonate, in position sn-2; there are few differences among the three lipids in this instance.

Fatty acids	PI		PI monophosphate		PI diphosphate
Table 2. Distribution of fatty acids (mol % of the total) in positions sn‑1 and sn‑2 in phosphatidylinositol (PI) and the phosphatidylinositol mono- and diphosphates of ox brain.
Fatty acids	sn-1	sn-2	sn-1	sn-2	sn-1	sn-2

16:0	15		9		7
18:0	74		69		69
18:1	10	10	20	13	21	10
18:2	1	2	trace	1	1	1
20:3(n-9)		5		10		10
20:3(n-6)		5		11		12
20:4(n-6)		67		49		52
22:3		7		10		7
22:6(n-3)		trace		trace		trace
Data from Holub, B.J. et al., J. Lipid Res., 11, 558-564 (1970); DOI. Molecular species data, see Traynor-Kaplan, A. et al., Biochim. Biophys. Acta, 1862, 513-522 (2017); DOI.

Biosynthesis: Phosphatidylinositol per se is the ultimate precursor of all phosphoinositides, the head groups of which have different charges and structures that impact directly on membrane properties and via metabolic interactions can function as chemical switches. The individual phosphoinositides are maintained at steady state levels in membranes by a continuous and sequential series of phosphorylation and dephosphorylation reactions by specific kinases, phosphatases and phospholipase C enzymes, which are regulated and/or relocated with multiple interdependencies through cell surface receptors for extracellular ligands, i.e., the phosphoinositide cycle. While this has been termed a ‘futile cycle’, which can consume a significant proportion of cellular ATP production, it is only part of a wider pattern of reactions - the phosphatidylinositol cycle (see below). Controlled synthesis of these different phosphoinositides occurs in different intracellular compartments for distinct and independently regulated functions with spacially distinct target enzymes or receptors. In mammals, the complexity is such that 18 phosphoinositide inter-conversion reactions have been identified to date, and these are mediated by at least 20 phosphoinositide kinases and 34 phosphoinositide phosphatases that span 8 and 10 classes, respectively; some have yet to be characterized. Most of these enzymes are conserved across the eukaryota, and each has distinct functions and specificities that cannot be replaced by the activity of related isoforms.

As a generality, most mono-phosphorylations occur in endomembranes, such as the endosomes and the Golgi network, while second and third phosphorylations occur primarily at the plasma membrane, and this is reflected in the lipid composition of each membrane. While these enzymes are believed to work independently and sequentially to produce a specific product, there remains a possibility that some participate in protein complexes to coordinate their activities. Specific transporters, especially the 'Nir2' protein, facilitate the exchange of phosphoinositides between membranes. It should be noted that there are links to the metabolism of phosphatidylcholine, which can be hydrolysed by phospholipase D to phosphatidic acid, an important activator of key kinases.

Polyphosphoinositide metabolism in animal tissues

Thus, as an example, phosphatidylinositol 4-phosphate (PI(4)P) is produced by the action of a phosphatidylinositol 4-kinase (PI4K) in the Golgi, and this is in turn phosphorylated by a phosphatidylinositol phosphate 5-kinase (PIPK I) to form phosphatidylinositol 4,5-bisphosphate (PI(4,5)P) at the plasma membrane, although the latter can also be formed by phosphorylation of phosphatidylinositol 5‑phosphate by a specific 4-kinase (PIPK II). Four isoforms of PI4K in two structural families are known that each operate in different subcellular membrane compartments to produce phosphatidylinositol 4-phosphate for particular signalling functions. Some selectivity in the formation of molecular species or remodelling may occur to further enrich the arachidonic acid content.

Subsequently, it was discovered that phosphatidylinositol is phosphorylated by a 3-kinase (PI3K III or the VPS 34 complex) to produce phosphatidylinositol 3-phosphate (PI(3)P) in the early endosomes. Three phosphatidylinositol 3-kinases families (eight isoforms) have been described to date, each with distinct substrate specificities. A second phosphoinositide signalling pathway involves activation of two of these 3‑kinases, stimulated by growth factors and hormones, which phosphorylate phosphatidylinositol 4,5‑bisphosphate (by PI3K I - four isoforms) and phosphatidylinositol 4‑phosphate (by PI3K II - three isoforms) to produce phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P) and phosphatidylinositol 3,4‑bisphosphate (PI(3,4)P), respectively. While phosphatidylinositol 3-phosphate and other 3‑phosphorylated metabolites amount to only about 0.5% of the total phosphoinositides in resting mammalian cells, they are now recognized to be of profound importance for cellular metabolism.

In addition to the activity of kinases, the amounts of these various metabolites are regulated by the activities of specific phosphoinositide phosphatases, which are highly conserved in eukaryotes and dephosphorylate phosphoinositides at the 3, 4 and 5 positions of the inositol ring. For example, so-called ‘SHIP’ phosphatases convert phosphatidylinositol 4,5‑bisphosphate back to phosphatidylinositol 4‑phosphate by hydrolysis of the 5-phosphate group. 3‑Phosphorylated phosphoinositides are only degraded by phosphatases, especially those of the PTEN family, and not by phospholipase C (see below).

