Stimulation- and palmitoylation-dependent changes in oligomeric conformation of serotonin 5-HT1A receptors.
In the present study we analyzed the oligomerization state of the serotonin 5-HT1A receptor and studied oligomerization dynamics in living cells. We also investigated the role of receptor palmitoylation in this process. Biochemical analysis performed in neuroblastoma N1E-115 cells demonstrated that both palmitoylated and non-palmitoylated 5-HT1A receptors form homo-oligomers and that the prevalent receptor species at the plasma membrane are dimers. A combination of an acceptor-photobleaching FRET approach with fluorescence lifetime measurements verified the interaction of CFP- and YFP-labeled wild-type as well as acylation-deficient 5-HT1A receptors at the plasma membrane of living cells. Using a novel FRET technique based on the spectral analysis we also confirmed the specific nature of receptor oligomerization. The analysis of oligomerization dynamics revealed that apparent FRET efficiency measured for wild-type oligomers significantly decreased in response to agonist stimulation, and our combined results suggest that this decrease was mediated by accumulation of FRET-negative complexes rather than by dissociation of oligomers to monomers. In contrast, the agonist-mediated decrease of FRET signal was completely abolished in oligomers composed by non-palmitoylated receptor mutants, demonstrating the importance of palmitoylation in modulation of the structure of oligomers.
Kobe
Fritz
F
DFG-Research Center for the Molecular Physiology of the Brain (CMPB), University of Göttingen, Germany.
Renner
Ute
U
Woehler
Andrew
A
Wlodarczyk
Jakub
J
Papusheva
Ekaterina
E
Bao
Guobin
G
Zeug
Andre
A
Richter
Diethelm W
DW
Neher
Erwin
E
Ponimaskin
Evgeni
E
D000818:Animals; D045744:Cell Line, Tumor; D019281:Dimerization; D031541:Fluorescence Resonance Energy Transfer; D054878:Lipoylation; D021962:Membrane Microdomains / Q000737:chemistry; D051379:Mice; D038761:Photobleaching; D011487:Protein Conformation; D044282:Receptor, Serotonin, 5-HT1A / Q000737:chemistry* / Q000378:metabolism; D058825:Serotonin 5-HT1 Receptor Agonists; D013050:Spectrometry, Fluorescence
D058825:Serotonin 5-HT1 Receptor Agonists; D044282:Receptor, Serotonin, 5-HT1A