Interaction of purinergic receptors with GPCRs, ion channels, tyrosine kinase and steroid hormone receptors orchestrates cell function

Abstract

Extracellular purines and pyrimidines have emerged as key regulators of a wide range of physiological and pathophysiological cellular processes acting through P1 and P2 cell surface receptors. Increasing evidence suggests that purinergic receptors can interact with and/or modulate the activity of other classes of receptors and ion channels. This review will focus on the interactions of purinergic receptors with other GPCRs, ion channels, receptor tyrosine kinases, and steroid hormone receptors. Also, the signal transduction pathways regulated by these complexes and their new functional properties are discussed.

Introduction

Extracellular purines and pyrimidines have widespread and specific signalling actions in the regulation of a variety of functions in many tissues. They have emerged as physiological regulators of cell growth, differentiation, and death [1]. Moreover, they have been implicated in neoplastic transformation, embryogenesis, platelet aggregation, cardiovascular function, bone and muscle regeneration, insulin release, inflammation and immunomodulation, neuroprotection, and initiation of pain [2–5]. Taking these facts into account, there is increasing interest in the therapeutic potential of purinergic and pyrimidinergic compounds [1, 4].

Purinergic and pyrimidinergic nucleotides cannot be transported across the plasma membrane by simple diffusion, so they are released to the extracellular environment via lytic (diffusion through the damaged plasma membrane during trauma, injury, apoptosis, and necrosis) [6–10] and non-lytic mechanisms (mechanical distension, ATP release channels, ATP-binding cassette proteins, facilitated diffusion by nucleotide-specific transporters, and vesicular exocytosis) [11–19] either under physiological and pathophysiological conditions. However, some of the transport mechanisms involved in ATP release are controversial, for instance, it has been reported that cystic fibrosis transmembrane conductance regulator cannot carry this nucleotide [20].

Nucleotides have a short half-life due to the presence of ectonucleotidases that rapidly degrade them, so they can activate plasma membrane receptors, called purinergic receptors, in an autocrine and paracrine manner [5, 21]. Many receptor subtypes for purines and pyrimidines have been identified on the basis of cloning, signal transduction and pharmacology. They are divided into P1 adenosine receptors (A₁, A_2A, A_2B, and A₃ subtypes), P2Y metabotropic receptors (P2Y_{1, 2, 4, 6, 11–14}), and P2X ionotropic receptors (P2X_1–7 subtypes forming both homomultimers and heteromultimers) [22].

P1 receptors are all members of the rhodopsin-like family of G protein-coupled receptors (GPCRs). They have a short extracellular N-terminal domain, seven transmembrane domains, and a short intracellular C-terminal loop. They couple principally to adenylate cyclase, either negatively (A₁ and A₃) or positively (A_2A and A_2B). The human A_2B receptor has also been observed to couple through G_q/11 to regulate phospholipase C (PLC) activity [23, 24].

P2Y receptors are also members of the GPCR family; their structure consists of an extracellular N-terminal domain, seven transmembrane spanning regions that form the ligand binding pocket, and a C-terminal domain containing several binding/phosphorylation sites for protein kinases and G proteins. Particularly, the second and third intracellular loops (IL-2 and IL-3) of GPCRs are important for G protein coupling. Studies showed that when IL-2 and IL-3 are deleted, GPCRs are no longer able to couple to G proteins [25, 26].

Each P2Y receptor subtype is directly coupled to multiple G proteins triggering the activation of various intracellular signalling cascades. P2Y₁, P2Y₂, P2Y₄, P2Y₆, and P2Y₁₁ receptors couple to G_αq protein to induce the activation of PLC which catalyses the hydrolysis of phosphatidylinositol 4,5-biphosphate to generate inositol 1,4,5-triphosphate (IP₃) and diacylglycerol (DAG) [27]. DAG induces the activation of protein kinase C (PKC) leading to the stimulation of diverse downstream effectors; IP₃ stimulates intracellular calcium (Ca²⁺) mobilization. In addition, some of these P2Y receptors also couple to adenylyl cyclase inducing changes in intracellular cyclic adenosine monophosphate (cAMP) levels. P2Y₁₃ receptors can simultaneously couple to G_i and G_s inducing opposite effects on intracellular cAMP levels [28].

P2X receptors are trimers or hexamers formed by protein subunits, each consisting of intracellular N and C termini possessing consensus binding motifs for protein kinases, two transmembrane spanning regions involved in channel gating and ion pore lining, respectively, and a large extracellular loop containing the ATP-binding site [23]. Six homomultimers (P2X_{1, 2, 3, 4, 5, 7}) and three heteromultimers (P2X₂/P2X₃, P2X₄/P2X₆, and P2X₁/P2X₅) have been functionally characterized. These receptors trigger the activation of many intracellular signalling pathways by increasing the [Ca²⁺]i concentration [21].

Therefore, P1 and P2 receptors can lead to the activation of several signalling pathways such as the mitogen-activated protein kinase (MAPK) cascade [23, 27, 29–34], and the phosphatidylinositol-3 kinase (PI3K)/Akt signalling pathway [27, 34–39] to regulate cell survival, cell differentiation, programmed cell death, cell cycle progression, and cellular growth.

