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

Abstract

Abstract Background FFA1 is abundantly expressed in the liver, skeletal muscle, monocytes and nervous system, but is particularly abundant in pancreatic β 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 Gβγ 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.

Keywords

• FFA1
• Phosphatidylinositol-specific phospholipase C
• Extracellular signal-regulated kinase 1 and 2
• Gαq/11
• Gαi/o