Extracellular purine and pyrimidine nucleotides act as signaling molecules through the activation of P2X ion channels and P2Y G protein-coupled receptors (GPCRs) (Abbracchio et al. 2006). Among the eight members of the P2Y receptor family, four respond to extracellular uracil nucleotides: P2Y2, P2Y4, P2Y6, and P2Y14 receptors. None of the P2X ion channels are substantially activated by uracil nucleotides.
Originally, Chambers et al. (2000) cloned a human GPCR designated GPR105 or KIAA0001 that was found to respond to uracil nucleotide sugars, such as UDPG, but lacked a response to most other nucleotides. Soon thereafter this sequence was designated the P2Y14 receptor, because it was recognized to belong structurally and functionally to the P2Y receptor family, which was previously known to be activated by various nucleotides other than UDPG.
Structure and Signaling Pathways
X-ray crystallographic structural information is now available for the closely related P2Y12 receptor (Zhang et al. 2014), and sequence analysis and modeling studies have been used to predict the recognition features of the P2Y14 receptor. The P2Y14 receptor has a sequence identity of about 47% to the P2Y12 receptor (human subtypes, Communi et al. 2001). There are four cysteine residues in the extracellular region of the P2Y14 receptor forming two possible disulfide bridges: One disulfide bridge is highly conserved in the family A of GPCRs and it connects the extracellular part of TM3 with the EL2; and the other putative disulfide bridge connects the N-terminus to the EL3. A recent homology model of the P2Y14 receptor (Trujillo et al. 2015) supports the hypothesis of a conserved binding mode of nucleotides in the three P2Y12-like receptors involving functionally conserved residues. Basic residues of TM6 (Arg253) and TM7 (Lys277), conserved among the P2Y12-like family, are predicted to coordinate the phosphate groups of nucleotides agonists. The glucose moiety of UDP-glucose docked consistently with known structure–activity relationship in a secondary subpocket.
In model functional studies of the P2Y14 receptor activities of nucleotide analogs, it has been convenient to use the recombinant human or rat homologue expressed in cell lines, such as COS, CHO, or HEK-293 cells (Fricks et al. 2008; Carter et al. 2009). Heterologous coexpression of a chimeric G protein that responds to Gi-coupled receptors allows the use of phosphoinositide hydrolysis as assay readout, but this also can affect the observed agonist efficacy. Cell lines in which a native P2Y14 receptor occurs are: RBL-2H3 mast cells, C6 glioma, and A549 and BEAS-2B epithelial cells (Gao et al. 2010; Muller et al. 2005). Synergistic signaling of the P2Y14 receptor has been proposed in combination with the P2Y6 receptor to promote various cell functions including activation of the MAP kinase isozymes Erk1/Erk2 (Harden et al. 2010).
Identification of the physiological functions of the P2Y14 receptor has been difficult to establish, and isolated reports of the involvement in immune function, differentiation, and chemokine release have appeared P2Y14R activation includes chemotaxis of neutrophils (Barret et al. 2013; Azroyan et al. 2015; Sesma et al. 2016). Extracellular release of 1 upon trafficking of glycoproteins to the plasma membrane has been demonstrated, suggesting its widespread role in signaling (Lazarowski et al. 2003). UDP-sugars are required for glycosylation of proteins in the endoplasmic reticulum (ER) and Golgi apparatus as they are trafficked to the surface and are present there in high concentrations and dependent on specific nucleotide-sugar transporters (Sesma et al. 2009). Thrombin enhanced UDPG release (Kreda et al. 2008), and the P2Y14 receptor was detected in platelets, but its role is undetermined. P2Y14 receptor has been shown to be a novel modulator of insulin secretion (Meister et al. 2014) where P2Y14 KO mice exhibited reduced glucose tolerance, impaired insulin secretion, and altered smooth muscle function in the GI tract and airways. In the rodent stomach, Bassil et al. (2009) used agonists and P2Y14 knockout (KO) mice to demonstrate differential effects of the P2Y14 receptor in contractility and gastric emptying.
The P2Y14 receptor has been studied in epithelial tissues. In cultured mouse uterine cells, UDPG triggered innate immunity through the production of inflammatory cytokines (Arase et al. 2009), but not all of the pharmacological observations were consistent with the P2Y14 receptor.
The P2Y14 receptor is endogenously expressed in RBL-2H3 mast cells (Gao et al. 2010), where it mediates degranulation, suggesting it as a potential novel therapeutic target for allergic conditions. P2Y14 receptor agonists also induced [35S]GTPγS binding to RBL-2H3 cell membranes and phosphorylation of MAP kinases: Erk1/Erk2, P38, and JNK. UDPG 1 and selective agonist MRS2690 2 concentration-dependently enhanced hexosaminidase release with EC50 values of 1,150 and 103 nM, respectively, which was blocked by pertussis toxin and significantly diminished by P2Y14 receptor-specific siRNA. UDP was a partial agonist in RBL-2H3 cells. The P2Y14 receptor was recently identified to be an early pivotal regulator in mesenchymal stem cell commitment and was shown to be active in adipogenic differentiation (Zippel et al. 2011).
