Effect of docosahexaenoic acid, phorbol myristate acetate, and insulin on the interaction of the FFA4 (short isoform) receptor with Rab proteins.

Human embryonic kidney (HEK) 293 cells were co-transfected with plasmids for the expression of mCherry fluorescent protein-tagged FFA4 receptors and the enhanced green fluorescent protein-tagged Rab proteins involved in retrograde transport and recycling, to study their possible interaction through Förster Resonance Energy Transfer (FRET), under the action of agents that induce FFA4 receptor phosphorylation and internalization through different processes, i.e., the agonist, docosahexaenoic acid, the protein kinase C activator phorbol myristate acetate, and insulin. Data indicate that FFA4 receptor internalization varied depending on the agent that induced the process. Agonist activation (docosahexaenoic acid) induced an association with early endosomes (as suggested by interaction with Rab5) and rapid recycling to the plasma membrane (as indicated by receptor interaction with Rab4). More prolonged agonist stimulation also appears to allow the FFA4 receptors to interact with late endosomes (interaction with Rab9), slow recycling (interaction with Rab 11), and target to degradation (Rab7). Phorbol myristate acetate, triggered a rapid association with early endosomes (Rab5), slow recycling to the plasma membrane (Rab11), and some receptor degradation (Rab7). Insulin-induced FFA4 receptor internalization appears to be associated with interaction with early endosomes (Rab5) and late endosomes (Rab9) and fast and slow recycling to the plasma membrane (Rab4, Rab11). Additionally, we observed that agonist- and PMA-induced FFA4 internalization was markedly reduced by paroxetine, which suggests a possible role of G protein-coupled receptor kinase 2.

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