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Current Medicinal Chemistry, 2013, 20, 56-63
Metabotropic Purinergic Receptors in Lipid Membrane Microdomains N. D’ Ambrosi and C. Volonté* Cell Biology and Neurobiology Institute, Consiglio Nazionale delle Ricerche/IRCCS S. Lucia Foundation, Rome, Italy Abstract: There is broad evidence that association of transmembrane receptors and signalling molecules with lipid rafts/caveolae provides an enriched environment for protein-protein interactions necessary for signal transduction, and a mechanism for the modulation of neurotransmitter and/or growth factor receptor function. Several receptors translocate into submembrane compartments after ligand binding, while others move in the opposite direction. The role of such a dynamic localization and functional facilitation is signalling modulation and receptor desensitization or internalization. Purine and pyrimidine nucleotides have been viewed as primordial precursors in the evolution of all forms of intercellular communication, and they are now regarded as fundamental extracellular signalling molecules. They propagate the purinergic signalling by binding to ionotropic and metabotropic receptors expressed on the plasma membrane of almost all cell types, tissues and organs. Here, we have illustrated the localization in lipid rafts/caveolae of G protein-coupled P1 receptors for adenosine and P2Y receptors for nucleoside tri- and di-phosphates. We have highlighted that microdomain partitioning of these purinergic GPCRs is cell-specific, as is the overall expression levels of these same receptors. Moreover, we have described that disruption of submembrane compartments can shift the purinergic receptors from raft/caveolar to non-raft/non-caveolar fractions, and then abolish their ability to activate lipid signalling pathways and to integrate with additional lipid-controlled signalling events. This modulates the biological response to purinergic ligands and most of all indicates that the topology of the various purinergic components at the cell surface not only organizes the signal transduction machinery, but also controls the final cellular response.
Keywords: Adenosine, caveolae, extracellular ATP, GPCR, lipid rafts, membrane microdomains, purinergic receptors. 1. PURINERGIC RECEPTORS The metabotropic receptors for adenosine (classified as P1Rs or ARs, with four different subtypes cloned so far from mammalian species) [1] and the metabotropic P2Y receptors (P2YRs, with eight cloned different subtypes) [2] specific for extracellular nucleoside tri- and di-phosphates, belong to the vast family of purinergic receptors, which also comprises the ionotropic P2X receptors (P2XRs, with seven cloned distinct subtypes) [3] exclusively for extracellular ATP. These receptors are expressed on the plasma membrane of almost all cell types, tissues and organs, comprising very primitive organisms such as bacteria, diatoms, algae and slime mould [4]. For this reason, purine and pyrimidine derivatives have been viewed as primordial precursors in the evolution of all forms of intercellular communication. Both ARs and P2YRs propagate the purinergic signalling triggered by extracellular purines and pyrimidines concomitantly in the same cell type and tissue. Moreover, both ARs and P2YRs share the overall topological structure typical of G protein-coupled receptors (GPCRs) belonging to the rhodopsin A-like family, that is seven -helical trans-membrane domains crossing the lipid bilayer of the plasma membrane, three extracellular and three intracellular loops, N- terminus in the extracellular milieu and C-terminus in the intracellular portion of the cell. Nevertheless, from comprehensive sequence comparisons and phylogenetic analyses, it is clear that ARs and P2YRs belong to two different groups of the rhodopsin-like family of GPCRs, respectively the rhodopsin - and -group [5]. 1.1. Adenosine P1 Receptors P1Rs are further classified into four distinct subtypes, A1R, A2aR, A2bR, A3R, all coupled to G proteins. The majority of cell types express multiple ARs that moreover couple to different G proteins, thus activating various different intracellular signalling pathways. For instance, A1R and A3R subtypes are coupled to Gi proteins, A2 subunits are coupled to Gs, while A2b can also be coupled to Gq proteins. In addition, there is some evidence that ARs may signal via other G proteins, such as Go and G15/16. After activation of the G proteins, down-stream enzymes and ion *Address correspondence to this author at the Cell Biology and Neurobiology Institute, CNR/FSL, Via del Fosso di Fiorano 65, 00143 Rome, Italy; Tel: +39-06-501703084; Fax: +39-06-501703321; E-mail:
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channels are in turn affected: A1Rs mediate inhibition of adenylyl cyclase, activation of several K+-channel types, inactivation of N-, P-, and Q-type Ca2+ channels, activation of phospholipase C- (PLC-). The same appears to be true for A3Rs. Both A2aRs and A2bRs stimulate adenylyl cyclase and the formation of cAMP, but other actions, including elevation of phosphoinositides, mobilization of intracellular calcium and activation of PLC- and mitogenactivated protein kinase (MAPK), have also been described, particularly for A2bRs [6, 7]. AR subunits can moreover heterodimerize and, as a consequence, alter their receptor kinetic properties and cell signalling, thus rendering their final outcome of activation strictly dependent on the specific tissue context and cell type. Also the highly mutable extracellular environment and the presence of plasma membrane ecto-enzymes are of fundamental importance for the purinergic signalling mediated by P1Rs. Particularly the very heterogeneous class of ecto-nucleotidases can indeed very rapidly modulate the extracellular concentrations of ATP and adenosine targeting the receptors, and in turn differentially condition the cell response during health or pathological states as cancer, trauma, stress and neurodegeneration/inflammation [1]. In general, the effects of modulating adenosine receptors tend to be protective under a wide variety of physiological conditions, including immunomodulation and proper central nervous system (CNS) functioning, or also pathological events. These comprise for instance adenosinedependent effects as bradikardia to decrease energy demand, vasodilatation or angiogenesis to increase energy supply, ischemic preconditioning in the heart or brain, inhibition of the release of excitotoxic neurotransmitters, suppression of cytokine-induced apoptosis, or reduced inflammatory responses [8]. For this reason, ARs constitute a central target of therapeutic intervention against various pathologies, for instance inflammatory diseases such as asthma, ischemia, arthritis and sepsis, cardiovascular diseases, pain [9-11]. 1.2. Nucleoside di and tri-phosphate P2Y Receptors On the basis of the nucleotide that they are binding to, metabotropic P2YRs are classified as purinergic (P2Y1R, P2Y2R, P2Y11R, P2Y12R, P2Y13R subtypes) or pyrimidinergic (P2Y2R, P2Y4R, P2Y6R and P2Y13R subtypes). On the basis of the G protein that they are coupled to, i.e. Gq for P2Y1R, P2Y2R, P2Y4R, P2Y6R, and P2Y11R subtypes, or Gi for P2Y12R, P2Y13R, and P2Y14R, they respectively promote inositol lipid signalling and strongly activate PLC-, or they lead to inhibition of adenylyl cy© 2013 Bentham Science Publishers
