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J. Biol. Chem. 277 (4): 2534-2546

© 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

The 5-Hydroxytryptamine(4a) Receptor Is Palmitoylated at Two Different Sites, and Acylation Is Critically Involved in Regulation of Receptor Constitutive Activity*

Evgeni G. PonimaskinDagger , Martin HeineDagger , Lara Joubert§, Michèle Sebben§, Ulf BickmeyerDagger , Diethelm W. RichterDagger , and Aline Dumuis§

From the Dagger  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 of the 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-terminal end of the receptor was also found to be palmitoylated. Using pulse and pulse-chase labeling techniques, we demonstrated that palmitoylation of individual cysteines is a reversible process and that agonist stimulation of the 5-HT4(a) receptor independently increases the rate of palmitate turnover for both acylation sites. Analysis of acylation-deficient mutants revealed that non-palmitoylated 5-HT4(a) receptors were indistinguishable from the wild type in their ability to interact with Gs, to stimulate the adenylyl cyclase activity and to activate cyclic nucleotide-sensitive cation channels after agonist stimulation. The most distinctive finding of the present study was the ability of palmitoylation to modulate the agonist-independent constitutive 5-HT4(a) receptor activity. We demonstrated that mutation of the proximal palmitoylation site (Cys328 right-arrow Ser/Cys329 right-arrow Ser) significantly increases the capacity of receptors to convert from the inactive (R) to the active (R*) form in the absence of agonist. In contrast, the rate of isomerization from R to R* for the Cys386 right-arrow Ser as well as for the triple, non-palmitoylated mutant (Cys328 right-arrow Ser/Cys329 right-arrow Ser/Cys386 right-arrowSer) was similar to that obtained for the wild type.


* This work was supported by the fund of the Medical School at the University of Göttingen and by Deutsche Forschungsgemeinschaft Grant PO 732/1-1 (to E. G. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 49-551-395911; Fax: 49-551-396031; E-mail: d.richter@gwdg.de.


Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.


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