Báo cáo khoa học: Membrane compartments and purinergic signalling: the role of plasma membrane microdomains in the modulation of P2XR-mediated signalling pot

MINIREVIEW Membrane compartments and purinergic signalling: the role of plasma membrane microdomains in the modulation of P2XR-mediated signalling Mikel Garcia-Marcos1, Jean-Paul Dehaye2 and Aida Marino3 Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA, USA ´ Laboratoire de Biochimie et de Biologie Cellulaire, Institut de Pharmacie C.P 205 ⁄ 3, Universite libre de Bruxelles, Belgium Departamento de Bioquimica y Biologia Molecular, Universidad del Pais Vasco, Bilbao, Spain Keywords caveolae; compartmentalization; detergentresistant membrances; ectonucleotidases; lipid rafts; membrane fractionation; microdomains; P2X; purinergic receptors; purinoceptors Correspondence M Garcia-Marcos, 9500 Gilman Dr., Mailcode 0651, La Jolla, CA 92093-0651, USA Fax: +1 858 534 8649 Tel: +1 858 534 7713 E-mail: mgarciamarcos@ucsd.edu (Received 15 July 2008, accepted 29 September 2008) doi:10.1111/j.1742-4658.2008.06794.x Purinergic signalling is implicated in virtually any cellular and physiological function These functions are mediated through the activation of different receptor subfamilies, among which P2X receptors (P2XRs) are ligand-gated ion channels that respond mostly to ATP In addition to forming a nonselective cation channel, these receptors engage with a complex network of signalling pathways, including protein kinase cascades, lipid signal mediators and proteases It is poorly understood how P2XR stimulation couples to such a variety of intracellular pathways and how the outcome from this complex signalling network is tuned In this context, segregation of receptors and other signalling components at the plasma membrane is an attractive explanation Lipid rafts are microdomains of biological membranes with unique physicochemical properties that make them segregate from the bulk of the membrane, provoking the differential partition of receptors and signalling molecules among different domains of the plasma membrane Here we give an overview of the properties of lipid rafts and how they are studied, along with recent advances in the understanding of their role in modulating P2XR-mediated signalling ATP is the major energy reserve within cells, where its concentration is in the millimolar range Most of the energy needed by the cell is obtained through hydrolysis of the anhydride bond between the b and the c phosphate of the nucleotide This canonical feature of ATP in cellular function was probably the cause of the scientific community’s resistance to the ‘purinergic hypothesis’ proposed by Geoffrey Burnstock in the early 1970s [1,2] The regulation of cellular functions by extracellular purines had been reported as early as 1929 [3], but the idea of ATP (and its derivatives) working as a neurotransmitter was conceived as an attack on the rational conservation of energy by the cell First, why would a cell release ATP, and second, what would be the targets of its action? These questions have been answered to some extent by the uncovering of a myriad of roles for extracellular nucleosides and nucleotides in the control of multiple cellular and physiological functions It is now clearly established that adenine nucleotides can be released into the medium via a number of mechanisms, including release from dying cells or leakage from large pores or transport mechanisms in intact cells [4], and that cellular responses to these extracellular nucleotides are medi- Abbreviations ENaC, epithelial sodium channel; ERK ⁄ 2, extracellular signal-regulated kinase ⁄ 2; MAP, mitogen-activated protein; N-SMase, neutral sphingomyelinase; PKC, protein kinase C; PKD, protein kinase D; PLA2, phospholipase A2; PLC, phospholipase C; PLD, phospholipase D; RTK, receptor tyrosine kinase 330 FEBS Journal 276 (2009) 330–340 ª 2008 The Authors Journal compilation ª 2008 FEBS M Garcia-Marcos et al ated through the activation of plasma membrane receptors Many of these receptors have been cloned and characterized both functionally and pharmacologically [5] Two main classes of purinergic receptors are well characterized, i.e P1 receptors which bind adenosine, and P2 receptors which are responsive to phosphorylated nucleosides as ATP, ADP and other related nucleotides [5,6] P2 receptors have been further subdivided into the P2Y and P2X subfamilies [6] P2Y receptors, like P1 receptors, are members of the superfamily of G protein-coupled receptors (GPCR) and P2X receptors are ligand-gated ion channel receptors The overall structure of the seven P2X receptors cloned to date indicates a number of conserved features [4,7,8]: each subunit possesses two transmembrane-spanning regions and a large extracellular loop This extracellular domain contains conserved sequences for nucleotide binding as well as for possible modulation of receptor function