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-Marcos
1
, Jean-Paul Dehaye
2
and Aida Marino
3
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:
(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.
330 FEBS Journal 276 (2009) 330–340 ª 2008 The Authors Journal compilation ª 2008 FEBS
ated 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’ P2X
7
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 opening 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
PLA
2
, 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
M. Garcia-Marcos et al. P2X receptors and membrane microdomains
FEBS Journal 276 (2009) 330–340 ª 2008 The Authors Journal compilation ª 2008 FEBS 331
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 pack-
ing 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 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 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
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 4 °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 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
P2X receptors and membrane microdomains M. Garcia-Marcos et al.
332 FEBS Journal 276 (2009) 330–340 ª 2008 The Authors Journal compilation ª 2008 FEBS
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 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].
M. Garcia-Marcos et al. P2X receptors and membrane microdomains
FEBS Journal 276 (2009) 330–340 ª 2008 The Authors Journal compilation ª 2008 FEBS 333
Lipid 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 P2Y

2
receptors have also been mor-
phologically localized to caveolae at least in placenta
[80]. In platelets, the P2Y
12
-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 P2Y
12
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 P2Y
1
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 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 P2X
3
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 (P2X
1
, P2X
2
, P2X
4
, P2X
7
) 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 P2X
1

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
rafts ⁄ caveolae
Also found
in the non-raft
fraction?
a
Effect on P2XR function
observed upon lipid raft
disruption Ref.Detergent-based Detergent-free
P2X
1
No
b
Yes No Blockade of receptor-mediated
currents and artery contraction
[83,88]
P2X
2
No ND – –

P2X
3
Yes Yes Yes ND [87]
P2X
4
Yes
c
ND No ND [89]
P2X
5
ND ND – –
P2X
6
ND ND – –
P2X
7
No
a
Yes Yes Inhibition of receptor-mediated
activation of PLA
2
and 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.
c
However, it is only found when mild detergent

conditions (Brij 95 instead of Triton X-100) are used.
P2X receptors and membrane microdomains M. Garcia-Marcos et al.
334 FEBS Journal 276 (2009) 330–340 ª 2008 The Authors Journal compilation ª 2008 FEBS
cholesterol depletion delocalized P2X
1
receptors 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 P2X
3
receptor, no translocation upon receptor acti-
vation could be observed. More recently, Barth and
colleagues reported that only a minor fraction of the
P2X
4
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 P2X
4
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 P2X
7

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 P2X
7
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 P2X
7
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 P2X
7
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 P2X

7
receptor to caveolae is critical for its nor-
mal turnover in the cell [92]. In addition, the same
group has recently observed that the P2X
7
receptor
can form a protein complex with caveolin-1 [89]. This
supports the idea that the P2X
7
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 P2X
7
receptors in lipid rafts regard-
ing the regulation of intracellular signalling pathways.
Interestingly, the P2X
7
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 P2X
7
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
P2X
7
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 PLA
2
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 P2X
7
greatly depended on the topology of the receptor at
the cell surface. The P2X
7
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 P2X
7
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 P2X
7
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
M. Garcia-Marcos et al. P2X receptors and membrane microdomains
FEBS Journal 276 (2009) 330–340 ª 2008 The Authors Journal compilation ª 2008 FEBS 335

glycosylphosphatidylinositol-anchored proteins, which
are commonly found in lipid rafts. In fact, glycosyl-
phosphatidylinositol-anchored proteins are archetypi-
cal 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 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.
P2X receptors and membrane microdomains M. Garcia-Marcos et al.
336 FEBS Journal 276 (2009) 330–340 ª 2008 The Authors Journal compilation ª 2008 FEBS
single cells or the complex responses associated to
some receptors like the P2X
7
.
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
´
ny
Ciencia (MEC) to AM.
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