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

Abstract

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...

Role of the C-terminal di-leucine motif of 5-HT1A and 5-HT1B serotonin receptors in plasma membrane targeting

“…Furthermore, we found that the glycosylation state and the ligand-binding capacity of 5-HT 1A R were not dependent on the presence of the palmitoylated cysteines. These data are consistent with results obtained by Papoucheva et al (Papoucheva et al, 2004) who recently demonstrated the constitutive palmitoylation of cysteines 417 and 420 of this receptor. These authors also showed that palmitoylated cysteines 417 and 420 are necessary for the receptor coupling to G ␣i3 subunit in transfected insect Sf.9 cells, as well as for its capacity to inhibit adenylyl cyclase activity in NIH3T3 cells and to activate ERK in CHO cells.…”

“…This would suggest the need for another signal localized in a different domain of the receptor in addition to the di-leucine motif of the C-terminal tail, which would be present in the 5-HT 1A R but not in the 5-HT 1B R, thereby leading to plasma membrane targeting of the 1ActB but not the 1BctA chimera. Alternatively, it is possible Role of palmitoylated cysteines 5-HT 1A R and 5-HT 1B R were shown to contain palmitoylated cysteines in their C-terminal domain (Ng et al, 1993;Papoucheva et al, 2004). Substitution of these residues with serines did not affect the subcellular localization of either receptors or chimeras.…”

“…For numerous integral membrane proteins, such motifs have been shown to play a role in internalization and lysosomal or plasma membrane targeting (Hunziker and Fumey, 1994;Letourneur and Klausner, 1992;Schülein et al, 1998). On the other hand, the C-terminal domains of 5-HT 1A R and 5-HT 1B R contain palmitoylated cysteines (Ng et al, 1993;Papoucheva et al, 2004). In other GPCRs, these residues were found to be involved in several functional aspects, such as membrane targeting and receptor signaling (for a review, see Qanbar and Bouvier, 2003).…”

Molecular Determinants in the Second Intracellular Loop of the 5-Hydroxytryptamine-1A Receptor for G-Protein Coupling

“…The C-terminal domain of the 5-HT1A receptor also seems to be critical for coupling to both G␣ i and G␤␥ pathways. Unlike other receptors, the 5-HT1A receptor is constitutively palmitoylated at C-terminal cysteine residues (417 and 420), and palmitoylation is required for coupling to G i (i.e., loss of inhibition of AC activity) and G␤␥-mediated activation of extracellular signal-regulated kinases (Papoucheva et al, 2004). Thus, Ci2, Ni3, Ci3, and palmitoylated 5-HT1A C-terminal domains seem critical for G-protein coupling and may directly interact to form a Gprotein coupling interface.…”

“…Thus, the T149A residue of the Ci2 domain plays a crucial role in G␤␥-induced pathways of the 5-HT1A receptor. In addition to the i2 domain, the i3 and palmitoylated C-terminal domains of the 5-HT1A receptor have also been implicated in G-protein coupling (Varrault et al, 1994;Sun and Dale, 1999;Papoucheva et al, 2004;Turner et al, 2004). Peptides derived from the Ni3 or Ci3 domains of the 5-HT1A receptor can mimic G␣ i -mediated inhibition of adenylyl cyclase, whereas i2 peptides prevented this coupling (Varrault et al, 1994;Ortiz et al, 2000).…”