by cations; it also contains 10 conserved cysteine residues for disulfide bond bridging, and several glycosylation sites Regarding the intracellular extremities, conserved phosphorylation sites for protein kinases have been reported to play a role in receptor desensitization and in limited cases, such as for the ‘atypical’ P2X7 receptor which contains a larger C-terminal tail [9], defined motifs for binding to signalling adaptor proteins have been described The protein sequence identity between the different P2X receptors ranges from  30% to  50% The stoichiometry of a functional P2X receptor is believed to involve three identical or different subunits [7] Activation of a functional receptor by ligand binding leads to the opening of a channel pore, which behaves as a non-selective cation channel (typically for sodium, calcium and potassium ions as well as protons) P2X receptors are widely distributed across different tissues and organs [10] They are expressed in both excitable cells (such as neurons) and non-excitable cells (such as epithelial and immune cells) Some remarkable functions of P2XR include regulation of exocrine secretion, pain transmission, ion transport in the kidney, inflammatory response, homeostasis of the central nervous system and tumour development A detailed description of the tissue distribution and function of the P2X receptor in a subtype-specific way is given in more comprehensive reviews [10,11] The (physio)pathological implications of this subfamily of receptors in cellular signalling make them attractive therapeutic targets The coupling between stimulation of the P2X receptor and the generation of intracellular signalling cascades is still poorly understood Although P2XR activation can trigger the rapid elevation of intracellu- P2X receptors and membrane microdomains lar calcium ions as a second messenger, it is also coupled to a number of signalling molecules (which in many cases are not directly regulated by intracellular calcium) It has been reported that P2XR can activate several Ser ⁄ Thr kinases [such as protein kinase C (PKC), Akt ⁄ protein kinase B (PKB), protein kinase D (PKD), extracellular-signall regulated kinase ⁄ (ERK ⁄ 2), mitogen-activated protein kinase (MAPK) p38] [12–17], caspases [18], lipid kinases (such as phosphoinositide 3-kinase) [14,17] and phospholipases (such as PLA2, PLD and SMase) [19–24] This variety of signalling pathways that can be activated by the different P2XR members raises the question about how the correct coupling and fine tuning of the signalling in response to extracellular stimuli is achieved An attractive explanation is presented by the concept of plasma membrane compartmentation brought up by the ‘lipid raft’ hypothesis What is a lipid raft? The fluid–mosaic model proposed by Singer and Nicholson in 1972 depicted biological membranes as a homogeneous sea of lipids where proteins floated as icebergs [25] In this scenario, lipids formed a 2D milieu having little effect on protein function However, seminal biophysical studies on model lipid membranes indicated that biomembranes may be heterogeneous and that lipids could coexist in different physical phases [26] According to this hypothesis, cholesterol and phospholipids with saturated alkyl chains would form a tightly packed liquid-ordered phase, where lipids would have restrained mobility [26] Translating this concept to the cellular membranes was first attempted through formulation of the ‘lipid raft’ hypothesis in the context of membrane transport in polarized epithelial cells [27] It was postulated that domains rich in cholesterol and sphingolipids are preformed in the trans-Golgi network giving rise to the differential composition between apical and basolateral membranes of polarized epithelial cells The idea evolved, not without controversy, in the 1990s to be crystallized in the proposal of ‘lipid rafts’ as functional entities in the cellular membranes involved in both trafficking and cellular signalling processes [27–30] Recently, a definition of lipid rafts was proposed during a Keynote Symposium on Lipid Rafts and Cell Function held in Steamboat Springs, CO: ‘Membrane rafts are small (10–200 nm), heterogeneous, highly dynamic, sterol- and sphingolipidenriched domains that compartmentalize cellular processes Small rafts can sometimes be stabilized to form larger platforms through protein–protein and FEBS Journal 276 (2009) 330–340 ª 2008 The Authors Journal compilation ª 2008 FEBS 331 P2X receptors and membrane microdomains M Garcia-Marcos et al protein–lipid interactions’ [31] By extension, lipid rafts could be defined as localized rigid regions within the bulk of fluid membrane and which are enriched in cholesterol and (glycero-)sphingolipids In addition the fatty acid chains of the phospholipids composing these rigid domains are generally more saturated than those in the lipids of the surrounding membrane In