title: Quantification of Gi-mediated inhibition of adenylyl cyclase activity reveals that UDP is a potent agonist of the human P2Y14 receptor.
abstract: The P2Y14 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 P2Y14 receptor, allowing facile examination of its coupling to native Gi family G proteins and their associated downstream signaling pathways (J Pharmacol Exp Ther 330:162-168, 2009). In the current study, we examined P2Y14 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 P2Y14 receptor activated by UDP-glucose, but it also was activated by UDP. The apparent efficacies of UDP and UDP-glucose were similar, and the EC50 values (74, 33, and 29 nM) for UDP-dependent activation of the P2Y14 receptor in HEK293, CHO, and C6 glioma cells, respectively, were similar to the EC50 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 P2Y14 receptor-expressing HEK293 cells but not in wild-type HEK293 cells. A series of analogs of UDP were potent P2Y14 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 P2Y14 receptor over the UDP-activated P2Y6 receptor, and these molecules stimulated phosphorylation of ERK1/2 in differentiated human HL-60 promyeloleukemia cells, which natively express the P2Y14 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 P2Y14 receptor.
authors.affiliation: Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, NC, USA.
authors.forename: Rhonda L
authors.initials: RL
authors.lastname: Carter
authors.forename: Ingrid P
authors.initials: IP
authors.lastname: Fricks
authors.forename: Matthew O
authors.initials: MO
authors.lastname: Barrett
authors.forename: Lauren E
authors.initials: LE
authors.lastname: Burianek
authors.forename: Yixing
authors.initials: Y
authors.lastname: Zhou
authors.forename: Hyojin
authors.initials: H
authors.lastname: Ko
authors.forename: Arijit
authors.initials: A
authors.lastname: Das
authors.forename: Kenneth A
authors.initials: KA
authors.lastname: Jacobson
authors.forename: Eduardo R
authors.initials: ER
authors.lastname: Lazarowski
authors.forename: T Kendall
authors.initials: TK
authors.lastname: Harden
mesh_terms: D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000067956:Adenylyl Cyclase Inhibitors; D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology; D006224:Cricetinae; D003412:Cricetulus; D004789:Enzyme Activation / Q000187:drug effects; D048049:Extracellular Signal-Regulated MAP Kinases / Q000096:biosynthesis; D019204:GTP-Binding Proteins / Q000502:physiology*; D018922:HL-60 Cells; D006801:Humans; D058910:Purinergic P2 Receptor Agonists; D018048:Receptors, Purinergic P2; D015398:Signal Transduction / Q000187:drug effects; D014530:Uridine Diphosphate / Q000494:pharmacology*; D014532:Uridine Diphosphate Glucose / Q000494:pharmacology
chemical_list: D000067956:Adenylyl Cyclase Inhibitors; C518528:P2Y14 receptor, human; D058910:Purinergic P2 Receptor Agonists; D018048:Receptors, Purinergic P2; D005576:Colforsin; D014530:Uridine Diphosphate; D048049:Extracellular Signal-Regulated MAP Kinases; D019204:GTP-Binding Proteins; D015056:1-Methyl-3-isobutylxanthine; D014532:Uridine Diphosphate Glucose