Purinergic Receptors in Microdomains
clase and modulation of a vast range of effector proteins including inward rectifying K+ channels. Moreover, activation of several P2YR subtypes is commonly associated to stimulation of MAPK, in particular extracellular signal regulated protein kinases 1/2 (ERK1/2). Other classes of protein kinases, for instance protein kinase C (PKC), calcium and phosphoinositol-3-dependent protein kinase (PI3K), can also be involved to a variable extent [2]. As for ARs, P2YRs are widely distributed in all cells, organs and tissues, where they are involved in a broad range of physiological responses, ranging from Cl secretion by epithelia, aggregation of platelets, control of vascular and gastrointestinal functions, to neurotransmission, neuro- and immuno-modulation in the central and peripheral nervous system [12]. For these reasons, also P2YRs constitute an important target of therapeutic intervention against various diseases: for instance P2Y12R antagonists (as clopidogrel and prasugrel) are commonly used against stroke, thrombosis, cardiovascular diseases, chronic asthma; P2Y2R agonists are proposed for the treatment of cystic fibrosis and dry eye disease. Further studies are also prospecting the use of additional P2YRs as therapeutic targets especially in cardiovascular diseases and in pathological conditions characterized by neuroinflammatory components [13]. 1.3. Purinergic Cooperation and Membrane Domains The purinergic signalling begins when purines and pyrimidines are released extracellularly. In addition to P1 and P2 purinergic receptors, the targets for extracellular purine/pyrimidine molecules comprise nucleoside/nucleotide transporters and ectonucleotidases, proteins that are again widely distributed and co-expressed by many different cell types and tissues. Nucleoside (adenosine) transport is mediated by bidirectional equilibrative processes driven by chemical gradients, and by unidirectional concentrative processes driven by sodium (and proton) electrochemical gradients. These specialised concentrative and equilibrative transporters are thus responsible for the intracellular uptake of nucleosides, the event that at last limits or terminates the extracellular purinergic/pyrimidinergic signalling. In recent years, we have highlighted that all these purinergic elements are not physically separated units delivering a single task, rather they often associate with each other within multiprotein complexes and intermolecular interaction networks [14-16]. For instance, it is well known that ectonucleotidases form oligomeric complexes with P1R and P2R subtypes [17]; nucleoside and nucleotide transporters combine with ARs [18] and P2Rs [19], respectively; A1Rs and A2aRs associate with each other and form dimers, trimers and higher order complexes also with P2YRs [17]; finally, homo- and hetero-oligomeric complexes have been demonstrated within the P2XR and P2YR subfamilies [20, 21]. Molecular and/or functional associations often coupled to transactivation of receptors were also demonstrated between metabotropic purinergic receptors and growth factor receptors, for instance between P2Y1R and vascular endothelial growth factor receptor [22] or epidermal growth factor receptor [23], P2Y2R and epidermal growth factor receptor [24], or A2aR and tyrosine receptor kinase B [25]. In addition, metabotropic purinergic receptors can also interact with other proteins residing in the plasma membrane as the integrins, to further regulate the down-stream signalling pathways and diversify the consequent biological outcomes. This is known for instance for the P2Y2R [26, 27], the P2Y1R and P2Y12R [28], and for the A2aR [29]. As a result, other than being multipurpose at the cellular and functional level, the biological information delivered by purinergic and pyrimidinergic ligands can be also defined as “multidirectional” at the molecular level. This means that a single ligand can concomitantly or sequentially merge with various receptor subtypes (although with different molecular affinities), with different classes of ectonucleotidases and transporters, even with assorted oligomeric
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complexes forming among these same targets. Moreover, a single ligand can give rise to several diverse metabolic ligands, with the final aim of amplifying or attenuating the original purinergic signal. A network of overlapping, mutually not exclusive, biological reactions and a dynamic cross-regulation of signalling is thus generated. A precise space-time coincidence is then the only possible condition in which purinergic and pyrimidinergic signals can be integrated and become operative, i.e. perceived, discriminated, maintained and terminated. In this multipart context, a major role is played by the plasma membrane microenvironment, accommodating and integrating the different components of the purinergic machinery, the purinome [30]. This synergism can be more easily accomplished in specialized submembrane compartments (lipid rafts, raft-like structures, caveolae) that permit complex control systems involving molecular associations, cooperation, conformational or electronic state changes in enzymes, receptors or channels. Indeed, compartments either exclude or include certain proteins, separate unrelated reactions, favour proper cooperative behaviour by decreasing the search time for an enzyme to find a substrate, or for a ligand to find a receptor. It is well known that purinergic P2XR [31, 32], P2YR and AR proteins, as well as ectonucleotidases [33, 34] and nucleotide transporters [35, 36] localize in lipid rafts⁄caveolae. Often, these proteins can translocate out of the membrane microdomains upon stimulation, and this could be interpreted as a further mechanism for regulation, coupling to effectors, or desensitization and inhibition. For instance, disruption of lipid rafts by cholesterol sequestering agents can even shift the purinergic nucleotidases, transporters and receptors from raft to non-raft fractions, thus abolishing their ability to in turn activate lipid signalling pathways and to integrate with additional signalling events. This means that the exact topology of the purinergic components at the cell surface has also the key function to organize the signal transduction machinery and to contribute to its fine-tuning, by controlling for instance the local kinetics of extracellular agonist metabolism and the integration with different purinergic signal inputs to generate the final cellular outcome. In the present work, we will describe all the GPCR subtypes belonging to the AR and P2YR purinergic family that have been so far identified as actively residing and/or moving in and out from lipid membrane microdomains. 