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. 2020 May;97(5):304–313. doi: 10.1124/mol.119.117697

Fig. 3.

Fig. 3.

Heterologous phosphorylation of FFA4-L. (A) Whole cell phosphorylation assays in cells stimulated with the downstream PKA activator forskolin (FSK) (1 µM, 20 minutes) or PKA inhibitor H89 (10 µM, 20 minutes) did not demonstrate differences in phosphorylation of FFA4-L vs. control. Significant increases in FFA4-L phosphorylation was seen in cells stimulated with the PKC activator PMA (1 µM, 20 minute) (228% ± 0.8% of control), an effect that was inhibited by the PKC inhibitor BIMII (10 µM, 20 minute) (91% ± 2.4%). A representative autoradiograph (upper) is shown while the graph depicts pooled data from four independent experiments (lower). (B) Stimulation of cells expressing FLAG-FFA4-L/H1R with histamine (10 μM, 10 minutes) significantly increased FFA4-L phosphorylation (187% ± 7.6% of control), an effect that was blocked by BIMII pretreatment (51.28% ± 6.9%) (upper left and lower). Stimulation of cells expressing FLAG-FFA4/β2AR with isoproterenol hydrochloride (1 μM, 10 minutes) had no effect on FFA4-L phosphorylation in the absence or presence of H89 (upper right). Representative autoradiographs are shown while the graph depicts pooled data from two (for β2AR) or four (for H1R) independent experiments. ***P < 0.001 vs. unstimulated control; †††P < 0.001 vs. histamine-stimulated condition. (C) DHA-stimulated FFA4-L phosphorylation (150% ± 3.3% of control) is blocked by BIMII pretreatment (87% ± 3%). A representative autoradiograph is shown while the graph depicts pooled data from four independent experiments. **P < 0.01 vs. unstimulated control; †P < 0.05 vs. the DHA-stimulated condition. (D) Pooled results from three independent experiments show that basal phosphorylation of FFA4-S is significantly higher than that of FFA4-L as are the effects of PMA and BIMII alone and in combination. ***P < 0.001 vs. complimentary group in FFA4-L. (E and F) Homologous and heterologous phosphorylation of FFA4-L are inhibited upon truncation of the C-terminal tail following Pro356. Phosphorylation is induced in WT FFA4-L upon agonism with DHA or treatment with PMA (E) and upon treatment of cells coexpressing H1R/WT-FFA4-L with histamine (F), but these effects are completely absent in cells expressing FFA4-L-Δ356. UNTR represents parallel experiments in untransfected cells.