Scottish thistle The various organelles in cells have membranes with distinct functions and molecular compositions. Yet, all the phosphatidylinositol precursor is formed primarily at the endoplasmic reticulum, and the different membrane lipids must be transported between membrane sites via specific trafficking processes/proteins. There is selective recruitment of effector proteins to particular membranes by binding only to a single type of phosphoinositide, and this is followed by interactions between the phosphoinositide-binding proteins and various enzymes to channel phosphoinositide production to the required biological outcomes and to regulate signalling. For example, much of the phosphatidylinositol 4‑phosphate and phosphatidylinositol 4,5-bisphosphate involved in signalling is believed to be formed at contact sites between the endoplasmic reticulum and plasma membrane.

A concept has emerged in which each phosphoinositide has its own role, the ‘lipid code hypothesis’, in which defined lipids act as labels for each cellular membrane to organize cells into dynamic and responsive membrane-bound compartments and maintain the orderly flow required for the complexities of membrane trafficking and spatio-temporal signalling reactions. Thus, phosphatidylinositol 4‑phosphate, phosphatidylinositol 4,5‑bisphosphate, phosphatidylinositol 3-phosphate and phosphatidylinositol 3,5‑bisphosphate are found mainly on the Golgi, plasma membrane, early endosomes and late endocytic organelles, respectively, where they are sometimes regarded as landmarks for these compartments. Phosphatidylinositol 4,5‑bisphosphate is present throughout the plasma membrane and is considered a general marker for this, while phosphatidylinositol 3,4,5-triphosphate, is a characteristic component of the basolateral region of this membrane in a polarized cell but is absent from the apical part. On the other hand, it should be noted that this map of phosphoinositides to specific organelles is derived from their steady state distributions, but the highly dynamic generation and consumption of different phosphoinositides in response to different stimuli in the various sub-cellular compartments in living cells by the action of kinases and phosphatases together with lipase reactions, may lead to the formation of transient pools of distinct molecular forms. There must be a continuous replenishment of the precursors by new synthesis.

Function: The distinctive subcellular location of the different phosphoinositide species, together with the rapid and reversible nature of phosphorylation, gives them a central and general position in the fields of cell signalling cascades and intracellular membrane trafficking. The precise locations of particular phosphoinositides are factors that contribute a specific identity to each organelle and sometimes even to each face of an organelle, such as the cis and trans faces of the Golgi apparatus, and this enables directional transport of cellular constituents between organelles or membranes. Phosphoinositides can achieve signalling effects directly by binding to characteristic cytosolic domains of membrane proteins via their polar head groups, thereby triggering downstream signalling cascades, often in conjunction with an acidic phospholipid, such as phosphatidylserine or phosphatidic acid at an adjacent-binding site. The term 'lipidon' has been coined to describe the unique collection of co-located lipids that distinguish nano-environments of biological membranes and provide the context for PI recognition in vivo. In this way, they can regulate the function of innumerable proteins integral to membranes, for example by relocating a protein from one area of the cell to another, e.g., from the cytosol to the inner leaflet of the plasma membrane, or they can attract cytoskeletal and signalling components to the membrane. Among the proteins that bind to phosphoinositides in this way are phospholipases, protein kinases, regulators of membrane trafficking, and cytoskeletal, scaffold and ion channel proteins. Dysregulation of phosphoinositide metabolism and signalling is a factor many diseases, including cancer.

Binding usually involves electrostatic interactions with the negative charges of the phosphate groups on the inositol ring with characteristic clusters of basic amino acid residues in proteins to recruit them to intracellular membranes, while often leading to specific folding and thence increased activity of unstructured peptides. At least 70 distinct types of binding sites for phosphoinositides have been identified in proteins. In particular, a binding region termed the pleckstrin homology (PH) domain, consisting of ~100 amino acids, is the most abundant lipid-binding domain with more than 225 examples identified, and this can exhibit great specificity for particular polyphosphoinositides, often binding simultaneously with other proteins. While the interaction is driven by non-specific electrostatic interactions initially, it is followed by specific binding to increase the membrane residence time. The phox homology (PX) domain family with 49 members in humans is unique in that it can recognize all seven phosphoinositide forms, while proteins with a FYVE domain, which is enriched in cysteine and is stabilized by two zinc atoms, binds specifically to phosphatidylinositol 3-phosphate (PI(3)P). The protein kinase C family have C1 or C2 domains which recognize phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5‑trisphosphate specifically (and sometimes other lipids). The distinctive phosphoinositide composition of membranes in different organelles adds strength and specificity to the interactions by cooperative binding with other membrane proteins.

Phosphatidylinositol 3-phosphate and the other phosphatidylinositol monophosphates are present in cells at low levels only, and these do not appear to fluctuate greatly. PI(3)P is a marker lipid that recruits key cytosolic proteins to the early endosomes and defines their identity. In particular, it plays a pivotal role in the initiation of autophagy, i.e., the controlled internal degradation and turnover of cellular constituents, while PI(3,5)P₂ is important in the autophagosome-lysosome fusion step and in the subsequent acidification of this organelle. After sorting of the lysosomal contents, components of the internalized cargo are recycled to the plasma membrane and PI(3)P is dephosphorylated to phosphatidylinositol by a specific phosphatase, before this is in turn phosphorylated to PI(4)P. Thus, the processes of internalization, sorting and trafficking of membrane proteins depend on the interconversion of phosphoinositide species by coordinated phosphorylation-dephosphorylation reactions.