2. P1 RECEPTORS IN MEMBRANE MICRODOMAINS Significant evidence has accumulated over the past years that AR signalling is regulated via membrane microdomains and, indeed, all four human AR subtypes contain the caveolin binding domain (CBD) , where is an aromatic amino acid [37]. Moreover, palmitoylation sites well known to contribute to localize receptors to lipid rafts, have been identified only on A1R, but predicted also on A2bR and A3R [38, 39]. In Fig. (1), we aligned the aminoacid sequences at the junction between the seventh trans-membrane domain and the COOH-terminus tail of the human ARs, highlighting the CBD and the S-palmitoyl cysteines and illustrating with the cartoon their general localization on the ARs. The distribution of ARs in membrane microdomains was recently thoroughly reviewed by Lasley [40]. 2.1. A1 Receptor The AR subtype first reported to be localized in lipid rafts/caveolae, and for which the most evidence exists up to now, is the A1R which contains a caveolin binding domain at the junction between the seventh trans-membrane domain and the COOHterminus tail. It was moreover reported that recombinant human A1R expressed in HEK-293 cells also incorporates 3H palmitate on a cysteine residue present within the cytoplasmic carboxyl-terminal domain [38]. One of the main set of studies regarding the subcellu-
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Fig. (1). Representation of caveolin binding domains and S-palmitoyl cysteines on adenosine receptors. Aminoacid sequences between positions 281 and 326 and proximal to the COOH-tail (in bold) are represented for A1R, A2aR, A2bR and A3R. The CBD and the predicted S-palmitoyl cysteines are highlighted inside boxes. The cartoon is a general representation of the topological localization of CBD and Spalmitoyl cysteines on ARs.
lar distribution of this receptor is based on cardiomyocytes, where A1R exerts a potent inhibition of 1-adrenergic contractile and biochemical responses, referred to as the A1R antiadrenergic effect [41]. In detail, it was reported that A1R is concentrated in caveolae of adult rat ventricular cardiomyocytes and treatment with the A1R agonist 2-chloro-N6-cyclopentyladenosine (CCPA) results in the switch of A1R from caveolin-3-enriched domains to the bulk plasma membrane (this is moreover prevented by the A1R antagonist 8-cyclopentyl-1,3-dipropylxanthine) [42]. Moreover, A1R activation can also recruit down-stream signalling molecules such as specific PKC subunits into caveolae [43], and selectively modulate MAPK activities in caveolin-rich cardiac membrane fractions [44]. This pattern of receptor localization seems to be essential for the activation of K ATP channels by ARs, an action that has been linked for instance to cardioprotection against ischaemia. On this regard, Garg and colleagues (2009) reported that the disruption of cholesterol-rich microdomains by methy--cyclodextrin (MCD), or the knockdown of caveolin-3 by silencing RNA, significantly reduces the effect of adenosine receptors on K ATP channels, in rat ventricular myocytes [45]. Differently from what was reported in these works, Schwarzer and colleagues, by the use of radioligand binding, instead found A1Rs out of rafts/caveolae in the different cell system of HL-1 cells, one of the best models of native cardiac myocytes. Moreover, this same localization in the bulk membranes occurs in HEK293 cells transfected with A1R fused to yellow fluorescent protein (YFP), while A1R-YFP fused to caveolin-2 locates in caveolae/rafts. The A1R-YFP-caveolin2 receptor interestingly showed much slower activation kinetics and less desensitization compared with the A1R-YFP subtype. Moreover, the non-caveolar localization was described also for the subunits of the K ATP channel, while the inhibitory Gi protein distributes both to non-caveolar/nonraft and caveolar/raft domains. The authors concluded from these studies that active signalling to the K ATP channel by A1R involves interactions outside of caveolae and rafts, and that sequestration of the A1R away from the K ATP channel accrues a kinetic penalty since both the receptor or G protein subunits must leave caveolae to engage with and activate the K ATP channel [46].
In different cell types, particularly smooth muscle and epithelial cells, there is significant evidence that the internalization of A1R, which occurs rather slowly relative to other AR subtypes, implicates its localization in caveolae. In pig kidney LLC-PK1 epithelial cells and in hamster vas deferens smooth-muscle DDT1MF-2 cells, the A1R selective agonist N6-(R)-(phenylisopropyl) adenosine (RPIA) leads to aggregation of A1Rs in caveolae, which are then internalized in the endocytic compartment before being ultimately recycled back to the cell surface to a non-caveolar location. This process still occurs in the presence of inhibitors of the clathrinmediated endocytosis, further suggesting the involvement of lipid rafts/caveolae [47, 48]. Additional studies confirmed that A1Rs localize in and/or modulate signalling in lipid rafts/caveolae. In rabbit intestinal smooth muscle cells, also Gq/11, Gi1/2, and Gi3 proteins are present in caveolin-3 enriched fractions, as well as heavier fractions. Moreover, the A1R agonist cyclopentyladenosine (CPA) caused a significant increase in the amount of Gi3 subunit in these caveolin-enriched fractions. Pre-treatment with CPA finally reduced the subsequent response to this same agonist, probably due to receptor desensitization as well as binding of Gi3 to caveolin-3 [49]. 2.2. A2a Receptor Both Gs and adenylyl cyclase, to whom A2aRs are mainly coupled, are present in lipid rafts or caveolae, as thoroughly described in the literature [50]. In contrast, there are only a few reports showing A2aRs in microdomains. One study providing details for A2aR localization in lipid rafts is by Mojsilovic-Petrovic and colleagues. Interestingly, this work links the presence of adenosine A2aRs, brain derived neurotrophic factor (BDNF) receptor, the tyrosine receptor kinase B (TrkB), and src-family kinases in lipid raft complexes, to the neuroprotective effect of TrkB antagonism in motoneurons, then highlighting a role for such an association in amyotrophic lateral sclerosis [51]. Evidence was also provided for A2aR modulation of TrkB localization and signalling in rat cortical neurons in a way to amplify excitatory synaptic transmission [52].
