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

Abstract

BackgroundFFA1 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.MethodsA 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.ResultsWe 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.ConclusionsOur 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s11658-017-0043-3) contains supplementary material, which is available to authorized users.

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“…GPR43 exhibits dual coupling to the G I/O subunit and the G q/11 subunit but not to the G S subunit 4 . Specific activation of GPR43 subsequently influences cAMP and Ca 2+ and thus activates downstream signaling 4 , and the G q/11 subunit is capable of activating PLC and IP 3 to activate downstream signaling pathways 20,33,34 . Consistent with these conclusions, our results proved that acetate could decrease Ca 2+ levels after stimulation and that this process required IP 3 R participation, as the blockage of IP 3 R reversed the Ca 2+ decrease after acetate treatment.…”

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“…Elevated ROS also regulate signaling pathways by inhibiting activities of phosphatases and the consequent increase in protein tyrosine phosphorylation levels (22). In turn, redox signaling activates certain kinases, for example, p38 MAPK (234), JNK, PKC (166), and ERK (235). Such signaling often modifies ion channel function.…”

“…FAs were considered to only augment GSIS in pancreatic β-cells when exposed at low doses or for hours (49, 92, 99). The existence of FA-stimulated insulin secretion (FASIS) has not been widely recognized; despite it has been traditionally ascribed to the GPR40 pathway (96, 98, 248, 113, 122, 129, 139, 168, 171, 194, 235).…”

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“…LCFAs were supposed to directly activate the GPR40/FFA1 (free FA1) receptor, (i.e., another receptor of β-cells stimulating insulin granule exocytosis via the Gq protein), but also via the Gs and arrestin pathways [ 30 , 31 , 32 , 33 , 34 ]. Activation of GPR40 is further relayed to the signal-regulated kinase 1 and 2 (ERK1/2) [ 35 ]. The GPR40 ablation or point mutation in mice led to the impaired insulin secretion that was stimulated by FAs, while only mice with the point mutation had normal GSIS [ 36 ].…”