Scottish thistle In general, PI(3)P controls cellular processes by recruiting effector proteins through low to moderate affinity interactions with specific PI(3)P binding domains. A protein designated Akt (protein kinase B) is recognized as a direct effector of the PI3K signalling cascade with receptor tyrosine kinases as the main upstream activators, for example, but it is now known that every phosphatidylinositol phosphate has a specific set of effector proteins that are recruited to target membranes or are allosterically regulated by specific receptors; each function may require a different effector. A further function of PI(3)P is in the regulation of the final stage of cell division (cytokinesis), and the lipid is known to accumulate where cells divide. As the class I PI3K isoforms especially have been implicated in the aetiology and maintenance of various diseases and metabolic disorders, including cancer, inflammation and autoimmunity, drug companies are actively pursuing the development of inhibitors. In particular, these enzymes mediate insulin-independent glucose transport and many of the physiological actions of insulin. In relation to lung cancer especially, RAS proteins, which are key signalling switches essential for the control of proliferation, differentiation and survival of eukaryotic cells, regulate the activity of type I phosphatidylinositol 3-kinase (PI3K); this is essential for tumour initiation and maintenance.

Phosphatidylinositol 4-phosphate is the precursor for the 4,5-bisphosphate, but it binds to a protein on the cytoskeleton of the cell and has its own characteristic functions. It is the most widely distributed of the phosphoinositides, and as well as the Golgi and the plasma membrane, it is present in late endosomes, lysosomes, secretory vesicles and autophagosomes. As a part of protein-lipid complexes, it is believed to have a role in essential nuclear processes. In yeast, it has a function in the anterograde transport from the trans-Golgi and the retrograde transport from the Golgi to the endoplasmic reticulum, and it is necessary for the formation of secretory vesicles in the Golgi that are targeted to the plasma membrane. Some PI(4)P in the plasma membrane is exchanged for phosphatidylserine by the action of specific transport proteins at junctions with the endoplasmic reticulum. PI(4)P has been called the 'fuel' that drives cholesterol transport as its hydrolysis provides the energy that enables the establishment of active sterol concentration gradients across membrane-bound compartments with the aid of the oxysterol-binding protein (OSBP)-related protein (ORP) family, which is a key regulator of cholesterol, oxysterol and PI(4)P concentrations in membranes, as discussed in our web page on cholesterol.

PI(4)P is essential for the structure and function of the late endosomes, where it is required for the recruitment of proteins that control cargo exit (following hydrolysis of PI(3)P) and participate in vesicle formation. It accumulates rapidly in damaged lysosomes and is essential for their repair by recruiting multiple members of the OSBP family, which catalyse robust endoplasmic reticulum-to-lysosome transfer of phosphatidylserine and cholesterol to restore lysosomal integrity. PI(3)P initiates autophagy, but then PI(4)P, PI(4,5)P₂ and their binding proteins are modulators of the process at most stages.

In the plasma membrane, PI(4)P can support the functions of ion channels, and it contributes to the anchoring of proteins with polybasic domains, although it is not utilized for synthesis of PI(4,5)P₂ in this membrane. On the other hand, PI(4)P derived from PI(4,5)P₂ in the membrane of primary cilia in the retina is important for vision. PI(4)P has an important influence on the progression of many diseases, especially virus replication, cancer and various inflammatory diseases, and inhibitors of PI4-kinase are under study for their therapeutic potential.

While the biological properties of phosphatidylinositol 5-phosphate have taken longer to unravel, because of the difficulties of separation of this isomer, it is now apparent that it is involved in osmoregulation both in plants and animals. While it is the least abundant phosphatidylinositol monophosphate, it is involved in signalling at the nucleus and in the cytoplasm, modulating cellular responses to various stresses, hormones and growth factors. In the endosomes, it is a regulator of protein sorting.

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) is found primarily in the inner leaflet of the plasma membrane, where it may define membrane identity in eukaryotic cells, although it is present in endosomes, the endoplasmic reticulum and nucleus in small amounts. It plays a crucial role in the maintenance of epithelial characteristics and especially to provide the cell-cell adhesion that is essential for maintaining the integrity of multicellular organisms. Because of its large head group and multivalent negative charge, PI(4,5)P₂ has been described as an "electrostatic beacon" that interacts in various ways with membrane proteins, other lipids and cellular cations. In consequence and despite its relatively low concentration, it is a key regulator of innumerable events at the plasma membrane, including cell adhesion and motility, vesicle endocytosis and exocytosis, and the function of ion channels, especially those for potassium, calcium and sodium. With ion channels, for example, it appears to be an obligatory factor, increasing their activity by activating key proteins, while its hydrolysis by phospholipase C reduces such activity. It is an essential precursor of lipid second messengers such as diacylglycerols with vital signalling functions that operate through plasma membrane G-protein coupled receptors, receptor tyrosine kinases and immune receptors. On the other hand, another proposal is that PI(4,5)P₂ is not so much a signalling molecule but rather serves as a necessary cofactor to activate cellular processes selectively at the plasma membrane where it is the ‘master regulator’ of all functions.

