General

P2Y receptors are one of three families of extracellular receptors for purine and pyrimidine nucleotides involved in purinergic signalling [11]. Adenosine (previously known as P1) receptors and the P2X receptors are classified by separate NC-IUPHAR Subcommittees. The division of P2 nucleotide receptors into two families - a P2X ion channel family and a P2Y G protein-coupled receptor family - first proposed by Abbracchio and Burnstock [2], has been generally adopted with growing numbers of receptors recognised since that time [74].

P2Y receptors are a multigene family

The first genes for P2Y receptor subtypes were cloned in 1993 with the isolation of cDNAs from chick brain [86] and a murine neuroblastoma (NG108-15) cell line [54]. These two cDNAs encode structurally related metabotropic proteins that are activated either by adenine nucleotides (chick P2Y₁ receptor) or by adenine and uracil nucleotides (murine P2Y₂ receptor). The structural similarities between the chick P2Y₁ and mouse P2Y₂ receptor proteins provided the requisite tools for a wave of homology screening. Thereafter, the numbers of species orthologues of P2Y₁ and P2Y₂ receptors, and of novel P2Y-like sequences, grew at a rapid rate, with the corresponding need to periodically review classification of the family.

Current classification

The mammalian P2Y₁, P2Y₂, P2Y₄, P2Y₆, P2Y₁₁, P2Y₁₂, P2Y₁₃ and P2Y₁₄ receptors, and the non-mammalian receptors chick p2y₃, Xenopus p2y₈ and turkey p2y, are known to be functional nucleotide receptors [1,3,12,14,17,38,48,74]. Only the mammalian receptors are formally classified by NC-IUPHAR. There is much uncertainty about the human p2y₅ receptor and, for the moment, it should still be considered an orphan receptor. Also, p2y₉ and p2y₁₀ are orphan receptors (i.e. no known agonists) [42,74] while the receptor protein originally called p2y₇ is actually a leukotriene B₄ (LTB₄) receptor [89]. The long-sought platelet ADP receptor [originally called P2T [33], and also called P_2T [40], P2T_AC [44,51], P2Y_AC receptor [78], P2Y_cyc [35], P2Y_ADP [28], P2Y_?[13] has been cloned in 2001 (P2Y₁₂). P2Y₁₃ and P2Y₁₄ receptors have been characterized during a systematic study of orphan receptors and are now formally recognised [1,17]. In addition to these cloned P2Y receptors, another endogenous P2Y receptor is recognised: the dinucleotide receptor[73] [alternatively called P_2D [71], P2Y_ApnA [28], P4 [72]. There may exist additional yet-unidentified P2Y receptors [1,3].

Structural features of of nucleotide receptors

It is believed that the transmembrane regions TMI-VII of metabotropic proteins form the nucleotide binding pocket of P2Y receptors [41]. The extracellular N-terminus of the protein is not critically important for agonist binding, whereas the intracellular C-terminus is believed to influence signal transduction. The structural relatedness of nucleotide receptors can be compared by multiple alignments of the deduced amino acid sequence for TMI-VII for each receptor protein (Figure 1A). Alignment of human receptor sequences (P2Y₁, P2Y₂, P2Y₄, P2Y₆, P2Y₁₁, P2Y₁₂, P2Y₁₃ and P2Y₁₄) shows this subfamily of nucleotide receptors to be approximately 21-61% identical. Where conservative substitutions are taken into account (i.e., where amino acids have been exchanged for other amino acids of similar structure and charge), the degree of relatedness amongst human nucleotide receptors is much higher (37-69% identical) (Figure 1B). It might be expected that the nucleotide binding pocket created by the region TM1-VII should show a high degree of structural relatedness.

