Membrane compartments and purinergic signalling: the role of plasma membrane microdomains in the modulation of P2XR-mediated signalling Mikel Garcia-Marcos1, Jean-Paul Dehaye2and Aida Ma
Trang 1Membrane compartments and purinergic signalling: the role of plasma membrane microdomains in the modulation
of P2XR-mediated signalling
Mikel Garcia-Marcos1, Jean-Paul Dehaye2and Aida Marino3
1 Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA, USA
2 Laboratoire de Biochimie et de Biologie Cellulaire, Institut de Pharmacie C.P 205 ⁄ 3, Universite´ libre de Bruxelles, Belgium
3 Departamento de Bioquimica y Biologia Molecular, Universidad del Pais Vasco, Bilbao, Spain
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
hydroly-sis 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 ques-tions have been answered to some extent by the uncov-ering 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 med-ium via a number of mechanisms, including release from dying cells or leakage from large pores or trans-port mechanisms in intact cells [4], and that cellular responses to these extracellular nucleotides are
medi-Keywords
caveolae; compartmentalization;
detergent-resistant 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 nonse-lective cation channel, these receptors engage with a complex network of signalling pathways, including protein kinase cascades, lipid signal media-tors 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 recep-tors and other signalling components at the plasma membrane is an attrac-tive 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
Abbreviations
ENaC, epithelial sodium channel; ERK 1 ⁄ 2, extracellular signal-regulated kinase 1 ⁄ 2; MAP, mitogen-activated protein; N-SMase, neutral sphingomyelinase; PKC, protein kinase C; PKD, protein kinase D; PLA 2 , phospholipase A 2 ; PLC, phospholipase C; PLD, phospholipase D; RTK, receptor tyrosine kinase.
Trang 2ated through the activation of plasma membrane
receptors Many of these receptors have been cloned
and characterized both functionally and
pharmacologi-cally [5] Two main classes of purinergic receptors are
well characterized, i.e P1 receptors which bind
adeno-sine, and P2 receptors which are responsive to
phos-phorylated nucleosides as ATP, ADP and other related
nucleotides [5,6] P2 receptors have been further
subdi-vided into the P2Y and P2X subfamilies [6] P2Y
receptors, like P1 receptors, are members of the
super-family 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
fea-tures [4,7,8]: each subunit possesses two
transmem-brane-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
Regard-ing the intracellular extremities, conserved
phosphory-lation 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
bind-ing leads to the openbind-ing of a channel pore, which
behaves as a non-selective cation channel (typically for
sodium, calcium and potassium ions as well as
pro-tons) P2X receptors are widely distributed across
different tissues and organs [10] They are expressed in
both excitable cells (such as neurons) and
non-excit-able 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
func-tion 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
recep-tor and the generation of intracellular signalling
cascades is still poorly understood Although P2XR
activation can trigger the rapid elevation of
intracellu-lar calcium ions as a second messenger, it is also cou-pled 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 1⁄ 2 (ERK
1⁄ 2), mitogen-activated protein kinase (MAPK) p38] [12–17], caspases [18], lipid kinases (such as phosphoi-nositide 3-kinase) [14,17] and phospholipases (such as PLA2, PLD and SMase) [19–24] This variety of signal-ling pathways that can be activated by the different P2XR members raises the question about how the cor-rect coupling and fine tuning of the signalling in response to extracellular stimuli is achieved An attrac-tive 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 How-ever, seminal biophysical studies on model lipid mem-branes indicated that biomembranes may be heterogeneous and that lipids could coexist in differ-ent 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 mem-branes was first attempted through formulation of the
‘lipid raft’ hypothesis in the context of membrane transport in polarized epithelial cells [27] It was pos-tulated that domains rich in cholesterol and sphingoli-pids 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), hetero-geneous, highly dynamic, sterol- and sphingolipid-enriched domains that compartmentalize cellular processes Small rafts can sometimes be stabilized to form larger platforms through protein–protein and
Trang 3protein–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
pack-ing of phospholipids and cholesterol, increaspack-ing 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
defi-nition’ 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
pro-cess is that they can contain or exclude proteins such
as receptors, transducer⁄ adaptor proteins and
effec-tors, 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
trans-duction 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
deter-gents) 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
mem-brane systems to cell memmem-branes 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
membranes in a cell could be segregated into domains with different properties and resistance to solubiliza-tion by non-ionic detergents was initially established
as an operational definition of lipid rafts The use of this general biochemical approach has not been with-out 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 micros-copy 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 Laur-dan 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 immuno-electron microscopy has been another interesting approach to quantify the degree of clustering of mole-cules 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 micro-domains 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 solubili-zation of cellular membranes with 1% Triton at 4C and recovery of the buoyant membrane fractions from the interface of a sucrose density gradient (typically from the 5–35% sucrose interface) [29] Similar meth-ods have been described using lower detergent concen-trations 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 hetero-geneity in the population of lipid rafts Detergent-free
Trang 4methods 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
den-sity 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
per-forming 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
cyto-skeletal components The detergent extraction
condi-tions 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,
con-tain the majority of lipid raft⁄ caveolar markers
(caveo-lin-1, flotillins, etc.) and, importantly, be devoid of
protein markers for other organelles (Golgi,
endoplas-mic 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
