Characterization Of The Endocytosis Pathway And ERK1/2 Phosphorylation Signaling Of Free Fatty Acid Receptor 1(GPR40)

GPR40 that is abundantly expressed in pancreaticβ-cells, is activated by medium-and long-chain free fatty acids (FFAs) and is believed to be an attractive drug target for type 2 diabetes. GPR40 has been found to couple to Gq protein, leading to activation of phospholipase C and subsequent increases in the intracellular Ca2+level. However, the underlying mechanisms that regulate the internalization and desensitization of GPR40 remain to be elucidated. We have prepared a construct of GPR40 fused with enhanced green fluorescent protein (EGFP) at its C-terminus for direct imaging of the localization and internalization of GPR40 by confocal microscopy. In stable transfected HEK293 cells, GPR40-EGFP underwent rapid agonist-induced internalization and constitutive ligand-independent internalization. We demonstrated that the agonist-mediated internalization of GPR40 was significantly blocked by hypertonic sucrose treatment and by siRNA mediated depletion of the heavy chain of clathrin. In contrast, constitutive GPR40 internalization was not affected by hypertonic sucrose or by knock-down of clathrin expression, but it was affected by treatment with methyl-β-cyclodextrin (MβCD). We also performed an arrestin-3-EGFP redistribution assay and siRNA-mediated knock-down of arrestin-3 expression. We found that arrestin-3 plays a critical role in the regulation of agonist-mediated GPR40 internalization, but it has no role in the regulation of constitutive GPR40 internalization. Additionally, we observed that the internalized GPR40-EGFP co-localized extensively with Alexa Fluor 594-labeled transferrin in early endosomes and recycled to the cell plasma membrane. Because FFA receptors exhibit a high level of homology, our observations could be applicable to other members of this protein family.Considering insufficientβ-cell mass is one of the major contributors to type 2 diabetes, it is important to elucidate mechanisms closely linking cell proliferation and apoptosis which are mediated by GPR40. However, the underlying molecular mechanism of extracellular signal-regulated kinase 1/2 (ERK1/2) induced by activated GPR40 remains to be elucidated. For that reason we have chosen to focus our attention on characterizing the MAPK pathways mediated by GPR40. Many GPCRs regulate ERK cascades via distinct G proteins,β-arrestin-dependent and EGFR transactivation signaling pathway, leading to activation of the extracellular signal-regulated kinases (ERKs), which function as transcriptional regulators. Previous study have demonstrated that HEK-293 cells that stably expressing GPR40-EGFP chimera exhibited the same activities in response to linoleic acid (LA) and linolenic acid (LNA, 100μM) as wild-type GPR40. In the present study, we demonstrated that upon stimulation with LA, activated GPR40 signal to ERK1/2 via Ca2+/PKC and Gi-dependent, butβ-arrestin and EGFR-independent pathways. Interestingly, high concentrations of LA significantly reduced ERK1/2 phosphorylation inβ-TC-6 cells. Considering the high level of homology among FFA receptors, our findings could provide new insight into the other members of this family.