Organization of higher-order oligomers of the serotonin₁(A) receptor explored utilizing homo-FRET in live cells.
The serotonin₁(A) receptor is a representative member of the GPCR superfamily and serves as an important drug target. The possible role of GPCR oligomerization in receptor function is an active area of research. We monitored the oligomerization state of serotonin₁(A) receptors using homo-FRET and fluorescence lifetime measurements. Homo-FRET is estimated by a reduction in fluorescence anisotropy and provides a superior approach for exploring oligomerization. In addition, homo-FRET offers the possibility of detecting higher-order oligomers. On the basis of an observed increase in fluorescence anisotropy upon progressive photobleaching and analysis of the difference between the extrapolated anisotropy and the predicted anisotropy of an immobile monomer, we propose the presence of constitutive oligomers of the serotonin₁(A) receptor. To the best of our knowledge, these results constitute the first report of higher-order oligomers for the serotonin₁(A) receptor. We further show that cholesterol depletion and antagonist treatment result in a reduced population of higher-order oligomers. In contrast, agonist stimulation and destabilization of the actin cytoskeleton lead to an increased contribution from higher oligomers. These results provide novel insight into the oligomerization status of the serotonin₁(A) receptor that could enhance the ability to design better therapeutic strategies to combat diseases related to malfunctioning of GPCRs.
Centre for Cellular and Molecular Biology, Hyderabad, India.
Sourav
S
Ganguly
Andrew H A
AH
Clayton
Amitabha
A
Chattopadhyay
D000199:Actins / Q000378:metabolism; D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D002784:Cholesterol / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D005454:Fluorescence Polarization / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer / Q000295:instrumentation* / Q000379:methods; D051379:Mice; D038761:Photobleaching; D044282:Receptor, Serotonin, 5-HT1A / Q000737:chemistry* / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry / Q000378:metabolism
D000199:Actins; D043562:Receptors, G-Protein-Coupled; D044282:Receptor, Serotonin, 5-HT1A; D002784:Cholesterol
Biochim Biophys Acta. 2008 Aug;1783(8):1503-16
18381076
Anal Biochem. 2000 Sep 10;284(2):438-40
10964438
Annu Rev Pharmacol Toxicol. 2002;42:409-35
11807178
Biophys J. 2008 Jul;95(1):451-63
18339759
Curr Opin Pharmacol. 2010 Feb;10(1):53-8
19910252
Biophys J. 1995 Oct;69(4):1569-83
8534828
Biochemistry. 2004 Dec 21;43(50):15643-56
15595821
Biochim Biophys Acta. 2007 Apr;1768(4):825-35
17069751
J Fluoresc. 2005 Mar;15(2):153-60
15883770
Annu Rev Cell Dev Biol. 2004;20:839-66
15473862
Nat Cell Biol. 2007 Jan;9(1):7-14
17199125
J Biol Chem. 2000 Jun 9;275(23):17556-60
10748019
Biophys J. 2003 Jun;84(6):4078-86
12770911
Cytometry A. 2004 Apr;58(2):185-94
15057972
Biophys J. 2010 Sep 8;99(5):1397-407
20816051
Biochemistry. 2004 Dec 21;43(50):15852-62
15595840
Methods Enzymol. 2003;360:580-97
12622169
Nat Rev Drug Discov. 2002 Oct;1(10):808-20
12360258
Pharmacol Rep. 2007 Jul-Aug;59(4):379-92
17901566
Nat Methods. 2006 Dec;3(12):1001-6
17086179
Biophys J. 2007 May 1;92(9):3098-104
17416632
Biochemistry. 2007 Dec 18;46(50):14403-17
18027983
Proc Natl Acad Sci U S A. 2006 Feb 14;103(7):2138-43
16461466
J Phys Chem B. 2007 Dec 27;111(51):14436-9
18052368
Nature. 1998 Aug 20;394(6695):798-801
9723621
Cell Mol Neurobiol. 2005 Jun;25(3-4):553-80
16075379
J Biol Chem. 2002 May 3;277(18):15482-5
11854302
Anal Biochem. 2005 Aug 15;343(2):344-6
16005425
Science. 2007 Nov 23;318(5854):1258-65
17962520
Drug Discov Today. 2009 Mar;14(5-6):231-40
19121411
Growth Factors. 2008 Dec;26(6):316-24
18937111
Biochem Soc Trans. 2003 Oct;31(Pt 5):1020-7
14505472
Biochem Biophys Res Commun. 2009 Oct 2;387(4):661-5
19615977
Methods Cell Biol. 2008;85:415-30
18155473
Nature. 2009 May 21;459(7245):356-63
19458711
Biophys J. 1979 Jun;26(3):557-73
263688
Biophys J. 2002 Sep;83(3):1631-49
12202387
Chem Phys Lipids. 2009 Feb;157(2):61-77
18723009
Biophys J. 2009 Nov 4;97(9):2613-22
19883605