It is well known that interactions between GPCRs can modulate their activity either potentiating or inhibiting it. Such interactions can take place through the formation of a physical complex (receptor dimerization), or through receptor cross-talk, when second messengers integrate coincident signals from multiple receptors, which are not physically associated [4, 23]. Thus, P2Y receptors can interact and/or regulate the activity of other P2Y receptors and, also, of other GPCRs. In addition, P2Y receptors can also modulate the activity of P2X ion channels and receptor tyrosine kinases (RTKs), recently recognized as important in the regulation of signalling and cellular responses [40–44]. Related to this, it is known that purinergic receptors, particularly A₁, A_2A, P2X_1,3,4,7, and P2Y_1,2,4,6,12 subtypes as well as ectonucleotidases and nucleotide transporters are assembled in specialized sub-membrane compartments (lipid rafts, raft-like structures, and caveolae). Altogether, these reciprocal influences control the duration, magnitude, and/or direction of the signals triggered by purines and pyrimidines, and the impact that each single ligand has on a variety of short- and long-term functions [45]. However, the interaction of receptors for purines and pyrimidines with other receptor types is one issue that remains unresolved [22]. Thus, in this review, we focus on the interplay occurring between P2 receptors and other receptor families. Particularly, we discuss the relationship between purinergic receptors and other GPCRs and tyrosine kinase and steroid hormone receptors.

Interactions between purinergic receptors and other GPCRs

As previously mentioned in the introduction, GPCRs exist as dimers or higher-order oligomers that may modify their functions. P2Y metabotropic receptors tend to form homo- or heterodimers with GPCRs not only of different families but also of the same purinergic receptor families, leading to alterations in functional properties. Such dimerization occurs constitutively in the endoplasmic reticulum where it could have an important role in the quality control of newly synthesized receptors and specific subcellular localization [46]. Only limited information is available on the physiological relevance of the various GPCR dimers identified to date in cell culture systems and native tissues, because of difficulties associated with demonstrating any physiological significance of dimerization in the native systems [47].

P2Y homodimerization and homomultimerization

It has been reported that P2Y₁ receptors exist as dimers, in HEK293 cell membranes, in the resting state. Agonist exposure induces a rise in receptor dimerization. This effect follows desensitization and is fully reversible upon withdrawal of agonist. Both monomer and constitutive dimers are fully active [48]. In addition, this receptor may form oligomers in other types of cells [49]. Moreover, it was demonstrated that P2Y₂ receptors form homo-oligomeric assemblies and that the formation of P2Y₂ receptor oligomers does not depend on the presence of UTP as an agonist [50]. The P2Y₄ receptor subunit can also form higher-order complexes. These multimers appear stable, being to some extent resistant to denaturing and reducing conditions, thus indicating that they derive, at least in part, from covalent disulphide bonds occurring between the subunits. Moreover, both rat and human endogenous P2Y₄ receptors appear as stable dimers in cell lines or primary neurons from the peripheral and central nervous system. This also occurs for the heterologous P2Y₄ receptor transiently transfected in the neuroblastoma SH-SY5Y cell line [43]. Endogenously expressed P2Y₄ and P2Y₆ receptors form high-order complexes in neurons. The protomeric unit at the basis of the P2Y₆ receptor complex appeared to be the monomer while the dimer seems to be the unit for P2Y₄ subtypes. Moreover, dimeric P2Y₄ and monomeric P2Y₆ proteins display selective microdomain partitioning in lipid rafts from specialized subcellular compartments such as synaptosomes. Receptor activation by UTP induced the oligomerization of the P2Y₆ but not of the P2Y₄ receptor. Transfected P2Y₄ and P2Y₆ proteins homo-interact and possess the appropriate domains to associate with P2Y_{1, 2, 4, 6, 11} receptor subtypes as judged by the results obtained using a direct method of double co-transfection (i.e. co-transfection with Myc-P2Y₄ plus, respectively, FLAG-P2Y_{1, 2, 6, 11} or with FLAG-P2Y₆ plus, respectively, Myc-P2Y_{1, 2, 4, 11}); however, endogenous P2Y₄ form hetero-oligomers only with P2Y₆ receptors [51].

It has been established that P2Y₁₂ receptors exist predominantly as homo-oligomers, essential for their functionality, which are situated in lipid rafts of mammalian cells and in freshly isolated platelets. Upon in vitro treatment with the active metabolite of clopidogrel or in vivo oral clopidogrel administration to rats, the homo-oligomers are disrupted into non-functional dimers and monomers that are sequestered outside the lipid rafts [52].

