International Union of Pharmacology LVIII: Update on the P2Y G Protein-Coupled Nucleotide Receptors: From Molecular Mechanisms and Pathophysiology to Therapy

A. Nomenclature and Molecular History of P2Y Receptors

Regarding the currently used nomenclature, P2Y is used for functional mammalian receptor proteins and functional nonmammalian species. The lower case, p2y, is used for mammalian orphan receptors or functional nonmammalian receptors without a mammalian ortholog. The subscript number (1-n) following P2Y or p2y sequentially list proteins in their chronological order of cDNA cloning. The first P2 receptors were cloned in 1993 (Lustig et al., 1993; Webb et al., 1993). They corresponded to receptors previously characterized by pharmacological criteria: P2Y₁ (formerly P_2Y) and P2Y₂ (formerly P_2U). Since then several other subtypes were isolated by homology cloning and assigned a subscript on the basis of cloning chronology (P2Y₄, P2Y₆, and P2Y₁₁). The long-awaited G_i-coupled ADP receptor (P2Y₁₂) of platelets was finally isolated by expression cloning (Hollopeter et al., 2001), and P2Y₁₃ and P2Y₁₄ receptors were characterized during a systematic study of orphan receptors (Chambers et al., 2000; Communi et al., 2001a). As of today, there are eight accepted human P2Y receptors: P2Y₁, P2Y₂, P2Y₄, P2Y₆, P2Y₁₁, P2Y₁₂, P2Y₁₃, and P2Y₁₄ (Abbracchio et al., 2003) (Table 1; Fig. 1) (see also section IV.). The missing numbers represent either nonmammalian orthologs or receptors having some sequence homology to P2Y receptors but for which there is no functional evidence of responsiveness to nucleotides. In particular p2y3 (Webb et al., 1996a) may be a chicken ortholog of P2Y₆ (Li et al., 1998), whereas p2y8 (Bogdanov et al., 1997) and tp2y (Boyer et al., 2000) could be the Xenopus and turkey orthologs of P2Y₄, respectively. p2y7 (Akbar et al., 1996) is a leukotriene B₄ receptor (Herold et al., 1997; Yokomizo et al., 1997); however, recently cross-reaction between agonists for some leukotriene receptors and some P2Y receptors has been found (see section IX.C.1), requiring further investigation. p2y5 (Webb et al., 1996b; Li et al., 1997) and p2y10 (Rao et al., 1999) must be considered as orphan receptors, although it has been reported (King and Townsend-Nicholson, 2000) that human p2y5 expressed in oocytes gives functional responses to ATP. p2y9 was reported to be a novel receptor for lysophosphatidic acid, distant from the Edg family (Noguchi et al., 2003). P2Y₁₅ was recently introduced to designate the orphan receptor GPR80/GPR99 on the basis that it would be a receptor for adenosine 5′-monophospahte (AMP²) (Inbe et al., 2004), but it is now firmly established that it is actually a receptor for α-ketoglutarate (He et al., 2004; Qi et al., 2004; Suarez Gonzalez et al., 2004), as was also recently underlined in a report by the Subcommittee (Abbracchio et al., 2005).

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Fig. 1.

A phylogenetic tree (dendrogram) showing the relationships among the current members of the P2Y receptor family. The P2Y receptors can be divided into two subgroups, shown with green and blue backgrounds. Sequences were aligned using CLUSTALX, and the tree was built using the TREEVIEW software. Reprinted from Abbracchio et al. (2003) with permission from Elsevier.

B. Structural Aspects

In contrast with P2X receptors, P2Y receptor genes do not contain introns in the coding sequence, except for the P2Y₁₁ receptor. Site-directed mutagenesis of the P2Y₁ and P2Y₂ receptors has shown that some positively charged residues in transmembrane domains (TM) 3, 6, and 7 are crucial for receptor activation by nucleotides (Erb et al., 1995; Jiang et al., 1997b) (Fig. 2) (section V.). They probably interact with the negative charges of the phosphate groups of nucleotides, since it is known that the receptor ligands are nucleotide species uncomplexed to magnesium or calcium. Actually, the eight P2Y receptors identified so far have a H-X-X-R/K motif in TM6. The P2Y₁, P2Y₂, P2Y₄, P2Y₆, and P2Y₁₁ receptors share a Y-Q/K-X-X-R motif in TM7, whereas another motif, K-E-X-X-L is found in P2Y₁₂, P2Y₁₃, and P2Y₁₄ receptors (Abbracchio et al., 2003) (see also Fig. 2 and sections IV. and V.). More recently, for P2Y₁₂, P2Y₁₃, and P2Y₁₄ receptors, one additional K residue in extracellular loop 2 has been suggested to be particularly important for nucleotide binding (Costanzi et al., 2004) (see also below; section V.).

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Fig. 2.

Conserved residues among P2Y receptors are shown in a larger size font. The residues that have been mutated in the studies of Erb et al. (1995) and Jiang et al. (1997b) are underlined. Those residues that are crucial in the activation of those receptors are in bold. The corresponding sequences of orphan receptors structurally related to P2Y receptors of the second subgroup are also displayed.

C. Orphan Receptors Related to P2Y Receptors

The bioinformatic analysis of the human genome has revealed the existence of approximately 800 G proteincoupled receptors (GPCRs) (Fredriksson et al., 2003), of which 367 would be receptors for endogenous ligands, the remaining ones being olfactory and other chemosensory receptors (Vassilatis et al., 2003). Among the 367 “endoG-PCRs,” more than 150 remain orphans. Several of the currently available orphan GPCR sequences are structurally related to P2Y receptors (Lee et al., 2001; Wittenberger et al., 2001; Joost and Methner, 2002; Vassilatis et al., 2003): GPR87, H963, and GPR34. In particular, the sequence of those receptors contains the structural motifs in TM6 and TM7 described earlier (Fig. 2).

Human GPR87 has been shown to be highly expressed in placenta and thymus. The mouse ortholog has also been cloned and has been found to be expressed in brain and liver (Wittenberger et al., 2001).

Human H963 has been shown to be expressed in fibroblasts, human peripheral blood mononuclear cells, and T cells (S. Ferrario, D. Lecca, C. Volonté, and M. P. Abbracchio, unpublished data). Neither the mouse nor the rat orthologs have been cloned; however, a recent study has suggested that GPR34 is not a nucleotide receptor (Sugo et al., 2006).

Human GPR34 is expressed in brain, heart, placenta, small intestine, pancreas, spleen, thymus, kidney, and skeletal muscle (Schöneberg et al., 1999). The mouse ortholog has also been described, and prominent expression has been found in liver and testis (Schöneberg et al., 1999).

Despite active research in several laboratories, the ligands of these receptors have not been identified. For example, despite the demonstration that GPR34 and ADP-like receptors (e.g., P2Y₁₂ and P2Y₁₃) have a common evolutionary origin (Schulz and Schöneberg, 2003), in the inositol phosphate assay, Cos-7 cells coexpressing GPR34 and Gα_qi4 did not show any response to ADP application (Schöneberg et al., 1999).

A. Coupling to G Proteins and Intracellular Signaling Pathways

Coupling of the various P2Y receptors to specific G proteins was initially inferred from indirect evidence [measurement of intracellular levels of inositol phosphates, calcium, or cAMP and determination of pertussis toxin (PTX) sensitivity]. Direct evidence was recently obtained by measuring the effect of ADP on GTP hydrolysis in vesicles reconstituted with P2Y₁ and either Gα_qβ₁γ₂ or Gα₁₁β₁γ₂ (Waldo and Harden, 2004). Similar experiments demonstrated that P2Y₁₂ couples to Gα_i2 more effectively than to Gα_i1 and Gα_i3 and not at all to Gα_o or Gα_q (Bodor et al., 2003). One given P2Y receptor can couple to functionally distinct G proteins. For instance, in HEL cells, activation of phospholipase C (PLC) by the P2Y₂ receptor is inhibited completely by a Gα₁₆ antisense oligonucleotide but also partially by PTX (Baltensperger and Porzig, 1997). Similarly, in gastric smooth muscle cells, it appears that P2Y₂ couples to PLC-β₁ via Gα_q/11 and to PLC-β₃ via Gα_i3β₁γ₂-derived βγ subunits (Murthy and Makhlouf, 1998). The P2Y₂ receptor also has been shown to interact with α_v integrin to promote G_o-mediated chemotaxis in astrocytoma cells (Bagchi et al., 2005). The P2Y₁₂ receptor activates phosphatidylinositol 3-kinase (PI3-K) via Gα_i, but also RhoA and Rho kinase (Soulet et al., 2004). This action, which is insensitive to PTX, might be mediated by Gα_12/13, recently shown to play a critical role in platelet activation (Moers et al., 2003). Coupling of the same P2Y receptor to distinct G proteins and signaling pathways provides the possibility of agonist-specific signaling involving distinct active conformations of the receptor. For instance, activation of the P2Y₁₁ receptor by ATP leads to a rise in cAMP and in inositol trisphosphate (IP₃) and cytosolic calcium, whereas activation by uridine 5′-triphosphate (UTP) has been reported to produce calcium mobilization without IP₃ or cAMP increase (White et al., 2003). The P2Y₁₃ receptor can simultaneously couple to G₁₆, G_i, and, at high concentrations of ADP, to G_s (like other G_i-coupled receptors such as the α₂-adrenergic receptor): these three signaling pathways are characterized by different ratios of ADP to 2-methylthio-ADP (2-MeSADP) potency, suggesting the existence of ligand-specific conformations (Marteau et al., 2003). The activation of several P2Y receptors is commonly associated with the stimulation of several mitogen-activated protein (MAP) kinases, in particular extracellular signal-regulated protein kinase (ERK) 1/2. According to the cell context and the particular subtype, other classes of the MAP kinases, protein kinase (PK) C, calcium, and PI3-K are found to be involved to a variable extent (Soltoff et al., 1998; Huwiler et al., 2000; Communi et al., 2001a; Santiago-Pérez et al., 2001; Sellers et al., 2001).

B. P2Y Receptor Coupling to Ion Channels

C. Other Potential Interactions with Ion Channels

Little is known of these as yet. One clue to some other interactions of the P2Y₁ receptor comes from the recent finding (Fam et al., 2005) that it can bind strongly to the Na⁺/H⁺ exchanger regulatory factor 2 (NHERF-2) through the extreme C-terminal motif DTSL, which is specific to P2Y₁ in this family. For comparison, binding of the related NHERF-1 to P2Y₁ receptors (Hall et al., 1998) is negligible (Fam et al., 2005). The three membrane-located NHERF subtypes either activate or inhibit various Na⁺/H⁺ exchangers, but these actions are indirect since it is now known that NHERFs are actually scaffolding proteins, which can localize various exchangers in membrane microdomains with selected receptors and signaling intermediates, e.g., Gα_q, Src, and certain isoenzymes of PLC and of PKC (Donowitz et al., 2005). For example, the NHERF-2 scaffold can link a cGMP-dependent protein kinase or a protein kinase A anchoring protein and hence protein kinase A, to modulate a tethered Na⁺/H⁺ exchanger by specific phosphorylations thereon (Cha et al., 2005; Donowitz et al., 2005).

When the endogenous P2Y₁ receptor (as studied in C6 glioma cells) is linked through its tail to the NHERF-2 scaffold, the Ca²⁺ transients produced by its activation by 2-MeSADP become strongly prolonged (Fam et al., 2005). This will change the P2Y₁ selectivity for the various calcium-sensitive signaling cascades and for ion channel interactions. Another interaction of the P2Y₁ receptor is with the chloride channel of the cystic fibrosis transmembrane conductance regulator (CFTR); in renal epithelial cells, 2-MeSADP activation of native P2Y₁ receptors stimulates the chloride channel activity of the CFTR. This is again an indirect action arising from the NHERF-2 colocalization of this P2Y subtype and the CFTR; expression of a dominant negative NHERF-2 mutant blocks the CFTR regulation through P2Y₁, as does a blocker of the protein kinase A anchoring protein binding of PKA (Guerra et al., 2004). The evidence suggested that P2Y₁ receptor-mediated PKC activation leads to potentiation of PKA and its action on the associated CFTR channels.

Additionally, a highly unusual mode of GPCR interaction with an ion channel has been suggested for several P2Y receptors by Lee et al. (2003b). A novel, unidentified, voltage-gated channel of the Xenopus oocyte, T_in, was reported to be activated and modulated after expression of human P2Y_1,2,6,11, but not by P2Y₄ nor by other G_q/11-linked GPCRs. It was deduced that this does involve a direct receptor binding to the channel. However, expression of Gα also activates this channel and the mechanism and physiological significance are at present unclear.

A. Chemical Structure of Agonist and Antagonist Ligands

Most of the P2Y receptor subtypes are still lacking potent and selective synthetic agonists and antagonists. However, considerable progress in exploring structure-activity relationships (SARs) has been made for P2Y₁ and P2Y₁₂ receptors and to a lesser extent for the P2Y₂ receptor. Radioligand binding studies have been successfully carried out at the P2Y₁ and P2Y₁₂ receptors, but so far not at any other P2Y receptor subtype. Here we describe the current state of molecular probes known for the P2Y receptors, categorized by the chemical class of the endogenous agonists.

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Fig. 3.

Structures of adenine-derived nucleotide agonists of P2Y receptors.

1. ADP-Preferring P2Y Receptors:

P2Y₁, P2Y₁₂, and P2Y₁₃. ADP (1 in Fig. 3) is the endogenous agonist at the P2Y₁, P2Y₁₂, and P2Y₁₃ receptors, and it interacts at these subtypes with generally greater affinity than does ATP (3) (Palmer et al., 1998; Boeynaems et al., 2003; Marteau et al., 2003). At P2Y₁ receptors, derivatives of ADP tend to be full agonists (the EC₅₀ of ADP is ∼100 nM), whereas ATP appears to be a partial agonist. At P2Y₁₂ receptors, ADP derivatives activate (the EC₅₀ of ADP is ∼100 nM) and 5′-triphosphate derivatives antagonize (Gachet and Hechler, 2005). At P2Y₁₃ receptors, both ADP and ATP might act as full agonists, with EC₅₀ values of ∼100 nM. However, under some conditions, ATP can behave as a weak partial agonist, suggesting that, as described for the P2Y₁ receptor, the activity of ATP in recombinant systems may vary according to the level of expression of the P2Y₁₃ receptor (see also below).

Phosphate modifications among P2Y receptor ligands often serve to increase their stability toward ecto-nucleotidases. For example, the added stability of a terminal thiophosphate group resulted in its incorporation in some useful P2Y nucleotide agonists. One such analog, ADPβS (2), is a potent agonist of both P2Y₁ (EC₅₀ = 96 nM), P2Y₁₂ (EC₅₀ = 82 nM) (Jacobson et al., 2002), and P2Y₁₃ receptors (EC₅₀ = 42 nM) (Communi et al., 2001a). Although terminal thiophosphates are enzymatically more stable than the oxygen equivalents, they are subject to chemical oxidation reactions; thus, solutions of these compounds are prone to instability.

