游离脂肪酸受体1（GPR40）MAPK信号通路与内吞机制研究
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摘要
脂肪酸不仅仅是人体的能量来源,同时也参加一系列的生理活动,比如免疫反应和胰岛素分泌。2003年,三个独立的研究小组发现一系列的中长链脂肪酸能够激活孤儿G蛋白偶联受体GPR40。紧接着GPR40被命名为游离脂肪酸受体1 (FFAR1). GPR40仅在高浓度葡萄糖的存在下能够促进胰岛β细胞葡糖依赖性的胰岛素分泌,从而降低了低血糖的风险,因此成为非常重要的治疗2型糖尿病的靶标。了解调控GPR40的信号通路为GPR40生理功能的阐述及糖尿病的药物设计提供理论的基础。因此我们的研究着力于研究GPR40受体激活后内吞的转运机制。我们构建了融合GFP的表达载体,并筛选得到稳定表达GPR40-EGFP受体的单克隆HEK293稳定细胞株。荧光显微镜下观察发现,GPR40-EGFP主要分布在细胞膜上,也有很大部分位于胞内。在配体100μM LA的刺激下,受体分布发生改变,集中到细胞核周围,成点状聚集分布。后期研究发现GPR40的内吞主要通过clathrin依赖型的途径并受arrestin-3的调控。有趣的是,我们发现GPR40受体的本底细胞内表达水平很高,而clathrin和arrestin-3的knockdown并不影响GPR40受体的本底性内吞,暗示GPR40受体配体依赖性和不依赖性的内吞机制并不相同。
所有的G蛋白偶联受体都能够促进MAPK的信号级联反应,参与调控细胞增殖和凋亡。促分裂素原活化蛋白激酶(mitogen-activated protein kinases, MAP激酶,MAPK)链是真核生物信号传递网络中的重要途径之一,其中细胞外信号调节蛋白激酶(extracellular regulated protein, ERK)为MAPK家族重要成员。ERK1/2信号途径对细胞的分裂、迁移和细胞凋亡等起到调控作用,如GLP-1受体信号转导能够通过ERK1/2途径抑制胰岛β细胞的凋亡。糖尿病产生的重要原因之一是胰岛素β细胞的损伤,因此,研究GPR40如何调控ERK1/2途径以及ERK1/2途径是否参与胰岛β细胞的增殖和凋亡过程具有重要的生理意义。第三章的实验研究中,我们着力于解决GPR40介导的ERK1/2磷酸化。通过利用稳定转染GPR40受体的HEK293细胞和内源性表达的β-TC-6细胞,我们发现在HEK细胞中,LA能够通过Ca2+/ PKC和Gi途径激活ERK1/2。考虑到脂肪酸受体的同源性,我们的研究也会给脂肪酸受体家族的其他受体提供参考和依据。
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.
所有的G蛋白偶联受体都能够促进MAPK的信号级联反应,参与调控细胞增殖和凋亡。促分裂素原活化蛋白激酶(mitogen-activated protein kinases, MAP激酶,MAPK)链是真核生物信号传递网络中的重要途径之一,其中细胞外信号调节蛋白激酶(extracellular regulated protein, ERK)为MAPK家族重要成员。ERK1/2信号途径对细胞的分裂、迁移和细胞凋亡等起到调控作用,如GLP-1受体信号转导能够通过ERK1/2途径抑制胰岛β细胞的凋亡。糖尿病产生的重要原因之一是胰岛素β细胞的损伤,因此,研究GPR40如何调控ERK1/2途径以及ERK1/2途径是否参与胰岛β细胞的增殖和凋亡过程具有重要的生理意义。第三章的实验研究中,我们着力于解决GPR40介导的ERK1/2磷酸化。通过利用稳定转染GPR40受体的HEK293细胞和内源性表达的β-TC-6细胞,我们发现在HEK细胞中,LA能够通过Ca2+/ PKC和Gi途径激活ERK1/2。考虑到脂肪酸受体的同源性,我们的研究也会给脂肪酸受体家族的其他受体提供参考和依据。
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.
引文
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2. Leahy, J.L., Pathogenesis of type 2 diabetes mellitus. Arch Med Res,2005. 36(3):p.197-209.
3. Ahren, B., Glucagon-like peptide-1 (GLP-1):a gut hormone of potential interest in the treatment of diabetes. Bioessays,1998.20(8):p.642-51.
4. Chung, L.T.K., et al., Exendin-4, a GLP-1 receptor agonist, directly induces adiponectin expression through protein kinase A pathway and prevents inflammatory adipokine expression. Biochemical and Biophysical Research Communications,2009.390(3):p.613-618.
5. Marre, M., et al., Liraglutide, a once-daily human GLP-1 analogue, added to a sulphonylurea over 26 weeks produces greater improvements in glycaemic and weight control compared with adding rosiglitazone or placebo in subjects with Type 2 diabetes (LEAD-1 SU). Diabet Med,2009.26(3):p.268-78.
6. Ahren, B., Islet G protein-coupled receptors as potential targets for treatment of type 2 diabetes. Nat Rev Drug Discov,2009.8(5):p.369-85.
7. Ahren, B., Glucagon-like peptide-1 (GLP1):a gut hormone of potential interest in the treatment of diabetes. Bioessays,1998.20:p.642-651.
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