PI(4,5)P₂ interacts with cationic residues of a large array of proteins in concert with cholesterol to form localized membrane domains that are distinct from the sphingolipid-enriched rafts. Indeed, it has a much higher concentration than other phosphoinositide species in cells, although most of this is in effect sequestered by binding proteins. With its diacylglycerol metabolites, it is important for vesicle formation in membranes. For example, a major pathway in cells for internalization of cell surface proteins such as transferrin is the clathrin-coated vesicle pathway. PI(4,5)P₂ is essential to this process in that it binds to the machinery involved in the membrane, increasing the number of clathrin-coated pits and permitting internalization of proteins. It has a related function in caveolae, where it is concentrated at the rim.

Scottish thistle Through its attachment to the apical plasma membrane, phosphatidylinositol 4,5-bisphosphate is intimately involved in the development of the actin cytoskeleton and thereby controls cell shape, motility, division, and many other processes. In particular, it binds with high specificity to effectors such as vinculin, a membrane-cytoskeletal protein that is involved in linkage of integrin adhesion molecules to the actin cytoskeleton. Dysregulation of this function has been implicated in the migration and metastasis of tumour cells. It appears that the presence of stearic acid in position sn-1 is essential for this purpose in yeasts. In the cell nucleus, this lipid is believed to be involved in maintaining chromatin, the complex combination of DNA, RNA and protein that makes up chromosomes, in a transcriptionally active conformation, as well as being a precursor for further signalling molecules. It has a role in gene transcription and RNA processing, especially in the modulation of RNA polymerase activity, and in other nuclear processes.

Via its binding to specific proteins, the lipid is an essential component of the immune response of animal tissues to toxic bacterial lipopolysaccharides, and it is involved in the pathophysiology of the HIV virus via an interaction with the Tat protein secreted by infected cells.

PI(4,5)P₂ is the primary precursor of the endocannabinoid 2-arachidonoylglycerol in neurons. It is an essential cofactor for phospholipase D and so affects the cellular production of phosphatidic acid with its distinctive signalling functions. By binding to ceramide kinase, the enzyme responsible for the synthesis of ceramide-1-phosphate, it has an influence on sphingolipid metabolism. Like ceramide-1-phosphate, it binds to and activates the Ca²⁺-dependent phospholipase A₂, which generates the arachidonate for eicosanoid production. One molecule of phosphatidylinositol 4,5‑bisphosphate is bound to each subunit of the protein in the crystal structure of mammalian GIRK2 potassium channel, where it enables a conformational change that assists the transport function of the protein.

Perhaps, the best characterized of the phosphoinositide signalling functions results from the hydrolysis of phosphatidylinositol phosphates by phospholipase C isoforms, in this instance to produce sn-1,2-diacylglycerols and inositol 3,4,5-trisphosphate (see next section), which act as second messengers. Only those polyunsaturated diacylglycerol species derived from PI(4,5)P₂ are able to bind and activate protein kinase C (α, ε, δ) isoforms both in vitro and in vivo. This lipid is doubly important as it binds strongly to these enzymes via a basic patch distal to a Ca²⁺ binding site, and this targets them selectively to the plasma membrane. Aberrant expression of phospholipase Cγ2 may be a factor in neurodegenerative diseases. Via the action of PI3 kinase, PI(4,5)P₂ is the precursor of PI(3,4,5)P₃ with its own distinctive signalling properties.

Phosphatidylinositol 3,4-bisphosphate can be produced by two routes and regulates a variety of cellular processes with relevance to health and disease that include B cell activation and autoantibody production, insulin sensitivity, neuronal dynamics, endocytosis and cell migration. It is known to bind selectively to several proteins, and it acts as a secondary messenger by recruiting the protein kinases Akt (protein kinase B) and so may influence the cell cycle, cell survival, angiogenesis and glucose metabolism. During endocytosis in the endolysosomal system, it is produced from PI(4,5)P₂ and controls the maturation of endocytic coated pits. Its synthesis and turnover are spatially segregated within the endocytic pathway. In epithelial cells, it is located on the apical membrane, i.e., facing the lumen, as opposed to the basolateral membranes, and it is believed to be is a determinant of the identity and function of the apical membrane.

Phosphatidylinositol 3,5-bisphosphate is present at low levels only in cells (0.04-0.1% of the total phosphatidylinositides), unless stimulated by growth factors, but it is important in membrane and protein trafficking, especially in the late endosomes in eukaryotes and in yeast vacuoles. For example, conversion of PI(3)P to PI(3,5)P₂ promotes endosomal maturation and degradative sorting. It is involved in the mediation of signalling in response to stress and hormonal cues and in the control of ion transport in membranes, while genetic studies confirm that it is essential for healthy embryonic development, especially in the nervous system.

Phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P₃) is almost undetectable in quiescent cells, but its intracellular level rises very rapidly from synthesis at the plasma membrane in response to agonists such as extracellular growth factors and hormonal stimuli. By recruiting proteins with pleckstrin homology (PH) domains to the plasma membrane, it has been implicated in a variety of cellular functions that include growth, cell survival, proliferation, cytoskeletal rearrangement, intracellular vesicle trafficking and cell metabolism. In particular, it is an important component of a signalling pathway in the cell nucleus. In epithelial cells, it is located on the basolateral membrane, i.e., facing adjacent cells, where it may be a determinant of the identity and function of this membrane. In contrast to phosphatidylinositol 3-phosphate, it opposes autophagy by binding to and activating the PH domain of Akt, so inducing cell proliferation. During feeding, various physiological responses lead to the secretion of insulin, which stimulates the phosphorylation of phosphatidylinositol 4,5-bisphosphate to phosphatidylinositol 3,4,5-trisphosphate and triggers a signalling cascade that leads to the suppression of autophagy. When this pathway is impaired, it has deleterious effects upon the insulin resistance associated with various metabolic diseases including obesity and diabetes, and it has been implicated in tumour cell migration and metastasis. PI(3,4,5)P₃ is also present in the nucleus and nucleoli of cells where it is believed to have functions in RNA processing/splicing, cytokinesis, protein folding and DNA repair. In contrast, like phosphatidylserine, it is reportedly transferred to the outer leaflet of the plasma membrane in aged or damaged cells as an 'eat‑me' signal for phagocytes and apoptosis.

The human immune system utilizes neutrophils, which are highly mobile cells, to eliminate pathogens from infected tissue. The first step is to track and then pursue molecular signals, such as cytokines, emitted by pathogens. It has been established that two phospholipids operate in sequence to point the neutrophils in the correct direction. The first of these is phosphatidylinositol 3,4,5-trisphosphate, which binds to a specific protein DOCK2 and enables it to translocate to the plasma membrane. Then phosphatidic acid, generated by the action of phospholipase D on phosphatidylcholine, takes over and directs the DOCK2 to the leading edge of the plasma membrane. This causes polymerization of actin within the cell and in effect reshapes the neutrophil and points it in the direction from which the pathogens signals are coming. On the other hand, Mycobacterium tuberculosis can subvert phosphoinositide signalling to arrest phagosome maturation by dephosphorylation of phosphatidylinositol 3-phosphate.

3. Water-Soluble Inositol Phosphates

As mentioned briefly above, hydrolysis of phosphatidylinositol phosphates by calcium-dependent phospholipase C (or 'phosphoinositidase C') leads to generation of sn‑1,2‑diacylglycerols (see the specific web page), which act as second messengers in animal cells and are of enormous metabolic importance. There are many different enzymes of this type, but the activity of the phosphoinositide-specific phospholipase C constitutes an essential step in the inositide signalling pathways. The enzyme exists in six families consisting of at least 13 isoenzymes, all of which have conserved regions such as the plekstrin homology (PH) binding domain. Each one has a distinctive role and can have a characteristic cell distribution that is linked to a specific function. Activity of these enzymes is stimulated by signalling molecules such as G-protein coupled receptors, receptor tyrosine kinases, Ras-like GTPases and calcium ions, thus linking the hydrolysis of phosphatidylinositol phosphates to a wide range of other cellular signals. As phospholipase C is a soluble protein located mainly in the cytosol, translocation to the plasma membrane is a crucial step in signal transduction. Regulation of these isoenzymes, of which PLCβ is most active, is vital for health as they are associated with the activation or inhibition of important pathophysiological processes, especially in relation to cancer (form PLCγ1).

The other products of the phospholipase C reaction that are of special relevance because of their many essential functions are water-soluble inositol phosphates. Up to 60 different compounds of this type are possible, and at least 37 of these have been found in nature at the last count, all of which are extremely important biologically. However, polyphosphoinositides with a phosphate in position 3 are not substrates for phospholipase C.

Generation of water-soluble inositol phosphates

For example, under the action of various physiological stimuli in animals, including sphingosine-1-phosphate, and acting via various G‑protein-coupled receptors, phosphatidylinositol 4,5-bisphosphate in the plasma membrane is hydrolysed to release inositol 1,4,5‑trisphosphate, an important cellular messenger that diffuses into the cytosol and stimulates calcium release from an ATP-loaded store in the endoplasmic reticulum via ligand-gated calcium channels (the diacylglycerols remain in the membrane to recruit and activate members of the protein kinase C family). The increase in calcium concentration, together with the altered phosphorylation status, activates or de-activates many different protein targets to enable cells to respond in an appropriate manner to the extracellular stimulus. For rapid replenishment of the phosphatidylinositol 4,5‑bisphosphate used in this way, a cycle of reactions - the phosphatidylinositol cycle - must occur (see below). On the other hand, a recent publication suggests that phosphatidylinositol 4-phosphate in the plasma membrane may be a more important source of diacylglycerols following stimulation of G protein–coupled receptors.

All the various inositol phosphates appear to be involved in the control of cellular events in very specific ways, but especially in the organization of key signalling pathways, the rearrangement of the actin cytoskeleton and intracellular vesicle trafficking. They have been implicated in gene transcription, RNA editing, nuclear export and protein phosphorylation. As these remarkable compounds can be rapidly synthesised and degraded in discrete membrane domains or even sub-nuclear structures, they are considered to be ideal regulators of dynamic cellular mechanisms. From structural studies of inositol polyphosphate-binding proteins, it is believed that the inositides may act in part at least by modifying protein function by acting as structural cofactors, ensuring that proteins adopt their optimum conformations. Phosphoinositides and the inositol polyphosphates are key components of the nucleus of the cell, where they have many essential functions, including DNA repair, transcription regulation and RNA dynamics. It is believed that they may be activity switches for the nuclear complexes responsible for such processes, with the phosphorylation state of the inositol ring being of primary importance. As different isomers appear to have specific functions at each level of gene expression, extracellular events must coordinate the production of these compounds in a highly synchronous manner.