fact, this fatty acid composition favors a tighter packing of phospholipids and cholesterol, increasing the rigidity of these domains [32–35] This composition accounts for their insolubility in non-ionic detergents, a hallmark that has been used as an ‘operational definition’ of these domains and which has been exploited to study them from a biochemical point of view (see below) [29,35,36] Probably the most relevant reason for the implication of lipid rafts in the signalling process is that they can contain or exclude proteins such as receptors, transducer ⁄ adaptor proteins and effectors, which are directly involved in signal transduction [35,37,38] The clustering of signalling molecules within rafts provides a rational explanation for the high efficiency and specificity observed in signal transduction processes which otherwise would be difficult to explain in a model in which the different signalling components localize and interact randomly across the plane of a fluid plasma membrane Interestingly, caveolae are also considered to be plasma membrane domains with the property of organizing signalling molecules [35,37] Caveolae were originally described by Palade in 1953 as flask-shaped plasma membrane invaginations [39] and were later found to be enriched in the structural protein caveolin-1 [40,41] Caveolae are often studied as a subset of lipid rafts because they are also enriched in cholesterol and in (glycero)sphingolipids with saturated fatty acids and are less fluid than the surrounding bulk of membranes In fact, many methods designed to isolate lipid rafts (such as resistance to solubilization in non-ionic detergents) cannot discriminate for caveolae How are lipid rafts studied? One of the biggest challenges in the field of lipid raft research has been the transposition from model membrane systems to cell membranes It was originally reported that lipids can coexist in model membranes in liquid-disordered and liquid-ordered states and that the latter have the property to resist solubilization by non-ionic detergents (i.e Triton X-100) [26,42] These results were later used to explain how a subset of membranes acquired resistance to solubilization by detergents during its transport from the Golgi to the plasma membrane [27,29] Thus, the proposal that 332 membranes in a cell could be segregated into domains with different properties and resistance to solubilization by non-ionic detergents was initially established as an operational definition of lipid rafts The use of this general biochemical approach has not been without controversy [43] The existence of lipid rafts was questioned and some naysayers pretended they were merely an artefactual consequence of the methodology used to isolate them The existence of lipid rafts in vivo is now supported by a number of studies For example, it has been shown by direct live cell microscopy that ‘raft-resident’ clusters of proteins segregate from ‘non-raft’ proteins in the cell membrane [44–46] The same technique combined with the use of Laurdan as a fluorescent probe has also demonstrated that the lipid components of the membrane can segregate into domains with different physical properties (i.e more versus less ordered) [47] The use of immunoelectron microscopy has been another interesting approach to quantify the degree of clustering of molecules in ‘ripped-off’ plasma membrane sheets [48–50] Using this technique, several protein and lipid markers have been studied for their ability to cluster in response to extracellular stimuli and the size of the domains containing those clusters has been estimated In summary, multiple lines of evidence have helped to argue in favour of the existence of lipid rafts in cell membranes and the use of detergent-resistance and other alternatives (see below) as a biochemical approach to their study are considered to be valid if performed carefully Isolation of lipid rafts ⁄ caveolae is very often the first step in the biochemical analysis of the role of these domains in cellular signalling After disruption of cells, lipid rafts ⁄ caveolae can be extracted by virtue of their resistance to solubilization in non-ionic detergents or even using non-detergent methods These microdomains can then be isolated by ultracentrifugation in density gradients because they are highly buoyant due to a relatively high lipid ⁄ protein ratio and can be recovered from the lower density fractions [29,35] The ‘canonical’ detergent-based method consists of solubilization of cellular membranes with 1% Triton at °C and recovery of the buoyant membrane fractions from the interface of a sucrose density gradient (typically from the 5–35% sucrose interface) [29] Similar methods have been described using lower detergent concentrations and other non-ionic detergents such as NP-40, Chaps, Lubrol, Brij-98 or octyl-glucoside [36,51] The fact that the fractions isolated using different methods contain not only a