Species homology

The degree of structural relatedness between P2Y receptor proteins has been viewed as a means to identify species homologues, particularly where non-mammalian and human P2Y receptors have been compared[12]. Thus, it has been proposed that chick p2y₃ is the species homologue of h P2Y₆ on the basis of sequence identity (66%), whilst Xenopus p2y₈ and turkey p2y have been viewed as species homologues of human P2Y₄ (61% and 62% identical, respectively). However, there are no clear guidelines to say what level of sequence identity is categorically predictive. For example, chick P2Y₁ and turkey P2Y₁ are much more closely related to human P2Y₁ (both 89% identical) and, therefore, levels of homology in the 60--80% range might not be so meaningful. Furthermore, some closely related species homologues (human P2Y₄ and rat P2Y₄; 90% identical) actually have different pharmacological profiles, so that phenotype alone is not predictive of relatedness. The P2Y₂ and P2Y₄ proteins (62% identical), encoded by different genes (on chromosomes 11 and X, respectively), are structurally related and phenotypically different. Thus, it is a difficult process to successfully predict which P2Y receptor proteins are true species homologues.

Nucleotide binding sites

Regarding the molecular nature of the nucleotide binding pocket, useful information has been made available from SAR studies of human P2Y₁ and mouse P2Y₂ receptors [23,41,43]. Analysis of agonist activity at mutant P2Y₁ receptors revealed that TM6 and TM7 lie close to the adenine ring, TM3 and TM6 lie close to the ribose moiety, and key residues in TM3, TM5, TM6 and TM7 lie near the triphosphate chain[41]. Positively charged residues in TM3 and TM7 may interact with the α-phosphate, and TM5 residues with the γ-phosphate of nucleotidic agonists [43]. Two disulphide bridges in the extracellular domain were identified as essential (perhaps to stabilise the receptor structure), whilst positively charged lysine and arginine residues in the second and third extracellular loop (EL) are essential for high agonist potency [41]. Site-directed mutagenesis of mouse P2Y₂ receptors also reveals the importance of positively charged residues in TM6 (H-X-X-R/K) and TM7 (Y-Q/K-X-X-R) in nucleotide triphosphate binding [23], consistent with findings on the human P2Y₁ receptor [41]. In P2Y₁₂, P2Y₁₃ and P2Y₁₄ receptors, the (Y-Q/K-X-X-R) motif in TM7 is substituted by the highly conserved motif K-E-X-X-L which might affect ligand binding characteristics [1]. More recently, for these receptors, one additional K residue in EL2 has been suggested to be particularly important for nucleotide binding [21].

P2Y receptor signal transduction

In heterologous expression systems, recombinant P2Y₁, P2Y₂, P2Y₄, P2Y₆ receptors couple to G proteins (mainly G_q) which, in turn, activate intracellular signalling cascades - mainly the phospholipase C_β (PLC_β) isoform to mobilise intracellular Ca²⁺ ions[12]. Some recombinant P2Y receptors (e.g. P2Y₁₁) also affect intracellular cAMP levels [9,12,18] while P2Y₁₂, P2Y₁₃, P2Y₁₄ mainly couple to G_i/o proteins and inhibit cAMP formation. Hydrophobicity plots of deduced amino acid sequences for functional P2Y receptors indicate that seven segments (TMI-VII) of the receptor protein cross the plasmalemma, while the N-terminus of the protein resides on the extracellular side and C-terminus on the intracellular side of the cell [54,86]. Each transmembrane region is believed to form an α-helix and, accordingly, P2Y receptors are sometimes described as heptahelical proteins. Therefore, P2Y receptors are part of the Rhodopsin superfamily [81].

Recombinant P2Y receptors affect a narrow range of signalling pathways (mainly PLC_β and adenylate cyclase, AC) in heterologous expression systems, whereas endogenous metabotropic P2 receptors affect a much wider range of intracellular signalling cascades (PLC_β, PLD, PLA₂, AC, MEK/MAP kinase system, Rho-dependent kinase). The reasons for the narrow selectivity of signal transduction by recombinant P2Y receptors have not been explained but, in part, may be related to a limited or insufficient availability of the relevant effector targets in the expression systems so far utilised.

