Abstract
In the present study, we have used wild-type and palmitoylation-deficient mouse 5-hydroxytryptamine1A receptor (5-HT1A) receptors fused to the yellow fluorescent protein- and the cyan fluorescent protein (CFP)-tagged αi3 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 Gi-proteins. Our data demonstrate that activation of the 5-HT1A receptor caused a partial release of Gαi protein into the cytoplasm and that this translocation is accompanied by a significant increase of the intracellular Ca2+ concentration. In contrast, acylation-deficient 5-HT1A mutants failed to reproduce both Gαi3-CFP relocation and changes in [Ca2+]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.
Footnotes
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These studies were supported by the fund of the Medical School at the University of Göttingen and by the Deutsche Forschungsgemeinschaft through the Center of Molecular Physiology of the Brain (to E.G.P.) and grant PO 732.
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U.R. and K.G. contributed equally to this work.
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Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
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doi:10.1124/mol.107.037085.
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ABBREVIATIONS: GPCR, G-protein-coupled receptor; 5-HT1A, mouse 5-hydroxytryptamine1A receptor; CTX, cholera toxin; CFP, cyan fluorescent protein; DRM, detergent-resistant membrane fraction; MβCD, methyl-β-cyclodextrin; PTx, pertussis toxin; YFP, yellow fluorescent protein; lo, liquid-ordered; HA, hemagglutinin; WT, wild-type; PBS, phosphate-buffered saline; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; GFP, green fluorescent protein; Erk, extracellular signal-regulated kinase; AM, acetoxymethyl ester; CHO, Chinese hamster ovary; Mut, acylation-deficient; GTPγS, guanosine 5′-3-O-(thio)triphosphate; 8-OH-DPAT, 8-hydroxy-2-dipropylaminotetralin; KGlu buffer, potassium glutamate, potassium acetate, and HEPES; TMA-DPH, 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene-p-toluenesulfonate; WAY100635, [O-methyl-3H]-N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridinyl)cyclohexanecarboxamide trihydrochloride.
- Received April 13, 2007.
- Accepted May 31, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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