Purinergic Receptors in Microdomains
In particular, the A2aR agonist CGS21680 increased the levels of TrkB receptors in the lipid raft fraction and over-increased BDNFinduced TrkB phosphorylation in this membrane microdomain. This effect was not altered by a clathrin-dependent endocytosis inhibitor, but it was blocked after treatment of the cells with the cholesterol reducing agent MCD [25]. In another study, the heteromerization between adenosine A2aR and dopamine D2 receptors (which display a strong reciprocal antagonistic interaction) and the possible role of caveolin-1 in the co-trafficking of these molecular complexes, was analyzed. In particular, in A2aR–D2 receptor cotransfected CHO cells, caveolin-1 was found to co-localize with both A2aR and D2 receptors, and either CGS21680 or quinpirole (a D2 receptor agonist) induced internalization of caveolin-1 with A2aR and D2 receptors. These results suggest that A2aR and D2 receptors are, at least in part, localized in caveolae forming a macro-complex, and displaying caveolin-dependent and clathrinindependent endocytosis upon agonist treatment [53]. Additional studies provided evidence that A2aR downstream signalling is modulated by or involves the membrane microdomain compartments. It has been suggested that the mode of coupling of A2aRs to G proteins is different from that of other adenosine receptor subtypes, with A2aR forming a tight interaction with G proteins, referred to as “restricted collision coupling” or “pre-coupling” [37]. This appears to restrict agonist-induced movement of the receptor in the cell membrane, due to the location of the receptor in a cholesterol rich region, probably caveolae/lipid rafts, rather than by attachment to the actin cytoskeleton [54]. Consistent with these observations, cholesterol stabilizes helix II of the human A2aR [55], rationalizing the observation that the A2aR couples to G proteins only in the presence of cholesterol [56]. Consistently, additional studies also reported that reducing cholesterol or disrupting lipid rafts can alter A2aR receptor signalling. In mouse colon epithelial cells, cholesterol reduction with MCD indeed enhanced adenosine A2aR-activated trans-epithelial short circuit current. Moreover, the cholesterol content in colonic epithelial cells affected adenosinemediated anion secretion, by controlling agonist-selective signalling [57]. On the other hand, it was reported that in human erythrocyte membranes, the disassembling of lipid rafts, without reducing the levels of cholesterol, reversibly blocked the effects of 5'-Nethylcarboxamidoadenosine on cAMP accumulation [58]. 2.3. A2b Receptor There is only one report supporting possible A2bR localization in lipid rafts or caveolae. In the T84 intestinal epithelial cell line, the stimulation with adenosine induced a redistribution of A2bRs to the plasma membrane and caveolar fractions, where they interact with the (Na+/H+) exchanger-3 kinase A regulatory protein and ezrin scaffolding proteins. This would result in the anchoring of the A2bR protein to the plasma membrane, and in the stabilization of the receptor in a specific signalling complex [59]. 2.4. A3 Receptor Although A3R has a predicted site for post-translational attachment of palmitate [39], the only study about A3R localization in membrane rafts or caveolae performed up to date is by Cordeaux and colleagues, who studied CHO cells transfected with human A3R [60]. Using fluorescence correlation spectroscopy, they observed that following exposure to a fluorescent A3R agonist, ABEA-X-BY630, on the basis of membrane diffusion coefficients there appeared to be two populations of agonist-occupied receptors. They postulated that A3R complexes with the slowest mobility could be localized in caveolae. 3. P2Y RECEPTORS IN MEMBRANE MICRODOMAINS Much evidence has accumulated about the functional localization of several P2YRs in membrane microdomains, and particularly
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P2Y1R, P2Y2R, P2Y4R, P2Y6R and P2Y12R subunits. Nevertheless, the potential localization of some receptor subtypes that have been cloned more recently, the P2Y11R and P2Y14R, have not been investigated yet. 3.1. P2Y1 Receptor Although myristoylation and/or palmitoylation which govern the localization of proteins in raft domains have not been described for the P2YRs so far, analysis of the amino acid sequences of P2YRs shows that only the P2Y1R has a potential site for this lipidic modification [61]. As a matter of fact, the presence and the role of P2Y1Rs in membrane microdomains has been very well characterized in both endothelial and vascular smooth muscle cells, where nucleotides are responsible for the regulation of vascular tone [62, 63]. Indeed, one of the first evidence of the localization of P2Y GPCRs in cholesterol-rich signalling microdomains comes from guinea pig aortic endothelial cells. Endothelial cells respond to hormonal stimuli and shear stress by releasing UTP and ATP into the lumen of vessels. ATP and UTP, per se, or their metabolites ADP and UDP can then act on the endothelium at the site of release or downstream, in order to signal vasodilatation by P2YRs. It was therefore supposed that endothelial cells may organize different P2YRs into different signalling domains, in order to distinguish diverse stimuli and to regulate the coupling to second messengers. This hypothesis comes from the observations that P2Y1R-mediated endothelial cell calcium mobilization, and the resulting relaxation of vascular smooth muscle, are sensitive to both the removal of membrane cholesterol by MCD, and the sequestration of cholesterol by filipin [64]. In addition, the presence of P2Y1R in caveolar structures was demonstrated with immunogold electron microscopy, in the basal part of sinus endothelial rat spleen cells [65], and in human endothelial placenta, in association with ecto-nucleoside triphosphate diphosphohydrolase1 [66]. The microdomain partitioning of P2Y1R might thus be important for the control of blood cell passage through the splenic cord beneath endothelial cells in one case, and