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J. Biol. Chem., Vol. 279, Issue 5, 3280-3291, January 30, 2004

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The 5-Hydroxytryptamine(1A) Receptor Is Stably Palmitoylated, and Acylation Is Critical for Communication of Receptor with G_i Protein^*

Ekaterina Papoucheva, Aline Dumuis¶, Michèle Sebben¶, Diethelm W. Richter, and Evgeni G. Ponimaskin||

From the Abteilung Neuro- und Sinnesphysiologie, Physiologisches Institut, Universität Göttingen, Humboldtallee 23, D-37073 Göttingen, Germany and ^¶UPR CNRS 9023, 141 Rue de la Cardonille, 34094 Montpellier Cedex 5, France

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 C-terminal 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.

Received for publication, July 27, 2003 , and in revised form, November 4, 2003.

^* 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 732/1-2. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Recipient of a Georg-Christoph Lichtenberg doctoral fellowship from Lower Saxony, Germany.

^|| To whom correspondence should be addressed. Tel.: 49-551-395939; Fax: 49-551-396031; E-mail: evgeni{at}ukps.gwdg.de.

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