substantial overlap but also major differences has been interpreted as pre-existing heterogeneity in the population of lipid rafts Detergent-free FEBS Journal 276 (2009) 330–340 ª 2008 The Authors Journal compilation ª 2008 FEBS M Garcia-Marcos et al methods have also been described, where the critical step is a fine disruption of the membranes by extensive sonication A popular version of this method utilizes a high pH buffer to strip peripheral proteins [52]; another common method is performed in neutral pH buffers but includes several density-gradient steps to obtain the desired purified fraction [53] More recently, a simplified version of the latter protocol was described by MacDonald and Pike, which yields a membrane fraction enriched in lipid raft markers by a one-step density gradient ultracentrifugation [54] Methods developed to specifically isolate caveolae and not other membrane microdomains have also been developed Oh and Schnitzer used the silica-coating technique originally described for the isolation of endothelial membranes to subsequently disrupt them and obtain a caveolar fraction by floatation in a density gradient [55,56] Immunoisolation of the caveolar fraction from a detergent-resistant preparation of membranes using caveolin Ig has also been successful [57] None of the methods described above is flawless and the best way to achieve a meaningful result is by performing rigorous controls and using complementary approaches to validate the results For example, detergent-based methods can abolish the interaction of proteins weakly associated with lipid rafts or provoke the loss of raft proteins that also tightly bind to cytoskeletal components The detergent extraction conditions can also lead to the artefactual formation of membrane domains and promote lipid mixing between different membrane fractions [42,51] ‘Detergent-free’ methods might seem to interfere less with the native properties of the membranes, but are less reproducible and more likely to contain contaminants because any lipid-rich membranous fraction can potentially float in a density gradient [35,54,58] Normally, the lipid raft fractions should contain < 5% of the total protein; they should be relatively enriched in cholesterol, contain the majority of lipid raft ⁄ caveolar markers (caveolin-1, flotillins, etc.) and, importantly, be devoid of protein markers for other organelles (Golgi, endoplasmic reticulum, nucleus) or for non-raft membrane domains (adaptins, transferrin receptor) Another way to study the role of lipid rafts is to analyze the impact on cell functions of the manipulation of their constituents Methyl-b-cyclodextrins, which sequester cholesterol from membranes, or filipin, a cholesterol-binding antibiotic, are used extensively to interfere with the ability of cholesterol to maintain the structure of lipid rafts [35,37] The role of lipid rafts can be first tested by analysing isolated raft fractions from pre-treated cells and subsequently use these P2X receptors and membrane microdomains agents to uncover the effect of lipid raft disassembly in signalling functions of intact cells Lipid rafts in cellular signalling One crucial role attributed to lipid rafts is their ability to organize signalling molecules in an environment proper for efficient and fine-tuned signal transduction [37] The ability of lipid rafts to recruit some molecules and exclude others can help, for example, to couple receptor ⁄ transducer ⁄ adaptor ⁄ effectors and exclude enzymes that contribute to turn off the signal The dynamic nature of lipid rafts might also contribute to regulate the duration of a response Lipid rafts ⁄ caveolae have been shown to be implicated in a myriad of signalling pathways For example, receptor tyrosine kinase (RTK) for epidermal growth factor, insulin, nerve growth factor or platelet-derived growth factor have been shown to localize to lipid rafts [59–61] However, upon stimulation with the respective agonists, the localization of these receptors with regard to raft versus non-raft domains varies from case to case, implying different mechanisms of regulation A substantial number of studies have investigated Ras signalling in the context of lipid rafts Ras is a small GTPase that is activated downstream of many RTKs and mediates signalling to MAPK and phosphatidylinositol 3-kinase from the plasma membrane Specifically, the H-ras isoform is recruited to lipid rafts upon activation, which triggers intracellular signalling [49,62] Another molecule that mediates signalling from RTKs and is found in lipid rafts is the lipid phosphatidylinositol 4,5-bisphosphate [63,64] This is a substrate for both PLCc and phosphatidylinositol 3-kinase, two enzymes usually coupled to RTKs The intermediate phosphatidylinositol 4,5-bisphosphate is also shared with certain signalling pathways coupled to GPCRs Many members of this family of receptors (b-adrenergic receptors, muscarinic