The P2Y 14 receptor was initially identified as a G protein-coupled receptor activated by UDP-glucose and other nucleotide sugars. We have developed several cell lines that stably express the human P2Y 14 receptor, allowing facile examination of its coupling to native G i family G proteins and their associated downstream signaling pathways (J Pharmacol Exp Ther 330: [162][163][164][165][166][167][168] 2009). In the current study, we examined P2Y 14 receptor-dependent inhibition of cyclic AMP accumulation in human embryonic kidney (HEK) 293, C6 glioma, and Chinese hamster ovary (CHO) cells stably expressing this receptor. Not only was the human P2Y 14 receptor activated by UDP-glucose, but it also was activated by UDP. The apparent efficacies of UDP and UDP-glucose were similar, and the EC 50 values (74, 33, and 29 nM) for UDP-dependent activation of the P2Y 14 receptor in HEK293, CHO, and C6 glioma cells, respectively, were similar to the EC 50 values (323, 132, and 72 nM) observed for UDP-glucose. UDP and UDP-glucose also stimulated extracellular signal-regulated kinase (ERK) 1/2 phosphorylation in P2Y 14 receptor-expressing HEK293 cells but not in wild-type HEK293 cells. A series of analogs of UDP were potent P2Y 14 receptor agonists, but the naturally occurring nucleoside diphosphates, CDP, GDP, and ADP exhibited agonist potencies over 100-fold less than that observed with UDP. Two UDP analogs were identified that selectively activate the P2Y 14 receptor over the UDP-activated P2Y 6 receptor, and these molecules stimulated phosphorylation of ERK1/2 in differentiated human HL-60 promyeloleukemia cells, which natively express the P2Y 14 receptor but had no effect in wild-type HL-60 cells, which do not express the receptor. We conclude that UDP is an important cognate agonist of the human P2Y 14 receptor.The metabotropic P2Y receptors include a subgroup of five receptors, the P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , and P2Y 11 receptors, that primarily signal through G q -activated signaling pathways and a subgroup of three receptors, the P2Y 12 , P2Y 13 , and P2Y 14 receptors, that primarily signal by activating heterotrimeric G proteins of the G i family (Abbracchio et al., 2006;Burnstock, 2007). The human P2Y 1 , P2Y 11 , P2Y 12 , and P2Y 13 receptors are activated by adenine nucleotides. The human P2Y 4 and P2Y 6 receptors are activated by uridine nucleotides, and the P2Y 2 receptor is activated by both ATP and UTP.

Background:PhoP is global regulator of Mycobacterium tuberculosis physiology. However, the role of phosphorylation of PhoP remains unknown. Results: PhoP activates complex lipid biosynthesis only upon phosphorylation. Conclusion: PhoP regulates lipid biosynthesis by a phosphorylation-dependent mechanism to contribute to morphology of the bacilli. Significance: This study sheds light on the unexplored role of phosphorylation of PhoP in regulating biosynthesis of lipids unique to M. tuberculosis.

The P2Y 14 receptor, a nucleotide signaling protein, is activated by uridine-5′-diphosphoglucose 1 and other uracil nucleotides. We have determined that the glucose moiety of 1 is the most structurally permissive region for designing analogues of this P2Y 14 agonist. For example, the carboxylate group of uridine-5′-diphosphoglucuronic acid proved to be suitable for flexible substitution by chain extension through an amide linkage. Functionalized congeners containing terminal 2-acylaminoethylamides prepared by this stratgegy retained P2Y 14 activity, and molecular modeling predicted close proximity of this chain to the 2nd extracellular loop of the receptor. In addition, replacement of glucose with other sugars did not diminish P2Y 14 potency. For example, the [5″] ribose derivative had an EC 50 of 0.24 μM. Selective monofluorination of the glucose moiety indicated a role for the 2″-and 6″-hydroxyl groups of 1 in receptor recognition. The β-glucoside was 2-fold less potent than the native α-isomer, but methylene replacement of the 1″-oxygen abolished activity. Replacement of the ribose ring system with cyclopentyl or rigid bicyclo[3.1.0]hexane groups abolished activity. Uridine-5′-diphosphoglucose also activates the P2Y 2 receptor, but the 2-thio analogue and several of the potent modified-glucose analogues were P2Y 14 -selective.

Uridine-5′-diphosphoglucose (UDPG) activates the P2Y14 receptor, a neuroimmune system GPCR. P2Y14 receptor tolerates glucose substitution with small alkyl or aryl groups or its truncation to uridine-5′-diphosphate (UDP), a full agonist human P2Y14 receptor expressed in HEK-293 cells. 2-Thiouracil derivatives displayed selectivity for activation of the human P2Y14 vs. the P2Y6 receptor, such as 2-thio-UDP 4 (EC50 1.92 nM at P2Y14, 224-fold selectivity vs. P2Y6) and its β-propyloxy ester 18. EC50 of β-methyl ester of UDP and its 2-thio analogue were 2730 and 56 nM, respectively. β-t-Butyl ester of 4 was 11-fold more potent than UDPG, but β-aryloxy or larger, branched β-alkyl esters, such as cyclohexyl, were less potent. Ribose replacement of UDP with a rigid North or South methanocarba (bicyclo[3.1.0]hexane) group abolished P2Y14 receptor agonist activity. α,β-Methylene and difluoromethylene groups were well tolerated at the P2Y14 receptor and are expected to provide enhanced stability in biological systems. α,β-Methylene-2-thio-UDP 11 (EC50 0.92 nM) was 2160-fold selective versus P2Y6. Thus, these nucleotides and their congeners may serve as important pharmacological probes for the detection and characterization of the P2Y14 receptor.