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The 5-Hydroxytryptamine(4a) Receptor Is Palmitoylated at Two
Different Sites, and Acylation Is Critically Involved in Regulation of
Receptor Constitutive Activity*
Evgeni G.
Ponimaskin,
Martin
Heine,
Lara
Joubert§,
Michèle
Sebben§,
Ulf
Bickmeyer,
Diethelm W.
Richter¶, and
Aline
Dumuis§
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
We have reported recently that the mouse
5-hydroxytryptamine(4a) (5-HT4(a)) receptor undergoes
dynamic palmitoylation (Ponimaskin,E. G., Schmidt, M. F.,
Heine, M., Bickmeyer, U., and Richter,D. W. (2001) Biochem.
J. 353, 627-663). In the present study,conserved cysteine
residues 328/329 in the carboxyl terminus ofthe 5-HT4(a)
receptor were identified as potential acylation sites.In contrast to
other palmitoylated G-protein-coupled receptors,the additional
cysteine residue 386 positioned close to the COOH-terminalend of the
receptor was also found to be palmitoylated. Usingpulse and
pulse-chase labeling techniques, we demonstrated thatpalmitoylation of
individual cysteines is a reversible processand that agonist
stimulation of the 5-HT4(a) receptor independentlyincreases the rate of palmitate turnover for both acylation sites.Analysis of acylation-deficient mutants revealed that non-palmitoylated5-HT4(a) receptors were indistinguishable from the wild
type intheir ability to interact with Gs, to stimulate the
adenylyl cyclaseactivity and to activate cyclic nucleotide-sensitive
cation channelsafter agonist stimulation. The most distinctive finding
of thepresent study was the ability of palmitoylation to modulate theagonist-independent constitutive 5-HT4(a) receptor
activity. Wedemonstrated that mutation of the proximal palmitoylation
site(Cys328 Ser/Cys329 Ser)
significantly increases the capacity of receptors to convertfrom the
inactive (R) to the active (R*) form in the absence ofagonist. In
contrast, the rate of isomerization from R to R* forthe
Cys386 Ser as well as for the triple,
non-palmitoylated mutant (Cys328 Ser/Cys329 Ser/Cys386Ser) was similar to that obtained for the
wildtype.
Restricted Lateral Diffusion of Luteinizing Hormone Receptors in Membrane Microdomains.
A. L. Wolf-Ringwall, P. W. Winter, J. Liu, A. K. Van Orden, D. A. Roess, and B. G. Barisas (2011)
J. Biol. Chem.
286, 29818-29827
|Abstract »|Full Text »|PDF »
Differential Regulation of Two Palmitoylation Sites in the Cytoplasmic Tail of the {beta}1-Adrenergic Receptor.
D. M. Zuckerman, S. W. Hicks, G. Charron, H. C. Hang, and C. E. Machamer (2011)
J. Biol. Chem.
286, 19014-19023
|Abstract »|Full Text »|PDF »
G-protein-coupled receptors, cholesterol and palmitoylation: facts about fats.
Alternative splicing and exon duplication generates 10 unique porcine 5-HT4 receptor splice variants including a functional homofusion variant.
J. H. De Maeyer, J. Aerssens, P. Verhasselt, and R. A. Lefebvre (2008)
Physiol Genomics
34, 22-33
|Abstract »|Full Text »|PDF »
Functional and Structural Roles of Conserved Cysteine Residues in the Carboxyl-Terminal Domain of the Follicle-Stimulating Hormone Receptor in Human Embryonic Kidney 293 Cells.
A. Uribe, T. Zarinan, M. A. Perez-Solis, R. Gutierrez-Sagal, E. Jardon-Valadez, A. Pineiro, J. A. Dias, and A. Ulloa-Aguirre (2008)
Biol Reprod
78, 869-882
|Abstract »|Full Text »|PDF »
Distinct Subcellular Localization for Constitutive and Agonist-modulated Palmitoylation of the Human {delta} Opioid Receptor.
U. E. Petaja-Repo, M. Hogue, T. T. Leskela, P. M. H. Markkanen, J. T. Tuusa, and M. Bouvier (2006)
J. Biol. Chem.
281, 15780-15789
|Abstract »|Full Text »|PDF »
Carboxyl Tail Cysteine Mutants of the Thyrotropin-Releasing Hormone Receptor Type 1 Exhibit Constitutive Signaling: Role of Palmitoylation.
D. Du, B. M. Raaka, H. Grimberg, M. Lupu-Meiri, Y. Oron, and M. C. Gershengorn (2005)
Mol. Pharmacol.
68, 204-209
|Abstract »|Full Text »|PDF »
Palmitoylation of the 5-Hydroxytryptamine4a Receptor Regulates Receptor Phosphorylation, Desensitization, and {beta}-Arrestin-Mediated Endocytosis.
E. Ponimaskin, A. Dumuis, F. Gaven, G. Barthet, M. Heine, K. Glebov, D. W. Richter, and M. Oppermann (2005)
Mol. Pharmacol.
67, 1434-1443
|Abstract »|Full Text »|PDF »
New sorting nexin (SNX27) and NHERF specifically interact with the 5-HT4(a) receptor splice variant: roles in receptor targeting.
L. Joubert, B. Hanson, G. Barthet, M. Sebben, S. Claeysen, W. Hong, P. Marin, A. Dumuis, and J. Bockaert (2004)
J. Cell Sci.
117, 5367-5379
|Abstract »|Full Text »|PDF »
The 5-Hydroxytryptamine(1A) Receptor Is Stably Palmitoylated, and Acylation Is Critical for Communication of Receptor with Gi Protein.
E. Papoucheva, A. Dumuis, M. Sebben, D. W. Richter, and E. G. Ponimaskin (2004)
J. Biol. Chem.
279, 3280-3291
|Abstract »|Full Text »|PDF »
Construction and characterization of a kappa opioid receptor devoid of all free cysteines.
D. Ott, R. Frischknecht, and A. Pluckthun (2004)
Protein Eng. Des. Sel.
17, 37-48
|Abstract »|Full Text »|PDF »
Truncation of the A1 Adenosine Receptor Reveals Distinct Roles of the Membrane-proximal Carboxyl Terminus in Receptor Folding and G Protein Coupling.
H. Pankevych, V. Korkhov, M. Freissmuth, and C. Nanoff (2003)
J. Biol. Chem.
278, 30283-30293
|Abstract »|Full Text »|PDF »
Palmitoylation of the Human Prostacyclin Receptor. FUNCTIONAL IMPLICATIONS OF PALMITOYLATION AND ISOPRENYLATION.
S. M. Miggin, O. A. Lawler, and B. T. Kinsella (2003)
J. Biol. Chem.
278, 6947-6958
|Abstract »|Full Text »|PDF »
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Ser/Cys329 