receptors, endothelin receptors, rhodopsin) are located in lipid rafts [65–70] where they are in close proximity to their transducing GTP-binding protein (Gs, Gi, Go, Gq and transducin alpha subunits) [53,69,71–73] and to some effectors like adenylyl cyclases or the guanosine 3¢,5¢cyclic monophosphate-dependent phosphodiesterase in retinal cells [65,66,69,72] In addition to all the components listed above some regulators of G-protein signalling (RGS proteins), responsible for turning off the Ga subunit, have also been found in lipid rafts [74,75] Another major signalling pathways found to operate in lipid rafts include immune system-related receptors, such as immunoglobulin E receptors (FceRI) [76] or T-cell antigen receptors [77] FEBS Journal 276 (2009) 330–340 ª 2008 The Authors Journal compilation ª 2008 FEBS 333 P2X receptors and membrane microdomains M Garcia-Marcos et al Lipid rafts and P2X receptors Purinergic receptors have also been found to localize to lipid rafts ⁄ caveolae Considering that many components of the signalling machinery coupled to GPCRs are targeted to lipid rafts, it is not surprising that P1 and P2Y receptors have been found to compartmentalize in the plasma membrane One of the earliest findings in this regard was the enrichment of the adenosine receptor in caveolar fractions of cardiomyocytes [78] This receptor was found to translocate out of caveolae upon stimulation, contrary to other cardiac receptors such as the M2 muscarinic receptor [68] This observation could be interpreted as a different mechanism for the regulation of coupling to effectors and ⁄ or desensitization It was also shown that signalling through P2Y subfamily receptors in endothelial cells was compartmentalized within cholesterol-rich domains [79] and P2Y2 receptors have also been morphologically localized to caveolae at least in placenta [80] In platelets, the P2Y12-mediated decrease in cAMP levels is sensitive to lipid raft disruption [81,82] This receptor forms functional homo-oligomers in platelet membrane rafts; clopidogrel (an antithrombotic drug) and its active compound derivative probably block the P2Y12 receptor by disassembling these oligomers and displacing them to non-raft domains [82] In contrast to these results, the depletion of cholesterol by methyl-b-cyclodextrins does not affect the increase of calcium levels in response to the activation of platelet P2Y1 receptors [83] As previously mentioned, P2X receptors are not coupled to G proteins but form non-selective cation channels with structural similarities to the sodium channels encoded by the ENaC ⁄ degenerins gene [84] Some voltage-regulated ion channels have been reported to function via lipid rafts [85], a property shared with some ligand-gated channels like nicotinic, AMPA, NMDA, GABA and ATP receptors [86] However, localization in membrane microdomains is not a general property of P2X receptors and the experimental demonstration of their localization depends on the methodology to isolate rafts (see Table for a summary of the different P2XR found in lipid rafts and the methodology used in each case) The first evidence of a P2X receptor in lipid rafts was provided by Vacca et al who reported that the P2X3 receptor (endogenous or exogenously expressed) localized to lipid rafts in neuronal cells regardless of the isolation method used (detergent-based or detergent-free) and the activation status of the receptor [87] In the same study, these authors also reported that other P2X receptors (P2X1, P2X2, P2X4, P2X7) did not localize in lipid rafts prepared using a Triton X-100 extraction method The presence of these receptors was not investigated in lipid rafts prepared by a detergent-free protocol Yet further studies confirmed that this method was the most appropriate considering that the localization of P2X receptors in lipid rafts was sensitive to detergent extraction For example, the P2X1 receptor could be isolated in raft fraction prepared by a detergent-free protocol but increasing concentrations of Triton X-100 (0.1–1%) led to a shift of the protein to high-density detergent-soluble fractions [88] This receptor was found in lipid rafts when heterologously expressed in HEK 293 cells or when investigated in smooth muscle cells and platelets that constitutively express the receptor Disruption of lipid rafts by Table P2XR localization in lipid rafts ⁄ caveolae ND, not determined Method used to isolate lipid rafts ⁄ caveolae Receptor Detergent-based Detergent-free Also found in the non-raft fraction?a P2X1 Nob Yes No P2X2 P2X3 P2X4 P2X5 P2X6 P2X7 No Yes Yesc ND ND Noa ND Yes ND ND ND Yes – Yes No – – Yes Effect on P2XR function observed upon lipid raft disruption Blockade of receptor-mediated currents and artery contraction – ND ND – – Inhibition of receptor-mediated activation of PLA2 and SMase Ref [83,88] [87] [89] [21,89–92] a Defined only for those cases where a significant