Figure 3 shows the structures of adenine-derived nucleotide agonists of P2Y receptors. The structure of the nucleobase of adenine nucleotides has been extensively probed for effects at P2Y receptors. 8-Aza and 1-deaza modifications are generally tolerated. A fluorescent adenine-modified derivative of ATP (5) behaved as a potent P2Y₁ receptor agonist (Sharon et al., 2004). The 2-position of the adenine ring can accommodate a wide variety of substituents, with resultant activation of both P2Y₁ and P2Y₁₂ receptors. Long-chain and sterically bulky groups may be accommodated at the 2-position. In particular, 2-alkylthio ethers (Fischer et al., 1993; Brown et al., 2000b) appear to provide high potency at these subtypes when bonded to a variety of alkyl or alkylaryl groups. Notably, the smallest member of this class is 2-MeSADP [6, which is a potent agonist (EC₅₀) at P2Y₁ (6 nM), P2Y₁₂ (1 nM), and P2Y₁₃ (1 nM) receptors (Jacobson et al., 2002; Marteau et al., 2003); however, see also above and individual receptor subsection] and is highly selective in comparison with other P2Y receptor subtypes. For example, at the P2Y₂ receptor compound 6 is inactive at 100 μM. The corresponding triphosphate, 2-MeSATP (7), is less potent and selective as a P2Y receptor agonist, since it also activates P2X receptors (King, 1998). The sterically bulky p-aminophenylethylthio analog (2-[2-(4-aminophenyl)ethylhio]adenosine 5′-triphosphate) (8), potently activated the P2Y₁ receptor (EC₅₀ = 1 nM) (Fischer et al., 1993). The SAR of alkynyl substitutions at the 2-position of P2Y₁ receptor agonists has been explored (Cristalli et al., 2005).

Although AMP is inactive at the P2Y₁ receptor, addition of a 2-thioether substituent as a receptor “anchor” causes AMP analogs to activate P2Y₁ receptors. Among these derivatives, 2-hextylthioadenosine 5′monophosphate (9) was especially potent, with an EC₅₀ of 59 nM at the turkey P2Y₁ receptor (Boyer et al., 1996b). Certain 2-thioether derivatives of AMP derivatives also activate the P2Y₁₂ receptor in C6 glioma cells in the micromolar range. The α-thio modification of AMP analogs (e.g., 10) increases potency at the P2Y₁ receptor (Fischer et al., 1999). Such monophosphate derivatives have also been reported to inhibit ecto-nucleotidases, which complicates their use as P2Y receptor agonists.

A BH₂ moiety may serve as a substitute for an ionized oxygen atom of the α-phosphate of ATP derivatives in promoting binding to the P2Y₁ receptor binding site. Thus, 5′-(1-boranotriphosphate) derivatives such as the 2-methylthio derivative (11) have been found to potently activate the P2Y₁ receptor (Nahum et al., 2002). Because the 1-boranotriphosphate moiety is chiral, it was possible to separate two stable isomers in this series. The more potent isomer of 11 displayed an EC₅₀ of 2.6 nM at the rat P2Y₁ receptor.

The ribose moiety of nucleotide derivatives was also modified, resulting in enhanced potency at the P2Y₁ and P2Y₁₂ receptors. Simple carbocyclic (cyclopentyl) analogs of ATP were found to enhance antagonist affinity at the P2Y₁₂ receptor (see below) (van Giezen and Humphries, 2005). Similarly, at the P2Y₁ receptor, carbocyclic and even acyclic substitutions of ribose were studied. In general, carbocyclics and ring-constrained nucleotide analogs were able to maintain agonism at the P2Y₁ receptor, whereas acyclic derivatives proved to be exclusively antagonists (see below). A ring-expanded, yet nonglycosidic, dehydroanhydrohexitol analog MRS2255 (12) activated the P2Y₁ receptor with an EC₅₀ of 3.0 μM (Nandanan et al., 2000).

Among the more successful examples of the use of carbocyclic or sterically constrained carbocyclic substitution of the ribose moiety for P2Y receptor interactions are the “methanocarba” analogs (Nandanan et al., 2000; Kim et al., 2002). These analogs incorporate a conformationally fixed bicyclic ring system, consisting of fused cyclopentane and cyclopropane rings, in place of the ribose moiety. Depending on the position of fusion, the resulting nucleotides may adopt one of two conformations: (N), north, or (S), south. Correlation of ring geometry with the biological activities helped define the conformational requirements of the ribose moiety in P2Y receptor binding and led to pharmacological probes of unusual selectivity and affinity. For example, the two isomeric methanocarba equivalents of ATP indicated a strong preference (ratio of potency >100-fold) at the P2Y₁ receptor for the (N)-isomer 13 over the (S)-isomer 14 (Kim et al., 2002).

Combination of this ring system with other favorable modifications of ADP or ATP resulted in large qualitative differences from the native nucleotides in receptor activation. For example, whereas β,γ-methylene ATP (β,γ-meATP) is a weak partial agonist at the human P2Y₁ receptor, the corresponding (N)-methanocarba-β,γ-meATP (15) was a full agonist with an EC₅₀ of 158 nM (Ravi et al., 2002). MRS2365 (16), the most potent known agonist of the human P2Y₁ receptor, with an EC₅₀ of 0.4 nM, induces platelet shape change without aggregation (Chhatriwala et al., 2004). In addition, the high selectivity of 16 for the P2Y₁ receptor in comparison to its inactivity at P2Y₁₂ and P2Y₁₃ receptors was striking, in contrast to the relatively nonselective 2-MeSADP (2). Thus, the P2Y₁₂ and P2Y₁₃ receptors have very different conformational preferences within the ribose-binding region than does the P2Y₁ receptor. At P2Y₁₃ receptors, under optimal experimental conditions, ATP (2) and 2-MeSATP (5) are equipotent as agonists.

Figure 4 shows the structures of nucleotide-based antagonists of P2Y receptors. A successful approach to the development of potent and selective P2Y₁ receptor antagonists was made possible by the observation by Boyer et al. (1996a) that naturally occurring adenosine bisphosphate derivatives such as A3P5P (17) act as partial agonists or antagonists of the receptor. Thus, the splitting and repositioning of the phosphate groups of the 5′-diphosphate group of ADP to separate ribose positions (5′- and either 3′- or 2′-) reduces efficacy at the P2Y₁ receptor. Removal of the 2′-hydroxyl group and addition of the potency-enhancing N⁶-methyl group resulted in MRS2179 (18), and the corresponding 2-chloro analog MRS2216 (19), which became full antagonists at the P2Y₁ receptor with IC₅₀ values of 300 and 100 nM, respectively (Nandanan et al., 1999; Brown et al., 2000b). The SAR of alkyl, thioether, and other substitutions at the 2-position of bisphosphate antagonists has been explored (Nandanan et al., 1999, Raboisson et al., 2002a). Raboission et al. (2002b) synthesized a C-nucleotide bisphosphate derivative (20) that antagonized P2Y₁ receptors. In addition, the adenine N9 nitrogen is not essential in P2Y₁ receptor interaction, and similarly the N1 nitrogen was found to be unnecessary through the evaluation of 1-deaza analogs (Nandanan et al., 1999).

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Fig. 4.

Structures of nucleotide-based antagonists of P2Y receptors.

Upon introduction of the conformationally preferred (N)-methanocarbo ring system into this series of bisphosphate nucleotide antagonists, the P2Y₁ receptor affinity was further enhanced. Thus, MRS2279 (21), the (N)-methanocarbo equivalent of the riboside (19), and the corresponding 2-iodo derivative MRS2500 (22) demonstrated high affinity in competitive antagonism at the human, turkey, rat, and mouse P2Y₁ receptors (Nandanan et al., 2000; Boyer et al., 2002; Waldo et al., 2002; Cattaneo et al., 2004). The K_i value of MRS2500 was 0.78 nM, as determined in inhibition binding experiments at the human P2Y₁ receptor (Ohno et al., 2004), and the compound was highly specific for this subtype. MRS2279 or related antagonists were demonstrated to be inactive at P2Y_{2,4,6,11,12,13} and P2X_2,3,4,7 receptors (Boyer et al., 2002; Marteau at al., 2003). Weak antagonism by MRS2279 of the rat P2X₁ receptor expressed in Xenopus oocytes was observed (Brown et al., 2000b). However, the potency of many of the known P2 receptor antagonists is magnified in this assay; thus, MRS2279 may still be considered highly selective for the P2Y₁ receptor. In platelet studies, for example, antagonism of the P2X₁ receptor by this compound and related P2Y₁ receptor antagonists is not observed (Baurand et al., 2001). The K_i value of MRS2500 at the P2Y₁ receptors was found to be 0.79 nM, and it was consistently potent in inhibiting the ADP-induced aggregation of human and rat platelets. [³³P]MRS2179, [³H]MRS2279, and [³²P]MRS2500 were prepared and shown to be effective as radioligand probes in platelets and in other tissue (Baurand et al., 2001; Waldo et al., 2002; Houston et al., 2005). A novel ring system was incorporated in a carbocyclic locked nucleic acid derivative MRS2584 (23), which acted as a P2Y₁ receptor antagonist with a binding K_i of 23 nM (Ohno et al., 2004).

In addition to the approach of rigidifying the ribose moiety in a conformation that approximates the conformation preferred in receptor binding, the opposite approach, which used a flexible acyclic ribose equivalent, also produced bisphosphate antagonists of the P2Y₁ receptor of intermediate affinity. The acyclic nucleotide analog MRS2298 (24) represents a bisphosphate structure that was optimized for affinity at the P2Y₁ receptor, with an IC₅₀ in inhibition of PLC of 200 nM (Cattaneo et al., 2004). A related analog containing the metabolically stable phosphonate linkage, MRS2496 (25), displayed an IC₅₀ at the rat platelet P2Y₁ receptor of 0.68 μM (Xu et al., 2002a).

Extensive structure-activity studies of ATP derivatives as antagonists of the platelet P2Y₁₂ receptor resulted in high-affinity, selective antagonists of interest as antithrombotic agents. Several such 5′-triphosphate derivatives, including AR-C67085MX (26) and AR-C69931MX (27) (Ingall et al., 1999), were used in clinical trials, with the recognition that triphosphate derivatives would not be suitable for oral administration. In this series, it was also possible to substitute the unwieldy triphosphate group with uncharged moieties such as short alcohols or esters, thus proving that a highly anionic moiety is not needed for recognition by the P2Y₁₂ receptor. This discovery led to compounds such as the carbocyclic nucleoside derivative AZD6140 (28), which is an orally active P2Y₁₂ receptor antagonist of nanomolar affinity that inhibits platelet aggregation up to 8 h after administration (Springthorpe, 2003). The presence of the 3,4-difluorophenyl group limits the metabolism of compound 28.

Analogs of ADP having neutral, hydrophobic substitutions at the ribose 2′- and 3′-hydroxyl groups and at the adenine NH₂ position were found to antagonize the P2Y₁₂ receptor. One such analog is INS49266 (29a), which displayed a K_B of 361 nM in the inhibition of ADP-induced platelet aggregation (Douglass et al., 2002). The agonist potencies of compound 29a at P2Y₁ and P2Y₂ receptors were >10 and 14 μM, respectively. A related analog in this series, INS50589 (29b), has entered clinical trials as a platelet aggregation inhibitor with a rapid onset and offset mechanism of action that is intended for intravenous administration.

The acyclic template used in MRS2298 (24) has been adapted to antagonists of the P2Y₁₂ receptor (Xu et al., 2002a). Upon replacement of the two phosphate groups with hydrophobic esters, such as in the dipivaloate MRS2395 (30), the selectivity shifted entirely from the P2Y₁ receptor to the P2Y₁₂ receptor. MRS2395 displayed an IC₅₀ of 3.6 μM in the inhibition of ADP-induced aggregation of rat platelets by antagonizing the P2Y₁₂ receptor.

The action of the successful antithrombotic drug clopidogrel (31 in Fig. 5) is also dependent on the P2Y₁₂ receptor present on platelets. Clopidogrel (31) produces a metabolite (32), which acts as an irreversible P2Y₁₂ receptor antagonist (Savi et al., 2000). CS-747 (33) (also known as prasugrel or LY640315) is acting with a similar mechanism (Sugidachi et al., 2001; Niitsu et al., 2005). CT50547 (34)isaP2Y₁₂ antagonist characterized by a non-nucleotide (and not highly charged) structure (Scarborough et al., 2001). Recently, a novel P2Y₁₂ receptor antagonist structure in the form of a pyrazolidine-3,5-dione derivative (35) was presented (Fretz et al., 2005). The search for more drug-like (nonphosphate-containing or uncharged), and structurally novel antagonists of the P2Y₁ receptor has so far had limited success. A dipeptide conjugate of adenosine, which bore carboxylate groups, was found to antagonize hP2Y₁ receptor responses with a K_B value of 4.0 μM (Sak et al., 2000). Metabolites of the hypolipidemic drug nafenopin may act as P2Y₁ receptor antagonists, including a coenzyme A conjugate that displayed a K_B value of 58 nM. A cyclic depsipeptide YM-254890 (36), a fermentation product of a Chromobacterium isolated from soil, inhibited the ADP-induced aggregation of platelets with an IC₅₀ of 31 nM and was inactive at P2Y₁₂ receptors (Uemura et al., 2006). The mechanism was found to be inhibition of Gα_q rather than direct interaction with P2Y₁ receptors.

Figure 5 shows the structures of non-nucleotide antagonists of P2Y receptors. Known non-nucleotide antagonists of P2 receptors were also modified in an effort to achieve P2Y₁ receptor selectivity. Many of the classic antagonists of P2 receptors are highly negatively charged polycyclic compounds and tend to block certain P2Y as well as P2X responses. Although no antagonists are totally nonselective with respect to the entire P2 superfamily (such an antagonist would be a useful tool), these polyanions are the least selective and often have activities beyond the P2 receptors. For example, the nonselective P2X/P2Y antagonist Reactive blue 2 (37) and its derivatives (such as Acid Blue 80, Acid Blue 129, and Acid Violet 34) have been shown to block action at P2Y₁ receptors; however, high potency and selectivity have not been achieved (Brown and Brown, 2002; Jacobson et al., 2002). The polysulfonate suramin (38) and its derivatives, in addition to displaying trypanocidal drug properties, are relatively nonselective P2 antagonists with, in general, reversibility upon washout (King, 1998). Within the P2Y family, suramin has been characterized as an antagonist of P2Y₂ receptors (Wildman et al., 2003) and P2Y₁₁ (Communi et al., 1999b) receptors. Derivatives of the pyridoxal phosphate derivative PPADS (39) have been shown to antagonize P2Y₁ receptor effects in a competitive fashion, although at μM concentrations (Lambrecht et al., 2002). Extensive SAR manipulations within these families have not resulted in antagonists of nanomolar affinity.

