Identification of the orphan G protein-coupled receptor GPR31 as a receptor for 12-(S)-hydroxyeicosatetraenoic acid - PubMed (original) (raw)

GPR31 transduces 12-(S)-HETE signaling. A, CHO cells transiently transfected with GPR31, HA epitope tagged-GPR31 or pcDNA were treated with 12-(S)-HETE (300 n

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). Cell lysates were immunoblotted with phosphospecific ERK1/2 antibody (_upper panel_s). Lower panels, nitrocellulose membranes were reprobed with ERK1/2. B, COS-7 cells were stably transfected with pcDNA and pNFκB-Luc, or GPR31 and pNFκB-Luc. Luciferase activity was measured after 12-(S)-HETE (300 n

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) treatment for 6 h. *, p < 0.05 (t test). C, CHO cells transiently transfected with GPR31 were treated with various concentrations of 12-(S)-HETE for 10 min in the presence or absence of pertussis toxin (PTx). ERK1/2 activation was measured. In a control, cells were treated with 300 n

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20-(S)-HETE. The ERK1/2 activation was quantitated by a densitometer (lower panel). D, NFκB activation was measured in COS-7 stably expressing GPR31 treated with various concentrations of 12-(S)-HETE. *, p < 0.05, versus no treatment control, t test. E, CHO cells were transiently transfected with GPR31, 5-oxoR, and HM74, together with pNFκB-Luc vector. Luciferase activity was measured after stimulation with or without 12-(S)-HETE. *, p < 0.05, versus GPR31-transfected cells without 12-(S)-HETE treatment, t test. F, PC3 cells stably transfected with pcDNA or GPR31 were treated with 12-(S)-HETE (300 n

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). Upper panel, lysates was probed with phosphospecific MEK antibody; lower panel, membrane was reprobed with MEK antibody. G, left panel, RT-PCR (upper) and real-time PCR quantitation (lower) of GPR31 in PC-3M cells stably transfected with different shRNA constructs. Fold change was normalized to PC-3M cells transfected with the # 5 construct (control empty vector). Right panel, PC-3M cells stably transfected with #5 (control empty vector) were stimulated with various concentrations of 12-(S)-HETE for 10 min. The activation of MEK, indicated by the intensity of phospho-MEK Western blot, was quantitated by a densitometer (lower). H, MEK activation was measured in PC-3M cells stably transfected with si-GPR31 #2 or #4 constructs following 12-(S)-HETE stimulation.