Correspondence of native and cloned P2Y receptors

A major challenge is understanding the correspondence between native and recombinant nucleotide-activated G protein-coupled receptors [48]. For a number of reasons, it has proved exceedingly problematic to associate particular recombinant P2Y receptors with metabotropic P2 receptors found in mammalian tissues. It is often the case that several P2Y receptors are co-expressed in any one tissue or cell type. With the exception of P2Y₁ and P2Y₁₂, there is a paucity of selective agonists and antagonists to clearly distinguish one P2Y receptor from another. Furthermore, pharmacological analyses of metabotropic P2 receptors have often been hampered by the presence of ecto-enzymes (surface enzymes), which i) remove one or more phosphate groups attached to the ribose moiety of the nucleotide to change the nature of the agonist (e.g. ATP into ADP); and ii) exchange phosphates from an exogenously applied nucleotide to an endogenous nucleotide released by the host cell thereby generating another agonist (e.g. generating UTP from ATP, via locally released UDP). In addition to those G protein-coupled receptors activated principally or exclusively by dinucleotides (e.g. Ap_nAs), there may be additional P2Y receptors on the human genome, as suggested by the presence of yet-uncharacterized "P2Y-like" receptors [1,3]. A further problem involves the disparities in the types of signalling cascades utilised by native and recombinant P2 receptors. Lastly, the notion has been raised that P2Y receptors may in theory dimerize, to generate homodimeric P2Y receptors with significantly different pharmacological profiles from the related monomeric receptor[25], or heterodimeric P2Y receptors with novel phenotypes [58]. By analogy, the β₂-adrenoceptor has been reported to form homodimeric receptors [34], whereas opioid receptors are capable of forming heterodimeric complexes [45].

Pharmacological properties of P2Y receptors

Mammalian Receptors

The P2Y₁ receptor is activated by adenine nucleotides and not by uracil nucleotides. Avian P2Y₁ receptors are activated by ATP and ADP [26,86], whereas mammalian P2Y₁ receptors are potently activated mainly by ADP and related diphosphate analogues[77]. Studies of the human P2Y₁ receptor indicate that ATP and related triphosphates can act in some cases as receptor antagonists [36,56] whilst, in other cases, ATP and related triphosphates can act as partial agonists when receptor reserve is high and as antagonists when receptor reserve is low [68]. Additionally, ATP and related triphosphates may act as full agonists when P2Y₁ receptor number is in excess and, theoretically, when homodimeric P2Y₁ receptors are present [25]. Homologues of P2Y₁ receptors are blocked by PPADS, a substance originally proposed to be selective for P2X receptors, and also are blocked by suramin. However, adenosine bisphosphate derivatives are much more potent antagonists (MRS2179 and MRS2279) [8,63-64]. A viable P2Y₁ knockout mouse model has been developed [24,55].

The P2Y₂ receptor is activated by both adenine and uracil nucleotides, and by their triphosphates but not diphosphates [54,67]. Species homologues of the P2Y₂ receptor are not known to possess different pharmacological profiles. Other uracil dinucleotides, such as INS 365 (Up₄U) and INS 37217 (Up₄dC), also potently activate the P2Y₂ receptor [79,88]. The P2Y₂ receptor is blocked by suramin but is insensitive to PPADS.

The human P2Y₄ receptor is activated by uridine nucleotidic triphosphates (not diphosphates) and is considered to be a pyrimidinoceptor [20,66]. ATP is reported to be an antagonist at the human P2Y₄ receptor [47]. However, the rat P2Y₄ receptor, like the P2Y₂ receptor, is activated by both ATP and UTP [7,46-47,84]. The rat P2Y₄ receptor is blocked by Reactive blue 2 but is insensitive to suramin and PPADS [7]. This suramin-insensitivity distinguishes the rat P2Y₄ from the rat P2Y₂ receptor which is blocked by suramin.

P2Y₆ receptor is activated by uracil 5'-diphosphate derivates, while MRS2567 and MRS2578 act as antagonists both at the human and rat P2Y₆ receptor [57]. RT-PCR and Northern Blotting studies show an high expression of this receptor in spleen and placenta, followed by kidney, adipose, bone, lung, heart, brain and skeletal muscle [19,61].

The human P2Y₁₁ receptor is activated by adenine nucleotides, principally ATP and weakly by ADP, but not by uracil nucleotides [18,58]. This ATP receptor is unique in promoting the elevation of both IP₃ and cAMP levels with agonist activation in heterologous expression systems. A similar behaviour has been demonstrated for turkey p2y receptor, which is activated by all naturally occurring nucleoside triphosphates, resulting in activation of PLCβ and inhibition of adenylate cyclase [9] (see also below). Thus, the ability of human P2Y₁₁ and turkey p2y to modulate cAMP levels distinguishes these proteins from other recombinant P2Y receptors.