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Fig. 5.

Structures of non-nucleotide antagonists of P2Y receptors.

A variety of structurally diverse, non-nucleotide antagonists of the P2Y₁ receptor have been demonstrated to be noncompetitive inhibitors. For example, pyridyl isatogen tosylate (41), which has been explored as a possible allosteric modulator of the receptor (King et al., 1996), was found to be a P2Y₁-selective antagonist in recombinant P2Y receptor systems (Gao et al., 2004). It reduced the maximal effect of 2-MeSADP in stimulation of PLC, with an IC₅₀ of 0.14 μM, but had no effect on the binding of [³H]MRS2279.

Two nucleotide 5′-triphosphate derivatives (26 and 27) were shown to potently antagonize the P2Y₁₃ receptor (Kim et al., 2005), but in a noncompetitive manner. The following compounds were found to antagonize action at the P2Y₁₃ receptor (IC₅₀): suramin (38) (2.3 μM), PPADS (39) (11.7 μM), Ap₄A (0.216 μM), and AR-C69931MX (27) (0.004 μM) (Marteau et al., 2003). Recently, a derivative of PPADS, MRS2211 (40), was shown to selectively antagonize the human P2Y₁₃ receptor (Kim et al., 2005). The antagonism of MRS 2211 of agonist-induced PLC was competitive with a pA₂ value of 6.3.

3. UTP-Recognizing P2Y Receptors:

P2Y₂ and P2Y₄. The P2Y₂ receptor is activated nearly equipotently by UTP (47 in Fig. 6) and ATP (3) but is not activated by the corresponding 5′-diphosphates, i.e., UDP (46) and ADP (1). The P2Y₄ receptor is primarily activated by uracil nucleotides, depending on species. In the rat, ATP is also a P2Y₄ agonist, but in humans it acts as a P2Y₄ antagonist. Uridine β-thiodiphosphate (UDPβS, 48) and the γ-thiophosphate (UTPγS, 49) are selective agonists for P2Y₆ and P2Y₂/P2Y₄ receptors, respectively (Malmsjö et al., 2000). Numerous substitutions of the uracil ring of UTP have been reported to reduce potency at the P2Y₂ receptor (Müller, 2002). The 5-bromo derivative of UDP (50) retains potency at the P2Y₆ receptor.

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Fig. 6.

Structures of uracil-derived nucleotide agonists of P2Y receptors.

The adenine dinucleotide Ap₄A is a potent agonist at the rat P2Y₄ receptor and is less potent than ATP at the P2Y₂ receptor. Other uracil dinucleotides, such as INS365 (Up₄U, 51), also potently activate the P2Y₂ receptor (Shaver et al., 2005). The dependence of potency at various P2Y receptors on the number of bridging phosphate units in the dinucleotide series indicates an optimum at the tetraphosphate. Newer-generation P2Y₂ receptor agonists, such as INS37217 (Up₄dC, 52), have been reported (Pendergast et al., 2001; Yerxa et al., 2002). INS37217 is less prone to enzymatic hydrolysis than more common dinucleotide agonists. P2Y₂ receptor agonists are of clinical interest for the treatment of pulmonary and ophthalmic diseases and possibly cancer.

Ribose substitution with the (N)-methanocarba ring system has been shown to preserve the potency of both adenine and uracil nucleotides at the P2Y₂ receptor and UTP (e.g., MRS2341, 53) at the P2Y₄ receptor (Kim et al., 2002). However, inclusion of the same (N)-methanocarba ring system in the corresponding 5′-diphosphate prevented activation of the P2Y₆ receptor. Therefore, enzymatic cleavage of compound 53 to the diphosphate does not have complicating actions at the P2Y₆ subtype.

Figure 6 shows the structures of uracil-derived nucleotide agonists of P2Y receptors. Suramin (38) is a weak antagonist at the P2Y₂ receptor with an IC₅₀ of 48 μM (Müller, 2002). A family of selective, heterocyclic antagonists of the P2Y₂ receptor containing a thiouracil moiety, including AR-C126313 (42) and the related aminotetrazole derivative AR-C118925 (43), has been reported (Meghani, 2002). Reactive Blue 2 (37) at a concentration of 100 μM effectively blocks rat P2Y₄ receptors but only partially blocks human P2Y₄ receptors. ATP antagonizes the human but not rat P2Y₄ receptor (Kennedy et al., 2000).

Flavonoids have been identified as a new lead for the design of P2Y₂ receptor antagonists (Kaulich et al., 2003). Tangeretin (44) is a potent, noncompetitive antagonist with an IC₅₀ of 12 μM.

B. Molecular Modeling Studies

The two distinct subgroups of P2Y receptors were successfully modeled by homology modeling, with the high-resolution structure of bovine rhodopsin serving as a template (Moro and Jacobson, 2002). The putative TM binding site and other regions of the human P2Y₁ receptor have been extensively studied by means of mutagenesis (Fig. 2). To ascertain which residues of the P2Y₁ receptor are involved in ligand recognition, individual residues of the TMs and extracellular loops were mutated to Ala and other amino acids (see also Table 4).

Recent computational models of all of the P2Y receptors were derived from a multiple-sequence alignment based on a combined manual and automatic approach, which takes into account not only the primary structure of the proteins but also the three-dimensional information deducible from the secondary and tertiary structures of the template (Costanzi et al., 2004). The receptors display the general motif of a single-polypeptide chain forming seven helical TMs, which are connected by three extracellular and three intracellular loops. The ends of the chain form an extracellular amino-terminal region and a cytoplasmic carboxyl-terminal region, as shown for the P2Y₁ and P2Y₁₂ receptors (Fig. 7). According to the models, at the cytoplasmic end of TM7 both the receptors fold at an angle of ∼90° to form a helical segment that is homologous to H8 in rhodopsin and runs parallel to the plane of the cell membrane (Hoffmann et al., 1999).

Figure 7 shows the theoretical structures of the putative nucleotide binding sites of P2Y₁ (A) and P2Y₁₂ (B) receptors, based on mutagenesis and molecular modeling experiments as described in Costanzi et al. (2004). The large figures show the binding sites as viewed from the plane of the plasma membrane with docked nucleotide ligands (the antagonists MRS2500 for P2Y₁ and AZD6140 for P2Y₁₂). Key residues found to interact with the ligand in the human P2Y₁ and P2Y₁₂ receptors are indicated. To the left of each detailed structure is a smaller three-dimensional representation of the receptor including seven TMs (color coded: cyan, TM1; orange, TM2; green, TM3; magenta, TM4; blue, TM5; red, TM6; gray, TM7) and the connecting loops. The orientation of the entire receptor relative to the membrane is the same as for each detailed binding site model.

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Fig. 7.

Theoretical structures of the putative nucleotide binding sites of P2Y₁ (A) and P2Y₁₂ (B) receptors based on mutagenesis and molecular modeling experiments as described in Costanzi et al. (2004).

Table 4 summarizes the effects and structural role of specific amino acid residues of the human P2Y₁ receptor, deduced from site-directed mutagenesis, molecular modeling, and homology to other GPCRs. Ligand docking modeling was performed on the P2Y₁ and P2Y₁₂ receptor models. The results suggested that ADP binds to the P2Y₁ and P2Y₁₂ receptors on the exofacial side of the cavity delimited by TM1, TM2, TM3, TM6, and TM7 and capped with extracellular loop 2 (Fig. 7). Two different sets of three basic amino acids for each of the two subgroups are involved in coordination of the phosphate moiety of agonists (Costanzi et al., 2004). Molecular recognition in the P2Y1receptor of non-nucleotide antagonists, such as derivatives of PPADS (compound 39), was also studied.

A cluster of positively charged amino acid side chains in TMs 3, 6, and 7 was proposed to form the counterions to the negatively charged 5′-di- or triphosphate moiety at the P2Y₁ receptor. Site-directed mutagenesis validated this prediction and further indicated several uncharged hydrophilic residues that may coordinate the nucleobase. Thus, the agonist 2-MeSADP (compound 6) was inactive at R128(3.29)A and R310(7.39)A and S314(7.43)A mutant P2Y₁ receptors and had a markedly reduced potency at K280(6.55)A and Q307(7.36)A mutant P2Y₁ receptors.

In the P2Y₁₂ subgroup, the role of R3.29 in TM3 seemed to be fulfilled by a Lys residue in extracellular loop 2, whereas the residue R7.39 in TM7 seemed to be substituted by K7.35, located within the same TM but at a distance of four residues, i.e., one helical turn in the exofacial direction. Only R6.55 was common to the essential cationic residues of the two subclasses of P2Y receptors (Costanzi et al., 2004).

A. P2Y₁

Human (Ayyanathan et al., 1996; Janssens et al., 1996; Léon et al., 1996; Schacter et al., 1996), rat (Tokuyama et al., 1995), mouse (Tokuyama et al., 1995), cow (Henderson et al., 1995), chick (Webb et al., 1993), turkey (Filtz et al., 1994), and Xenopus (Cheng et al., 2003) P2Y₁ receptors have been cloned and characterized. In most species, ADP is a more potent agonist than ATP and their 2-methylthio derivatives are more potent than the parent compounds. UTP, UDP, CTP, and GTP are inactive (Waldo et al., 2002; Waldo and Harden, 2004). At present, the most potent agonist known is the N-methanocarba analog of 2-MeSADP, MRS2365 (Chhatriwala et al., 2004). ATP is, in fact, a partial agonist at the P2Y₁ receptor (Palmer et al., 1998) and so at low levels of receptor expression will act as an antagonist (Léon et al., 1997; Hechler et al., 1998b). Extracellular acidification and alkalinization do not appear to modify the activity of ATP (King et al., 1996). The first antagonists to display selectivity for the P2Y₁ receptor were A3P5P and A3P5PS (Boyer et al., 1996a), but these have been superseded by the highly selective and more potent MRS2179 (Boyer et al., 1998; Camaioni et al., 1998) and MRS2279 (Boyer et al., 2002). More recent studies showed that modification of both MRS2179 (Mathieu et al., 2004) and MRS2279 (Kim et al., 2003a; Cattaneo et al., 2004) at the 2-position of the adenine moiety further increases antagonist potency. The increased potency and selectivity of MRS2279 has been used in binding studies showing that [³H]MRS2279 bound specifically to the human P2Y₁ receptor, with a K_d of 3.8 nM. The binding was displaced by 2-MeSADP > ADP = 2-MeSATP > ATP and by MRS2279 = MRS2179 > A3P5P (Waldo et al., 2002; Waldo and Harden, 2004).

Site-directed mutagenesis studies on the human P2Y₁ receptor have produced a binding pocket model in which amino acid residues in TM3, 6 and 7 are critical determinants in the binding of ATP and other nucleotide derivatives (Jiang et al., 1997b; Moro et al., 1998). Arginine 128 (TM3) and lysine 280 (TM6) are proposed to interact with the α and β phosphate groups of ATP, arginine 310 (TM7) also with the β phosphate, and threonine 222 (TM5) with the γ phosphate. Additionally, glutamine 307 (TM7) and serine 314 (TM7) may interact with the adenine ring. Alanine scanning mutagenesis studies revealed that four cysteine residues in the extracellular loops, which are conserved in P2Y receptors, are essential for proper trafficking of the human P2Y₁ receptor to the cell surface (Hoffmann et al., 1999). These studies also showed that several charged residues in the extracellular loops are essential for P2Y₁ activation.

A recent study on the purified and reconstituted human P2Y₁ receptor showed that it couples to Gα_q and Gα₁₁ but not to Gα_i1, Gα_i2, Gα_i3, or Gα_o (Waldo and Harden, 2004). This is consistent with a large body of studies showing that activation of this receptor evokes an increase in intracellular IP₃ levels and the release of intracellular Ca²⁺ stores, in a PTX-insensitive manner.

Northern blotting and RT-PCR revealed P2Y₁ receptor mRNA in most human tissues, including the brain, heart, placenta, lungs, liver, skeletal muscle, kidneys, pancreas, and various blood cells (Ayyanathan et al., 1996; Janssens et al., 1996; Léon et al., 1996). Two mRNA bands of 7.0 to 7.5 and 4.4 kb, whose expression pattern varied with the tissue examined, were seen (Ayyanathan et al., 1996; Léon et al., 1996). Quantitative RT-PCR indicated that expression was highest in the brain, prostate gland, and placenta and was also detected at varying levels in the pituitary gland, lymphocytes, spleen, heart, lung, liver, kidney, stomach, intestine, skeletal muscle, adipose tissue, and pancreas (Moore et al., 2001). In the study within the brain, P2Y₁ mRNA was highest in the nucleus accumbens, putamen, caudate nucleus, and striatum, with lower levels seen in the hippocampus, parahippocampal gyrus, globus pallidus, cingulate gyrus, and hypothalamus. Histochemical studies with a specific antibody also showed widespread distribution of the P2Y₁ receptor throughout the brain, with notable staining in the cerebral cortex, cerebellar cortex, hippocampus, caudate nucleus, putamen, globus pallidus, subthalamic nucleus, red nucleus, and midbrain (Moore et al., 2000a). In addition, in post-mortem brain sections from persons with Alzheimer's disease, the P2Y₁-like immunoreactivity in the hippocampus and entorhinal cortex was localized to neurofibrillary tangles, neuritic plaques, and neuropil threads, which are characteristic Alzheimer's structures (Moore et al., 2000b). Western blots showed bands of 45, 90 and 180 kDa in the smooth muscle cells of the human left internal mammary artery and of 90 and 180 kDa in human umbilical vein endothelial cells (Wang et al., 2002).

A similar pattern of mRNA distribution is seen in rat tissues by Northern blotting (Tokuyama et al., 1995; Nakamura and Strittmatter, 1996). Mapping of the P2Y₁ mRNA by in situ hybridization has been performed across the chick brain (Webb et al., 1998), showing an abundant expression in many regions; at the cellular level this was located in many of the cell bodies of neurons, as well as in astrocytes. In rat brain, there is prominent P2Y₁-like immunoreactivity in neurons in Purkinje cells, the cerebellar cortex and hippocampus (Moran-Jiminez and Matute, 2000), ventral tegmentum (Krügel et al., 2001), midbrain, brainstem, and medulla (Fong et al., 2002). Glial cells in the brain also stain positive (Moran-Jiminez and Matute, 2000; Fong et al., 2002; Weick et al., 2003; Franke et al., 2004). The receptor is also expressed in sensory neurons (Nakamura and Strittmatter, 1996; Fong et al., 2002; Ruan and Burnstock, 2003; Gerevich et al., 2004), consistent with a potential role for P2Y₁ receptors in sensory reception. The presence of P2Y₁-like immunoreactivity (Fong et al., 2002) and mRNA (Buvinic et al., 2002; Kaiser and Buxton, 2002) in rat arterial endothelial cells is also consistent with the well-characterized vasodilatory activity of P2Y₁ receptors. P2Y₁-like immunoreactivity is also present in rat uterus epithelial cells (Slater et al., 2002), the pancreas (Coutinho-Silva et al., 2003), and glomeruli of rat kidney (Bailey et al., 2004). Northern blot analysis in chick embryos during the first 10 days of development showed that expression of P2Y₁ mRNA varied in a regulated manner in the limb buds, mesonephros, somites, brain, and facial primordia (Meyer et al., 1999). This suggests that the receptor may play a role in the development of each of these systems.