manipula-tion 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
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⁄ caveo-lae 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 agon-ists, the localization of these receptors with regard to raft versus non-raft domains varies from case to case, implying different mechanisms of regulation A sub-stantial number of studies have investigated Ras sig-nalling in the context of lipid rafts Ras is a small GTPase that is activated downstream of many RTKs and mediates signalling to MAPK and phosphatidyl-inositol 3-kinase from the plasma membrane Specifi-cally, 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, endo-thelin 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 compo-nents listed above some regulators of G-protein signal-ling (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]
Trang 5Lipid rafts and P2X receptors
Purinergic receptors have also been found to localize
to lipid rafts⁄ caveolae Considering that many
compo-nents 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
compartmen-talize in the plasma membrane One of the earliest
findings in this regard was the enrichment of the
aden-osine 1 receptor in caveolar fractions of
cardiomyo-cytes [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
mecha-nism 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
mor-phologically 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
antithrom-botic drug) and its active compound derivative
prob-ably 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
activa-tion of platelet P2Y1receptors [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 exper-imental demonstration of their localization depends on the methodology to isolate rafts (see Table 1 for a summary of the different P2XR found in lipid rafts and the methodology used in each case) The first evi-dence 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 inves-tigated in lipid rafts prepared by a detergent-free pro-tocol Yet further studies confirmed that this method was the most appropriate considering that the localiza-tion 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 deter-gent-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 1 P2XR localization in lipid rafts ⁄ caveolae ND, not determined.
Receptor
Method used to isolate lipid
in the non-raft fraction? a
Effect on P2XR function observed upon lipid raft
Detergent-based Detergent-free
currents and artery contraction
[83,88]
activation of PLA2and SMase
[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.cHowever, it is only found when mild detergent conditions (Brij 95 instead of Triton X-100) are used.
Trang 6cholesterol depletion delocalized P2X1receptors out of
lipid rafts and greatly impaired the increase in
intra-cellular calcium concentrations and the muscle
con-traction in response to receptor occupancy [88] As for
the P2X3receptor, no translocation upon receptor
acti-vation 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
recruit-ment to raft fractions were promoted upon ATP
stim-ulation [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
informa-tion 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
deter-gent 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
detergent-free method preserved all the biochemical and
biophys-ical properties argues in favour of an effect of the
detergent on the interaction that maintains the
recep-tor associated to these fractions rather than a
conse-quence of a methodological artefact [91] Importantly,
in the work published by Barth et al the P2X7
recep-tor 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 P2X7receptor to caveolae is critical for its
nor-mal 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
dur-ing lipid raft isolation Moreover, these studies have
provided some insights into the significance of the
localization of the P2X7 receptors in lipid rafts
regard-ing the regulation of intracellular signallregard-ing pathways
Interestingly, the P2X7 receptor is a substrate for
ART 2.2, a glycosylphosphatidylinositol-anchored
ADP-ribosyltransferase that is also enriched in lipid rafts [90] The fact that the P2X7 can be activated by ADP-ribosylation 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 activa-tion 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 seques-tering agents shifts the receptor from raft to non-raft fractions and abolishes its ability to activate lipid sig-nalling 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 intracellu-lar 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 inter-acted with several intracellular proteins via its C-termi-nal end [95,96] These features raise the possibility that the localization of the P2X7 receptor to lipid rafts pro-motes 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 microdo-mains of the plasma membrane, the topology of puri-nergic receptors is important for modulating signalling triggered by P2X receptors, and also for its integration with other purinergic signalling events (Fig 1) Extra-cellular levels of nucleotides are regulated by ectonu-cleotidases [97] Considering the exquisite specificity of different purinergic receptors for different purinergic compounds [6], this extracellular processing of nucleo-tides 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 signal-ling would shift to P2Y receptors and in a similar fash-ion to P1 receptors once adenosine is generated by subsequent enzymatic processing Nucleotides can be hydrolysed by enzymes with a broad spectrum of sub-strates, such as ecto-alkaline phosphatases (hydrolysis
of nucleoside-5¢-tri-, di- and monophosphates) or ecto-5¢-nucleotidases (hydrolysis of nucleoside-5¢-mono-phosphates) [97] Interestingly, these enzymes are not localized randomly at the plasma membrane but are
Trang 7glycosylphosphatidylinositol-anchored proteins, which
are commonly found in lipid rafts In fact,
glycosyl-phosphatidylinositol-anchored proteins are
archetypi-cal markers for lipid rafts and have historiarchetypi-cally 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
signal-ling [97] and to localize to lipid rafts⁄ caveolae via
palmitoylation [80,100–102] In this scenario, local
con-centrations 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
con-centration 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
recep-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 com-partmentalized entity that organizes the signal trans-duction 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 unre-solved issues regarding purinergic signalling [103], such
as the functional interaction of multiple receptors in
Fig 1 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 acceler-ated 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.
Trang 8single 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 BFU2007-62728 Direccio´n
General de Investigacio´n Ministerio de Educacio´n y
Ciencia (MEC) to AM
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