P2Y heterodimerization

P2Y₁ and P2Y₁₁ receptors were found to associate together when co-expressed in HEK293 cells. The hetero-oligomer formation promotes agonist-induced internalization of the P2Y₁₁ receptor, which by itself is unable to undergo endocytosis. This interaction and subsequent internalization has an important impact on P2Y₁₁ receptor desensitization. Co-internalization of these receptors was also seen in 1321N1 astrocytoma cells upon stimulation with ATP or with the P2Y₁ receptor-specific agonist 2-MeS-ADP. In addition, the association of P2Y₁ with the P2Y₁₁ receptor influences the ligand selectivity of the P2Y₁₁ receptor. In this way, the specific P2Y₁ receptor antagonist MRS2179 inhibited both the rise in [Ca²⁺]i induced by the potent P2Y₁₁ receptor agonist 2′,3′-O-(4-benzoyl-benzoyl)-ATP (BzATP) and the internalization of the P2Y₁₁ receptor in response to ATP, whereas the highly potent P2Y₁₁ receptor antagonist NF157 was not able to inhibit any of these effects. Thus, the hetero-oligomerization of these receptors allows novel functions of the P2Y₁₁ receptor in response to extracellular nucleotides [53]. Heterodimerization also takes place between purinergic receptors and other types of GPCRs. For instance, it has been reported that adenosine A₁ and P2Y₁ receptors can form constitutive hetero-oligomers in co-transfected cells. This process is promoted by the simultaneous activation of both receptors [54–56]. Oligomeric association of A₁ and P2Y₁ receptors generates P2Y₁-like agonistic pharmacology and provides a molecular mechanism for an increased diversity of purine signalling [55, 57]. Co-localization of A₁–P2Y₁ receptors at glutamatergic synapses and surrounding astrocytes has also been demonstrated in rat hippocampus. P2Y₁ receptor stimulation impaired the potency of A₁ receptor coupling to G protein, whereas the stimulation of A₁ receptors increased the functional responsiveness of P2Y₁ receptors. This may be particularly important during pathological conditions, when large amounts of these mediators are released. The same complex was also demonstrated in human astroglial cells [58, 59]. A₁ and P2Y₂ receptors can also associate in co-transfected HEK293T cells and intact rat brain. This heterodimerization affects the receptor binding site attenuating A₁ agonist binding by P2Y₂ receptor agonists in membranes of co-transfected cells. Moreover, A₁ receptor activity is suppressed and P2Y₂ receptor activity synergistically enhanced, upon simultaneous addition of A₁ and P2Y₂ receptor agonists [60].

Cross-talk between purinergic receptors and other GPCRs

Hypertonic stress-induced cell shrinkage releases ATP from polymorphonuclear neutrophils (PMNs), released ATP augments PMNs functions through P2 receptors and p38 MAPK activation, or ATP is converted to adenosine, which suppresses PMNs functions via A₂ receptors that activate cAMP/PKA signalling. This bidirectional control by released ATP allows PMNs to register and differentially respond to osmotic changes in their extracellular environment [61]. Cross-talk between A₁ and P2Y₂ receptors has additionally been reported to function in local regulation of water transport and homeostasis by the kidney [62]. There is evidence supporting cross-talk between P2Y₁₂ and P2Y₁ receptors in platelets. There, P2Y₁₂ receptor activation by ADP positively modulates the P2Y₁-dependent calcium response, whereas P2Y₁ receptor activation negatively modulates P2Y₁₂ receptor function through Src kinase activation. Moreover, modulation of both receptors is mediated by PI3K and inhibition of adenylate cyclase. In turn, a negative feedback pathway from P2Y₁ receptors, mediated by Src tyrosine kinase, inhibits the PI3K-dependent signalling component. Ca²⁺ signalling, therefore, represents a point of cross-talk between these receptors and a key regulator of platelet response to ADP [63]. On the other hand, the calcium response evoked by P2Y₁ receptors is potentiated by the activity of P2Y₁₂ receptor-dependent signalling pathways in glioma C6 cells. There, Ca²⁺ influx, enhanced by the cooperation of P2Y₁ and P2Y₁₂ receptor activities, directly depends on the capacitative calcium entrance mechanism [64]. Simultaneous activation of P2Y and adenosine A1 receptors synergistically increases Ca²⁺ transients and translocation of PKC to the plasma membrane in DDT1 MF-2 cells [65].

P2Y₂ receptor activation by ATP decreases angiotensin type 1 receptor density through nitric oxide (NO)-mediated S-nitrosylation of nuclear factor κB in rat cardiac fibroblasts [66]. In transfected CHO cells, the G_i/G_o protein-coupled adenosine A₁ receptor activates MAPK via a pathway which is independent of PKC but involves tyrosine kinase, PI3K and MEK1 activation. Moreover, co-activation of adenosine A₁ and P2Y₂ receptors induces synergistic increases in MAPK activity [67]. This effect may be related to the enhancement of G_q/11/[Ca²⁺] signalling observed upon the simultaneous activation of these receptors [60]. In this way, the PKC/Raf-1 upstream mediators of the MAPK cascade may synergistically increase MAPK signalling. In addition, simultaneous activation of endogenous A₁ and P2Y₂ receptors in DDT1 MF-2 cells synergistically increases translocation of PKC to the plasma membrane [65]. However, identifying the mechanism(s) underlying the synergistic increases in MAP kinase activity will require further research.

Channel regulation by P2 receptors

Growing evidence implicates a key role for extracellular nucleotides in the regulation of ion channels, but the mechanism for such action is poorly defined. ATP and other nucleotides, including UTP, decrease epithelial Na⁺ channel (ENaC) activity via apical P2Y₂ receptors. P2Y₂ receptors couple to ENaC via PLC. In this way, locally released ATP acts in an autocrine/paracrine manner to tonically regulate ENaC in mammalian collecting duct. Loss of this intrinsic regulation leads to ENaC hyperactivity and contributes to hypertension that occurs in P2Y₂ receptor−/− mice. P2Y₂ receptor activation by nucleotides thus provides physiologically important regulation of ENaC and electrolyte handling in mammalian kidney [68]. A paracrine regulation of ENaC by UTP has also been reported in lung epithelia of mice infected with the respiratory syncytial virus (RSV). RSV infection resulted in higher levels of pyrimidines and purines in the alveolar space which mediated, at least in part, the harmful effects of RSV on lung epithelia [69].