P2Y₁ receptor knockout mice have been generated by homologous recombination in two separate laboratories, and the reported phenotypes are identical (Fabre et al., 1999; Léon et al., 1999a) (see also Section. VII.J.). These mice are viable with no apparent abnormalities affecting their development, survival, and reproduction. Platelet counts and morphology are normal. In contrast, platelet shape change and aggregation to usual concentrations of ADP are completely abolished in these mice, whereas the ability of ADP to inhibit cAMP formation is maintained (Léon et al., 1999a). At higher concentrations of ADP (>10 μM), aggregation is observed without shape change, which is entirely due to P2Y₁₂ (Kauffenstein et al., 2001). Their bleeding time is mildly prolonged. These mice display resistance to systemic thromboembolism and to localized arterial thrombosis (Léon et al., 1999b, 2001; Lenain et al., 2003). Conversely, transgenic mice overexpressing the P2Y₁ receptor specifically in the megakaryocytic/platelet lineage have also been generated using the promoter of the tissue-specific platelet factor 4 gene (Hechler et al., 2003). This led to a phenotype of platelet hyper-reactivity in vitro. Moreover, overexpression of the P2Y₁ receptor enabled ADP to induce granule secretion, unlike in wild-type platelets, which suggests that the level of P2Y₁ expression is critical for this event and that the weak responses of normal platelets to ADP are due to a limited number of P2Y₁ receptors rather than to activation of a specific transduction pathway. In addition, transgenic mice display a shortened bleeding time and an increased sensitivity to in vivo platelet aggregation induced by infusion of a mixture of collagen and adrenaline (Hechler et al., 2003).

The P2Y₁ receptor is broadly expressed throughout the body. Thus, its gene knockout could have phenotypic consequences other than the sole hemostasis system. This is indeed the case with glucose homeostasis, since it has been found that the knockout mice display higher glucose levels as well as higher weight than wild-type mice (Léon et al., 2005)

For more information on the P2Y₁ receptor, see Table 5.

B. P2Y₂

P2Y₂ receptors, previously known as P_2U, have been cloned and pharmacologically characterized from human, rat, mouse, canine, and porcine cells or tissues (Lustig et al., 1993; Parr et al., 1994; Bowler et al., 1995; Rice et al., 1995; Chen et al., 1996; Zambon et al., 2000; Shen et al., 2004). P2Y₂ receptors are fully activated by equivalent concentrations of ATP and UTP, whereas ADP and UDP are less effective agonists (Lustig et al., 1993; Parr et al., 1994; Lazarowski et al., 1995a). An exception is the porcine P2Y₂ receptor, which is relatively insensitive to ATP (Shen et al., 2004). UTPγS has been shown to be a potent hydrolysis resistant agonist of P2Y₂ receptors (Lazarowski et al., 1995b). Suramin acts as a competitive antagonist of human and rat P2Y₂ receptors (Charlton et al., 1996; Lambrecht et al., 2002; Wildman et al., 2003). P2Y₂ receptors can directly couple to PLCβ₁ via Gα_q/11 protein to mediate the production of IP₃ and diacylglycerol (DAG), second messengers for calcium release from intracellular stores and PKC activation, respectively. Coupling of P2Y₂ receptors to other G protein subtypes has been reported (Baltensperger and Porzig, 1997; Murthy and Makhlouf, 1998; Weisman et al., 1998; Bagchi et al., 2005). The P2Y₂ receptor is partially sensitive to PTX, a G_i/G_o protein inhibitor, and studies indicate that access to G_o protein is dependent upon association of the P2Y₂ receptor with α_Vβ₃/β₅ integrins (Erb et al., 2001) and regulates nucleotide-induced chemotaxis (Bagchi et al., 2005). Expression of P2Y₂ receptor mRNA has been detected in human skeletal muscle, heart, brain, spleen, lymphocytes, macrophages, bone marrow, and lung, with lower expression levels detected in liver, stomach, and pancreas (Moore et al., 2001). Functional P2Y₂ receptors are expressed in epithelial, smooth muscle, and endothelial cells and in leukocytes, cardiomyocytes, osteoblasts, and cells derived from the peripheral and central nervous system, including Schwann cells, rat cortical neurons, oligodendrocytes, dorsal horn and cortical astrocytes, immortalized astrocytes, astrocytoma cells, and NG108-15 neuroblastoma × glioma hybrid cells (Bowler et al., 1995; Ho et al., 1995; Kirischuk et al., 1995; Rice et al., 1995; Berti-Mattera et al., 1996; Kim et al., 1996; Kunapuli and Daniel, 1998; Weisman et al., 1999; Pillois et al., 2002; Seye et al., 2002; Gendron et al., 2003; Kumari et al., 2003).

Site-directed mutagenesis of the P2Y₂ receptor has been used to demonstrate that replacement of positively charged amino acids in TM helices 6 and 7 with neutral amino acids decreases the potencies of ATP and UTP, suggesting that these domains play a role in binding the negatively charged moieties of nucleotide agonists (Erb et al., 1995) (see above). The P2Y₂ receptor undergoes agonist-induced desensitization in several cell types (Wilkinson et al., 1994; Garrad et al., 1998; Clarke et al., 1999; Otero et al., 2000; Velázquez et al., 2000; Santiago-Pérez et al., 2001), and mutagenesis studies indicate that deletion of structural motifs in the intracellular C-terminal domain of the P2Y₂ receptor that contain putative phosphorylation sites for GPCR kinase diminishes agonist-induced desensitization and internalization of the P2Y₂ receptor (Garrad et al., 1998). The P2Y₂ receptor also contains the consensus integrin-binding motif, Arg-Gly-Asp (RGD) in its first extracellular loop that facilitates P2Y₂ receptor colocalization with α_Vβ₃/β₅ integrins when the P2Y₂ receptor is expressed in human 1321N1 astrocytoma cells that are devoid of endogenous G protein-coupled P2Y receptors (Erb et al., 2001). A mutant P2Y₂ receptor in which the RGD motif was replaced with Arg-Gly-Glu, a sequence that does not have high affinity for integrins, exhibited an EC₅₀ for nucleotide-induced calcium mobilization that was ∼1000-fold greater than that for the wild-type P2Y₂ receptor (Erb et al., 2001). The α_Vβ₃/β₅ integrins are known to regulate angiogenesis and inflammatory responses including cell proliferation, migration, adhesion, and infiltration (Zhang et al., 2002b; Hutchings et al., 2003; Kannan, 2003; Li et al., 2003; Pidgeon et al., 2003), responses also mediated by P2Y₂ receptor activation (Wilden et al., 1998; Seye et al., 2002; Greig et al., 2003a,b; Schafer et al., 2003; Bagchi et al., 2005; Kaczmarek et al., 2005), suggesting that nucleotides may transactivate integrin signaling pathways by virtue of P2Y₂ receptor binding to integrins. The RGD sequence in the P2Y₂ receptor also has been shown to play an integrin-independent role in targeting of the receptor to the apical membrane of Madin-Darby canine kidney cells (Qi et al., 2005).

P2Y₂ receptor activation increases the synthesis and/or release of arachidonic acid (AA), prostaglandins and nitric oxide (NO) (Lustig et al., 1992; Pearson et al., 1992a,b; Xing et al., 1999; Xu et al., 2002b, 2003; Welch et al., 2003). In primary murine astrocytes, P2Y₂ receptors mediate the activation of calcium-dependent and calcium-independent PKCs and ERK1/2 that can activate cytosolic phospholipase A₂, leading to production of AA (Gendron et al., 2003; Xu et al., 2003), the precursor of eicosanoids, prostaglandins, and leukotrienes (Balsinde et al., 2002). Activation of P2Y₂ receptors in isolated UTP- or ATP-perfused rat hearts induces pronounced vasodilatation (Godecke et al., 1996), consistent with the role of P2Y₂ receptors in relaxation of smooth muscle through the endothelium-dependent release of NO and prostacyclin (Lustig et al., 1992; Pearson et al., 1992a,b). P2Y₂ receptor expression in smooth muscle cells is up-regulated by agents that mediate inflammation, including interleukin (IL)-1β, interferon-γ, and tumor necrosis factor-α (Hou et al., 1999, 2000) and P2Y₂ receptor up-regulation has been shown to promote nucleotide-induced activation of PKC, cyclooxygenase, and MAPK (Koshiba et al., 1997; Turner et al., 1998; Seye et al., 2002). In addition to ERK1/2, P2Y₂ receptor activation can induce the phosphorylation of the stress-activated kinases JNK and p38 (Gendron et al., 2003). P2Y₂ receptor activation also induces p38- and ERK1/2-dependent up-regulation of genes that regulate cell survival in human astrocytoma cells (i.e., Bcl-2 and Bcl-xl) and genes that regulate neurite outgrowth in PC-12 cells (Chorna et al., 2004). Human neutrophil P2Y₂ receptors have been shown to regulate neutrophil degranulation induced by fibrinogen, independent of AA metabolites (Meshki et al., 2004), and P2Y₂ receptors have been suggested to play a role in the wound healing process (Burrell et al., 2003; Greig et al., 2003a,b).

Studies have indicated that P2Y₂ receptor-mediated MAPK activation in rat-1 fibroblasts and PC12 cells is dependent upon transactivation of the epidermal growth factor receptor (EGFR) via a Src/Pyk2-dependent pathway (Soltoff, 1998; Soltoff et al., 1998). In contrast, embryonic fibroblasts derived from Src^-/-, Pyk2^-/-, or Src^-/-Pyk2^-/- mice have been used to demonstrate that Src and Pyk2 were essential for GPCR-mediated transactivation of the EGFR but not for GPCR-mediated MAPK activation (Andreev et al., 2001). Gβγ subunits have been shown to regulate Src-mediated transactivation of growth factor receptors (Luttrell et al., 1997), which may represent a common pathway whereby GPCRs stimulate cell proliferation. Other studies have identified two SH3-binding domains (i.e., PXXP motifs in which P is proline and X is any amino acid) in the intracellular carboxyl-terminal tail of the human P2Y₂ receptor that are necessary for transactivation of epidermal or platelet-derived growth factor (PDGF) receptors by ATP or UTP (Liu et al., 2004). Deletion of these SH3-binding domains inhibited nucleotide-induced P2Y₂ receptor colocalization with EGFR and UTP-induced EGFR transactivation (i.e., phosphorylation) but did not suppress ERK1/2 activation when the mutant receptors were expressed in 1321N1 astrocytoma cells (Liu et al., 2004), most likely because of the ability of the P2Y₂ receptor to also activate Src and ERK1/2 via PLC and integrin signaling pathways (Erb et al., 2001). Furthermore, Src coimmunoprecipitated with the P2Y₂ receptor in UTP-treated cells expressing the wild-type P2Y₂ receptor, but not the SH3-binding domain deletion mutant (Liu et al., 2004), strongly suggesting that activation of the P2Y₂ receptor promotes Src binding.

Activation of P2Y₂ receptors causes proliferation and/or migration of human epidermal keratinocytes, lung epithelial tumor cells, glioma cells, and smooth muscle cells (Wilden et al., 1998; Tu et al., 2000; Seye et al., 2002; Schafer et al., 2003, Greig et al., 2003b). P2Y₂ receptor activation also can induce cell cycle progression in smooth muscle cells from G₁ to S and M phases (Malam-Souley et al., 1996; Miyagi et al., 1996). HeLa cell proliferation in response to P2Y₂ receptor activation is associated with the PI3-K- and ERK1/2-dependent expression of the early response protein c-fos (Muscella et al., 2003). Consistent with a role for P2Y₂ receptors in cell proliferation, P2Y₂ receptor mRNA expression is down-regulated during cell differentiation (Martin et al., 1997). The P2Y₂ receptor is up-regulated in thymocytes as an immediate early gene response (Koshiba et al., 1997) and in injured and stressed tissues (Turner et al., 1997; Seye et al., 2002), and nucleotides induce ERK-dependent astrocyte proliferation under a variety of conditions including stretch- or stab-induced injury (Neary et al., 1994, 1996, 1999, 2003; Franke et al., 1999). Up-regulation of P2Y₂ receptors occurs in arteries after balloon angioplasty (Seye et al., 1997), in rat submandibular gland cells after short-term culture or upon duct ligation in vivo (Turner et al., 1997, 1998), and in a mouse model of Sjögren's syndrome (Schrader et al., 2004). Placement of a vascular collar around rabbit carotid arteries increases expression of P2Y₂ receptors in smooth muscle and endothelium that is associated with neointimal hyperplasia, monocyte infiltration, and smooth muscle cell expression of the proliferative protein osteopontin, responses that were enhanced by application of the P2Y₂ receptor agonist UTP (Seye et al., 2002). In addition, P2Y₂ receptor up-regulation in endothelial cells increases the binding of monocytes to endothelial cells due to P2Y₂ receptor-mediated increases in the endothelial expression of vascular cell adhesion molecule-1 (Seye et al., 2003), a process that promotes vascular inflammation in atherosclerosis. Up-regulation of vascular cell adhesion molecule-1 was shown to be dependent on P2Y₂ receptor-mediated transactivation of vascular endothelial growth factor receptor-2 (KDR/Flk-1), a response that was inhibited by deletion of the SH3 binding motifs from the P2Y₂ receptor, demonstrating a mechanism whereby P2Y₂ receptors can cause inflammatory responses (Seye et al., 2004).

P2Y₂ receptor activation increases Cl^- secretion and inhibits Na⁺ absorption in epithelial cells, which has potential relevance for the treatment of cystic fibrosis, a disease that is caused by genetic defects in the gene for the CFTR, a major epithelial anion channel (Clarke and Boucher, 1992; Parr et al., 1994; Clarke et al., 2000; Kellerman et al., 2002). The synthetic P2Y₂ receptor agonist dCp₄U (INS37217) has been used to promote chloride and water secretion in tracheal epithelium and increase ciliary beat frequency and mucin release in human airway epithelium (Yerxa et al., 2002) and to stimulate subretinal fluid reabsorption in a rabbit model of retinal detachment (Meyer et al., 2002).