In organisms from plants to mammals, an extra tier of regulatory mechanisms is produced by kinases that generate energetic diphosphate (pyrophosphate)-containing molecules from inositol phosphates. Conversely, these can by dephosphorylated by polyphosphate phosphohydrolase enzymes to recover the original inositol phosphates. These inositol pyrophosphates and the enzymes involved in their metabolism are likewise involved in the regulation of cellular processes by modulating the activity of proteins by a variety of mechanisms.

It should be noted that the phospholipase C isoenzymes regulate the concentration of phosphatidylinositol 4,5-bisphosphate and related lipids and thence their activities in addition to the generation of new biologically active metabolites. Some phosphatidic acid is synthesised from the diacylglycerols produced within the plasma membrane through the activity of diacylglycerol kinases, and this is transported back to the endoplasmic reticulum and ultimately can be re-utilized for phosphatidylinositol biosynthesis.

4. Phosphatidylinositides in Plants

In plants as in animals, phosphatidylinositol and polyphosphoinositides have essential biological functions, exerting their regulatory effects by acting as ligands that bind to protein targets via specific lipid-binding domains and so alter the location of proteins and their enzymatic activities. However, it appears that polyphosphoinositide metabolism developed in different ways after the divergence of the animal and plant kingdoms so the details of the processes in each are very different, not least because the subcellular locations of phosphoinositides differ appreciably between plants and animals. Phosphatidylinositol per se is of course the precursor of the phosphorylated forms and determines their fatty acid compositions. It has a role in inhibiting programmed cell death by acting as the biosynthetic precursor of the sphingolipid ceramide phosphoinositol and so reducing the levels of ceramide.

As in animals, the various phosphoinositides (five in total) are produced and inter-converted rapidly by a series of kinases and phosphatases (in many isoforms) in different cellular membranes in response to environmental or developmental cues. For example, phosphatidylinositol is generated mainly in the endoplasmic reticulum, while PI 4-kinases and their product are located in the trans-Golgi network and nucleus, and PI4P 5-kinases and product are present in the plasma membrane. During the biosynthesis of polyphosphoinositides, the first phosphorylation occurs at the hydroxyl groups at positions 3 or 4 of the inositol ring, catalysed by the appropriate kinases, while the second phosphorylation then takes place at position 5; PI 5-phosphate is produced by the action of a phosphatase on PI 3,5‑bisphosphate. Most other metabolites are produced via phosphatidylinositol 3-phosphate, and reports that some phosphatidylinositol 3,4,5-trisphosphate may be produced from phosphatidylinositol 4,5‑bisphosphate require confirmation. In contrast to mammalian phosphatidylinositol 3-kinases, which accept both phosphatidylinositol and its monophosphates as substrates, the plant enzyme acts only on the former.

Polyphosphoinositide metabolism in plants

The reverse reaction in plants is accomplished by phosphoinositide phosphatases, which can be grouped into three main families with many members and remove individual 4- or 5-phosphates by phosphatases of the suppressor of actin (SAC) family and the 5-phosphatase (5-Ptase) family. 3-Phosphates by are hydrolysed by the phosphatase and tensin homolog deleted on chromosome ten (PTEN) family. Each enzyme within these families can have differing subcellular locations, substrate specificities and regulatory mechanisms.

Although what might be considered normal levels of phosphatidylinositol 4-phosphate are present, the concentrations of phosphatidylinositol 4,5‑bisphosphate and other phosphoinositides are extremely low in plants (10 to 20-fold lower than in mammalian cells), although they still have vital functions. There are differences between cell types, but in Arabidopsis epidermal root cells, PI(4,5)P₂ is present at highest concentration in the plasma membrane (apex region) and nucleus, while PI4P slowly distributes between the plasma membrane and Golgi with the highest concentration in the former. Multivesicular bodies/late endosomes accumulate both PI3P and PI(3,5)P₂, and the tonoplasts and autophagosomes contain PI3P. How the various metabolites are transported between membranes has yet to be determined, but non-vesicular transport is believed to occur at membrane contact sites and vesicular transport probably occurs also.

Highly polarized distributions of phosphoinositides are found within membranes, in general oriented toward the cytosolic leaflet, and they are believed to be organized in nanoclusters together with other lipids and proteins. For example, phosphatidylinositol-4-phosphate is an important constituent of the plasma membrane in plant cells, where it controls the electrostatic state and is involved in cell division. It is the only phosphoinositide present at the cell plate, i.e., the membrane separating two daughter cells during cell division. PI(4)P may control the location and function of many membrane proteins, including those required for development, reproduction, immunity, nutrition and signalling, and it may interact with salicylic acid in the plant immune response, and it is produced during salt stress. However, specific functions are now being discovered for each of the plant phosphoinositides, which are produced rapidly in response to osmotic and heat stress, and it has become evident that a continuous turnover is essential for cell growth and development. For example, they have marked effects on the growth of many cell types and on guard cell function. In the nucleus, proteins have been identified that bind to phosphoinositides via the acyl chains, leaving the head group exposed for enzymatic modifications and signal transduction.