In layer V pyramidal neurons of the prefrontal cortex post-synaptically localized P2Y receptors interact with NMDA receptor channels [70].

Activation of neuronal P2Y₁ receptors may gate calcium-dependent K⁺ channels (K(Ca)2 channels) via PLC-dependent increases in intracellular Ca²⁺, thereby defining an additional class of neuronal ion channels as novel effectors for P2Y receptors. This mechanism may form the basis for the control of synaptic plasticity via P2Y₁ receptors [71]. P2Y₁ receptors can transduce information from central sensory neurons through regulation of hyperpolarization-activated cation channel activities [72].

P2 receptors and RTKs

As previously mentioned, in addition to ion channel activity and GPCRs, P2Y receptors can modulate the activity of RTKs [73]. This latter family comprises high-affinity cell surface receptors for many polypeptide growth factors, cytokines, and hormones, which regulate normal cellular processes and also have a critical role in the development and progression of many types of cancer. At least 20 classes of RTKs have been identified, including the epidermal growth factor receptor (EGFR) family, the insulin receptor family, the platelet-derived growth factor receptor (PDGFR) family, the fibroblast growth factor receptor (FGFR) family, the nerve growth factor receptor (NGFR) family, and the vascular endothelial growth factor receptor (VEGFR) family [74].

Human carcinomas frequently express high levels of receptors of the EGFR family, and overexpression of at least two of these receptors has been associated with a more aggressive clinical behaviour [75, 76]. This could be explained by the fact that the EGFR function is transregulated by a variety of stimuli, including agonists of certain GPCRs [77]. Different P2Y receptor subtypes have been involved in the transactivation of the EGFR in normal and cancer cells. For example, in the PC12 cell line, derived from a pheochromocytoma of the rat adrenal medulla, P2Y₂ receptors mediate EGFR transactivation to finally induce MAPK activation. This occurs downstream of related adhesion focal tyrosine kinase (RAFTK, a member of the focal adhesion PTK family). As a consequence, although P2Y₂ and EGFRs may both activate a similar multiprotein signalling cascade immediately upstream of MAPK, the P2Y₂ receptor appears to uniquely utilize [Ca²⁺]i, PKC, and, subsequently, RAFTK [73]. Also in the human colonic cancer cell line, Caco-2, ATP-mediated stimulation of MAPKs involves cross-communication between P2Y_2/4 receptor subtypes and EGFR signalling systems [32]. Furthermore, in tumoral HeLa cells and normal female reproductive tract epithelial cells, cell-released nucleotides stimulate P2Y₁ receptors to trigger mitogenic signals by transactivating the EGFR. The pathway involves PKC, Src, and cell surface metalloproteases. Strikingly, the canine kidney epithelial cell line which ectopically expresses P2Y₁ receptors displays a highly proliferative phenotype that depends on EGFR activity associated with an increased level of EGFR. This discloses a novel aspect of GPCR-mediated regulation of EGFR function [77]. Similarly, an in vitro wound healing assay performed in human corneal and BEAS 2B (human bronchial) epithelial cells suggested that ATP released as a consequence of the wound triggers EGFR transactivation resulting in the stimulation of the PI3K and ERK signalling pathways to lead wound closure [78]. Moreover, ATP, acting through P2Y receptors, transactivates both PDGFR and EGFR leading to the activation of ERK1/2 and PI3K and to an increase in the proliferation rate of Müller glial cells. PDGF-induced proliferation may depend on transactivation of the EGFR kinase while metalloproteinase 9 was implicated in the signal transfer from P2Y to EGFRs [79]. As can be inferred, many reports do not determine the P2Y receptor subtype/s involved in EGFR transactivation. On the other hand, metalloproteinase-dependent transactivation of the EGFR is stimulated by ATP-induced ERK1/2 phosphorylation through P2Y₂/P2Y₄ receptors in bovine adrenal chromaffin cells [80]. In astrocytes, P2Y₂ receptors are also involved in the phosphorylation of the EGFR. This occurs due to cell stress-released nucleotides which induce the activation of P2Y₂ receptors leading to pro-inflammatory responses that can protect neurons from injury, including the stimulation and recruitment of glial cells. P2Y₂ receptor activation induces the phosphorylation of the EGFR, a response dependent upon the presence of SH₃ binding domains in the intracellular C terminus of the P2Y₂ receptor that promote Src binding and transactivation of EGFR, a pathway that regulates the proliferation of cortical astrocytes [81].

P2X receptors have also been recently implicated in the transactivation of the EGFR. In HEK 293 human embryonic kidney cells, transactivation of the EGFR by BzATP is essential for P2X₇ receptor-induced expression of Egr-1 [82].