A P2Y₂ receptor knockout mouse that is defective in nucleotide-stimulated ion secretion in airway epithelial cells has been produced, confirming a role for the P2Y₂ receptor in the regulation of epithelial transmembrane ion transport (Cressman et al., 1999). In addition, P2Y₂ receptors have been shown to inhibit bone formation by osteoblasts (Hoebertz et al., 2002), and N-type calcium currents in neurons (Filippov et al., 1997; Brown et al., 2000a). P2Y₂ receptors also can induce α-secretase-dependent amyloid precursor protein processing in astrocytoma cells, suggesting a neuroprotective role (Camden et al., 2005). Collectively, studies with the P2Y₂ receptor and its signaling pathways have elucidated potential pharmacological targets in atherosclerosis, inflammation, cystic fibrosis, eye disease, osteoporosis, cancer, and neurodegenerative disorders.

For more information on the P2Y₂ receptor, see Table 6.

C. P2Y₄

Human (Communi et al., 1995; Nguyen et al., 1995; Stam et al., 1996), rat (Bogdanov et al., 1998b; Webb et al., 1998), and mouse (Lazarowski et al., 2001b; Suarez-Huerta et al., 2001) P2Y₄ receptors have been cloned and characterized (Communi et al., 2005b). UTP is the most potent activator of the recombinant human P2Y₄ receptor (Nicholas et al., 1996). GTP and ITP are approximately 10 times less potent than UTP (Communi et al., 1996a). ATP behaves as a competitive antagonist (Kennedy et al., 2000). Up₄U (INS365) (Pendergast et al., 2001) and dCp₄U (INS37217) (Yerxa et al., 2002) are agonists of the human P2Y₄ receptor, whereas Ap₄A is inactive. On the contrary, the recombinant rat and mouse P2Y₄ receptors are activated equipotently by ATP and UTP and also with a lower potency by ITP, GTP and CTP (Bogdanov et al., 1998b; Webb et al., 1998). Pharmacological discrimination between rodent P2Y₄ and P2Y₂ receptors is thus difficult. No selective antagonist is available. Extracellular acidification enhanced the potency of ATP and UTP at rat P2Y₄, but not at rat P2Y₂ (Wildman et al., 2003). Zn²⁺ inhibited the ATP response at the rat P2Y₄ receptor but had no effect on rat P2Y₂ (Wildman et al., 2003). A study of chimeric human/rat P2Y₄ receptors showed that the structural determinants of agonism versus antagonism by ATP are located in the N-terminal domain and the second extracellular loop. Mutational analysis revealed that three residues in the second extracellular loop contribute to impart agonist property to ATP: Asn-177, Ile-183, and Leu-190 (Herold et al., 2004). Although their sequence is only distantly related to mammalian P2Y₄, Xenopus p2y8 (Bogdanov et al., 1997) and a turkey p2y (Boyer et al., 2000) could represent orthologs. They are activated with similar potencies by ATP, UTP, GTP, ITP, and CTP. The tp2y receptor is coupled to the stimulation of PLC and inhibition of adenylyl cyclase, indicating a dual coupling to G_q/11 and G_i/o.

In the absence of reconstitution data, there is no hard molecular evidence of P2Y₄ receptor interaction with specific G proteins. The IP₃ response to UTP of the recombinant human P2Y₄ receptor is partially inhibited by PTX, at early times after agonist addition but not later (Communi et al., 1996a). This finding suggests that the P2Y₄ receptor couples mainly to a G_q/11 protein and accessorily to a G_i/o protein. In sympathetic neurons injected with P2Y₄ cDNA, UTP inhibited the N-type Ca²⁺ current. This inhibition was relieved by PTX and by expression of the βγ subunits of transducin (Filippov et al., 2003). These observations are consistent with the involvement of βγ subunits released from G_i/o proteins in the function of the P2Y₄ receptor. In the same model, UTP inhibited the M-type K⁺ current in a PTX-insensitive way.

Among human organs, P2Y₄ mRNA was detected in placenta by Northern blotting (Communi et al., 1995) and was most abundant in the intestine, according to TaqMan quantitative RT-PCR (Moore et al., 2001). RT-PCR revealed the presence of P2Y₄ message in human umbilical vein endothelial cells, peripheral blood leukocytes (Jin et al., 1998b), fetal cardiomyocytes (Bogdanov et al., 1998a), and various cell lines derived from the human lung (Communi et al., 1999a). In the airway submucosal cell line 6CFSMEo-, an IP₃ response to UTP but not to ATP suggests a functional expression of the P2Y₄ receptor (Communi et al., 1999a). In the rat, the expression of P2Y₄ message in heart and brain was much higher in neonates than in adults (Webb et al., 1998; Cheung et al., 2003). In in situ hybridization on the adult rat brain neuronal P2Y₄ receptor mRNA was not detectable, it being present in the ventricular/choroid plexus system and strongly in the pineal gland, as well as apparently in astrocytes (Webb et al., 1998). In mouse, RT-PCR revealed that P2Y₄ message was the most abundant in stomach, liver, and intestine (Suarez-Huerta et al., 2001). Several histochemical studies have been performed using commercially available anti-rat P2Y₄ polyclonal rabbit antibodies, the specificity of which remains questionable (as is illustrated by Tung et al., 2004). Those studies suggest expression of the P2Y₄ receptor in rat dorsal root and trigeminal ganglia neurons (Ruan and Burnstock, 2003) and in the proximal convoluted tubular epithelium of the rat kidney (Turner et al., 2003). A combination of RT-PCR, Western blotting, immunohistochemistry, and pharmacology suggests that the P2Y₄ receptor is expressed and plays a role in the inner ear of rat, guinea pig, and gerbil (organ of Corti, stria vascularis marginal cells, and vestibular dark cells) (Teixeira et al., 2000; Sage and Marcus, 2002; Parker et al., 2003). These data are consistent with the hypothesis that nucleotides could play a role in the adaptation of the cochlea to noise exposure (Housley et al., 2002). Noise would increase the release of ATP and UTP in the scala media, leading to the inhibition of K⁺ release at the apical surface of the stria vascularis, presumably via activation of P2Y₄ receptors (Marcus and Scofield, 2001). In conclusion, a major site of P2Y₄ expression appears to be the intestine. Indeed RT-PCR has revealed that P2Y₄ message was particularly abundant in the human and murine intestine. This is consistent with the loss of the chloride secretory response to apical UTP in the jejunal epithelium of P2Y₄-null mice (Robaye et al., 2003).

P2Y₄-null mice have apparently normal behavior, growth, and reproduction (Robaye et al., 2003). The proportion of genotypes is consistent with X-linked Mendelian transmission. The chloride secretory response of the jejunal epithelium to apical UTP and ATP, measured in Ussing chambers, is abolished in P2Y₄-null mice. At the basolateral side, both P2Y₄ and P2Y₂ receptors are involved (Ghanem et al., 2005). In the colon, the epithelial secretion of chloride in response to UTP is mediated exclusively by the P2Y₄ subtype (Ghanem et al., 2005), whereas both P2Y₂ and P2Y₄ are involved in the UTP-induced secretion of potassium (Matos et al., 2005). Thus, in the intestine, the epithelial response to nucleotides is mediated mainly by the P2Y₄ receptor, but with a contribution of the P2Y₂ subtype, whereas in airways it involves mainly the P2Y₂ receptor (Cressman et al., 1999).

For more information on the P2Y₄ receptor, see Table 7.

D. P2Y₆

The mouse (Lazarowski et al., 2001b), rat (Chang et al., 1995; Nicholas et al., 1996), and human (Communi et al., 1996b) P2Y₆ receptors are UDP receptors. The p2y3 receptor is the avian ortholog of the mammalian P2Y₆ receptor and also displays selectivity for UDP (Webb et al., 1996a; Li et al., 1998). At the human P2Y₆ receptor, the rank order of potency of various nucleotides is as follows: UDP > UTP > ADP > 2-MeSATP >> ATP (Communi et al., 1996b). Adenine dinucleotides have little effect on P2Y₆, whereas diuridine triphosphate is a selective agonist of P2Y₆ (Pendergast et al., 2001). No selective competitive antagonist is available. Some aryl diisothiocyanate derivatives behave as potent insurmountable antagonists and exhibit selectivity for P2Y₆ compared with P2Y₁, P2Y₂, P2Y₄, and P2Y₁₁ receptors (Mamedova et al., 2004).

In the absence of reconstitution data, there is no hard molecular evidence of P2Y₆ interaction with specific G proteins. The IP₃ response to UDP of the recombinant P2Y₆ receptor is insensitive to PTX inhibition, suggesting a coupling to G_q/11 (Chang et al., 1995; Robaye et al., 1997). A P2Y₆-mediated increase in cAMP has been reported, but it is probably an indirect effect mediated by prostaglandins, since it was at least partially inhibited by indomethacin (Köttgen et al., 2003). A unique feature of the P2Y₆ receptor compared with other P2Y subtypes is its slow desensitization and internalization (Robaye et al., 1997; Brinson and Harden, 2001). This can be explained by the short C-terminal sequence of P2Y₆ that contains a single threonine and misses the Ser333 and Ser334 that play a key role in the UTP-dependent phosphorylation, desensitization, and internalization of P2Y₄ (Brinson and Harden, 2001).

Northern blotting has revealed a rather wide tissue distribution of P2Y₆ mRNA. In particular, the P2Y₆ transcript has been found in human spleen, thymus, placenta, intestine, and blood leukocytes (Communi et al., 1996b) and in rat lung, spleen, stomach, intestine, and aorta (Chang et al., 1995). Consistent with those initial observations, the expression and potential role of the P2Y₆ receptor has been documented in placenta (Somers et al., 1999), vascular smooth muscle, epithelia, and immune cells. P2Y₆ message is present in smooth muscle cells cultured from the rat aorta (Chang et al., 1995; Hou et al., 2002). UDP acts as a growth factor for these cells (Hou et al., 2002). In P2X₁-deficient mice, the vasoconstrictor effect of ATP on mesenteric arteries is abolished (Vial and Evans, 2002), but a contractile effect of UTP and UDP is maintained and probably involves the P2Y₆ receptor, although the pharmacological profile of that response, characterized by the equipotency of UTP and UDP, is atypical. UDP and UDPβS induced the contraction of other vessels such as rat (Malmsjö et al., 2003a) and human (Malmsjö et al., 2003b) cerebral arteries. The expression of P2Y₆ receptors on the basolateral side of rat colonic epithelial cells was demonstrated by immunochemistry and is involved in a sustained stimulatory effect of UDP on chloride secretion (Köttgen et al., 2003). In mouse gallbladder, apical UDP stimulates secretion of chloride, an effect that is maintained in P2Y₂-null mice and is likely to involve the P2Y₆ receptor (Cressman et al., 1999; Lazarowski et al., 2001b). Expression of the P2Y₆ receptor has also been demonstrated by RT-PCR and immunochemistry in human nasal epithelial cells (Kim et al., 2004), in which apical UDP stimulates chloride secretion (Lazarowski et al., 1997c). In human monocytic THP-1 cells, UDP stimulates the production of IL-8 (Warny et al., 2001). Furthermore, endogenous UDP released from those cells contributes to the IL-8 secretion induced by lipopolysaccharide (LPS), which is indeed partially decreased by P2Y₆ antisense oligonucleotides. UDP also acts on murine and human dendritic cells, where it increases cytosolic calcium, induces chemotaxis, and stimulates the release of chemokine CXCL8 (Marriott et al., 1999; Idzko et al., 2004). Northern blots revealed the expression of P2Y₆ mRNA in activated but not resting human CD4⁺ and CD8⁺ T cells (Somers et al., 1998). This expression was associated with a UDP-induced rise of [Ca²⁺]_i in the activated T cells only. In situ hybridization showed the presence of P2Y₆ mRNA in T cells infiltrating the lesions of patients with inflammatory bowel disease (IBD).

For more information on the P2Y₆ receptor, see Table 8.

E. P2Y₁₁

Among P2Y receptors, the human P2Y₁₁ has a unique profile: 1) it is the only P2Y receptor gene that contains an intron in the coding sequence; 2) the potency of its natural agonist ATP is relatively low; and 3) it is dually coupled to PLC and adenylyl cyclase stimulation. There does not seem to be a rodent ortholog of P2Y₁₁, confirming that this receptor subtype is quite different from the other members of the family. At the recombinant human P2Y₁₁ receptor, the rank order of potency with which nucleotides increase either cAMP or IP₃ is AR-C67085MX ≥ ATPγS ≈ BzATP > dATP > ATP > ADP (Communi et al., 1997, 1999b; Qi et al., 2001a). UTP, GTP, CTP, TTP, ITP, and dinucleotides (Ap₄A, Ap₅A, and Ap₆A) were completely inactive (although UTP has been claimed to be an agonist in some studies; see below and White et al., 2003). The EC₅₀ of ATP is in the 5 to 100 μM range, whereas in the same expression systems (i.e., 1321N1 or CHO cells), the EC₅₀ characterizing the activation of the other P2Y subtypes by their respective ligands is in the 10 to 500 nM range. Suramin behaves as a competitive antagonist of the human (h) P2Y₁₁ receptor with a K_i close to 1 μM (Communi et al., 1999b). The dog P2Y₁₁ receptor, the sequence of which shares only 70% amino acid identity with hP2Y₁₁, has a completely different profile characterized by a greater potency of diphosphates compared with triphosphates (Zambon et al., 2001). Indeed, the rank order of agonist potency is as follows: 2-MeSADP > ADPβS > ADP > ATP. A mutational analysis revealed that the change of Arg-265, located at the junction between TM6 and the third extracellular loop in the hP2Y₁₁, to Gln in cP2Y₁₁ is at least partially responsible for the diphosphate selectivity of the canine receptor (Qi et al., 2001b). Although the existence of a functional P2Y₁₁ receptor in Xenopus embryos was recently reported (C. Drew, unpublished data), an ortholog gene could not be detected in the murine genome, and there is no evidence of a functional P2Y₁₁ receptor in rat or mouse.

The hP2Y₁₁ gene differs from other P2Y genes by the presence in the coding sequence of a 1.9-kb intron that separates an exon encoding the first six amino acid residues from a second exon encoding the remaining part of the protein (Communi et al., 2001b). The hP2Y₁₁ gene is adjacent to the gene encoding the human ortholog of Ssf1, a nuclear protein playing an important role in Saccharomyces cerevisiae mating. Chimeric mRNA resulting from the cotranscription and intergenic splicing of the two genes is ubiquitously present in human organs. However, the fusion protein could only be detected after recombinant overexpression. Only half a dozen cases of intergenic splicing have been described in mammalian cells, and they are likely to represent an evolutionary tool to create new function via fusion of preexisting protein domains (Finta et al., 2002).