The long-sought platelet receptor for ADP is the P2Y₁₂ receptor, which is activated by adenine nucleotidic disphosphates (2-MeSADP>>ADP>ADPβS) [32]. The rat [38] and mouse [27] receptors have also been identified and characterized. P2Y₁₂ receptor is primarily coupled to G_i/o protein and is highly expressed in platelets where it plays a role in amplification of platelet activation and aggregation. The active metabolite of clopidogrel and ticlopidine act as potent P2Y₁₂ antagonists and are efficient antithrombotic drugs [76]. The AR-C compounds, including AR-C69931MX, which are all ATP analogues [39] act as potent direct competitive P2Y_12M antagonists. The P2Y₁₂ receptor has also been shown to be expressed in subregions of the brain [38]. Glial cells [5,30,75], brain capillary endothelial cells [80] and, recently reported, smooth muscle cells [87] and chromaffin cells [22], express P2Y₁₂ receptors, although the precise role of this receptor subtype in these cells is still under study. P2Y₁₂ knockout mice [4,27] display the phenotype of clopidogrel-treated animals i.e. prolonged bleeding time and inhibition of platelet aggregation to ADP.

The human [17,90], mouse [90] and rat [31] receptors have been identified and characterized. ADP is the naturally agonist of the P2Y₁₃ receptor, but the relative potencies of ADP and 2-MeSADP differed according to the assays used. AR-C69931MX is also an antagonist of the human and rat P2Y13 receptors [31,59]. Ap4A and 2-MeSAMP are two other P2Y₁₃ antagonists [59]. P2Y₁₃ receptor is primarily coupled to a G_o/i protein [17,59]. P2Y₁₃ mRNA is highly expressed in the spleen followed by placenta, liver, bone marrow, lung and various brain regions [90]. A significant expression in human monocytes or bone marrow, but not in human platelets [82,90] has also been found.

The human P2Y₁₄ receptor (previously known as GPR105 or UDP-glucose receptor) has been cloned by Chambers and coworkers and the complete sequence of the rat (VTR-15-20, [16,29]) and mouse orthologs [29] have been also described. The P2Y₁₄ receptor is activated by UDP-glucose as well as UDP-galactose, UDP-glucuronic acid and UDP-N-acetylglucosamine but not by uracil or adenine nucleotides [14]. At present, no selective antagonists are available, although it has to be underlined that the currently available P2 receptor antagonists have not been tested on this receptor. P2Y₁₄ receptor couples to the G_i/o family of G proteins. P2Y₁₄ mRNA is widely distributed in the human body, with moderate to high levels observed in placenta, adipose tissue, stomach, intestine, selected brain regions (e.g. c. striatum, cerebellum, caudate nucleus, hippocampus, hypothalamus), spleen, lung, heart, bone marrow and thymus. RT-PCR revealed also expression in brain glial cells [5,30] and prominent expression in neutrophils, lymphocytes and megakaryocytic cells [14,62].

Non-mammalian Receptors

The current NC-IUPHAR policy is to classify receptors from mammalian systems, but this does not fully recognise the importance of data gleaned from non-mammalian receptors. The first cloned P2Y receptor, P2Y₁, was cloned from chick brain and, only through homology screening, was the P2Y₁ receptor extended to mammalian receptors. Other non-mammalian receptors have since been cloned from Xenopus laevis (p2y₈) and chick (p2y₃ and p2y₅). In most cases, the discovery of non-mammalian receptors has led to the discovery of mammalian counterparts. Although no mammalian orthologue has been investigated for p2y₈, this receptor is clearly of the P2Y family and sufficiently novel to be of pharmacological interest, as well as appearing to subserve an important role in neurogenesis in frog [6] (see also below). Inclusion of this and other non-mammalian receptors would provide a fuller account for investigators interested in all aspects of the P2Y receptor family.