P2 receptors can potentiate or synergize with growth factors to regulate a cellular response. In the human breast cancer cell line MCF-7, ATP-γ-S, or EGF lead to ERK activation and phosphorylation of the transcription factors CREB and Elk-1. Co-stimulation synergistically activated c-Fos expression and notably increased the phosphorylation of ERK, CREB, and EGFR. Nevertheless, the ERK pathway does not fully account for this synergy since Fos induction was differentially sensitive to the MEK inhibitor U0126, indicating that ATP and EGF signal differently to c-Fos. Thus, extracellular nucleotides cooperate with growth factors to activate genes linked to the proliferative response in MCF-7 cells [83]. ATP, ADP, and UTP acting through P2Y₁ and P2Y₂ receptors, and low concentrations of adenosine, augmented adult multipotent neural stem cell proliferation in the presence of growth factors. This result infers nucleotide receptor-mediated synergism that augments growth factor-mediated cell proliferation, supporting the notion that extracellular nucleotides contribute to the control of adult neurogenesis [84]. In addition, Grimm and collaborators established that nucleotides and EGF, acting in a paracrine or autocrine manner, both induce converging intracellular signalling pathways (Akt and focal adhesion kinase) that carry potential for synergism in the control of neural stem cell proliferation and cell survival [85]. ATP and insulin act synergistically to stimulate the activation of ERK1/2, and also induce an additive activation of Raf and Ras in coronary artery smooth muscle cells (CASMCs), leading to synergistic stimulation of CASMCs proliferation [86]. Opposite, UTP or UDP significantly reduced the proliferative response to PDGF in vascular smooth muscle cells [87]. The mechanism underlying these opposite effects of P2Y receptor activation is not known. More than one P2Y receptor subtype may contribute and also P2Y receptors can respond differently depending on the expression of effector proteins and on the cross-talk occurring between different signalling pathways and receptors in a particular cell type. Therefore, interactions between P2Y receptors and RTKs can be complex [1].

Recently, it has been shown that plasma membrane distribution of P2Y₂ receptors is transregulated by the EGFR in smooth muscle cells isolated from human chorionic arteries. There, the use of AG1478, a selective and potent inhibitor of the EGFR tyrosine kinase activity, not only blocked the UTP-induced vasomotor activity but also abrogated both RhoA and Rac1 activation, the P2Y₂ receptor association with membrane rafts, and its internalization. These results reveal an unsuspected functional interplay that controls both the membrane distribution and the vasomotor activity of the P2Y₂ receptor in intact human blood vessels [88].

Extracellular purines can stimulate the synthesis and release of nerve growth factor (NGF) [89], which is essential for neuronal growth and differentiation, and they can also act in combination with this factor to regulate differentiation and growth of various cell lines [90].

It has been reported that the use of P2 receptor antagonists reversibly prevents diverse NGF-dependent responses in PC12 cells. Furthermore, NGF modulates extracellular release of ATP and also the expression levels of P2X₂ receptor protein [91]. These authors established that P2 receptor agonists can behave as neurotrophic factors for neuronal cells. They reported that ATP and 2-Cl-ATP promote neurite regeneration after priming of PC12 cells with NGF, whereas various P2 receptor antagonists were inhibitory. Moreover, NGF and ATP induced the expression of P2X₂, P2X₃, P2X₄ and P2Y₂ receptor proteins under neurite-regenerating conditions in PC12 cells [92]. On the other hand, the induction of PC12 cell differentiation by NGF altered mRNA expression of several P2Y and P2X receptors, but only increased P2X_1–4 protein expression. NGF enhanced the ability of the non-hydrolyzable ATP analog ATPγS to stimulate catecholamine (norepinephrine) release. These responses characterize sympathetic neuronal differentiation and appear to be physiologically important [93]. Additionally, both ATP and NGF enhanced the expression of the stress-induced heat shock proteins 70 and 90 [94], as well as the phosphorylation of ERK1/2 in PC12 cells [92]. In parallel with NGF, ATP prevented the cleavage and activation of caspase-2 and inhibits the release of cytochrome c from mitochondria into the cytoplasm. Finally, neither NGF nor ATP modulated the expression of P2 receptors suggesting a potential interaction between ATP and NGF signalling in the neuritic outgrowth and survival of PC12 cells [94]. Therefore, extracellular ATP potentiates the neurite outgrowth induced by NGF. On the other hand, it was shown that ATP and BzATP acting through P2X₇ receptors can induce biochemical and/or morphological changes characteristic of apoptotic cell death in some cell types [95–97]. These opposite effects exerted by ATP on cell apoptosis may be due to the interaction between purinergic and growth factor signalling. However, different expression of P2 receptors should also be considered.

The neurotrophic effect of ATP and other nucleotides was determined in the NGFR-negative mouse neuroblastoma neuro2a cell line. There, ATP stimulated neurite outgrowth, apparently, via P2Y₁₁ receptors as determined by the potency order of the P2 agonists ATP = ATPγS > ADP >> 2Me-S-ATP on the neuritogenic effect, the insensibility to UTP and to the antagonist PPADS. This neurotrophic effect was mediated by Src kinase, PLC and ERK1/2 MAPK, suggesting that ATP can stimulate neurite outgrowth independent of other neurotrophic factors and can be an effective trophic agent [98].

