The 5-hydroxytryptamine(1A) receptor is stably palmitoylated, and acylation is critical for communication of receptor with Gi protein.
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 alpha(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 beta gamma-mediated pathway, in addition to the G alpha(i)-mediated signaling.
Papoucheva
Ekaterina
E
Abteilung Neuro- und Sinnesphysiologie, Physiologisches Institut, Universität Göttingen, Humboldtallee 23, D-37073 Goettingen, Germany.
Dumuis
Aline
A
Sebben
Michèle
M
Richter
Diethelm W
DW
Ponimaskin
Evgeni G
EG
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000378:metabolism; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D003513:Cycloheximide / Q000494:pharmacology; D003545:Cysteine / Q000737:chemistry; D003593:Cytoplasm / Q000378:metabolism; D004247:DNA / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D000939:Epitopes; D004952:Esters / Q000737:chemistry; D005227:Fatty Acids / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D019811:Hydroxylamine / Q000494:pharmacology; D007313:Insecta; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D041681:NIH 3T3 Cells; D019308:Palmitic Acid / Q000737:chemistry; D010169:Palmitic Acids / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D044282:Receptor, Serotonin, 5-HT1A / Q000737:chemistry* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction
D000939:Epitopes; D004952:Esters; D005227:Fatty Acids; D010169:Palmitic Acids; D011500:Protein Synthesis Inhibitors; D044282:Receptor, Serotonin, 5-HT1A; D019811:Hydroxylamine; D019308:Palmitic Acid; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D004247:DNA; D003513:Cycloheximide; D000242:Cyclic AMP; D020928:Mitogen-Activated Protein Kinases; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D003545:Cysteine