The chick p2y₃ receptor activated by adenine and uracil nucleotides, (mostly by their diphosphates and much less so by triphosphates) likely represents the species ortholog of hP2Y₆ [83]. The pharmacological profile of the chick p2y₃ receptor when expressed in Jurkat cells (UDP-preferring) is different from that seen when the receptor is expressed in Xenopus oocytes (ADP-preferring). The chick p2y₃ receptor is blocked by suramin in both expression systems, and by Reactive blue 2 in the oocyte. The turkey p2y₃ homologue, expressed in human astrocytoma cells, is UDP-preferring [52]. Thus, the chick p2y₃ and turkey p2y₃ phenotypes observed in mammalian expression systems show a reasonable similarity to the pharmacological profile of rat and human P2Y₆ receptors which, themselves, are considered to be UDP receptors [15,19]. This similarity in phenotype provides a measure of support for the notion that avian p2y₃ and mammalian P2Y₆ receptors are species homologues.

Avian and mammalian homologues of the so-called p2y₅ receptor have been cloned [37,53,85], but there is scant evidence to show these proteins are true nucleotide receptors. Radiolabelled nucleotides bind to chick p2y₅ receptors expressed in COS-7 cells [85], whereas the turkey homologue failed to alter IP₃ and cAMP levels when expressed in human astrocytoma cells [53]. Further work is required to fully assess mammalian p2y₅ homologues as nucleotide receptors.

As mentioned above, an avian receptor, turkey p2y, is activated by adenine and uracil nucleotides and shows a marked preference for triphosphates [10]. The turkey p2y receptor is activated by all naturally occurring nucleoside triphosphates (ATP, CTP, GTP, ITP, UTP, XTP) resulting in the activation of PLCβ and inhibition of adenylate cyclase [9].

A novel non-mammalian P2Y receptor, p2y₈, was cloned from Xenopus laevis embryos and shown to be activated by all naturally occurring nucleoside triphosphates (ATP, CTP, GTP, ITP and UTP) but not by inorganic polyphosphates [6]. Expression of Xenopus p2y₈ in the amphibian neural plate is temporally related with neurogenesis, indicating a possible role for P2Y receptors in early neuronal development. The P2Y₁ receptor is also involved in chick embryonic development, although mainly in non-neural tissues [60]. P2Y₁ receptors have also been implicated in the development of neonatal rat salivary glands [69], whilst P2Y₂ receptors appear to be involved in mouse T cell differentiation [50]. ATP acting via endogenous P2Y receptors has profound trophic actions on neurones and astrocytic cells in culture [65], and the role of nucleotides in cell development, differentiation and growth represents an important area for future research.

Receptor nomenclature

P2Y is used for functional mammalian receptor proteins and functional receptor proteins from non-mammalian species. The lower-case, p2y, is used for non-mammalian receptors without a mammalian homologue. The subscript numbers (1-n) following P2Y or p2y sequentially list proteins in their chronological order of cDNA cloning. An example of this nomenclature is c P2Y₁, which is a functional chick P2Y receptor with functional mammalian homologues (h P2Y₁, b P2Y₁, m P2Y₁, r P2Y₁) and also the first cDNA cloned. Another example is x p2y₈, a functional Xenopus laevis P2Y receptor with no known mammalian homologue and the eighth cDNA cloned. A final example is h p2y₉, a human P2Y-like protein that does not function as a nucleotide receptor and, chronologically, was cloned after x p2y₈.

Non-mammalian receptors

The current NC-IUPHAR policy is to classify receptors from mammalian systems, but this does not fully recognise the importance of data gleaned from non-mammalian receptors. The first cloned P2Y receptor, P2Y₁, was cloned from chick brain and, only through homology screening, was the P2Y₁ receptor extended to mammalian receptors. Other non-mammalian receptors have since been cloned from Xenopus laevis (p2y₈) and chick (p2y₃ and p2y₅).

In most cases, the discovery of non-mammalian receptors has led to the discovery of mammalian counterparts. Although no mammalian orthologue has been investigated for p2y₈, this receptor is clearly of the P2Y family and sufficiently novel to be of pharmacological interest, as well as appearing to subserve an important role in neurogenesis in frog [6]. Inclusion of this and other non-mammalian receptors would provide a fuller account for investigators interested in all aspects of the P2Y receptor family.

P2 purinergic receptors and inflammation

ATP acting via P2 receptors (P2Y and P2X family members) serves as an essential mediator of functional responses in human eosinophils [49], responses which may be implicated in the pathogenesis of various forms of airway inflammation [70].

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