Received for publication April 13, 2007.
Revised May 31, 2007.
Accepted for publication May 31, 2007.

Localization of the 5-HT1A receptor in lipid microdomains depends on its palmitoylation and is involved in receptor-mediated signalling

Abstract

In the present study we have utilized wild-type and palmitoylation-deficient 5-HT1A receptors fused to the yellow fluorescent protein (YFP) as well as cyan fluorescent protein (CFP)-tagged alpha_i3-subunit of heterotrimeric G-protein to study spatio-temporal 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 Galphai protein into the cytoplasm and that this translocation is accompanied by significant increase of the intracellular Ca²⁺ concentration. In contrast, acylation-deficient 5-HT1A mutants failed to reproduce both Galphai3-CFP relocation as well as changes in [Ca²⁺]_i upon agonist stimulation. By using gradient centrifugation and co-patching assays, we also demonstrate that a significant fraction of the 5-HT1A receptor resides in membrane rafts, while the yield of the palmitoylation-deficient receptor in these membrane microdomains is considerably reduced. 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 appears to be involved in receptor-mediated signaling.

Key words: Serotonin, Gi family, Lipid rafts/microdomains