Phosphatidylinositol(4,5)bisphosphate: diverse functions at the plasma membrane

Abstract

Phosphatidylinositol(4,5) bisphosphate (PI(4,5)P2) has become a major focus in biochemistry, cell biology and physiology owing to its diverse functions at the plasma membrane. As a result, the functions of PI(4,5)P2 can be explored in two separate and distinct roles - as a substrate for phospholipase C (PLC) and phosphoinositide 3-kinase (PI3K) and as a primary messenger, each having unique properties. Thus PI(4,5)P2 makes contributions in both signal transduction and cellular processes including actin cytoskeleton dynamics, membrane dynamics and ion channel regulation. Signalling through plasma membrane G-protein coupled receptors (GPCRs), receptor tyrosine kinases (RTKs) and immune receptors all use PI(4,5)P2 as a substrate to make second messengers. Activation of PI3K generates PI(3,4,5)P3 (phosphatidylinositol(3,4,5)trisphosphate), a lipid that recruits a plethora of proteins with pleckstrin homology (PH) domains to the plasma membrane to regulate multiple aspects of cellular function. In contrast, PLC activation results in the hydrolysis of PI(4,5)P2 to generate the second messengers, diacylglycerol (DAG), an activator of protein kinase C and inositol(1,4,5)trisphosphate (IP3/I(1,4,5)P3) which facilitates an increase in intracellular Ca2+. Decreases in PI(4,5)P2 by PLC also impact on functions that are dependent on the intact lipid and therefore endocytosis, actin dynamics and ion channel regulation are subject to control. Spatial organisation of PI(4,5)P2 in nanodomains at the membrane allows for these multiple processes to occur concurrently.

Keywords: endocytosis; exocytosis; phosphatidylinositol; phospholipases.

Figure 1. Phosphatidylinositol(4,5)bisphosphate (PI(4,5)P₂)

(A) Multiple functions of PI(4,5)P₂ at the plasma membrane. PI(4,5)P₂ is a substrate for two signalling pathways, phospholipase C (PLC) and phosphoinositide 3-kinase (PI3K). PI(4,5)P₂ also functions as an intact lipid to regulate ion channels, membrane dynamics and the actin cytoskeleton. Three pathways can deplete PI(4,5)P₂ levels, marked with yellow arrows – PLC, PI3K and 5-phosphatase. Abbreviations: DAG, diacylglycerol; GPCR, G-protein coupled receptor; IP₃, inositol(1,4,5)trisphosphate; PI, phosphatidylinositol; PI4K, PI 4-kinase; PI4P, phosphatidylinositol 4-phosphate; PI4P5K, PI4P 5-kinase; PKC, protein kinase C; RTK, receptor tyrosine kinase. (B) Structure of PI(4,5)P₂. PI(4,5)P₂ comprises a glycerol backbone with an inositol headgroup which is phosphorylated at the 4 and 5 positions on the inositol ring. The fatty acid composition of PI(4,5)P₂ is distinctive; stearic acid (C18:0) at the sn-1 position and arachidonic acid (C20:4) at the sn-2 position of the glycerol backbone. (C) PI(4,5)P₂ can bind domains such as PH or by electrostatic interactions to basic residues of arginines and lysines. PI(4,5)P₂ can bind to structured domains such as PH domains or it can bind to unstructured clusters of positively charged lysine and arginine residues in proteins due to electrostatic interactions. Abbreviation: PH domain, pleckstrin homology domain.

Figure 2. Synthesis and degradation of PI(4,5)P₂ – phospholipase C cycle

PLC hydrolyses PI(4,5)P₂ resulting in the formation of the second messengers, IP₃ and DAG. DAG is phosphorylated to PA at the plasma membrane by DAG kinase (DAGK). PA is transferred to the ER via lipid transfer proteins. In the ER, PA is converted into CDP-DAG catalysed by CDS enzymes (CDS1 and CDS2). In the final step, inositol and CDP-DAG are synthesised into PI catalysed by the enzyme, PI synthase (PIS). The newly synthesised PI is transferred to the plasma membrane for phosphorylation to PI(4,5)P₂ by the resident enzymes, PI4KIIIα and PIP5K. Abbreviations: CDP-DAG, cytidine diphosphate diacylglycerol; CDS, CDP-DAG synthase; IP₃, inositol(1,4,5)triphosphate; PA, phosphatidic acid; PI, phosphatidylinositol; PIS, PI synthase; PITP, phosphatidylinositol transfer protein; PI4P, PI 4-phosphate; PI(4,5)P₂, phosphatidylinositol (4,5) bisphosphate.