population of receptors is found in lipid rafts ⁄ caveolae b This is considered negative when the amount of receptors is considerably lower when compared to detergent-free methods c However, it is only found when mild detergent conditions (Brij 95 instead of Triton X-100) are used 334 FEBS Journal 276 (2009) 330–340 ª 2008 The Authors Journal compilation ª 2008 FEBS M Garcia-Marcos et al cholesterol depletion delocalized P2X1 receptors out of lipid rafts and greatly impaired the increase in intracellular calcium concentrations and the muscle contraction in response to receptor occupancy [88] As for the P2X3 receptor, no translocation upon receptor activation could be observed More recently, Barth and colleagues reported that only a minor fraction of the P2X4 receptors of lung epithelial cells were located in rafts isolated with 1% Triton X-100 but that these receptors were prominently in lipid rafts prepared with Brij-95, a less stringent detergent Interestingly, in these cells the P2X4 receptor expression and recruitment to raft fractions were promoted upon ATP stimulation [89] However the role of P2X receptor partition between different membrane domains in the coupling to specific downstream signalling pathways is still poorly understood In this regard, some information has been made available for the P2X7 receptor which has been reported to be localized, at least in part, to lipid rafts by three different groups [21,89–92] Working on lymphoma cells, submandibular gland cells or lung epithelial cells these authors concluded that the P2X7 receptor could be found in raft-like membranes isolated in detergent-free or in mild detergent conditions (such as 0.05% Triton X-100) but not in the traditional 1% Triton X-100 conditions The fact that the raft fractions obtained by the detergentfree method preserved all the biochemical and biophysical properties argues in favour of an effect of the detergent on the interaction that maintains the receptor associated to these fractions rather than a consequence of a methodological artefact [91] Importantly, in the work published by Barth et al the P2X7 receptor was morphologically localized to caveolae by immunoelectron microscopy [92] The integrity of caveolae was shown to be critical for the normal expression of P2X7 receptors, because cells either depleted from caveolin-1 by siRNA or obtained from caveolin-1 knock-out mice expressed lower levels of the receptor This result suggests that the localization of the P2X7 receptor to caveolae is critical for its normal turnover in the cell [92] In addition, the same group has recently observed that the P2X7 receptor can form a protein complex with caveolin-1 [89] This supports the idea that the P2X7 receptor localizes to lipid rafts ⁄ caveole via a protein–protein interaction that might be destabilized by detergent extraction during lipid raft isolation Moreover, these studies have provided some insights into the significance of the localization of the P2X7 receptors in lipid rafts regarding the regulation of intracellular signalling pathways Interestingly, the P2X7 receptor is a substrate for ART 2.2, a glycosylphosphatidylinositol-anchored ADP- P2X receptors and membrane microdomains ribosyltransferase that is also enriched in lipid rafts [90] The fact that the P2X7 can be activated by ADPribosylation as an alternative to ligand binding [93,94], strongly suggests that the receptors localized in lipid rafts could be biased to this alternative way of activation which in turn could activate a specific downstream signalling pathway In fact, the distribution of the P2X7 among raft and non-raft fractions seems to dictate the signalling pathway to which the receptor couples Disruption of lipid rafts by cholesterol sequestering agents shifts the receptor from raft to non-raft fractions and abolishes its ability to activate lipid signalling pathways such as ceramide production upon N-SMase activation or downstream PLA2 activation; it does not affect its ability to form a non-selective cation channel [21] These observations indicated that the specific signalling pathway activated by the P2X7 greatly depended on the topology of the receptor at the cell surface The P2X7 receptor is much longer than the other P2X receptors It has a long intracellular C-terminal tail (150 amino acids versus 30 for the other receptors) which contributes to some additional features unique to the P2X7 receptor (ability to increase plasma membrane permeability > 900 Da molecules, to induce apoptosis, etc.) [7] The group of Surprenant clearly established that this receptor interacted with several intracellular proteins via its C-terminal end [95,96] These features raise the possibility that the localization of the P2X7 receptor to lipid rafts promotes its interaction with proteins coupled to specific signalling pathways Given that both P2X and other purinergic receptors (i.e P1 and P2Y) can localize