Internalization of the human nicotinic acid receptor GPR109A is regulated by G(i), GRK2, and arrestin3.
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.
Li
Guo
G
Institute of Biochemistry, College of Life Science, Zijingang Campus, Zhejiang University, Hangzhou, Zhejiang 310058, China.
Shi
Ying
Y
Huang
Haishan
H
Zhang
Yaping
Y
Wu
Kuangpei
K
Luo
Jiansong
J
Sun
Yi
Y
Lu
Jianxin
J
Benovic
Jeffrey L
JL
Zhou
Naiming
N
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
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
Mol Pharmacol. 1997 Jul;52(1):105-13
9224819
J Biol Chem. 1993 Apr 5;268(10):6862-5
8463213
Br J Pharmacol. 2003 Jun;139(4):695-704
12812992
Science. 1992 Aug 28;257(5074):1264-7
1325672
J Cell Sci. 2004 Feb 15;117(Pt 5):723-34
14734649
Am J Cardiol. 2007 Dec 3;100(11 A):S53-61
18047854
J Biol Chem. 2005 Jan 21;280(3):2197-204
15542600
J Clin Endocrinol Metab. 2004 Sep;89(9):4649-55
15356076
N Engl J Med. 1999 Aug 12;341(7):498-511
10441607
Pharmacol Rev. 2001 Mar;53(1):1-24
11171937
Biochem Biophys Res Commun. 2003 Mar 28;303(1):364-9
12646212
J Biol Chem. 1995 Oct 20;270(42):24782-9
7559596
J Biol Chem. 2000 Apr 7;275(14):10443-52
10744734
J Cell Biol. 1984 Apr;98(4):1163-9
6715403
Diabetes. 1999 Sep;48(9):1836-41
10480616
Trends Endocrinol Metab. 2006 May-Jun;17(4):159-65
16595179
Annu Rev Physiol. 2007;69:451-82
17037978
Trends Pharmacol Sci. 2000 May;21(5):175-80
10785651
J Biol Chem. 1999 Jun 4;274(23):16287-94
10347185
Mol Biol Cell. 1995 May;6(5):509-24
7545030
Curr Opin Neurobiol. 1997 Oct;7(5):631-9
9384543
Mol Pharmacol. 2001 Feb;59(2):349-57
11160872
J Clin Invest. 1979 May;63(5):858-67
221531
Traffic. 2007 May;8(5):462-70
17376169
J Clin Invest. 2009 May;119(5):1312-21
19349687
FASEB J. 1990 Aug;4(11):2881-9
2165947
J Neurochem. 1999 Aug;73(2):493-501
10428044
J Leukoc Biol. 1992 Apr;51(4):393-9
1564402
J Biol Chem. 1998 Sep 18;273(38):24592-602
9733754
J Med Chem. 2006 Feb 23;49(4):1227-30
16480258
J Intern Med. 2005 Aug;258(2):94-114
16018787
J Clin Invest. 2005 Dec;115(12):3634-40
16322797
J Biol Chem. 1995 Dec 1;270(48):29004-11
7499433
J Clin Invest. 1977 Oct;60(4):795-807
197124
Nat Med. 2003 Mar;9(3):352-5
12563315
Biochem Biophys Res Commun. 2005 Aug 26;334(2):729-32
16018973
J Biol Chem. 1987 Dec 15;262(35):17046-50
2824511
Mol Pharmacol. 1995 Apr;47(4):666-76
7723728
J Biol Chem. 1985 Mar 25;260(6):3440-50
3838314
Biochem Biophys Res Commun. 2004 Aug 13;321(1):226-33
15358239
J Biol Chem. 1994 Dec 23;269(51):32522-7
7798253
J Clin Invest. 1991 Oct;88(4):1282-90
1918378
J Biol Chem. 1994 Jan 7;269(1):37-40
8276821
Pharmacol Ther. 2003 Nov;100(2):101-18
14609715
J Biol Chem. 1993 Apr 15;268(11):8256-60
8463335
Diabetes. 1998 Oct;47(10):1609-12
9753299
J Biol Chem. 2003 Mar 14;278(11):9869-74
12522134
J Biol Chem. 1987 Jun 25;262(18):8743-7
3036825
J Biol Chem. 2007 Jun 22;282(25):18028-18036
17452318
Atherosclerosis. 2007 Jun;192(2):253-8
16945375
Mol Pharmacol. 2008 Aug;74(2):338-47
18388243
J Biol Chem. 1999 Oct 22;274(43):31076-86
10521508
Proc Natl Acad Sci U S A. 1988 Sep;85(18):6662-6
2842787
Mol Cell Biol. 1989 Dec;9(12):5537-47
2511437
Trends Cell Biol. 1999 Mar;9(3):107-12
10201076
J Cell Biol. 1983 Jun;96(6):1824-8
6304116
J Biol Chem. 1992 Feb 5;267(4):2222-7
1733928
EMBO J. 1997 Aug 1;16(15):4606-16
9303305
Mol Pharmacol. 1997 May;51(5):800-8
9145918
Traffic. 2006 Oct;7(10):1420-31
16899088