Constitutive Gs-mediated, but not G12-mediated, activity of the 5-hydroxytryptamine 5-HT7(a) receptor is modulated by the palmitoylation of its C-terminal domain.
The 5-HT(7) receptor is the most recently described member of the serotonin receptor family. This receptor is mainly expressed in the thalamus, hypothalamus as well as in the hippocampus and cortex. In the present study, we demonstrate that the mouse 5-hydroxytryptamine 5-HT(7(a)) receptor undergoes post-translational modification by the palmitate, which is covalently attached to the protein through a thioester-type bond. Analysis of protein-bound fatty acids revealed that the 5-HT(7(a)) receptor predominantly contains palmitic acid. Labelling experiments performed in the presence of agonists show that the 5-HT(7(a)) receptor is dynamically palmitoylated in an agonist-dependent manner and that previously synthesized receptors may be subjected to repeated cycles of palmitoylation/depalmitoylation. Mutation analysis revealed that cysteine residues 404 and 438/441 located in the C-terminal receptor domain are the main palmitoylation sites responsible for the attachment of 90% of the receptor-bound palmitate. Analysis of acylation-deficient mutants revealed that non-palmitoylated 5-HT(7(a)) receptors were indistinguishable from the wild-type for their ability to interact with G(s)- and G(12)-proteins after agonist stimulation. However, mutation of the proximal palmitoylation site Cys404-Ser (either alone or in combination with Cys438/441-Ser) significantly increased the agonist-independent, G(s)-mediated constitutive 5-HT(7(a)) receptor activity, while the activation of Galpha(12)-protein was not affected. This demonstrates a functional importance of 5-HT(7(a)) dynamic palmitoylation for the fine tuning of receptor-mediated signaling.
Kvachnina
Elena
E
Department Neuro- and Sensory Physiology, University of Göttingen, Göttingen, Germany.
Dumuis
Aline
A
Wlodarczyk
Jakub
J
Renner
Ute
U
Cochet
Maud
M
Richter
Diethelm W
DW
Ponimaskin
Evgeni
E
D000818:Animals; D002460:Cell Line; D044365:GTP-Binding Protein alpha Subunits, G12-G13 / Q000378:metabolism*; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000378:metabolism*; D054878:Lipoylation / Q000502:physiology*; D051379:Mice; D010169:Palmitic Acids / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000502:physiology; D011985:Receptors, Serotonin / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*
D010169:Palmitic Acids; D011985:Receptors, Serotonin; C104425:serotonin 7 receptor; D044365:GTP-Binding Protein alpha Subunits, G12-G13; D019205:GTP-Binding Protein alpha Subunits, Gs