ATPγS in the presence of NGF leads to phosphorylation of tyrosine receptor kinase A (TrkA, high-affinity nerve growth factor receptor) and to the co-localization (determined by immunocytochemistry) and association (determined by immunoprecipitation) of TrkA with P2Y₂ receptors; these events are required to enhance neuronal differentiation [99]. The use of Src family kinase inhibitor blocked ATPγS/P2Y₂ receptor-promoted enhancement of NGF/TrkA signalling and neuronal differentiation in PC12 cells, abrogated the enhancement by ATPγS of neurite outgrowth in primary cultures of dorsal root ganglion neurons, and also blocked co-immunoprecipitation of TrkA, P2Y₂ receptors, and Src family kinases. Thus, Src family kinases regulate P2Y₂ receptor-TrkA molecular cross-talk suggesting that they are key convergence points between RTKs and GPCRs [42]. Furthermore, ATPγS promotes phosphorylation of ERK1/2 and p38, thereby enhancing sensitivity to NGF and accelerating neurite formation in both PC12 cells and dorsal root ganglion neurons. In conclusion, the interactions of tyrosine kinase- and P2Y₂ receptor-signalling pathways provide a paradigm for the regulation of neuronal differentiation and suggest a role for P2Y₂ as a morphogen receptor that potentiates neurotrophin signalling in neuronal development and regeneration [99].

The GPR17 is a new P2Y-like receptor, responsive to uracil nucleotides and cysteinyl-leukotrienes (cysLTs), which may have a potential role in the regulation of both cell viability and differentiation state of central nervous system cells [100]. To distinguish GPR17 functions from other P2Y receptor activities, Daniele et al. [101] have demonstrated that the expression of GPR17 mRNA is selectively induced during PC12 cell differentiation to neuronal cells, whereas P2Y_{2, 4, 6, 12, 13, 14} receptors are constitutively expressed in PC12 cells and do not undergo modulation following NGF treatment. In addition, the specificity of GPR17 ligands (UDP glucose and LTD₄) was evaluated by the use of the GPR17 selective antagonists cangrelor and montelukast. Furthermore, to unequivocally prove a role for GPR17 some experiments were performed in PC12-differentiated cells following silencing of the receptor upon incubation of cells with small interfering RNAs. Thus, in NGF-differentiated PC12 cells, GPR17 ligands induced a significant pro-survival effect. They activated the intracellular phosphorylation of both ERK1/2 and p38 MAPKs, which have been identified as important signalling pathways for neurotrophins in PC12 cells. Additionally, GPR17 agonists promoted, both alone and synergistically with NGF, neurite outgrowth in PC12 cells, suggesting a possible interplay between endogenous uracil derivatives, cysLTs and NGF in the signalling pathways involved in neuronal survival and differentiation. GPR17 ligands were also able to confer a NGF-like activity to the EGF which also promoted cell differentiation and neurite elongation. Thus, GPR17, like other P2Y receptors, can act as a neurotrophic regulator for neuronal-like cells [101].

P2Y₂ receptors have been shown to transactivate VEGFR in human coronary endothelial cells. In these cells, P2Y₂ receptor activation by UTP induces rapid tyrosine phosphorylation of the VEGFR-2, and co-localization of both receptors. Consequently, the expression of the pro-inflammatory vascular cell adhesion molecule-1 (VCAM-1) augments through RhoA activation. Deletion or mutation of two Src homology-3-binding sites in the C-terminal tail of P2Y₂ receptors, or inhibition of Src kinase activity abolishes P2Y₂ receptor-mediated transactivation of VEGFR-2 and subsequently inhibits UTP-induced VCAM-1 expression. These data indicate a novel mechanism whereby a nucleotide receptor transactivates a receptor tyrosine kinase to generate an inflammatory response associated with atherosclerosis [102].

P2Y₁ receptors have also been found to transactivate the VEGFR in vascular endothelial cells. It was found that P2Y₁ receptor stimulation of VEGFR phosphorylation by 2-methyl-thio-ATP (2Me-S-ATP) was suppressed by the VEGFR-2 tyrosine kinase inhibitor, SU1498. In addition, phosphorylation of VEGFR-2 by VEGF was comparable with 2Me-S-ATP stimulation of the P2Y₁ receptor, and both 2Me-S-ATP and VEGF stimulation increased tyrosine phosphorylation of VEGFR-2 at Tyr 1175 [103, 104].

As previously mentioned, extracellular nucleotides can also stimulate the release of growth factors. In platelets, for example, activation of P2Y₁ and P2Y₁₂ receptors by ADP results in an increase in soluble VEGF concentrations. This suggests that ADP release in the tumour microenvironment may be, on balance, pro-angiogenic. P2Y receptor antagonism abrogates ADP-mediated pro-angiogenic protein release and thus may represent a potential pharmacologic strategy for regulating platelet-mediated angiogenesis [105]. It was reported that VEGF is released from primary human monocytes through P2X₇ receptor stimulation by ATP. This effect is calcium-dependent and is associated with reactive oxygen species production. Thus, P2X₇ receptors are also likely to be important in the control of angiogenesis and wound repair [106]. P2Y₂ receptor activation in human salivary gland cells promotes the formation of EGFR/ErbB3 heterodimers and metalloprotease-dependent neuregulin 1 release, resulting in the activation of both EGFR and ErbB3 [107]. P2X₇ receptors were also implicated in VEGF release in rat C6 glioma cells. Cell exposure to BzATP augmented P2X₇ receptor expression, increased intracellular calcium [Ca²⁺]i mobilization, induced the formation of large pores, and enhanced the expression of pro-inflammatory factors including MCP-1, IL-8, and VEGF [108].