for the vascular tone, blood fluidity and the placental circulation in general, in the other. Consistently, by separation of membrane fractions and comigration of P2Y1R with flotillin-1, caveolin-1 and caveolin-3, it was demonstrated that P2Y1R is present in membrane rafts from human chorionic artery-derived smooth muscle cells, mammary artery and the chorionic and saphenous vein [24]. In these cells, the association of P2Y1R to membrane rafts is linked to vasoconstriction, because disruption of these microdomains by lowering tissue cholesterol with MCD, selectively abolished the contractions elicited by P2Y1R agonists. Moreover, P2Y1R association to rafts seems to be regulated by agonists. Indeed, agonist administration resulted in the partition of the receptor out of lipid rafts, and in a subsequent rapid internalization and relatively slow cell surface recycling, a finding apparently correlating with a prolonged fading of the vasomotor responses. In addition, MRS2179, a selective P2Y1R antagonist, blocked both the exit of the receptor from the raft domains, and the vasomotor response induced by the receptor agonist. These results highlight the relevance of raft domains and the sequestration of the receptor from the cell surface, as a substantial mechanism for P2Y1R signalling in the vascular district. Additional evidence of P2Y1R presence in membrane microdomains comes from cells belonging to the CNS. In isolated membranes from rat oligodendrocyte progenitor cells and in astrocytes, western blot analysis showed co-localization of caveolin-1 with proteins involved in the Ca2+ signalling cascade and, in particular, with P2Y1R. Purinergic stimulation of astrocytes with the P2Y1R agonist 2-methyl-thioATP (2-Me-SATP) resulted in an increased recruitment to the raft/caveolin-rich membrane fractions of several components of the receptor down-stream signalling pathway, such as the receptor for inositol 1,4,5-trisphosphate (IP3) R2, PKC-,
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and phospholipase C-1. The biological correlate of these observations is found when analysing intracellular Ca2+ transients: astrocytes treated with 2-Me-SATP responded with a propagated Ca2+ wave while, in cholesterol depleted cells, wave propagation was either absent or significantly slowed. These data clearly indicate that the organization of P2Y1R into membrane microdomains is essential for long distance propagation of Ca2+ waves in astrocytes [67].
D’Ambrosi and Volonté
3.3. P2Y4 and P2Y6 Receptors
Moreover, Amadio and colleagues demonstrated that localization and distribution in vivo of the P2Y1R in rat cerebellum changes during development. When examined in juvenile rats at postnatal day 7, the receptor is found absent from membrane microdomains and predominantly expressed only by Bergmann and astroglial cells. At postnatal day 21, the receptor is instead found enriched in noradrenergic fibers and in membrane microdomains, as demonstrated by flotillin-2 co-localization and presence in low buoyant density detergent-resistant membranes from both total cerebellum and cerebellar synaptosomes [68].
Only a few indications are available up to date on P2Y4R and P2Y6R localization in membrane microdomains. One work concerns the distribution of both receptors on synaptosomes purified from rat cerebellar tissue [76]. P2Y4R and P2Y6R subtypes are found in monomeric, dimeric and traces of oligomeric forms in the sucrose high-density fractions from synaptosomes, devoid of lipid rafts. In the flotillin and GM1 enriched fractions, the receptors appear instead respectively only in the dimeric or monomeric molecular forms. It was therefore hypothesized that the different distribution of protomers into membrane microdomains might distinguish between functional and non-functional units of these two receptor subtypes particularly in synaptosomes, since none of these receptors are enriched in lipid raft fractions from total cerebellar extracts and PC12 cells. Moreover, the P2Y6R was localized by immunogold analysis in caveolae in the basal part of endothelial cells lining the splenic sinuses, where it probably contributes to control the blood cell passage through the splenic cord [65].
3.2. P2Y2 Receptor
3.4. P2Y12 Receptor
One of the most important physiological functions described for P2Y2R is cell migration. This receptor is known to work as a sensor for released UTP, which acts as a “find me” signal and therefore has a significant role in physiological and pathological processes such as brain injury [69, 70]. The regulation of this P2Y2Rdependent function by localization in lipid rafts was very well documented by Ando and colleagues [71]. By sucrose density gradient centrifugation they demonstrated that in NG 108-15 cells a part of P2Y2R, together with a main fraction of the coupled Gq/11 protein, are localized in lipid rafts with typical lipid rafts markers such as cholesterol, flotillin-1, and ganglioside GM1. Importantly, depletion of cholesterol by MCD suppressed different cellular pathways connected to UTP-induced migration. Phosphoinositide hydrolysis, Ca2+ elevation, Rho-A activation, stress fiber formation and cofilin phosphorylation were all clearly inhibited and migration was completely abolished. Vice versa, several of these parameters were restored by the addition of exogenous cholesterol. These data clearly demonstrate that the P2Y2R is localized in lipid rafts that are essential for effective signal transduction mechanisms leading to cell migration. Consistently, it was previously reported that MCD inhibited P2Y2R-mediated Ca2+ increase and phosphoinositide hydrolysis also in rat phaeochromocytoma PC12 cells, therefore confirming an essential function in signalling transduction for lipid raft-residing P2Y2R [72]. Moreover, short interfering RNAmediated knockdown of flotillin-2 or treatment with MCD to disrupt cholesterol in HeLa cells attenuated the UTP-induced activation of p38 MAPK but not ERK1/2, suggesting that Gq-coupled P2YRs (likely P2Y2R and/or P2Y4R) localization into lipid rafts specifically activates p38 MAPK [73].