Activation of recombinant hP2Y₁₁ or cP2Y₁₁ receptors leads to an increase of both cAMP and IP₃, presumably via the dual activation of G_s and G_q/11 (Communi et al., 1997, 1999b). Indeed, the use of various pharmacological tools (inhibition of PLC or prostaglandin synthesis, chelation of intracellular calcium, and down-regulation of PKC) has demonstrated that the cAMP increase is not merely an indirect consequence of rises in IP₃, [Ca²⁺]_i, and PKC activity (Suh et al., 2000; Qi et al., 2001a). However, PKC activation plays some role and amplifies the stimulation of adenylyl cyclase. In addition, it has been claimed that UTP acts via the hP2Y₁₁ receptor to induce an IP₃-independent Ca²⁺ mobilization that is sensitive to PTX inhibition, whereas the effect of ATP is not (White et al., 2003). This finding illustrates the fact that different agonists can recruit distinct signaling pathways via the same receptor.

Northern blotting has revealed a significant expression of P2Y₁₁ mRNA in human spleen and to a lesser extent intestine and liver (Communi et al., 1997, 2001b). According to a quantitative RT-PCR study, P2Y₁₁ message is most abundant in human brain and pituitary (Moore et al., 2001). It is also present in B lymphocytes from patients with chronic lymphocytic leukemia (Conigrave et al., 2001) as well as in human HL-60 (Communi et al., 1997, 2000) and NB4 (van der Weyden et al., 2000b) promyelocytic leukemia cells. In the HL-60 cell line, P2Y₁₁ message is up-regulated by all the agents that induce granulocytic differentiation, such as retinoic acid and granulocyte colony-stimulating factor (Communi et al., 2000). ATP has been shown to induce the differentiation of HL-60 cells into neutrophil-like cells (Jiang et al., 1997a). This action was associated with a rise in cAMP and the rank order potency of various nucleotides is consistent with that of recombinant P2Y₁₁ (Conigrave et al., 1998). P2Y₁₁ mRNA is also expressed in human monocyte-derived dendritic cells, in which ATP induces a semimaturation state characterized by increased surface expression of costimulatory molecules, inhibition of the production of proinflammatory cytokines such as IL-12, stimulation of IL-10 production, and modification of the repertoire of chemokine and chemokine receptor expression, resulting in a change of migratory behavior (Wilkin et al., 2001, 2002; La Sala et al., 2002; Schnurr et al., 2003). Semimature dendritic cells can orient CD4⁺ T lymphocytes toward a Th2 rather than a Th1 response or induce tolerance.

For more information on the P2Y₁₁ receptor, see Table 9.

F. P2Y₁₂

The human (Hollopeter et al., 2001; Savi et al., 2001; Zhang et al., 2001), rat (Hollopeter et al., 2001), and mouse (Foster et al., 2001) P2Y₁₂ receptors have been identified and characterized. ADP is the natural agonist of this receptor, whereas conflicting results were reported concerning the effects of ATP and its triphosphate analogs. For diphosphates, the rank order of agonist potency in all cases reported is 2-MeSADP >> ADP > ADPβS. Concerning ATP and its analogs, they were found to be agonists either in native P2Y₁₂-expressing cells (Simon et al., 2001; Unterberger et al., 2002) or in some heterologously transfected cells (Takasaki et al., 2001; Zhang et al., 2001; Simon et al., 2002). However, in platelets there have been reports over a long period that ATP and a wide range of its triphosphate analogs behave as antagonists of ADP-induced adenylyl cyclase inhibition (reviewed by Gachet, 2001). This has recently been confirmed: ATP and its triphosphate analogs are antagonists of the P2Y₁₂ receptor both in human and mouse platelets, provided care is taken to remove contaminants and to prevent enzymatic production of ADP or 2-MeSADP (Kauffenstein et al., 2004). There are currently, therefore, two alternative interpretations extant of the action of adenosine triphosphates on the P2Y₁₂ receptor (either native or recombinant). One (Kauffenstein et al., 2004) is that, both in the platelet and in nucleated (nonplatelet) cells, these agents are antagonists: an apparent agonist action of triphosphates would then be due in all cases to a secondary introduction of diphosphate, as just noted. The second (Barnard and Simon, 2001; Simon et al., 2002) is that the triphosphates are intrinsically agonistic at the P2Y₁₂ receptor generally, but that they behave as apparent antagonists in the platelet, due to the much lower receptor density there (or possibly to an absence in the platelet of some modifying component involved in nucleated cells). The latter interpretation has been made also (see section VI.A.) for a parallel difference in adenosine triphosphate behavior of the P2Y₁ receptor in the platelet and in most other cell types tested.

Behavior of ATP and ATPγS rather similar to that reported for these agents at P2Y₁₂ in the platelet can be obtained in a system reconstituted with purified P2Y₁₂ receptor protein (see section III.A.), in which enzymatic degradation or interconversion of the nucleotides is not possible (Bodor et al., 2003). There, also, one of the same explanations just given for the platelet case could hold, e.g., the effective density of functional P2Y₁₂ receptors in the vesicle surface may be too low for those agents to show intrinsic agonism. The general dichotomy discussed here need not apply to all adenosine triphosphates: ATPγS may behave at P2Y₁₂ receptors in general as an antagonist, that action being shown clearly in transfected 1321 N1 cells as well as in the platelet (Kauffenstein et al., 2004) (ATPγS has not yet been tested with full precautions elsewhere).

A clear agonist action of adenosine triphosphates of P2Y₁₂ receptors other than in platelets was measured for adenylate cyclase inhibition in the cases noted above (Simon et al., 2001, 2002; Unterberger et al., 2002) with all of the precautions used by Kauffenstein et al. (2004) and with a demonstration of negligible loss of ATP occurring during the assay. Furthermore, in the direct transduction by the P2Y₁₂ receptor in neurons through the N-type Ca²⁺ channel (see section III.B.3.), in which a fast application of pure 2-MeSATP is made with constant perfusion flow, in contact with only a low cell density and with an assay time of 50 ms, 2-MeSATP (EC₅₀ 0.042 nM) is a definite agonist, 2.5 times more potent than 2-MeSADP (Simon et al., 2002). Clearly this effect cannot be due to an artifactual introduction of the diphosphate. In conclusion, the situation concerning the agonist selectivity of the P2Y₁₂ receptor is controversial, may vary with the cell type, and is at present left open here.

The P2Y₁₂ receptor is mostly expressed in the megakaryocyte/platelet lineage in which it is the molecular target of the active metabolite of the antiplatelet drug clopidogrel (Savi and Herbert, 2005; see also below). This metabolite covalently binds cysteine residues of the extracellular loops resulting in inhibition of ligand binding (Savi et al., 2001). Ticlopidine and clopidogrel are efficient antithrombotic drugs of the thienopyridine family of compounds. A third antithrombotic thienopyridine, CS-747 or prasugrel, is currently under clinical evaluation (Niitsu et al., 2005). Potent direct competitive P2Y₁₂ antagonists also exist, including the AR-C69931MX compound named cangrelor as well as other AR-C compounds, which are all ATP analogs (Ingall et al., 1999). Of these, AZD6140 is a nonphosphorylated and orally active compound currently under clinical evaluation (Peters and Robbie, 2004).

In addition to the platelet lineage, the P2Y₁₂ receptor has also been shown to be expressed in subregions of the brain (Hollopeter et al., 2001), but its function there is not yet known. Glial cells (Fumagalli et al., 2003; Sasaki et al., 2003; Bianco et al., 2005), brain capillary endothelial cells (Simon et al., 2001), smooth muscle cells (Wihlborg et al., 2004), and chromaffin cells (Ennion et al., 2004) express P2Y₁₂ receptors. The precise role in these locations is still under study.

The platelet P2Y₁₂ receptor is coupled to Gα_i2 (see also section VII.J.), as was first been shown by photolabeling with radiolabeled GTP (Ohlmann et al., 1995) and confirmed in Gα_i2-deficient mouse platelets (Jantzen et al., 2001) as well as in reconstituted systems (Bodor et al., 2003). In the latter case, Gα_i2 was found to be the preferred Gα subunit, whereas Gα_i1 and Gα_i3 were poorly effective. No coupling with G_z was observed.

P2Y₁₂ knockout mice have been generated (Foster et al., 2001; André et al., 2003; see also section VII.J.), which display the phenotype of clopidogrel-treated animals, i.e., prolonged bleeding time, inhibition of platelet aggregation to ADP, and resistance to arterial thrombosis in various models (Conley and Delaney, 2003). In humans, molecular defects of this receptor exist, which result in hemorrhagic syndromes. Four families of patients have been described so far with essentially the same phenotype. Among these, three have a defect in receptor expression (Cattaneo et al., 1992, 1997; Nurden et al., 1995), whereas in one family, a mutant form of the receptor is expressed with defective function (for a recent review, see Cattaneo, 2005; Cattaneo et al., 2003).

For more information on the P2Y₁₂ receptor, see Table 10.

G. P2Y₁₃

The human (Communi et al., 2001a; Zhang et al., 2002a), mouse (Zhang et al., 2002a), and rat (Fumagalli et al., 2004) P2Y₁₃ receptors have been identified and characterized (see also Communi et al., 2005a). ADP and Ap₃A are naturally occurring agonists of the P2Y₁₃ receptor. IDP is also a potent agonist of the murine P2Y₁₃ receptor, but is 10-fold less potent than ADP on the human one (Zhang et al., 2002a). Ap₄A, Ap₅A, and Ap₆A are inactive. When contaminating ADP was enzymatically removed and testing was performed over a short period, ATP behaved as a weak partial agonist (Marteau et al., 2003). As described for the P2Y₁ receptor, the activity of ATP may vary according to the level of expression of the P2Y₁₃ receptor in different recombinant systems. The relative potencies of ADP and 2-MeSADP differed according to assays used. 2-MeSADP was more potent than ADP in competing with [³³P]2-MeSADP on intact 1321N1 cells expressing hP2Y₁₃ and in stimulating binding of GTPγ[³⁵S] to membranes of the same cells, whereas ADP was more potent than 2-MeSADP on the rat P2Y₁₃ (Fumagalli et al., 2004). In CHO-K1 cells expressing hP2Y₁₃, ADP and 2-MeSADP produced an equipotent inhibition of cAMP accumulation. These discrepancies suggest that the P2Y₁₃ receptor might exist in multiple active conformations characterized by differences in affinity for 2-MeSADP versus ADP, kinetics, and preference for G proteins. The antiplatelet and antithrombotic action of clopidogrel is mediated by an active metabolite. That metabolite has been shown to inhibit the binding of [³³P]2-MeSADP to hP2Y₁₂ with an IC₅₀ of 100 nM (Savi et al., 2001), but it had no effect on hP2Y₁₃ up to 2 μM (Marteau et al., 2003). Cangrelor (AR-C69931MX) is currently in development as an antiplatelet and antithrombotic agent. It is an ATP derivative that inhibits platelet aggregation by ADP at nanomolar concentrations (Ingall et al., 1999). It was previously believed to be a selective antagonist of the hP2Y₁₂ receptor (IC₅₀ = 2.4 nM) (Takasaki et al., 2001), but in the same range of nanomolar concentrations, it is also an antagonist of human and rat P2Y₁₃ receptors (Marteau et al., 2003; Fumagalli et al., 2004). Two other P2Y₁₂ antagonists, Ap₄A and 2-MeSAMP, are also antagonists of the P2Y₁₃ receptor (Marteau et al., 2003).

The effects of ADP mediated by the recombinant P2Y₁₃ receptor were all inhibited by PTX: increased binding of GTPγ[³⁵S], inhibition of cAMP formation, ERK1/2 phosphorylation, and accumulation of IP₃ in cells coexpressing Gα₁₆ (Communi et al., 2001a; Marteau et al., 2003). This finding suggests that the P2Y₁₃ receptor is primarily coupled to a G_i/o protein. The only exception was the increased cAMP formation observed at high ADP concentrations, and that presumably results from promiscuous coupling to G_s, a phenomenon observed with other recombinant G_i/o-coupled receptors, such as the α₂-adrenergic receptor (Communi et al., 2001a). More direct evidence for the coupling to G_i/o derives from the measurement of [Ca²⁺]_i increases in HEK cells coexpressing various chimeric G proteins (Zhang et al., 2002a). A significant stimulation by ADP was obtained in cells expressing either Gα_q/i or Gα_q/i3, that is, Gα_q in which the five C-terminal residues have been replaced by the corresponding sequence in either Gα_i1/2 or Gα_i3, respectively.

P2Y₁₃ mRNA was amplified by RT-PCR in several human organs. Signals were the most intense in spleen and brain (Communi et al., 2001a). In dot blot analysis, the spleen gave the most intense positive signal, followed by placenta, liver, bone marrow, lung, and various brain regions (Zhang et al., 2002a). Quantitative RT-PCR revealed a significant expression in human monocytes, T cells, and dendritic cells derived from blood monocytes or bone marrow, but not in human platelets (Zhang et al., 2002a; Wang et al., 2004a). Northern blots were positive for murine spleen, brain, liver, and heart. In the rat, again the RT-PCR signal was the most intense in the spleen (Fumagalli et al., 2004).

P2Y₁₃-null mice have been generated recently (A. Ben Addi and B. Robaye, unpublished data). No phenotype has been characterized so far.

For more information on the P2Y₁₃ receptor, see Table 11.

H. P2Y₁₄

From a phylogenetic and structural point of view, the P2Y₁₄ receptor (previously known as GPR105 or UDP-glucose receptor) lies with the P2Y₁₂ and P2Y₁₃ receptors in the second main branch of the P2Y receptor family and is 47% identical to these receptors. The gene for this receptor has been found in human chromosome 3q24-3q25 where a cluster of other related GPCRs, consisting of P2Y₁-P2Y₁₂, and P2Y₁₃ receptors and the orphan receptors GPR87, GPR91, and H963 have been found (Abbracchio et al., 2003).

The P2Y₁₄ receptor is activated by UDP-glucose as well as UDP-galactose, UDP-glucuronic acid, and UDP-N-acetylglucosamine but not by uridine or adenine nucleotides (Chambers et al., 2000; Harden, 2004). Of these endogenous ligands, to date only UDP-glucose has been shown to be released extracellularly by a variety of cell lines (Lazarowski et al., 2003b). 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. No radioligand binding assay is available for quantification of P2Y₁₄ receptor binding sites.

The complete sequences of the rat (VTR-15-20; Charlton et al., 1997; Freeman et al., 2001) and mouse orthologs (Freeman et al., 2001) have also been described. The rat and mouse orthologs show 80 and 83% amino acid identity, respectively, with human P2Y₁₄ and show similar agonist pharmacology (Freeman et al., 2001).

The P2Y₁₄ receptor couples to the G_i/o family of G proteins. In particular, data obtained on the recombinant receptor in HEK-293 cells show coupling to Gα subunits of the G_i/o family (Gα₁₆, Gα_qo5, and Gα_qi5), but not to G_s family or to endogenous G_q/11 proteins (Moore et al., 2003). Stimulation of native P2Y₁₄ receptors in primary rat cortical astrocytes as well as in murine N9 and rat primary microglial cells results in transient intracellular calcium increases (Fumagalli et al., 2003; Bianco et al., 2005), although the mechanisms at the basis of this transductional effect are not known yet.

