Hamel Martine, Henault Martin, Hyjazie Huda, Morin Nicolas, Bayly Christopher, Skorey Kathryn, Therien Alex G, Mancini Joseph, Brideau Christine, Kargman Stacia
Department of Biochemistry and Molecular Biology, Merck Frosst Canada, Inc., Kirkland, Quebec, Canada.
J Biomol Screen. 2011 Oct;16(9):1098-105. doi: 10.1177/1087057111415525. Epub 2011 Aug 5.
P2Y14 is a member of the pyrimidinergic GPCR family. UDP-Glc has been previously shown to activate human P2Y14, whereas UDP was unable to activate the receptor. In this study, the authors used conventional and nonconventional methods to further characterize P2Y14 and its ligands. Conventional calcium mobilization and nonconventional cellular impedance functional assays revealed that UMP and UDP selectively activated HEK cells coexpressing P2Y14 and Gα(qi5). In the impedance assays, the presence of exogenous Gα(qi5) resulted in agonist-induced Gq signaling, whereas in the absence of exogenous Gα(qi5), the signal was indicative of Gi. The authors established the first P2Y14 membrane filtration binding assay using a novel optimized expression vector and [(3)H]UDP as radioligand. UDP-Glc, UMP, and UDP dose dependently inhibited [(3)H]UDP binding in the binding assay, and saturation analysis revealed that UDP bound P2Y14 with a K(D) = 10 nM and a B(max) = 110 pmol/mg. The authors screened a phosphonate library and identified compound A, which inhibited UDP-Glc-mediated calcium signaling in the fluorometric imaging plate reader assay (IC(50) = 2.3 µM) and competed for [(3)H]UDP binding in the novel binding assay with a K(i) = 1280 nM.
P2Y14是嘧啶能G蛋白偶联受体（GPCR）家族的一员。先前已证明UDP-葡萄糖（UDP-Glc）可激活人P2Y14，而UDP无法激活该受体。在本研究中，作者使用传统和非传统方法进一步表征P2Y14及其配体。传统的钙动员和非传统的细胞阻抗功能测定表明，尿苷一磷酸（UMP）和UDP选择性激活共表达P2Y14和Gα（qi5）的人胚肾（HEK）细胞。在阻抗测定中，外源性Gα（qi5）的存在导致激动剂诱导的Gq信号传导，而在没有外源性Gα（qi5）的情况下，信号表明是Gi。作者使用新型优化表达载体和[³H]UDP作为放射性配体建立了首个P2Y14膜过滤结合测定法。在结合测定中，UDP-Glc、UMP和UDP剂量依赖性地抑制[³H]UDP结合，饱和分析表明UDP以K(D)=10 nM和B(max)=110 pmol/mg的亲和力结合P2Y14。作者筛选了一个膦酸酯文库并鉴定出化合物A，其在荧光成像板读数器测定中抑制UDP-Glc介导的钙信号传导（IC(50)=2.3 µM），并在新型结合测定中以K(i)=1280 nM竞争[³H]UDP结合。