Interactions between P2Y, P2X, and polypeptide growth factor signalling pathways may have important implications for CNS development as well as injury and repair. Besides, reports suggest that fibroblast growth factor 2 (FGF-2) is increased after injury and can stimulate astrocyte proliferation. It has been shown that extracellular nucleotides can potentiate FGF-2-mediated signalling. In primary cultures of rat cortical astrocytes, for example, extracellular ATP enhances FGF-2-induced proliferation in a process mediated by P2Y receptors, phosphorylation of ERK1/2 MAPK and increased cyclin expression. However, when P2X₇ receptors are activated, FGF-2-dependent proliferation is inhibited shifting cells to a state of reversible growth arrest that may involve phosphorylation of p38 and JNK MAPKs. Thus, P2Y and P2X₇ receptors mediate opposing effects on FGF-2-induced mitogenesis [109–111]. Furthermore, in adult mouse olfactory epithelium ATP also induces cell proliferation by promoting FGF-2 and TNF-α synthesis and activation of their receptors (FGFR and EGFR, respectively) [112].

P2 receptors and steroid hormone receptors

Purinoceptors are widely expressed in endocrine glands. For instance, in testicular Sertoli and in Leydig cells, they are involved in estradiol and testosterone secretion and are also expressed in the ovary where they mediate the antagonism of estradiol and progesterone secretion from granulosa cells [3, 4]. Recently, it has been found that 17β-estradiol acting via estrogen receptor alpha promotes proliferation of MCF-7 breast cancer cells by down-regulating P2Y₂ receptor expression and attenuating P2Y₂ receptor-induced increase of [Ca²⁺]i [113]. A similar P2 receptor down-regulation mechanism by this female gonadal hormone was determined in dorsal root ganglion (DRG) primary sensory neurons. In these cells, P2X₃ receptor subunit mRNA was significantly decreased by the application of 17β-estradiol in a concentration-dependent manner. The use of the estrogen receptor antagonist, ICI 182,780, blocked the reduction in the receptor subunit protein level. Thus, 17β-estradiol participates in the control of peripheral pain signal transduction by modulating the expression of the P2X₃ subunit and, consequently, P2X₃ receptor-mediated events [114]. On the other hand, P2X₃ receptor mRNA was significantly decreased in DRG neurons of ovariectomized rats. However, estrogen replacement could reverse this effect [115]. 17β-Estradiol may then participate in the regulation of P2 receptors, either decreasing or increasing their expression, to control cell signalling pathways.

Besides, it has been shown that estrogens can modulate cell events in a non-genomic manner by affecting signalling mediated by P2 receptors. For instance, purinergic agonists, acting mainly through P2Y₂ receptors, potently stimulate HCO⁻₃ secretion in highly differentiated cultures of monkey oviductal epithelium. When phenol red (an estrogen) is removed from the culture medium, ATP-dependent HCO⁻₃ secretion is markedly reduced but could be restored by treatment with estradiol. Therefore, estradiol induces changes in HCO⁻₃ concentration by mediating purinergic signalling pathways or ATP secretion [116]. In normal human cervical epithelial cells, apoptosis is mediated predominantly through P2X₇ receptors. In this case, estradiol inhibited the apoptotic effect induced by ATP or BzATP independent of its mitogenic function, implying a novel anti-apoptotic mechanism exerted by estradiol which antagonizes P2X₇ receptor-induced apoptosis [117]. Another example of the antagonistic effect of estradiol on P2X₇ receptors was established in CV-1 monkey kidney cells transformed by SV40 (COS cells) expressing the human P2X₇ receptor (hP2X7). ATP or BzATP induced a cation current through hP2X₇ receptor which was rapidly and reversibly inhibited by 17β-estradiol, in a concentration-dependent and non-genomic manner [118].

Although no reports suggest a role for progesterone in the regulation of the expression of P2 receptors, some authors showed that the hormone can act in a non-genomic manner to antagonize or potentiate ATP-mediated signalling. For instance, progesterone can selectively potentiate homomeric P2X₂ receptor cation influx [119]. On the other hand, in T47D-Y cells, a breast cancer cell line lacking expression of the classical nuclear progesterone receptors, progesterone can act in a rapid non-nuclear manner to inhibit extracellular ATP effects on intracellular calcium mobilization and ERK activation [120]. In addition, in human granulosa-luteal cells, human corionic gonadotrophin (hCG)-induced progesterone production was reduced by ATP treatment. Additionally, PD98059, an ERK1/2 MAPK inhibitor, reversed the inhibitory effect of ATP on hCG-induced progesterone production, suggesting that extracellular ATP inhibits progesterone production by hCG through ERK1/2 MAPK [121].

Androgens have important physiological effects, not only are they the precursors for steroid hormone biosynthesis in gonadal and extragonadal tissues, but also act directly via androgen receptors throughout the body [122]. Little is known about the regulation of P2 receptors by androgens or vice versa. In Leydig cells, ATP induces an increase in [Ca²⁺]i and testosterone secretion, supporting the hypothesis that Ca²⁺ signalling through purinergic receptors contributes to the process of testosterone secretion in these cells [123]. The receptors involved in this response were investigated. The presence of P2X₂, P2X₄, P2X₆, and P2X₇ receptor subunits was demonstrated, but functional results suggested that a heteromeric channel, possibly P2X_2/4/6, is responsible for testosterone secretion in Leydig cells [124]. In addition, sustaining the regulation of P2 receptors by androgens, it was determined that testosterone administration to adult hypogonadal mice restored purinergic excitatory transmission and P2X₁ receptor immunofluorescence of vasa deferentia [125].