The requirement for P2YR signalling in lipid rafts is very well exemplified and demonstrated in platelets with the P2Y12R, which is the target of the antithrombotic drug clopidogrel, a compound on top of standard treatment for cardiovascular diseases [77]. When platelets are stimulated with ADP they aggregate, release their granule contents and generate thromboxane A2. Two receptors for ADP, the P2Y1R and P2Y12R have been identified on platelets: they respectively stimulate the Gq and Gi protein signalling pathways and their co-stimulation is needed for ADP-induced primary aggregation [78]. Activation of the Gq pathway stimulates PLC that, in turn, leads to activation of PKC and increase in intracellular calcium; on the other hand, activation of the Gi pathway leads to inhibition of cyclic AMP formation and activation of PI3K [79]. It was demonstrated that the ability of Gi to potentiate ADPmediated platelet aggregation is highly dependent upon its localization to lipid rafts, while Gq signalling downstream of P2Y1R has not such a microdomain partitioning requirement [80]. In particular, aggregation, dense granule secretion and inhibition of cAMP production induced by ADP were significantly reduced after cholesterol depletion with MCD. This would suggest the direct involvement in these functions of P2Y12R-Gi signalling cascade, because platelet shape change and calcium mobilization (both of which are downstream of P2Y1R-Gq activation) were instead unaffected by cholesterol removal [80, 81]. Moreover, it was directly demonstrated that Gi protein subunit preferentially localizes in lowdensity platelet membrane fractions, together with the lipid raft marker flotillin-2. Consistently, Savi and colleagues demonstrated that P2Y12Rs (in platelets or in HEK293 transfected cells) exist predominantly as homo-oligomers located in lipid rafts and that this state of aggregation and distribution is essential for their functionality. Upon treatment in vitro and in rat in vivo with Act-Met, the active metabolite of the specific antagonist clopidogrel, the homooligomers are disrupted into non-functional dimers and monomers that are sequestered outside the lipid rafts [82]. This mechanism can therefore account for the in vivo anti-aggregating and antithrombotic activities of clopidogrel.
The direct association of a P2YR coupled to Gq/11 protein subunit, likely the P2Y2R, with caveolae was demostrated in rat C6 glioma cells, where the knockdown of Cav-1 by RNA interference significantly attenuated purinergic receptor-mediated signalling in terms of calcium transients, again highlighting that the functional activity of the receptor seems to be strictly regulated by membrane microdomains [74]. However, how the P2Y2R is localized in membrane microdomains is still controversial, and it might involve the presence of glycosylation sites in the P2Y2R sequence resulting from asparagine-linked high mannose carbohydrates, as well as hybrid and complex oligosaccharides [75]. Nevertheless, the localization of P2Y2R in membrane microdomains seems not to be a rule for all cell types, since it was also documented that in smooth muscle cells this subtype (differently from the P2Y1R) did not partition to lipid rafts [24].
3.5. P2Y13 Receptor The only evidence about P2Y13R presence in microdomains comes again from Savi and colleagues, who reported that in HEK293T cells the exogenously transfected HA-P2Y13R existed as monomeric and oligomeric species, which were mostly associated with lipid rafts, as demonstrated by sucrose gradient centrifugation and lipid fraction separation [82]. Differently from P2Y12R, cell treatment with the clopidogrel active metabolite Act-Met did not change the quaternary structure and membrane distribution of the receptor.
Purinergic Receptors in Microdomains
Table 1.
Current Medicinal Chemistry, 2013, Vol. 20, No. 1
61
Metabotropic Purinergic Receptors in Membrane Microdomains
Receptor
Tissue/Cell
Localization
Ligand
Ligand Effect
Reference
A1
Cardiomyocytes
Caveolae
CCPA
Out of caveolae
[42]
A1
HL-1 cells
Bulk
nd
nd
[46]
A1
HEK 293 cells
Bulk
nd
nd
[46]
A1
LLC-PK1 cells
Bulk
R-PIA
To caveolae
[48]
A1
DDT1MF-2 cells
Bulk
R-PIA
To caveolae
[47]
A2a
Cortical neurons
Lipid rafts
nd
nd
[25, 51]
A2a
CHO cells
Caveolae
CGS21680
Increased internalization via caveolae
[53]
A2b
T84 cells
Bulk/Caveolae
Adenosine
Increased in caveolae, bulk
[59]
A3
CHO cells
Bulk/Rafts
nd
nd
[60]
P2Y1
Vascular endothelial, smooth muscle cells
Rafts
ADP
Out of rafts
[24]
P2Y1
Astrocytes
Rafts
nd
nd
[67]
P2Y1
Platelets
Bulk
nd
nd
[80]
P2Y1
Cerebellum/synaptosomes
Rafts
nd
nd
[80]
P2Y2
NG108-5 cells
Rafts
nd
nd
[71]
P2Y2
PC12 cells
Rafts
nd
nd
[72]
P2Y4
Cerebellar synaptosomes
Rafts
nd
nd
[76]
P2Y6
Cerebellar synaptosomes
Rafts
nd
nd
[76]
P2Y6
Splenic endothelial cells
Caveolae
nd
nd
[65]
P2Y12
Platelets
Rafts
Clopidogrel (Act-Met)
Out of rafts
[80, 82]
P2Y13
HEK 293 cells
Rafts
Clopidogrel (Act-Met)
No effect
[82]
