title: Internalization of the human nicotinic acid receptor GPR109A is regulated by G(i), GRK2, and arrestin3.
abstract: Nicotinic acid (niacin) has been widely used as a favorable lipid-lowering drug for several decades, and the orphan G protein-coupled receptor GPR109A has been identified to be a receptor for niacin. Mechanistic investigations have shown that as a G(i)-coupled receptor, GPR109A inhibits adenylate cyclase activity upon niacin activation, thereby inhibiting free fatty acid liberation. However, the underlying molecular mechanisms that regulate signaling and internalization of GPR109A remain largely unknown. To further characterize GPR109A internalization, we made a construct to express GPR109A fused with enhanced green fluorescent protein (EGFP) at its carboxyl-terminal end. In stable GPR109A-EGFP-expressing HEK-293 cells, GPR109A-EGFP was mainly localized at the plasma membrane and was rapidly internalized in a dose- and time-dependent manner upon agonist stimulation. GPR109A internalization was completely blocked by hypertonic sucrose, indicating that GPR109A internalizes via the clathrin-coated pit pathway. Further investigation demonstrated that internalized GPR109A was recycled to the cell surface after the removal of agonist, and recycling of the internalized receptors was not blocked by treatment with acidotropic agents, NH(4)Cl and monensin. Pertussis toxin pretreatment not only inhibited forskolin-induced cAMP accumulation and intracellular Ca(2+) mobilization; it also significantly attenuated agonist-promoted GPR109A internalization. Moreover, RNA interference experiments showed that knockdown of GRK2 (G protein-coupled receptor kinase 2) and arrestin3 expression significantly impaired receptor internalization. Taken together, these results indicate that the agonist-induced internalization of GPR109A receptors is regulated by GRK2 and arrestin3 in a pertussis toxin-sensitive manner and that internalized receptor recycling is independent of endosomal acidification.
authors.affiliation: Institute of Biochemistry, College of Life Science, Zijingang Campus, Zhejiang University, Hangzhou, Zhejiang 310058, China.
authors.forename: Guo
authors.initials: G
authors.lastname: Li
authors.forename: Ying
authors.initials: Y
authors.lastname: Shi
authors.forename: Haishan
authors.initials: H
authors.lastname: Huang
authors.forename: Yaping
authors.initials: Y
authors.lastname: Zhang
authors.forename: Kuangpei
authors.initials: K
authors.lastname: Wu
authors.forename: Jiansong
authors.initials: J
authors.lastname: Luo
authors.forename: Yi
authors.initials: Y
authors.lastname: Sun
authors.forename: Jianxin
authors.initials: J
authors.lastname: Lu
authors.forename: Jeffrey L
authors.initials: JL
authors.lastname: Benovic
authors.forename: Naiming
authors.initials: N
authors.lastname: Zhou
mesh_terms: D000818:Animals; D019390:Arrestins / Q000378:metabolism*; D002460:Cell Line; D002966:Clathrin / Q000378:metabolism; D003034:Coated Pits, Cell-Membrane / Q000187:drug effects / Q000378:metabolism; D034281:Dynamins / Q000378:metabolism; D004705:Endocytosis / Q000187:drug effects; D011992:Endosomes / Q000187:drug effects / Q000378:metabolism; D054769:G-Protein-Coupled Receptor Kinase 2 / Q000378:metabolism*; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism*; D044387:GTP-Binding Protein beta Subunits / Q000378:metabolism; D044388:GTP-Binding Protein gamma Subunits / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism; D008954:Models, Biological; D037342:Pertussis Toxin / Q000494:pharmacology; D021381:Protein Transport / Q000187:drug effects; D043562:Receptors, G-Protein-Coupled / Q000819:agonists / Q000378:metabolism*; D011978:Receptors, Nicotinic / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D019061:src-Family Kinases / Q000378:metabolism
chemical_list: D019390:Arrestins; D002966:Clathrin; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; C530636:HCAR2 protein, human; D043562:Receptors, G-Protein-Coupled; D011978:Receptors, Nicotinic; D011993:Recombinant Fusion Proteins; C082333:arrestin3; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D037342:Pertussis Toxin; D019061:src-Family Kinases; C496567:GRK2 protein, human; D054769:G-Protein-Coupled Receptor Kinase 2; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D034281:Dynamins
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