Glucocorticoids are essential for stress responses. Also ATP released from stressed cells is implicated in inflammation. However, little is known about the effects of glucocorticoids on ATP-induced inflammation. In a human microvascular endothelial cell line, dexamethasone enhanced ATP-induced interleukin 6 (IL-6) secretion through PLC and p38 MAPK. In addition, dexamethasone induced P2Y₂ receptor mRNA expression, and when the P2Y₂ receptor was silenced by its small interfering RNA, ATP-induced IL-6 production decreased [126]. Dexamethasone also enhanced the ATP-induced [Ca²⁺]i increase and nitric oxide (NO) production in type I spiral ganglion neurons of the guinea pig cochlea. These effects were dependent on the presence of extracellular Ca²⁺ thereby suggesting that dexamethasone may rapidly enhance the Ca²⁺ influx through the activation of ionotropic P2X receptors which may interact with glucocorticoid receptors [127]. Different results were obtained in HT4 mouse neuroblastoma cells, where ATP-induced elevation of [Ca²⁺]i was inhibited by corticosterone, cortisol and dexamethasone. Both extracellular Ca²⁺ influx through P2X receptors, and internal Ca²⁺ release were attenuated. Therefore, glucocorticoids modulate P2X receptor-medicated Ca²⁺ influx through a membrane-initiated, non-genomic pathway in HT4 cells [128]. Besides, corticosterone inhibited ATP-induced cation currents through P2X₃ receptors in rat DRG neurons. These effects diminished after adding protein kinase A inhibitor H89. Thus, glucocorticoid hormones might participate in the modulation of P2X₃ receptor-associated events in sensory neurons, and the effect is mediated by glucocorticoid receptors and the downstream activation of protein kinase A [129].

It has been reported that P2Y₂ receptors contribute to NaCl homeostasis and blood pressure regulation in aldosterone-sensitive distal nephron [130]. In addition, the same role for P2Y₂ receptors was established in knockout mice lacking P2Y₂ receptors, which showed salt-resistant arterial hypertension linked to an inhibitory influence on renal Na⁺ and water reabsorption [131]. However, there are no reports suggesting a relation between mineralocorticoid receptors and P2 receptors.

Retinoids, vitamin A derivatives, are important regulators of the growth and differentiation of skin cells. It was established that, in normal human epidermal keratinocytes (NHEKs), all-trans-retinoic acid (ATRA) and 9-cis-retinoic acid, agonists to retinoic acid receptor, enhanced the expression of the P2Y₂ receptor mRNA and receptor function. So, retinoids, at least in part, exert their proliferative effects by up-regulating P2Y₂ receptors in NHEKs [132]. Besides, ATRA and 9-cis-retinoic acid significantly increased the mRNA and protein levels of P2X₂ receptors in rat pheochromocytoma PC12 cells [133]. On the other hand, retinoic acid (RA) induces neuronal differentiation and down-regulates P2X₇ receptor expression in human SH-SY5Y neuroblastoma cells, thus protecting them from extracellular nucleotide-P2X₇ receptor-induced neuronal death [134]. Similar results were obtained in the case of Neuro-2a cells, where RA-induced neuronal differentiation associated with decreased expression and function of P2X₇ receptors [135]. Together, these evidences suggest that retinoids can transcriptionally regulate the expression and function of P2 receptors, at least, in the skin and nervous system. However, in contrast with these results, RA-induced human neuroblastoma SK-N-BE(2)C cell differentiation did not alter the expression level of P2Y₆ receptors [136].

The existence of an interaction between P2 receptors and the vitamin D receptor (VDR) has not been studied yet. However, it was found that 1α, 25(OH)₂ vitamin D₃ induces ATP exocytosis in static ROS 17/2.8 and SAOS-2 cells and primary calvarial osteoblasts expressing VDR; this effect was abolished by inhibitors of vesicular exocytosis. Furthermore, silencing of VDR by siRNA prevented 1α, 25(OH)₂ vitamin D₃ stimulation of ATP exocytosis in ROS 17/2.8 and SAOS-2 cells. Similarly, 1α, 25(OH)₂ vitamin D₃ failed to activate ATP secretion in primary osteoblasts from a VDR knockout mouse. Thus, 1α, 25(OH)₂ vitamin D₃ stimulation of ATP exocytosis involves non-transcriptional VDR functions in osteoblasts [137].

Concluding remarks

Extracellular nucleotides can regulate many cellular effects through activation of P2 receptors. Nevertheless, it seems that these receptors can form membrane complexes with other P2 receptors or other classes of receptors. P2 receptors can homodimerize, heterodimerize, and even modulate the expression and/or activity of other GPCRs, receptor tyrosine kinases and steroid hormone receptors. This clearly affects intracellular signalling pathways either in physiological or pathophysiological conditions. Thus, P2 receptors should be viewed as components of homo/heteroreceptor complexes rather than self-dependent entities, although it remains unclear to what extent they can associate with each other to form signalling units. In addition, several metabolites and agonists can play a potential role in purinergic signalling. Therefore, P2 receptors can be considered as attractive targets for novel drug development.