Scottish thistle Phosphoinositides are of special importance in microdomains at the tip of growing tissues such as the shoot apical meristem, pollen tubes and root hairs where phosphatidylinositol 4,5-bisphosphate functions in stem cell maintenance and organogenesis. In the plasma membrane, it is enriched in the detergent-resistant component commonly equated with 'rafts'. Although its concentration is low, PI(4,5)P₂ has been shown to have signalling functions by binding to a number of different target proteins, which have characteristic binding domains. For example, together with phosphatidic acid, PI(4,5)P₂ regulates the activity of a number of actin-binding proteins, which in turn control the activity of the actin cytoskeleton. This has a key role in plant growth, the movement of subcellular organelles, cell division and differentiation and plant defence. In addition, this lipid exerts a control over ion channels, ATPases and phospholipase C-mediated lipid degradation and the production of further second messengers. It is an important factor in both clathrin-mediated endocytosis and in exocytosis. The specificity of the interactions may be dependent on the fatty acid composition of the lipid and on the activity of phosphatidylinositol 4-phosphate 5-kinase.

As in animals, phosphoinositides have a role in endosomal sorting but through the central vacuole, which is a plant specific organelle with both lytic and storage functions. Phosphatidylinositol 3,5-bisphosphate is the least abundant of the phosphoinositides, but it is a crucial lipid for membrane trafficking systems. The PI to PI(3)P to PI(3,5)P₂ cascade, the second step requiring a kinase designated FAB1, is required for endosomal sorting events leading to membrane protein degradation or retrieval, vacuolar morphogenesis and autophagy. PI(3,5)P₂ is involved in stomatal closure and the growth of root hairs, and it is induced in salt stress.

Many different enzymes of the phospholipase C type that are specific for polyphosphoinositides have been isolated from higher plants; they are activated by Ca²⁺ and unlike their mammalian counterparts, they are not regulated by G proteins. It is not certain whether phosphatidylinositol is itself a substrate for these enzymes in vivo. Less is known of the metabolism of the water-soluble inositol phosphates produced in comparison to animals, and plants appear to lack a receptor for inositol 1,4,5-trisphosphate (IP₃), although it is the most abundant metabolite of this type and is reported to induce release of calcium ions to trigger stomatal closure. However, there is increasing evidence for lipid signalling mediated by phospholipase C in abiotic stress tolerance and development in plants. There is a general if contested belief that inositol hexakisphosphate (phytic acid or IP₆), produced at least in part by sequential phosphorylation of inositol 1,4,5-trisphosphate, is a more important cellular messenger in plants and mobilizes an endomembrane store of calcium ions. Inositol-1,2,4,5,6-pentakisphosphate (IP₅) is a structural co-factor of the jasmonic acid receptor coronatine insensitive 1, linking phosphoinositide signalling with phytohormone-controlled pathways.

In plants in contrast to animals, diacylglycerols, the other product of phospholipase C hydrolysis of phosphoinositides, are rapidly converted to phosphatidic acid by diacylglycerol kinases and have not been considered important in signal transduction. Plants lack protein kinase C, but they do have proteins with related properties that appear to be influenced by diacylglycerols. Via the action of phospholipase D, inositol phospholipids are a further source of phosphatidic acid with its well-characterized signalling functions in plants, especially in defence.

Yeasts and protozoa: Yeasts produce only five phosphoinositides with more phosphatidylinositol 4,5-bisphosphate than plants but no detectable phosphatidylinositol 3,4‑bisphosphate or phosphatidylinositol 3,4,5-trisphosphate. They are produced rapidly in response to nitrogen starvation, and phosphatidylinositol 3,5-bisphosphate synthesis in particular is induced by osmotic stress. In contrast, parasitic protozoa, such as Trypanosoma brucei, appear to have a cycle of phosphoinositide biosynthesis and presumably function that is close to that in animals. The Amoebozoa, such as Dictyostelium discoideum, possess Class I PI3Ks, which produce phosphatidylinositol 3,4,5‑trisphosphate directly at the plasma membrane from 1‑hexadecyl-2-(11Z-octadecenoyl)-sn-glycero-3-phospho-(1'-myo-inositol), i.e., with an ether-linked 16:0 chain at the sn-1 position.

5. Lyso-Phosphoinositides

Lysophosphatidylinositols: Formula of lysophosphatidylinositol Lysophosphatidylinositols (LPI), i.e., with a single fatty acid linked to the glycerol moiety, are formed as intermediates in the remodelling of the fatty acid compositions of the diacyl-lipids by the action of phospholipase A₁ or phospholipase A₂ (e.g., cPLA₂α), and when arachidonic acid is released for eicosanoid biosynthesis (see above). In ovarian cancer, LPI is elevated appreciably to around 15µM in ascites, and it is present at high levels in obese subjects.

It has become apparent relatively recently that like other lysophospholipids, lysophosphatidylinositol and the polyphospho-analogues may have messenger functions. For example, it has long been known to stimulate the release of insulin from pancreatic cells, suggesting a role in glucose homeostasis. sn-2-Arachidonoyl-lysophosphatidylinositol is an endogenous ligand for a G protein-coupled receptor GPR55, and thereby can induce rapid phosphorylation of certain enzymes, including a protein kinase, which promote cancer cell proliferation, migration and metastasis. Indeed, lysophosphatidylinositol is a biomarker for poor prognosis in cancer patients, and its concentration is elevated significantly in highly proliferative cancer cells in vitro. GPR55 is expressed in many regions of the brain, the intestines, endocrine pancreas and islets (where it may stimulate insulin release). It has been implicated in macrophage activation and inflammation and in several metabolic diseases. It is reported to be a precursor of the endocannabinoid 2‑arachidonoylglycerol by the action of human glycerophosphodiesterase 3 as a lysophospholipase C. This enzyme suppresses the receptor for lysophosphatidylinositol, and so acts as a switch between GPR55 and endocannabinoid (CB2) signalling.