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., corpus striatum, cerebellum, caudate nucleus, hippocampus, and hypothalamus), spleen, lung, heart, bone marrow, and thymus. RT-PCR also revealed expression in brain glial cells and prominent expression in neutrophils, lymphocytes and megakaryocytic cells (Chambers et al., 2000; Moore et al., 2003). P2Y₁₄ receptor mRNA was high in immature monocyte-derived and low in mature monocyte-derived dendritic cells, suggesting a role for the receptor and its agonists in dendritic cell activation (Skelton et al., 2003).

Antiserum to a sequence in the first extracellular domain of P2Y₁₄ was used to isolate a population of hematopoietic cells restricted to bone marrow. Conditioned media from bone marrow stroma induce receptor activation and chemotaxis, suggesting a role for P2Y₁₄ as a chemoattractant receptor (Lee et al., 2003a). Antibody against the carboxyl terminus of the P2Y₁₄ receptor was used to demonstrate broad distribution in postmortem human brain, with glial cells as the primary site of expression, suggesting a role for this receptor in neuroimmune function (Moore et al., 2003). In the rat, P2Y₁₄, which is abundantly expressed in brain, has been reported to be regulated by immunological challenge (Charlton et al., 1997; Moore et al., 2003), suggesting that this receptor may link the humoral and nervous system responses to infection and inflammation. Consistent with this result, exposure of murine N9 cells to LPS resulted in a highly significant increase of receptor function, as suggested by the increase in the percentage of cells responding to UDP-glucose with intracellular calcium transients (Bianco et al., 2005).

For more information on the P2Y₁₄ receptor, see Table 12.

A. Excitable Cells, Nerves, Glial Cells, and Muscle

P2Y₁ receptors are widespread in many regions of the brain, whereas the P2Y₂ receptors have been localized on pyramidal neurons in the hippocampus and prefrontal cortex, on supraoptic magnocellular neurosecretory neurons in the hypothalamus, and on neurons in the dorsal horn of the spinal cord. In addition, mRNA but not protein has been reported for P2Y₄ and P2Y₆ receptor subtypes in the cerebellum and hippocampus, whereas P2Y₁₂ receptor mRNA has also been described in the cerebellum and P2Y₁₃ in the cortex. In the periphery, P2Y_1,2,4,6 receptors on subpopulations of sympathetic neurons, P2Y₂ and P2Y₄ receptors in intracardiac ganglia, and P2Y₁ and P2Y₂ receptors on sensory neurons (although P2Y₄ and P2Y₆ mRNA have also been reported) have been described, whereas P2Y₁ receptors appear to be the dominant subtype on enteric neurons.

P2Y_1,2,4,6 functional receptors have been found on astrocytes in the central nervous system (Neary et al., 1994, 1996, 1999, 2003; Bolego et al., 1997; Centemeri et al., 1997; Fumagalli et al., 2003) and also on microglia where functional P2Y₁₂ receptors have also been identified (for review, see Abbracchio and Verderio, 2006). P2Y₁ and P2Y₂ receptors have been located in Schwann cells and oligodendrocytes, in which functional P2Y₁₂ receptors also appear to be present. P2Y₂ (and/or P2Y₄) receptors are expressed on enteric glial cells. There is also emerging evidence for P2Y receptors on stem cells.

All cloned P2Y receptors have been found in both healthy and failing human hearts (Banfi et al., 2005) to support functional roles in myocardial function (Vassort, 2001). Whereas the dominant P2 receptor subtype on smooth muscle is P2X₁, P2Y receptor subtypes are also present, notably P2Y₁ and/or P2Y₂ receptors on visceral smooth muscle and P2Y_1,2,4,6 receptors on vascular smooth muscle. P2Y₁ receptors are present in developing skeletal muscle with evidence for P2Y₂ receptors on the myotube C2C12 cell line.

B. Immune Cells

ATP stimulates production of prostaglandin and has mitogenic actions on thymocytes largely via P2Y₂ receptors, although P2Y₁ receptor mRNA is also present. P2Y_2,6,11,13 receptor mRNAs have been identified in whole spleen.

P2Y₂ receptors are the dominant subtype in macrophages, although the presence of P2Y_1,4,12 receptors on alveolar macrophages and P2Y₁₁ receptors on human macrophages has also been noted. ATP and UTP acting via P2Y₁ and P2Y₂ receptors promote adhesion of neutrophils to endothelial cells.

P2Y_1,2,4,6,11 receptor mRNA have been identified with RT-PCR in both eosinophils and lymphocytes. Several P2Y subtypes are expressed in human monocyte-derived dendritic cells, in particular the P2Y₁₁ receptor that mediates the semimaturation of these cells in response to ATP (Wilkin et al., 2001; La Sala et al., 2002; Schnurr et al., 2003).

P2Y_1/2/4/6 receptors have been identified in monocytes, and UDP leads to production of IL-8 (Warny et al., 2001). ATP action via P2Y₁ and P2Y₂ receptors leads to degranulation and release of histamine from mast cells as well as cell migration and chemoattraction.

C. Endocrine, Adipose, and Exocrine Cells

The P2Y₂ receptors are dominant in the anterior pituitary in which they modulate prolactin release, and P2Y₁ receptors have been identified in the pineal gland. P2Y₂ receptors mediate regulation of aldosterone secretion in the adrenal gland. In the thyroid gland, P2Y_2,4,6 receptors mediate cell proliferation, whereas P2Y₂ receptors are involved in insulin release from pancreatic islet β cells.

P2Y₂ receptors are present in testicular Sertoli and in Leydig cells involved in estradiol and testosterone secretion. P2Y₂ receptors are expressed in ovary and placenta. P2Y₂ receptors mediate antagonism of estradiol and progesterone secretion from granulosa cells in the ovary.

Brown adipocytes express P2Y_1,2,4 receptors involved in regulation of lipogenesis. P2Y₂ receptors involved in regulation of ionic balance are dominant in salivary and lachrymal glands.

In sweat glands and exocrine pancreas, P2Y_1,2,4 receptors are present. They have been implicated in regulation of secretion.

D. Gut, Liver, and Biliary System

P2Y₁ and P2Y₁₁ receptors appear to mediate the purinergic component of nonadrenergic, noncholinergic relaxation of smooth muscle in the gastrointestinal tract, whereas P2Y_1,2,4,6 receptors on gut epithelial cells may mediate ion secretion, although P2Y₄ seems to play a major role, as demonstrated in knockout mice. P2Y₁ receptors have also been identified in intrinsic enteric neurons in both myenteric and submucosal plexuses. P2Y_1,2,4,6,13 receptors have been described in hepatocytes and may serve to regulate gluconeogenesis and glycolysis. P2Y_2,4,6 receptors are present on bile duct epithelium; in addition, P2Y₁ receptor mRNA is present in gallbladder epithelium. UTP has been show to stimulate Cl^- secretion and modulate bile release.

E. Kidney and Bladder

P2Y receptors are richly expressed in all regions of the kidney tubule and glomerulus. P2Y_1,4,6 receptors in the proximal convoluted tubule and loop of Henle are involved in reabsorption of water, ions, and nutrients. The P2Y₂ receptor is dominant in the distal convoluted tubule concerned with secretion of ions, acids, and toxins whereas P2Y_1,2,4,6 receptors appear to be involved in transport of water and ions in the collecting duct. In the glomerulus, P2Y_1,2,4,11,12 subtypes are all expressed on mesangial cells, P2Y_1,2,6 on podocytes, and P2Y₂ on endothelial cells. In rat bladder, ATP exerted an immediate and transient contraction, followed by a slower sustained relaxation, which was completely abolished by the G protein blocking agent, guanosine 5′-O-(2-thiodiphosphate), suggesting a functional role for P2Y receptors (Bolego et al., 1995). Evidence for purinergic functional regulation of human urinary bladder, which may have potential therapeutic implications for human incontinence, has been also provided (Palea et al., 1993).

F. Lung

P2Y₂ and P2Y₆ receptors on cultured smooth muscle cells from the lung induce an increase and a decrease in smooth muscle proliferation, respectively. P2Y₂ receptors are the predominant receptor subtype in lung epithelial cells and are involved in mucin secretion and mucociliary clearance. In addition, P2Y₁ and P2Y₆ receptor subtypes have been identified on respiratory epithelium cell lines.

G. Bone and Cartilage

P2Y₂ receptors on osteoblasts mediate inhibition of bone formation, whereas ADP acting via P2Y₁ receptors on osteoclasts increase bone resorption. Chondrocytes express P2Y₁ and P2Y₂ receptors, which mediate cartilage resorption and prostaglandin production.

H. Skin

P2 receptors play a major role in cell turnover of keratinocytes in stratified epithelium. P2Y₁ and P2Y₂ receptors modulate cell proliferation in basal cells.

I. Endothelial Cells

P2Y₁ and/or P2Y₂ receptors are dominant on vascular endothelial cells, where they mediate release of NO and subsequent vasodilatation as well as cell proliferation and the expression of adhesion proteins for monocytes. In some blood vessels, P2Y_4,6,11,12 receptors have also been found.

J. Special Senses

In the eye, P2Y₂ receptors mediate trophic events in the retina and cornea, whereas P2Y₁ receptors have been implicated in regulation of fluid secretion in the ciliary body. In the inner ear, P2Y receptors have been implicated in cochlear function, particularly P2Y₂ and P2Y₄ receptors in vestibular dark cells and stria vascularis marginal cells. P2Y receptors also appear to be involved in olfactory function, especially P2Y₂ and P2Y₆ receptors on nasal epithelium.

K. Platelets

Platelets express three nucleotide receptors: the P2X₁ cation channels activated by ATP, and two GPCRs, P2Y₁ and P2Y₁₂, both activated by ADP, which perhaps represent the most studied P2Y receptors in a native system (see also above). Each of these receptors has a selective role during platelet activation (Hechler et al., 2005), which has implications for their role in thrombosis (Gachet and Hechler, 2005). We will not expand here on the role of the P2X₁ receptor, which is not negligible, being involved in platelet activation by collagen and in the thrombosis of small arteries (Hechler et al., 2003; Mahaut-Smith et al., 2004). However, its deficiency does not induce a defect in normal hemostasis and does not modify the platelet responses to ADP. ADP-induced platelet aggregation is under the control of P2Y₁ and P2Y₁₂. Coactivation of both receptors is necessary for normal ADP-induced platelet aggregation since separate inhibition of each of them by selective antagonists results in dramatic inhibition of aggregation (Jin and Kunapuli, 1998; Gachet, 2001; Hechler et al., 2005).

A. Basal Unstimulated Nucleotide Release

B. ATP Release by Excitable and Secretory Tissues

Exocytosis of ATP-containing vesicles occurs in neurons, platelets, adrenal medullary chromaffin cells, neuroendocrine cells, and mast cells. Neuronal release of nucleotides was first identified 50 years ago by Holton and Holton (1954) and is now one of the best-understood sources of extracellular nucleotides (Bodin and Burnstock, 2001a). ATP and probably other nucleotides are stored at high concentrations in dense granules in platelets, synaptic vesicles in neurons, and chromaffin granules in adrenal glands. The concentration of nucleotides in these vesicles could reach up to 150 to 1000 mM. Stimulation of these cells results in the calcium-dependent fusion of these vesicles with the plasma membrane and the release of vesicular contents into the extracellular space. The specific mechanisms of exocytotic granule release have been extensively investigated (Sperlágh and Vizi, 1996).

C. ATP Release by Nonexcitatory Cells

Extracellular nucleotides are released by multiple nonexcitatory cell types such as endothelial and epithelial cells, smooth muscle cells, fibroblasts, astrocytes, red blood cells, lymphocytes, monocytes, and a number of transformed cell lines. The release of nucleotides by nonexcitatory cells is not completely understood and involves the participation of multiple mechanisms. No evidence for the existence of specialized ATP-containing vesicles in most of these cells has been demonstrated, and, therefore, alternative mechanisms for the release of nucleotides have been postulated. These mechanisms are outlined below in the context of 1) stress/hypoxia/mechanical stimulation, 2) vesicular trafficking, and 3) agonist-promoted stimulation.

D. ATP Release by Tissue Damage

Cell lysis or tissue damage results in the release of the intracellular nucleotide contents into the extracellular space providing nucleotide concentrations sufficient to activate nucleotide receptors. This source of extracellular nucleotides accounts for only some of the known physiological actions of ATP, including transmission of nociceptive signals and onset of inflammation.

E. Extracellular Nucleotide Metabolism

The physiological effect of extracellularly released nucleotides depends mainly on the particular receptor subtype(s) expressed in the tissue. In addition, an important modulatory effect of the physiological response is imposed by the presence of soluble and membrane-bound ecto-nucleotidases that rapidly metabolize the nucleotides with the potential to terminate or initiate receptor-specific signaling cascades.

Extracellular ecto-nucleotidases are expressed in most if not in all cell types. Several families of ectoenzymes participate in the degradation and interconversion of extracellular nucleotides. The most prominent enzyme classes are the ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase) family, the ecto-nucleotide pyrophosphatase/phosphodiesterase (E-NPP) family, ecto-5′-nucleotidase, alkaline phosphatase, the nucleotide-converting enzyme ecto-NDPK, and adenylate kinase. Ecto-nucleotidases exist as membrane-associated forms or soluble forms. Some of the soluble ecto-nucleotidases are produced by the proteolytic cleavage of the extracellular domains of membrane-bound forms or by the release from a glycosylphosphatidylinositol-anchoring phospholipid. The characterization of the ecto-nucleotidases has uncovered significant overlapping substrate specificities and tissue expression (Zimmermann, 2000; Goding et al., 2003).

A. Modes of Interaction between G Protein-Coupled Receptors

Individual GPCRs were long considered to preferentially activate specific intracellular signaling pathways and so act in a linear fashion to produce a change in cellular activity. However, it is now clear that they can also modulate the signals initiated by another GPCR to potentiate or inhibit the activity of these receptors. Such interactions can take place at the level of the GPCR themselves, through the formation of oligomers, or downstream of the receptor through the action of second messengers. The former process is commonly referred to as receptor dimerization and is characterized by the appearance of a physical receptor complex as demonstrated by coimmunoprecipitation or other methods, which displays novel pharmacological properties and/or interactions with second messenger systems. The latter process, known as receptor cross-talk, can have either positive or negative effects and serves to integrate coincident signals from multiple types of receptors, which are not physically associated.

