In the present study, we verified that the mouse 5-hydroxytryptamine(1A) (5-HT 1A ) receptor is modified by palmitic acid, which is covalently attached to the protein through a thioester-type bond. Palmitoylation efficiency was not modulated by receptor stimulation with agonists. Block of protein synthesis by cycloheximide resulted in a significant reduction of receptor acylation, suggesting that palmitoylation occurs early after synthesis of the 5-HT 1A receptor. Furthermore, pulse-chase experiments demonstrated that fatty acids are stably attached to the receptor. Two conserved cysteine residues 417 and 420 located in the proximal Cterminal domain were identified as acylation sites by site-directed mutagenesis. To address the functional role of 5-HT 1A receptor acylation, we have analyzed the ability of acylation-deficient mutants to interact with heterotrimeric G i protein and to modulate downstream effectors. Replacement of individual cysteine residues (417 or 420) resulted in a significantly reduced coupling of receptor with G i protein and impaired inhibition of adenylyl cyclase activity. When both palmitoylated cysteines were replaced, the communication of receptors with G␣ i subunits was completely abolished. Moreover, non-palmitoylated mutants were no longer able to inhibit forskolin-stimulated cAMP formation, indicating that palmitoylation of the 5-HT 1A receptor is critical for the enabling of G i protein coupling/effector signaling. The receptor-dependent activation of extracellular signal-regulated kinase was also affected by acylation-deficient mutants, suggesting the importance of receptor palmitoylation for the signaling through the G␤␥-mediated pathway, in addition to the G␣ i -mediated signaling. Serotonin (5-hydroxytryptamine or 5-HT)1 is a neuromodulator involved in the regulation of many different physiological functions of the central nervous system as well as the periphery by activating a large family of receptors. With the exception of the 5-HT 3 receptor, which is a transmitter-gated Na ϩ /K ϩ channel, all other 5-HT receptors belong to a large family of receptors that are coupled to different intracellular effectors via heterotrimeric guanine nucleotide-binding proteins (G proteins) (1, 2). Structurally, G protein-coupled receptors (GPCRs) possess seven transmembrane domains linked by alternating intracellular (i1-i3) and extracellular (e1-e4) loops. The extracellular receptor surface, including the N terminus, is known to be critically involved in ligand binding. The intracellular receptor surface, including the C-terminal domain and intracellular loops (in particular i2 and i3), is known to be important for G protein recognition and activation (3).The 5-HT 1A receptor is the most extensively characterized 5-HT receptor. This receptor is coupled to a variety of effectors via pertussis toxin-sensitive heterotrimeric G proteins of the G i/o families (2, 4, 5). Receptor-dependent activation of G␣ i subunits results in the inhibition of adenylate cyclase and subsequent decrease of cAMP lev...

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