CONCLUDING REMARKS There is broad evidence that association of transmembrane receptors and signalling molecules with lipid rafts and caveolae provides an enriched environment for protein-protein interactions necessary for signal transduction, and that the enrichment of these receptors within lipid membrane microdomains provides a mechanism for the modulation of neurotransmitter and/or growth factor receptor function. We have illustrated in this work that the localization of adenosine P1R, particularly A1R and A2aR, and metabotropic P2YR, particularly P2Y1R, P2Y2R, P2Y12R, in lipid rafts, raft-like structures and caveolae is cell-specific, and the overall expression levels of these same receptors differs in various cell types and tissues (Table 1). Moreover, these proteins often translocate in and out of these membrane micro-domains under specific physiological and/or pathological conditions, or upon stimulation. This permits complex control systems involving molecular associations, cooperation, conformational or electronic state changes of the receptors themselves. Indeed, compartments can either exclude or include certain proteins, separate unrelated reactions, favour proper cooperative behaviour for instance by decreasing the search time for a ligand to find a receptor. As a consequence the localization of purinergic receptors in membrane microdomains must be always examined both in the presence and in the absence of agonists and antagonists. This adds an additional level of complexity and diversification for triggering, propagating or terminating the purinergic response, i.e. a fine-tuned mechanism for the regulation, coupling to effectors, desensitization and inhibition of the receptors themselves. Moreover, the presence of purinergic GPCRs in membrane microdomains contributes to generate highly sophisticated cellular diversities in response to common epigenetic factors and/or modifications in the extracellular environment, and to model the cell architecture and biochemistry. In the present work, we have also highlighted that disruption of lipid rafts by pharmacologic (cholesterol sequestering agents) or
genetic approaches can even shift the purinergic receptors from raft/caveolar to non-raft/non-caveolar fractions, and then abolish their ability to specifically activate lipid signalling pathways and to integrate with additional lipid-controlled signalling events. This selectively adjusts the biological response to purinergic ligands and most of all indicates that the topology of the purinergic components at the cell surface is fundamental in order to organize the signal transduction machinery and to contribute to its fine-tuning, by controlling both local kinetics of extracellular agonist binding and the integration with different purinergic signal inputs to generate the final cellular response. CONFLICT OF INTEREST The author(s) confirm that this article content has no conflicts of interest. ACKNOWLEDGEMENTS Studies from the authors’ laboratory described in this work were supported by Ministero della Salute Progetto RC FSL-C: “Studio molecolare e funzionale dei recettori purinergici P2 nel sistema nervoso e nelle patologie neurodegenerative e neuroinfiammatorie”. ABBREVIATIONS ARs or P1Rs =
Adenosine Receptors
BDNF
=
Brain Derived Neurotrophic Factor
CBD
=
Caveolin Binding Domain
CNS
=
Central Nervous System
CCPA
=
2-chloro-N6-cyclopentyladenosine
CPA
=
Cyclopentyladenosine
62 Current Medicinal Chemistry, 2013, Vol. 20, No. 1
ERK1/2
=
D’Ambrosi and Volonté
Extracellular Receptor Kinase 1/2
IP3
=
Inositol 1,4,5-trisphosphate
2-MeS-ATP
=
2-methyl-thioATP
GPCRs
=
G Protein-Coupled Receptors
MAPK
=
Mitogen-Activated Protein Kinases
MCD
=
Methyl--cyclodextrin
R-PIA
=
N6-(R)-(phenylisopropyl) Adenosine
P2Rs
=
Purinergic Receptors
P2XRs
=
P2X Receptors
P2YRs
=
P2Y Receptors
PI3K
=
Phosphoinositol-Protein Kinase
PLC-
=
Phospholipase C-
PKC
=
Protein Kinase C
PKC-
=
Protein Kinase C-
TrkB
=
Tyrosine Receptor Kinase B
YFP
=
Yellow Fluorescent Protein
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
REFERENCES [27] [1]
[2]
[3] [4] [5]
[6]
[7]
[8]
[9]
[10] [11]
[12]
[13] [14]
[15] [16] [17]
[18]
Fredholm, B.B.; AP, I.J.; Jacobson, K.A.; Linden, J.; Muller, C.E., International Union of Basic and Clinical Pharmacology. LXXXI. Nomenclature and classification of adenosine receptors--an update. Pharmacol Rev, 2011, 63, (1), 1-34. Harden, T.K.; Sesma, J.I.; Fricks, I.P.; Lazarowski, E.R., Signalling and pharmacological properties of the P2Y receptor. Acta Physiol (Oxf), 2010, 199, (2), 149-160. Browne, L.E.; Jiang, L.H.; North, R.A., New structure enlivens interest in P2X receptors. Trends Pharmacol Sci, 2010, 31, (5), 229-237. Burnstock, G., Discovery of Purinergic Signalling, the Initial Resistance and Current Explosion of Interest. Br J Pharmacol, 2012. Bjarnadottir, T.K.; Gloriam, D.E.; Hellstrand, S.H.; Kristiansson, H.; Fredriksson, R.; Schioth, H.B., Comprehensive repertoire and phylogenetic analysis of the G protein-coupled receptors in human and mouse. Genomics, 2006, 88, (3), 263-273. Fredholm, B.B.; AP, I.J.; Jacobson, K.A.; Klotz, K.N.; Linden, J., International Union of Pharmacology. XXV. Nomenclature and classification of adenosine receptors. Pharmacol Rev, 2001, 53, (4), 527-552. Jacobson, K.A.; Balasubramanian, R.; Deflorian, F.; Gao, Z.G., G proteincoupled adenosine (P1) and P2Y receptors: ligand design and receptor interactions. Purinergic Signal, 2012. Muller, C.E.; Jacobson, K.A., Recent developments in adenosine receptor ligands and their potential as novel drugs. Biochim Biophys Acta, 2011, 1808, (5), 1290-1308. Hasko, G.; Linden, J.; Cronstein, B.; Pacher, P., Adenosine receptors: therapeutic aspects for