differentially in microdomains of the plasma membrane, the topology of purinergic receptors is important for modulating signalling triggered by P2X receptors, and also for its integration with other purinergic signalling events (Fig 1) Extracellular levels of nucleotides are regulated by ectonucleotidases [97] Considering the exquisite specificity of different purinergic receptors for different purinergic compounds [6], this extracellular processing of nucleotides is crucial in determining the final cellular response The ATP released to the media could act on P2X receptors but once degraded to ADP the signalling would shift to P2Y receptors and in a similar fashion to P1 receptors once adenosine is generated by subsequent enzymatic processing Nucleotides can be hydrolysed by enzymes with a broad spectrum of substrates, such as ecto-alkaline phosphatases (hydrolysis of nucleoside-5¢-tri-, di- and monophosphates) or ecto5¢-nucleotidases (hydrolysis of nucleoside-5¢-monophosphates) [97] Interestingly, these enzymes are not localized randomly at the plasma membrane but are FEBS Journal 276 (2009) 330–340 ª 2008 The Authors Journal compilation ª 2008 FEBS 335 P2X receptors and membrane microdomains M Garcia-Marcos et al Fig Schematic diagram of how the topological distribution of purinergic signalling components might regulate the final cellular response P2XR have been described as being localized in both raft and non-raft membrane fractions and to couple to different downstream signalling pathways depending on their location once activated by ATP The enrichment of ecto-nucleotidases in lipid rafts would promote the accelerated degradation of ATP to ADP and adenosine in the periphery of these microdomains ADP and adenosine would activate respectively P2Y and P1 receptors which are localized in lipid rafts and would engage their respective signalling pathways through G proteins The differential localization of receptors and ecto-nucleotidases would modulate the input signals in the form of ATP or its degradation products, as well as the specific intracellular signalling outputs from each subclass of receptors Finally, all these different intracellular signalling outputs would be integrated to provoke the cellular response glycosylphosphatidylinositol-anchored proteins, which are commonly found in lipid rafts In fact, glycosylphosphatidylinositol-anchored proteins are archetypical markers for lipid rafts and have historically been used as bona fide markers of lipid rafts [29,45,55,98] Nucleotides can also be hydrolysed by enzymes of the ecto-nucleoside triphosphate diphosphohydrolase family, which more specifically hydrolyse nuleotides tri- and diphosphate (ATP, ADP, UTP, UDP) [97,99] Among this family the CD39 ectonucleotidase has been extensively reported to regulate purinergic signalling [97] and to localize to lipid rafts ⁄ caveolae via palmitoylation [80,100–102] In this scenario, local concentrations of nucleotides surrounding lipid rafts are more tightly controlled than in the adjacent membrane and would promote a differential pattern of purinergic signalling in different microdomains For example, one can imagine that if starting from a homogeneous concentration of ATP in the media, the P2XR localized within lipid rafts would signal for a shorter time frame than the P2XR out of these microdomains given the relative enrichment of ectonucleotidases within rafts ⁄ caveolae At the same time, P2Y and P1 recep336 tors localized within lipid rafts would start to receive input signal in the form of ADP and adenosine as stimulation of P2XR fades Conclusions Signal transduction is a complex process by which membrane receptors couple to a variety of downstream effectors The idea of the plasma membrane as a compartmentalized entity that organizes the signal transduction machinery serves as a hypothesis to explain part of this complex process In the case of P2XR, this plasma membrane compartmentalization seems to determine coupling to different signalling pathways In addition, it also seems to contribute to the fine-tuning of P2XR-mediated signalling by controlling the local kinetics of extracellular agonist degradation and the integration with different purinergic signal inputs (via other purinoceptors such as P2Y or P1) to generate the final cellular response Further investigation of this topic might shed light on some controversial or unresolved issues regarding purinergic signalling [103], such as the functional interaction of multiple receptors in FEBS Journal 276 (2009) 330–340 ª 2008 The Authors Journal compilation ª 2008 FEBS M Garcia-Marcos et al P2X receptors and membrane microdomains single cells or the complex responses associated to some receptors like the P2X7 Acknowledgements This work was supported by grant no 3.4.528.07.F from the Fonds National de la Recherche Scientifique ´ to JPD and by grant 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