Figure 3. Mammalian phosphoinositide-specific phospholipase C (PLC) families

(A) Domain organisation of PLC enzymes. Domain organisation of PLC families, showing the PLC-core (green), that includes the catalytic βα−barrel domain (light green), and domains unique for each PLC family (pink). Some of the well-characterised regulatory interactions are indicated by symbols. Abbreviations: CDC25, cell cycle division 25 (Ras GEF domain); cSH2, C-terminal SH2; CTD; C-terminal domain; C2, protein kinase C conserved region 2; EF, EF-hands; nSH2, N-terminal SH2; PH, pleckstrin homology domain; RA, Ras-association domain; SH2, Src homology 2 domain; SH3, Src homology 3 domain; sPH, split PH; X and Y, conserved halves of the catalytic domain. (B) Mechanism of PLC activation. One common aspect of PLC activation involves the release of autoinhibition. In PLCγ enzymes, the activation is triggered by phosphorylation of a specific Tyrosine (Y) residue (yellow) within the regulatory region. In the inactive form, two domains within the regulatory region (cSH2 and sPH) directly contribute to autoinhibition. Following phosphorylation, the critical pY residue (yellow) binds to the cSH2 domain resulting in repositioning of the regulatory region and release of autoinhibition.

Figure 4. Class I Phosphoinositide-3-kinases (PI3K)

(A) Domain organisation of Class I PI3K. Domain organisation of Class I PI3Ks (IA and IB) showing the catalytic subunits (green), that include the kinase domain (light green), and regulatory subunits (pink). Heterodimers, comprising a specific combination of one catalytic and one regulatory subunit within each subclass, are commonly designated based on the identity of the catalytic subunit as PI3Kα, PI3Kβ, PI3Kγ and PI3Kδ. Abbreviations: ABD, adaptor-binding domain; BH, breakpoint cluster region homology; cSH2, C-terminal SH2; C2, protein kinase C conserved region 2; i-SH2, inter-SH2 domain; nSH2, N-terminal SH2. (B) Activation of PI3Kα. Schematic of the activation of PI3Kα (p110α/p85α heterodimer) downstream of RTKs and adaptors containing phosphorylated YXXM-motifs (pYXXM). The binding of PI3Kα to these proteins at the membrane proximity is mediated by the SH2 domains in p85α, resulting in disruption of inhibitory contacts with the p110α catalytic subunits. Ras also activates PI3Kα, with Ras activation being strongly synergistic with activation downstream of phosphorylated RTKs and adapters.

Figure 5. Actin cytoskeleton dynamics regulated by PI(4,5)P₂

Regulation of the actin-binding proteins, cofilin, N-WASP and ERM proteins by PI(4,5)P₂ levels. All these actin-binding proteins associate with PI(4,5)P₂ through similar multivalent electrostatic interactions, but have different affinities for P(4,5)P₂. Cofilin has low affinity, N-WASP has medium affinity and ERM proteins have high affinity. Cofilin is only bound to the membrane when PI(4,5)P₂ is present at high density. When PI(4,5)P₂ levels fall, cofilin is released into the cytosol to promote actin filament disassembly. In contrast, N-WASP interactions with PI(4,5)P₂ results in a change in confirmation leading to activation; this allows the binding of actin-related protein 2/3 (Arp2/3) to mediate actin filament nucleation at the plasma membrane. ERM proteins are stably attached to the membrane by PI(4,5)P₂ and link actin filaments to the plasma membrane. Cofilin and N-WASP require high PI(4,5)P₂ density for interactions with the membrane, whereas ERM remain bound to the membrane at low PI(4,5)P₂ density. Figure is adapted from [14]. Abbreviations: Arp2/2, actin-related protein 2/3; ERM, Ezrin, Radixin, Moesin; N-WASP, neural Wiskcott–Aldrich syndrome protein.

Figure 6. Examples of membrane peripheral and membrane integral proteins regulated by PI(4,5)P ₂

(A) Binding of PI(4,5)P₂ to the protein complex, AP2, changes its conformation to allow cargo and clathrin interactions. The adaptor protein, AP2 is a complex of four proteins consisting of a core comprising the N-terminal domains of the α-and β2-adaptins in complex with the μ2 and σ2 subunits. The α, β2 and μ2 subunits all contain PI(4,5)P₂ binding sites marked with pink stars. Long flexible linkers, referred to as hinge regions, connect the C-terminal appendage domains of α-and β2-adaptins to the core. AP2 exists in a closed conformation in the cytosol, in which the clathrin binding site is buried by interactions between the β2 hinge and the core and the cargo binding site on the μ2 subunit are also buried. Initially, the surface-exposed PI(4,5)P₂ binding site on the α-and β2-adaptin interact with the lipid triggering an allosteric conformational change to an open conformation. This exposes the clathrin binding site on the β2 hinge as well as the PI(4,5)P₂ and cargo binding sites of μ2. Figure adapted from [99]. (B) Regulation of potassium channels by PI(4,5)P₂ depletion by PLC. Potassium channels are maintained in the open state when bound to PI(4,5)P₂. Stimulation of the muscarinic M1 receptor by a cholinergic stimulus activates PLC to hydrolyse PI(4,5)P₂. PI(4,5)P₂ depletion results in closure of the ion channel. Abbreviations: M₁R, M₁ muscarinic receptor; PI(4,5)P₂, phosphatidylinositol(4,5,)bisphosphate. Figure adapted from [154].