inflammatory and immune diseases. Nat Rev Drug Discov, 2008, 7, (9), 759-770. Jacobson, K.A., Introduction to adenosine receptors as therapeutic targets. Handb Exp Pharmacol, 2009, (193), 1-24. Lopes, L.V.; Sebastiao, A.M.; Ribeiro, J.A., Adenosine and related drugs in brain diseases: present and future in clinical trials. Curr Top Med Chem, 2011, 11, (8), 1087-1101. Abbracchio, M.P.; Burnstock, G.; Boeynaems, J.M.; Barnard, E.A.; Boyer, J.L.; Kennedy, C.; Knight, G.E.; Fumagalli, M.; Gachet, C.; Jacobson, K.A.; Weisman, G.A., International Union of Pharmacology LVIII: update on the P2Y G protein-coupled nucleotide receptors: from molecular mechanisms and pathophysiology to therapy. Pharmacol Rev, 2006, 58, (3), 281-341. Jacobson, K.A.; Boeynaems, J.M., P2Y nucleotide receptors: promise of therapeutic applications. Drug Discov Today, 2010, 15, (13-14), 570-578. Volonté, C.; Amadio, S.; D'Ambrosi, N.; Colpi, M.; Burnstock, G., P2 receptor web: complexity and fine-tuning. Pharmacol Ther, 2006, 112, (1), 264280. Volonté, C.; Amadio, S.; D'Ambrosi, N., Receptor webs: can the chunking theory tell us more about it? Brain Res Rev, 2008, 59, (1), 1-8. Volonté, C.; D'Ambrosi, N.; Amadio, S., Protein cooperation: from neurons to networks. Prog Neurobiol, 2008, 86, (2), 61-71. Schicker, K.; Hussl, S.; Chandaka, G.K.; Kosenburger, K.; Yang, J.W.; Waldhoer, M.; Sitte, H.H.; Boehm, S., A membrane network of receptors and enzymes for adenine nucleotides and nucleosides. Biochim Biophys Acta, 2009, 1793, (2), 325-334. Escudero, C.; Casanello, P.; Sobrevia, L., Human equilibrative nucleoside transporters 1 and 2 may be differentially modulated by A2B adenosine receptors in placenta microvascular endothelial cells from pre-eclampsia. Pla-
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37] [38]
[39]
[40] [41]
[42]
[43]
centa, 2008, 29, (9), 816-825. Jiang, L.; Bardini, M.; Keogh, A.; dos Remedios, C.G.; Burnstock, G., P2X1 receptors are closely associated with connexin 43 in human ventricular myocardium. Int J Cardiol, 2005, 98, (2), 291-297. D'Ambrosi, N.; Iafrate, M.; Vacca, F.; Amadio, S.; Tozzi, A.; Mercuri, N.B.; Volonté, C., The P2Y receptor forms homo-oligomeric complexes in several CNS and PNS neuronal cells. Purinergic Signal, 2006, 2, (4), 575-582. Koles, L.; Gerevich, Z.; Oliveira, J.F.; Zadori, Z.S.; Wirkner, K.; Illes, P., Interaction of P2 purinergic receptors with cellular macromolecules. Naunyn Schmiedebergs Arch Pharmacol, 2008, 377, (1), 1-33. Rumjahn, S.M.; Yokdang, N.; Baldwin, K.A.; Thai, J.; Buxton, I.L., Purinergic regulation of vascular endothelial growth factor signaling in angiogenesis. Br J Cancer, 2009, 100, (9), 1465-1470. Buvinic, S.; Bravo-Zehnder, M.; Boyer, J.L.; Huidobro-Toro, J.P.; Gonzalez, A., Nucleotide P2Y1 receptor regulates EGF receptor mitogenic signaling and expression in epithelial cells. J Cell Sci, 2007, 120, (Pt 24), 4289-4301. Norambuena, A.; Poblete, M.I.; Donoso, M.V.; Espinoza, C.S.; Gonzalez, A.; Huidobro-Toro, J.P., P2Y1 receptor activation elicits its partition out of membrane rafts and its rapid internalization from human blood vessels: implications for receptor signaling. Mol Pharmacol, 2008, 74, (6), 1666-1677. Assaife-Lopes, N.; Sousa, V.C.; Pereira, D.B.; Ribeiro, J.A.; Chao, M.V.; Sebastiao, A.M., Activation of adenosine A2A receptors induces TrkB translocation and increases BDNF-mediated phospho-TrkB localization in lipid rafts: implications for neuromodulation. J Neurosci, 2010, 30, (25), 84688480. Kim, H.J.; Ajit, D.; Peterson, T.S.; Wang, Y.; Camden, J.M.; Gibson Wood, W.; Sun, G.Y.; Erb, L.; Petris, M.; Weisman, G.A., Nucleotides released from Abeta(1)(-)(4)(2) -treated microglial cells increase cell migration and Abeta(1)(-)(4)(2) uptake through P2Y(2) receptor activation. J Neurochem, 2012, 121, (2), 228-238. Peterson, T.S.; Camden, J.M.; Wang, Y.; Seye, C.I.; Wood, W.G.; Sun, G.Y.; Erb, L.; Petris, M.J.; Weisman, G.A., P2Y2 nucleotide receptor-mediated responses in brain cells. Mol Neurobiol, 2010, 41, (2-3), 356-366. Kamae, T.; Shiraga, M.; Kashiwagi, H.; Kato, H.; Tadokoro, S.; Kurata, Y.; Tomiyama, Y.; Kanakura, Y., Critical role of ADP interaction with P2Y12 receptor in the maintenance of alpha(IIb)beta3 activation: association with Rap1B activation. J Thromb Haemost, 2006, 4, (6), 1379-1387. Sullivan, G.W.; Lee, D.D.; Ross, W.G.; DiVietro, J.A.; Lappas, C.M.; Lawrence, M.B.; Linden, J., Activation of A2A adenosine receptors inhibits expression of alpha 4/beta 1 integrin (very late antigen-4) on stimulated human neutrophils. J Leukoc Biol, 2004, 75, (1), 127-134. Volonté, C.; D'Ambrosi, N., Membrane compartments and purinergic signalling: the purinome, a complex interplay among ligands, degrading enzymes, receptors and transporters. Febs J, 2009, 276, (2), 318-329. Vacca, F.; Amadio, S.; Sancesario, G.; Bernardi, G.; Volonté, C., P2X3 receptor localizes into lipid rafts in neuronal cells. J Neurosci Res, 2004, 76, (5), 653-661. Garcia-Marcos, M.; Dehaye, J.P.; Marino, A., Membrane compartments and purinergic signalling: the role of plasma membrane microdomains in the modulation of P2XR-mediated signalling. Febs J, 2009, 276, (2), 330-340. Kenworthy, A.K.; Edidin, M., Distribution of a glycosylphosphatidylinositolanchored protein at the apical surface of MDCK cells examined at a resolution of