In the central nervous system, P2Y14 receptors are associated with astrocytes. mRNA for the receptor is prominently expressed in immune cells including neutrophils, lymphocytes, and megakaryocytic cells. P2Y14 receptor expression in mouse microglial cells increases with age (Crain et al. 2009).
UDP also activates the P2Y6 receptor, and the relative degree of involvement of multiple native agonists of the P2Y14 receptor, various UDP-sugars that originate in a vesicular compartment and UDP, is still unclear. Nevertheless, this observation led to the synthesis of a variety of UDP analogues as selective agonists.
Structure–Activity Relationship (SAR)
Depending on the model examined, UDP has been reported to either be a full agonist, a partial agonist, or a competitive antagonist at the human P2Y14 receptor and a potent agonist at the rat P2Y14 receptor (Carter et al. 2009). In cellular assay systems utilizing a native G protein rather than a chimeric one, UDPG 1 (pIC50 6.5) and UDP 3 (pIC50 6.5) are both potent agonists of the human P2Y14 receptor.
The SAR of synthetic analogs of both UDP and UDPG has been probed at the P2Y14 receptor. The P2Y14 receptor appears to be one of the least permissive among P2Y receptors (Das et al. 2010). Most modifications of the nucleobase or ribose moieties abolished activity, but the glucose moiety was amenable to substitution with other sugars (e.g., UDP-galactose 4, pIC50 6.2). Stereochemistry of this moiety influenced potency, for example, the β-glucoside was only twofold less potent than the native α-isomer. One of the few modifications of the uracil ring possible was 2-thiol, which in the analog of UDPG (MRS2690, 2, pIC50 7.3) increased potency by sevenfold and prevented activation of the P2Y2 receptor. Stabilizing phosphonate groups have been introduced in analogs of UDP. For example, α,β-difluoromethylene-UDP, MRS2802 5 (pIC50 7.2) is inactive at the P2Y6 receptor and fully activates the human P2Y14 receptor. MRS2905 6 (pIC50 8.7) is >2,000-fold selective for the P2Y14 in comparison to the P2Y6 receptor.
The carboxylate group of uridine-5′-diphosphoglucuronic acid proved to be suitable for flexible substitution by chain extension through an amide linkage, leading to a high-affinity fluorescent agonist probe for the P2Y14 receptor (Kiselev et al. 2015).
Two classes of nonnucleotide antagonists of the P2Y14 receptor were recently identified in high throughput screening using a FLIPR-based calcium flux assay in HEK cells overexpressing the mouse or chimpanzee P2Y14 receptor. The dihydropyridopyrimidine derivative 7 is a P2Y14 antagonist with good oral bioavailability and potency (pIC50 8.0), but noncompetitive with UDP (Guay et al. 2011). The 4,7-disubstituted naphthoic acid antagonist 8 (PPTN, binding Ki values at the chimpanzee P2Y14 receptor of 1.9 nM) is competitive with UDP (Gauthier et al. 2011). 8 was also derivatized through its carboxylic acid as a prodrug 9 to greatly improve bioavailability in this hydrophobic series (Robichaud et al. 2011). 8 reduced inflammatory markers in sterile inflammation of kidney cells (Azroyan et al. 2015). 9 was derivatized with a functionalized chain at the piperidine nitrogen and coupled to a fluorophore to provide a high affinity fluorescent antagonist probe for the P2Y14 receptor that was used to screen new antagonists (Kiselev et al. 2015). A novel antagonist scaffold was reported, which included MRS4217 10 (Junker et al. 2016).
The P2Y14 receptor is a cell-surface signaling protein (G protein coupled) that is activated by extracellular uracil nucleotides and couples to inhibition of adenylyl cyclase and activation of many other enzymes, such as MAP kinases. It occurs in the immune cells, including T cells, dendritic cells, and mast cells and in various tissues, that is, placenta, adipose, stomach, intestine, spleen, lung, heart, and brain. In mast cells, this receptor promotes the release of inflammatory mediators, suggesting that it is a possible target for allergic diseases. Chemical modification of the native agonist uridine-5′-diphosphoglucose has led to novel, selective nucleotide agonists, and nonnucleotide antagonists have been derived by optimization of screening hits. UDP, which also activates the P2Y6 receptor, appears to be an endogenous P2Y14 agonist.
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