title: Differential requirements of arrestin-3 and clathrin for ligand-dependent and -independent internalization of human G protein-coupled receptor 40.
abstract: G protein-coupled receptor 40 (GPR40) is believed to be an attractive target to enhance insulin secretion in patients with type 2 diabetes. GPR40 has been found to couple to Gq protein, leading to the activation of phospholipase C and subsequent increases in the intracellular Ca(2+) level. However, the underlying mechanisms that regulate the internalization and desensitization of GPR40 remain to be elucidated. In the present study, a construct of GPR40 fused with enhanced green fluorescent protein (EGFP) at its C-terminus was constructed for direct imaging of the localization and internalization of GPR40 by confocal microscopy. In stably transfected HEK-293 cells, GPR40 receptors underwent rapid agonist-induced internalization and constitutive ligand-independent internalization. Our data 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) and nystatin. Furthermore, our results using an arrestin-3-EGFP redistribution assay and siRNA-mediated knock-down of arrestin-3 and GRK2 expression revealed that arrestin-3 and GRK2 play an essential role in the regulation of agonist-mediated GPR40 internalization, but are not involved in the regulation of constitutive GPR40 internalization. Additionally, our observation showed that upon activation by agonist, the internalized GPR40 receptors were rapidly recycled back to the plasma membrane via Rab4/Rab5 positive endosomes, whereas the constitutively internalized GPR40 receptors were recycled back to the cell surface through Rab5 positive endosomes. Because FFA receptors exhibit a high level of homology, our observations could be applicable to other members of this family.
authors.affiliation: Institute of Biochemistry, College of Life Science, Zijingang Campus, Zhejiang University, Hangzhou, Zhejiang 310058, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Qian
authors.affiliation: Institute of Biochemistry, College of Life Science, Zijingang Campus, Zhejiang University, Hangzhou, Zhejiang 310058, China.
authors.forename: Chun
authors.initials: C
authors.lastname: Wu
authors.affiliation: Institute of Biochemistry, College of Life Science, Zijingang Campus, Zhejiang University, Hangzhou, Zhejiang 310058, China.
authors.forename: Xiaopan
authors.initials: X
authors.lastname: Chen
authors.affiliation: College of Biology and Chemical Engineering, Jiangsu University of Science and Technology, Zhenjiang, Jiangsu 212003, China.
authors.forename: Xiangmei
authors.initials: X
authors.lastname: Li
authors.affiliation: Institute of Biochemistry, College of Life Science, Zijingang Campus, Zhejiang University, Hangzhou, Zhejiang 310058, China.
authors.forename: Guoyuan
authors.initials: G
authors.lastname: Ying
authors.affiliation: Institute of Biochemistry, College of Life Science, Zijingang Campus, Zhejiang University, Hangzhou, Zhejiang 310058, China.
authors.forename: Lili
authors.initials: L
authors.lastname: Jin
authors.affiliation: Institute of Biochemistry, College of Life Science, Zijingang Campus, Zhejiang University, Hangzhou, Zhejiang 310058, China.
authors.forename: Qiang
authors.initials: Q
authors.lastname: Ma
authors.affiliation: Institute of Aging Research, Hangzhou Normal University, Hangzhou, Zhejian, China.
authors.forename: Guo
authors.initials: G
authors.lastname: Li
authors.affiliation: Institute of Biochemistry, College of Life Science, Zijingang Campus, Zhejiang University, Hangzhou, Zhejiang 310058, China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Shi
authors.affiliation: College of Biology and Chemical Engineering, Jiangsu University of Science and Technology, Zhenjiang, Jiangsu 212003, China.
authors.forename: Guozheng
authors.initials: G
authors.lastname: Zhang
authors.affiliation: Institute of Biochemistry, College of Life Science, Zijingang Campus, Zhejiang University, Hangzhou, Zhejiang 310058, China. Electronic address: znm2000@yahoo.com.
authors.forename: Naiming
authors.initials: N
authors.lastname: Zhou
mesh_terms: D019390:Arrestins / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism*; D002966:Clathrin / Q000235:genetics / Q000378:metabolism*; D011992:Endosomes / Q000235:genetics / Q000378:metabolism*; D054769:G-Protein-Coupled Receptor Kinase 2 / Q000235:genetics / Q000378:metabolism; D055785:Gene Knockdown Techniques; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D009997:Osmotic Pressure / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D043562:Receptors, G-Protein-Coupled / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D013395:Sucrose / Q000494:pharmacology; D013549:Sweetening Agents / Q000494:pharmacology; D047392:beta-Cyclodextrins / Q000494:pharmacology; D020695:rab4 GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D020696:rab5 GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D019390:Arrestins; D002966:Clathrin; C473034:FFAR1 protein, human; D043562:Receptors, G-Protein-Coupled; D013549:Sweetening Agents; C082333:arrestin3; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D013395:Sucrose; C496567:GRK2 protein, human; D054769:G-Protein-Coupled Receptor Kinase 2; C427755:RAB5C protein, human; D020695:rab4 GTP-Binding Proteins; D020696:rab5 GTP-Binding Proteins
keywords: Arrestins; Constitutive activity; Free fatty acid; GPR40; Internalization; Recycling