B. Receptor Dimerization

Until recently, individual GPCRs were assumed to exist and function as monomers. However, in the mid-1990s it started to become clear that many GPCRs can coalesce into functional dimers or oligomers. Indeed, such aggregation may be the rule rather than the exception and may be essential for the correct trafficking and membrane expression of GPCRs (Bouvier, 2001; Milligan, 2004). At present, it is difficult to differentiate experimentally between dimers and oligomers and so the term dimer tends to be used. Within a receptor family, individual subtypes can form homodimers or different subtypes can form heterodimers. An increasing number of combinations are also being described for receptors from totally different families. As GPCRs are already major therapeutic targets, dimerization has important implications for the development of new drugs.

There is also evidence that the human P2Y₂ receptor forms homodimers. Expression in cell lines of constructs in which the receptor was tagged at the C terminus with the cyan or yellow variants of green fluorescent protein produced functional receptors that were activated by UTP and which coupled to endogenous G proteins to induce release of Ca²⁺ stores (Kotevic et al., 2005). When both variants were coexpressed in the same cells, fluorescence resonance energy transfer (FRET) imaging indicated that they were in close proximity, i.e., they formed constitutive receptor complexes. It was notable that UTP did not change the FRET signal, indicating that binding of an agonist does not induce dissociation of the oligomeric complex. Further studies are required to determine whether other P2Y subtypes similarly form homodimers.

To date, the interaction between the rat P2Y₁ receptor and adenosine A₁ receptor is the only example of dimerization involving P2Y receptors with non-P2Y receptors that has been characterized extensively. Yoshioka et al. (2001) coexpressed the rat P2Y₁ and adenosine A₁ receptors in HEK293 cells. Initial experiments showed that these receptors coimmunoprecipitated in Western blots of whole cell membrane lysates, indicating that they formed a heteromeric complex. Coexpressing the P2Y₁ receptor did not alter surface expression of the A₁ receptor, but it did inhibit the binding of radiolabeled A₁ agonists and antagonists in membrane preparations. This change was not seen in a mixture of membranes from cells expressing each receptor individually. Additionally, the binding of an A₁ agonist was displaced by the P2Y₁ agonist ADPβS and P2Y₁ antagonist MRS2179 in cotransfected cells, but not in cells expressing the A₁ receptor only. These data again indicate formation of a heteromeric complex.

A₁ receptors couple to G_i and so mediate depression of intracellular cAMP levels, whereas P2Y₁ receptors interact with G_q/11 and have no effect on cAMP. ADPβS inhibited cAMP production in cotransfected cells only, an effect that was antagonized by the A₁ antagonist 8-cyclopentyl-1,3-dipropylxanthine, but not by MRS2179, and abolished by pertussis toxin. Thus, ADPβS appears to have acted via the A₁ receptor ligand-binding site, i.e., the P2Y₁/A₁ dimer has novel pharmacological properties compared with the parent receptors. Interestingly, although ADPβS induced inositol phosphate synthesis, the A₁ agonist cyclopentyl adenosine (CPA) did not. Thus, dimerization did not lead to a complete change in pharmacological properties in this case.

Using confocal laser microscopy to study the subcellular distribution of the P2Y₁ and A₁ receptors, Yoshioka et al. (2001) showed that both were expressed mainly near the plasma membrane of HEK293 cells. Furthermore, there was a strong overlap in their distribution in individual cells. This was confirmed in a subsequent study using the biophysical technique of bioluminescence resonance energy transfer (Yoshioka et al., 2002b). In the absence of agonists, the receptors showed a homogeneous colocalization across the cells. Addition of ADPβS and CPA together, but not alone, induced an increase in the bioluminescence resonance energy transfer ratio over 10 min. Thus, although the receptors have a constitutive association, their coactivation increased the association. This association was also seen with native receptors in central neurons. Using confocal laser microscopy and double immunofluorescence, Yoshioka et al. (2002a) demonstrated that the P2Y₁ and A₁ receptors colocalized in neurons of the rat cortex, hippocampus, and cerebellum. A direct association was then shown by their coimmunoprecipitation in membrane extracts from these regions.

Together, these studies clearly indicate that the rat P2Y₁ and A₁ receptors physically interact to form a functional dimer with novel pharmacological properties. The structural requirements for this interaction are not known at present. The physiological roles of the P2Y₁/A₁ dimer also remain to be determined, although Nakata et al. (2003) have pointed out that its pharmacological properties resemble those of a presynaptic receptor that mediates inhibition of neurotransmitter release in some tissues. Finally, it is still not known whether other P2Y subtypes also form functional heterodimers, but this is likely to be the case as Yoshioka et al. (2001) reported that the rat P2Y₂ receptor also coimmunoprecipitated with the A₁ receptor when they were coexpressed in HEK293 cells. Thus, the formation of oligomers by P2Y receptors is likely to be widespread and to greatly increase the diversity of purinergic signaling.

Indeed, homodimers of the human P2Y₁ receptor have recently been shown to exist [Choi et al., 2005a (http://www.pa2online.org/abstracts/Vol3Issue2abst010P.pdf)]. Expressed receptors were labeled in culture, and FRET confocal microscopy was applied. This method can be used to report specifically on the receptors located at the cell membrane and measure the percentage dimerized. Constitutive P2Y₁ homodimers were found; dimerization was increased by exposure to agonist to 80 to 90%. An obligate determinant of the dimer formation resides in the tail of the sequence. P2Y₁ receptor agonist-induced internalization is preceded by the dimerization [Choi et al., 2005b (http://www.pa2online.org/abstracts/Vol3Issue2abst121P.pdf)].

C. Receptor Cross-Talk

The ability of GPCRs to modulate the signals initiated by other GPCRs, receptor tyrosine kinases and ligand-gated ion channels is widespread and has been studied extensively (Selbie and Hill, 1998; Hur and Kim, 2002). A variety of mechanisms can underlie these interactions, and here we will discuss a few examples of P2Y receptor-mediated cross-talk with these three different types of receptors.

A. Scope of the Gene Activations

GPCRs possess the important capability of maintaining signal transduction processes over extended periods where needed, allowing some transductions to proceed to the level of nuclear gene regulation and protein synthesis. Nearly all the cases for which this is known involve G_s-linked receptors, through their activation of the cAMP response element-binding transcription factor (CREB). Only one G_s-linked P2Y receptor is known: P2Y₁₁ (see section VI.E.); its responses in dendritic cells of the immune system (Wilkin et al., 2001; Marteau et al., 2004, 2005) suggest that it is likely there to promote transcription through the CREB pathway, since the activation of P2Y₁₁ led to production of specific cytokines via a cAMP increase. Furthermore, another study (van der Weyden et al., 2000a) made in renin-expressing cells has shown that activation of the P2Y₁₁ receptor therein increased renin mRNA and protein 3-fold over 36 h and gave strong luciferase-reporter activity when linked to a renin gene construct. This response was established to be through the CREB pathway, since the P2Y₁₁ activation stimulated CREB phosphorylation and its effects were blocked by mutation at the cAMP response element within the renin gene promoter.

There is a small amount of definitive information on gene transcription control by identified G_q-orG_i/o-linked P2Y receptors. One such case is represented by the P2Y₂ receptor. When stably expressed in 1321N1 cells, this receptor was found to signal through the p38 MAPK cascade to phosphorylate CREB, which then mediated cis-activation of target genes, including the antiapoptotic bcl-2 and bcl-xl genes (Chorna et al., 2004). UTP incubation also up-regulated expression of a range of genes for neurotrophins and neuropeptides and induced proliferation of the astrocytoma cells. It is interesting that P2Y₁ behaves differently from P2Y₂ in the same cells. In a study performed with the P2Y₁ receptor therein (Sellers et al., 2001), agonist treatment led, via a different intracellular cascade, to the activation instead of the transcription factor Elk-1. The outcome of that transduction route was apoptosis and inhibition of cell proliferation. In another study performed on rat astrocytes, activation of a native PTX-sensitive P2Y-like receptor increased the binding to DNA of the activator protein-1 and NF-κB transcription factors (Brambilla et al., 2003). The outcome in that case was the de novo synthesis of cyclooxygenase-2 (confirmed at the protein level), whose gene transcription has been shown to occur through these factors (Brambilla et al., 1999, 2000, 2002, 2003), followed by elongation of astrocytic processes, an index of reactive astrogliosis.

B. Synaptically Released ATP Can Act in the Control of Gene Transcription

At many synapses, ATP is stored and released with another transmitter throughout life; hence, it has the potential to serve as an additional nerve-released trophic factor, which would imply its regulation of gene transcription. This possibility has recently been explored using initially the neuromuscular junction (NMJ) of skeletal muscles; this type of synapse has the greatest accessibility and uniformity in its population and is currently the best understood of all types in structure, operation, and synaptogenesis. ATP is costored in vesicles of the nerve terminals with ACh and is coreleased quantally with it (at a ratio of approximately 1 ATP to 6 ACh) (Silinsky and Redman, 1996). Also, UTP is present in those vesicles at ∼10% of the ATP content (Zimmermann, 1994).

Functional postsynaptic P2Y₁ and P2Y₂ receptors colocalized at the NMJs with the nicotinic ACh receptors (AChRs) have been demonstrated in mammalian, chicken, and amphibian muscles (Choi et al., 2001; Tsim et al., 2003; Tung et al., 2004). There is earlier phenotypic evidence that muscle AChR and acetylcholinesterase (AChE) have some controls of their expression and clustering at the NMJ in common (Sanes and Lichtman, 1999); hence, the genes of both those effectors have been studied (Choi et al., 2001, 2003), using differentiated myotubes when the P2Y receptors are present throughout the cell surface before being clustered at nerve contact when in vivo. Exposure to 2-MeSADP or to UTP each produces an activation of the genes of the multiple subunits of the AChR and also of the AChE catalytic subunit gene. Properties of the gene activations mediated by the native P2Y₁ and P2Y₂ receptors in the muscle cells (Choi et al., 2001; Tung et al., 2004) are 1) a strong increase (up to 550%) in each mRNA (confirmed at the protein level where tested), related to the concentration of the P2Y agonists; 2) a dose-dependent agonist-induced increase in the activity of a promoter-luciferase reporter construct for each of the subunit genes; 3) approximate equivalence for the P2Y₁ and P2Y₂ receptor activities in stimulating the promoter, for each gene (one example is shown in Fig. 8C); 4) a total block of the P2Y₁-coupled action at the gene promoters by the specific P2Y₁ antagonist MRS2179 (Fig. 8C) or by suramin for both receptors; and 5) a great increase in the 2-MeSADP-activated promoter activities of the AChR genes when the P2Y₁ receptor protein content was boosted by a transfection of its cDNA.

Examination of several potential transcription factors for this action has identified Elk-1 as a candidate, which becomes maximally phosphorylated and nuclear-localized when myotubes are exposed for 30 min to 2-MeSADP or UTP. Four potential DNA binding sequences for Elk-1 were identified in the first intron of the AChE gene (Fig. 8A). Two were proven to be active by gel mobility shift assays, and each of these sites was used in the reporter assay, replacing the entire 2.2-kb DNA of the promoter-containing region by a 20-nucleotide Elk-1 binding site. Each again showed AChE gene promoter activity dependent on P2Y₁ or P2Y₂ receptor activation on the myotube (Fig. 8B). This was enhanced when the two sites were combined. In confirmation of their activity in the native gene, when the entire 2.2-kb promoter-containing DNA was mutated at six bases in those two sites, its P2Y₁/P2Y₂-mediated promoter activity was abolished.

The significance of the location at the synapse of two P2Y receptors that signal to the transcription of genes encoding effectors of the postsynaptic machinery should be considered in the context that a few subsynaptic nuclei at each junction become transcriptionally specialized during skeletal muscle development to sustain the local synthesis of the operational proteins of the junction, including the postsynaptic AChR and AChE (Duclert and Changeux, 1995). The P2Y₁ and P2Y₂ receptors at the synapse are thus part of a local circuit allowing efficient genetic control. ATP would not be the sole regulator of the expression of the AChR and AChE genes at the NMJ, since the neurally released trophic protein neuregulin is another major one, which also has its receptor, the ErbB protein, at the postsynaptic NMJ membrane (Fischbach and Rosen, 1997) and signals through a transduction cascade to activate a specific transcription factor in the nearby subsynaptic nuclei (Schaeffer et al., 2001). There is some evidence for an equivalent cascade route for the P2Y₁ receptor there (Choi et al., 2003), but this is not known fully yet, nor for the P2Y₂ receptor at this location. The primary action of neuregulin is in the development of the NMJ, although it later contributes to the maintenance of the NMJ (Buonanno and Fischbach, 2001). It is a protein secreted in low amount, and it may be that the constant large quantity of ATP released is also needed for an additional role in the maintenance, specifically, of synaptic AChRs and AChE.

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Fig. 8.

Examples of analysis of gene activations controlled through P2Y receptor action. A, promoter region of the gene for the catalytic subunit of AChE. The first exon is noncoding, within the 5′-untranslated region. Promoter sites lie on either side of that, illustrated for four of the transcription factors active in muscle cells. Shown are two of the four predicted sites there for the transcription factor Elk-1 (Elk-1 [1] and Elk-1 [3]), which were identified as specific Elk-1 binding sites by gel mobility shift analyses on extracts of myotube nuclei. DNA sequences at those sites were inserted upstream in a luciferase reporter vector for assays as in B. B, two reporter constructs controlled by the specific binding sites for Elk-1 from the AChE gene promoter are used to show that Elk-1 is activated when the P2Y₁ or P2Y₂ receptors in a muscle cell are stimulated; hence, Elk-1 binds to activate transcription of the AChE gene there. Mouse myotubes transfected with either construct were incubated at 37°C with the agonists shown, or with none (Basal), or with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), which produces a nonspecific activation of AChE synthesis in muscle cells. Both Elk-1 sites are seen to have receptor-stimulated promoter activity (and when combined in tandem they are synergistic: not shown). Elk-1 is activated by specific phosphorylation through P2Y₁ (2-MeSADP) or P2Y₂ (UTP) action. The effect of ATP is significantly higher than effects for the other two (with all three at a saturating concentration), since it acts at both subtypes. C, similar analysis performed using the promoter region of the ϵ subunit of the nicotinic AChR gene. Both the P2Y₁ and P2Y₂ receptors act through Elk-1 in activating the multiple genes for the AChR in muscle cells (α, δ, and ϵ subunit genes have been tested). Note that in confirmation, the P2Y₁-specific antagonist MRS2179 blocks this action by 2-MeSADP but not that by UTP (data from Choi et al., 2003; Tung et al., 2004).

In conclusion, for some P2Y receptors a contribution to gene transcription control over a longer time frame may commonly be a role. Synapses elsewhere should be examined for a situation similar to that at the NMJ. In short-term phenotypic analyses, it may be difficult to recognize all of the functions of those receptors. Cases in which a P2Y gene knockout seems to have no obvious effect, other than where that is shown to be by compensation by another subtype, may be in this category.