Localization of the mouse 5-hydroxytryptamine(1A) receptor in lipid microdomains depends on its palmitoylation and is involved in receptor-mediated signaling.
In the present study, we have used wild-type and palmitoylation-deficient mouse 5-hydroxytryptamine(1A) receptor (5-HT1A) receptors fused to the yellow fluorescent protein- and the cyan fluorescent protein (CFP)-tagged alpha(i3) subunit of heterotrimeric G-protein to study spatiotemporal distribution of the 5-HT1A-mediated signaling in living cells. We also addressed the question on the molecular mechanisms by which receptor palmitoylation may regulate communication between receptors and G(i)-proteins. Our data demonstrate that activation of the 5-HT1A receptor caused a partial release of Galpha(i) protein into the cytoplasm and that this translocation is accompanied by a significant increase of the intracellular Ca(2+) concentration. In contrast, acylation-deficient 5-HT1A mutants failed to reproduce both Galpha(i3)-CFP relocation and changes in [Ca(2+)](i) upon agonist stimulation. By using gradient centrifugation and copatching assays, we also demonstrate that a significant fraction of the 5-HT1A receptor resides in membrane rafts, whereas the yield of the palmitoylation-deficient receptor in these membrane microdomains is reduced considerably. Our results suggest that receptor palmitoylation serves as a targeting signal responsible for the retention of the 5-HT1A receptor in membrane rafts. More importantly, the raft localization of the 5-HT1A receptor seems to be involved in receptor-mediated signaling.
Renner
Ute
U
Abteilung Neuro- und Sinnesphysiologie, Physiologisches Institut, Universität Göttingen, Göttingen, Germany.
Glebov
Konstantin
K
Lang
Thorsten
T
Papusheva
Ekaterina
E
Balakrishnan
Saju
S
Keller
Bernhard
B
Richter
Diethelm W
DW
Jahn
Reinhard
R
Ponimaskin
Evgeni
E
D000818:Animals; D019921:Biotinylation; D002118:Calcium / Q000378:metabolism; D045744:Cell Line, Tumor; D004789:Enzyme Activation; D005456:Fluorescent Dyes / Q000378:metabolism; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D021962:Membrane Microdomains / Q000378:metabolism*; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D009154:Mutation; D041681:NIH 3T3 Cells; D009447:Neuroblastoma / Q000473:pathology; D019308:Palmitic Acid / Q000378:metabolism*; D010766:Phosphorylation; D011869:Radioligand Assay; D044282:Receptor, Serotonin, 5-HT1A / Q000172:deficiency* / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection
C485182:Cyan Fluorescent Protein; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D044282:Receptor, Serotonin, 5-HT1A; D049452:Green Fluorescent Proteins; D019308:Palmitic Acid; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D002118:Calcium