Glycerophosphoinositol: Sequential removal of both fatty acids from phosphatidylinositol by a specific phospholipase A₂ (PLA₂IVα) with both phospholipase A₂ and lysophospholipase activities releases water-soluble glycerophosphoinositol. While this can be hydrolysed by a glycerophosphodiester phosphodiesterase to inositol 1-phosphate, glycerophosphoinositol per se has distinctive biological activities and functions, as do related compounds derived from the phosphatidylinositol phosphates. Glycerophosphoinositol has anti-inflammatory activity in that it inhibits the inflammatory and thrombotic responses induced by bacterial lipopolysaccharides (endotoxins).

6. The Phosphatidylinositol Cycle

Phosphatidylinositol is at the centre of a cycle of reactions and intermediates that are involved in innumerable aspect of cellular signalling in animals (a similar cycle could be described for plants). These are discussed individually at length above, but it is useful to point out how each component forms part of a larger pattern. In brief as illustrated, the various synthetic and hydrolytic reactions involved in phosphoinositide metabolism can be considered to constitute a phosphatidylinositol cycle with enzymes located both in the endoplasmic reticulum and plasma membrane, so lipids have to be transferred across the cytosol in both directions between the two to complete the cycle, probably via adjacent membrane structures and facilitated by proteins of the phosphatidylinositol transfer protein membrane-associated family (PITPNM or nir2), which may channel phosphoinositide production to specific biological outcomes. Phospholipase C and phosphatidylinositol-4-phosphate 5-kinase (PI4P 5K) are located in the plasma membrane, while the cytidine diphosphate-diacylglycerol synthase (CDS2) and phosphatidylinositol synthase are in the endoplasmic reticulum. The epsilon isoform of diacylglycerol kinase (DGKε) is located at contact sites between the endoplasmic reticulum and plasma membrane, but there are nine further isoforms with differing cellular and subcellular locations that may be involved in the cycle. Each turn of the cycle uses a great deal of energy and consumes three moles of ATP, together with cytidine triphosphate and inositol. If it is assumed that the pyrophosphate is hydrolysed by endogenous pyrophosphatases to inorganic phosphate, the cycle can proceed in one direction only.

The phosphatidylinositol cycle

Factors such as membrane curvature must be considered, and the diagram is of necessity a considerable over-simplification. Many of the lipid intermediates in the cycle can be precursors for other lipids, and for example, diacylglycerols are potential precursors for triacylglycerols while phosphatidic acid is a precursor for phosphatidylcholine and phosphatidylethanolamine. Each lipid intermediate is subject to remodelling of the acyl chains via the Lands cycle, and polyunsaturated fatty acids released can be utilized for eicosanoid production. A further by-product of the cycle is inositol triphosphate, which contributes to the regulation of intracellular calcium levels.

It has been suggested that the unique molecular species composition of phosphoinositides (mainly 18:0-20:4) could influence their selective recycling back into phosphatidylinositol as many of the enzymes involved prefer this substrate. A further proposal is that the phosphatidylinositol cycle could act to enrich this species through multiple passages around the cycle.

7. Glycosyl-Phosphatidylinositol Anchors for Proteins, Lipophosphoglycans and Phosphatidylinositol Mannosides

Phosphatidylinositol is known to be the anchor that links a variety of proteins to the external leaflet of the plasma membrane via a glycosyl bridge (glycosyl-phosphatidylinositol(GPI)-anchored proteins). However, this was considered a sufficiently important topic for its own web page. Lipophosphoglycans (lipoarabinomannans and arabinogalactans) and phosphatidylinositol mannosides are important components of the membranes of parasitic protozoa and bacteria, but for convenience they too are discussed with the GPI-anchored proteins.

8. Analysis

Like all acidic phospholipids, phosphatidylinositol is not particularly easy to isolate in a pure state, special care being necessary to ensure that it is fully resolved from phosphatidylserine. However, this can be accomplished by adsorption TLC or HPLC with appropriate procedures. The phosphatidylinositol phosphates are a different matter, however, because of their high polarity and low abundance in tissues. It is necessary to used acidified solvents to extract them efficiently from tissues and to ensure that they are in a single salt form. For isolation of individual components, TLC methods are usually favoured, although detection can be a problem - one approach being to equilibrate with radioactive phosphorus to facilitate detection and quantification by liquid scintillation counting. HPLC with mass spectrometric detection is now showing great promise, although regiospecific analysis of fatty acid distributions on the glycerol moiety still seems to be fraught with difficulties. Analysis of the lipid-glycoconjugate-protein complexes and of the lipophosphoglycans is a rather specialized task for which modern mass spectrometric and NMR facilities are essential.

	© Author: William W. Christie
	Contact/credits/disclaimer	Updated: January 18^th, 2023