The Bile Acid Receptor TGR5 Does Not Interact with β-Arrestins or Traffic to Endosomes but Transmits Sustained Signals from Plasma Membrane Rafts
The Bile Acid Receptor TGR5 Does Not Interact with β-Arrestins or Traffic to Endosomes but Transmits Sustained Signals from Plasma Membrane Rafts
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DOI：
10.1074/jbc.m113.455774
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发表时间：
2013-08-09
影响因子：
4.8
通讯作者：
Corvera, Carlos U.
中科院分区：
文献类型：
--
作者：
Jensen, Dane D.;Godfrey, Cody B.;Corvera, Carlos U.
TGR 5是一种G蛋白偶联受体，介导胆汁酸（BA）对能量平衡、炎症、消化和感觉的影响。TGR 5信号传导的机制和时空控制知之甚少。我们研究了内源性表达TGR 5的转染HEK 293细胞和结肠细胞（NCM 460）中的TGR 5信号传导和运输。BA（脱氧胆酸（DCA），牛磺石胆酸）和选择性激动剂油酸和3-（2-氯苯基）-N-（4-氯苯基）-N，5-二甲基异恶唑-4-甲酰胺刺激cAMP的形成，但不诱导TGR 5内吞或募集β-抑制蛋白，如通过共聚焦显微镜评估。DCA，牛磺石胆酸，油酸没有刺激TGR 5协会与β-抑制蛋白1/2或G蛋白偶联受体激酶（GRK）2/5/6，确定由生物发光共振能量转移。3-（2-氯苯基）-N-（4-氯苯基）-N，5-二甲基异恶唑-4-甲酰胺刺激低水平的TGR 5与β-抑制蛋白2和GRK 2的相互作用。DCA诱导cAMP的形成在质膜和胞质溶胶中，如使用交换因子直接调节cAMP（Epac 2）为基础的报告，但cAMP信号没有脱敏。AG 1478，一种表皮生长因子受体酪氨酸激酶抑制剂，金属蛋白酶抑制剂巴马司他，甲基-β-环糊精和菲律宾，阻止脂筏形成，阻止DCA刺激ERK 1/2。生物发光共振能量转移分析显示，TGR 5和EGFR的相互作用被阻断的脂筏破坏。DCA刺激TGR 5重新分布到质膜微区，如通过免疫金电子显微镜定位的。因此，TGR 5不会与β-抑制蛋白相互作用、脱敏或运输到内体。来自质膜筏的TGR 5信号促进EGFR相互作用和反式激活。对TGR 5信号传导的时空控制的理解提供了对BA和治疗性TGR 5激动剂/拮抗剂的作用的见解。
TGR5 is a G protein-coupled receptor that mediates bile acid (BA) effects on energy balance, inflammation, digestion, and sensation. The mechanisms and spatiotemporal control of TGR5 signaling are poorly understood. We investigated TGR5 signaling and trafficking in transfected HEK293 cells and colonocytes (NCM460) that endogenously express TGR5. BAs (deoxycholic acid (DCA), taurolithocholic acid) and the selective agonists oleanolic acid and 3-(2-chlorophenyl)-N-(4-chlorophenyl)-N, 5-dimethylisoxazole-4-carboxamide stimulated cAMP formation but did not induce TGR5 endocytosis or recruitment of beta-arrestins, as assessed by confocal microscopy. DCA, taurolithocholic acid, and oleanolic acid did not stimulate TGR5 association with beta-arrestin 1/2 or G protein-coupled receptor kinase (GRK) 2/5/6, as determined by bioluminescence resonance energy transfer. 3-(2-chlorophenyl)-N-(4-chlorophenyl)-N, 5-dimethylisoxazole-4-carboxamide stimulated a low level of TGR5 interaction with beta-arrestin 2 and GRK2. DCA induced cAMP formation at the plasma membrane and cytosol, as determined using exchange factor directly regulated by cAMP (Epac2)-based reporters, but cAMP signals did not desensitize. AG1478, an inhibitor of epidermal growth factor receptor tyrosine kinase, the metalloprotease inhibitor batimastat, and methyl-beta-cyclodextrin and filipin, which block lipid raft formation, prevented DCA stimulation of ERK1/2. Bioluminescence resonance energy transfer analysis revealed TGR5 and EGFR interactions that were blocked by disruption of lipid rafts. DCA stimulated TGR5 redistribution to plasma membrane microdomains, as localized by immunogold electron microscopy. Thus, TGR5 does not interact with beta-arrestins, desensitize, or traffic to endosomes. TGR5 signals from plasma membrane rafts that facilitate EGFR interaction and transactivation. An understanding of the spatiotemporal control of TGR5 signaling provides insights into the actions of BAs and therapeutic TGR5 agonists/antagonists.