FIGURE 3.
Identification and characterization of a surrogate agonist for GPR84. A, effect of 6-OAU on the [35S]GTPγS binding assay. Membranes from Sf9 cells expressing human GPR84 (hGPR84), human GPR109A (hGPR109A), or OXER1 fused with bovine Gαi protein were incubated with 6-OAU for 1 h at room temperature. The data represent the means ± S.D. for quadruple determinations. B, effect of 6-OAU on GPR84 in the PI assay. HEK293 cells were transfected with human GPR84 or control vector, pcDNA3.1, in the presence of Gqi5 plasmid. Cells were incubated with various concentrations of 6-OAU or 3-OH-C12 for 2 h at 37 °C. The data represent the means ± S.D. for triplicate determinations. C, effect of 6-OAU on GPR40 in the PI assay. HEK293 cells were transfected with human GPR40 or control vector, pcDNA3.1. Cells were incubated with various concentrations of 6-OAU or C14 for 2 h at 37 °C. The data represent the means ± S.D. for triplicate determinations. D, receptor internalization assay. Internalization of stably expressing human GPR84-EGFP fusion protein in HEK293 cells was observed by fluorescence microscopy. Cells were treated without or with 0, 6.25, or 200 μm 6-OAU for 30 min.