Agonist-induced activation of human FFA1 receptor signals to extracellular signal-regulated kinase 1 and 2 through Gq- and Gi-coupled signaling cascades

Background: FFA1 is abundantly expressed in the liver, skeletal muscle, monocytes and nervous system, but is particularly abundant in pancreatic beta cells. It is widely believed that FFA1 exerts its regulatory roles in a variety of physiological and pathological functions. In response to oleic acid, FFA1 has been shown to induce the activation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) through a mechanism involving EGFR transactivation in a breast cancer cell line. However, the underlying molecular mechanism for ERK1/2 activation mediated by n-6 free fatty acid (LA) in HEK293 cells remains to be further elucidated. Methods: A FLAG-FFA1 vector was stably expressed in HEK293 cells. Western blot analysis was applied to investigate the change in LA-induced ERK1/2 phosphorylation change in response to kinase inhibitors. Arrestin-2/3-specific siRNA was used to analyze the effect of arrestin-2/3 knockdown on FFA1-mediated ERK1/2 activation. Results: We proved that activation of ERK1/2 by LA was rapid, peaking at 5 min. Further experiments proved that FFA1 couples to a Gq protein and activates PI-PLC, which induces the IP3/Ca2+ and DAG/PKC signal pathways, both of which are involved in ERK1/2 activation. We also showed that there is no EGFR transactivation, arrestin-2/3 or G beta gamma pathway participation in ERK1/2 phosphorylation. Treating cells with PTX abolished ERK1/2 activation at a late time point (>= 20 min), indicating a critical role for Gi subunits in FFA1-mediated ERK1/2 activation. Conclusions: Our study provides a detailed delineation of the LA- mediated activation of ERK1/2 in HEK293 cells that are stably transfected with human FFA1. We also present evidence of Gi/Gq-induced synergism in the regulation of ERK1/2 phosphorylation. These observations may provide new insights into the pharmacological effects of FFA1 and the physiological functions modulated by FFA1-mediated activation of ERK1/2.