The mouse 5-hydroxytryptamine 4(a) receptor [5-HT4(a)] was expressed with a baculovirus system in insect cells and analysed for acylation. [3H]Palmitic acid was effectively incorporated into 5-HT4(a) and label was sensitive to the treatment with reducing agents indicating a thioester-type bond. Analysis of protein-bound fatty acids revealed that 5-HT4(a) contains predominantly palmitic acid. Treatment of infected Sf9 (Spodoptera frugiperda) cells with BIMU8 {(endo-N-8-methyl-8-azabicyclo[3.2.1]oct-3-yl)-2,3-dehydro-2-oxo-3-(prop-2-yl)-1H-benzimid-azole-1-carboxamide}, a 5-HT4 receptor-selective agonist, generated a dose-dependent increase in [3H]palmitate incorporation into 5-HT4(a) with an EC50 of approx. 10nM. The change in receptor labelling after stimulation with agonist was receptor-specific and did not result from general metabolic effects. We also used both pulse labelling and pulse–chase labelling to address the dynamics of 5-HT4(a) palmitoylation. Incorporation studies revealed that the rate of palmitate incorporation was increased approx. 3-fold after stimulation with agonist. Results of pulse–chase experiments show that activation with BIMU8 promoted the release of radiolabel from 5-HT4(a), thereby reducing the levels of receptor-bound palmitate to approximately one-half. Taken together, our results demonstrate that palmitoylation of 5-HT4(a) is a reversible process and that stimulation of 5-HT4(a) with agonist increases the turnover rate for receptor-bound palmitate. This provides evidence for a regulated cycling of receptor-bound palmitate and suggests a functional role for palmitoylation/depalmitoylation in 5-hydroxytryptamine-mediated signalling.
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February 2001
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Research Article|
January 25 2001
5-Hydroxytryptamine 4(a) receptor expressed in Sf9 cells is palmitoylated in an agonist-dependent manner
Evgeni G. PONIMASKIN;
Evgeni G. PONIMASKIN
*Abteilung Neuro- und Sinnesphysiologie, Physiologisches Institut, Universität Göttingen, Humboldtallee 23, D-37073 Göttingen, Germany
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Michael F. G. SCHMIDT;
Michael F. G. SCHMIDT
†Institut für Immunologie und Molekularbiologie, Freie Universität Berlin, Philippstrasse 13, D-10115 Berlin, Germany
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Martin HEINE;
Martin HEINE
*Abteilung Neuro- und Sinnesphysiologie, Physiologisches Institut, Universität Göttingen, Humboldtallee 23, D-37073 Göttingen, Germany
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Ulf BICKMEYER;
Ulf BICKMEYER
*Abteilung Neuro- und Sinnesphysiologie, Physiologisches Institut, Universität Göttingen, Humboldtallee 23, D-37073 Göttingen, Germany
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Diethelm W. RICHTER
Diethelm W. RICHTER
1
*Abteilung Neuro- und Sinnesphysiologie, Physiologisches Institut, Universität Göttingen, Humboldtallee 23, D-37073 Göttingen, Germany
1To whom correspondence should be addressed (e-mail d.richter@gwdg.de).
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Publisher: Portland Press Ltd
Received:
August 25 2000
Revision Received:
October 16 2000
Accepted:
November 07 2000
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 2001
2001
Biochem J (2001) 353 (3): 627–634.
Article history
Received:
August 25 2000
Revision Received:
October 16 2000
Accepted:
November 07 2000
Citation
Evgeni G. PONIMASKIN, Michael F. G. SCHMIDT, Martin HEINE, Ulf BICKMEYER, Diethelm W. RICHTER; 5-Hydroxytryptamine 4(a) receptor expressed in Sf9 cells is palmitoylated in an agonist-dependent manner. Biochem J 1 February 2001; 353 (3): 627–634. doi: https://doi.org/10.1042/bj3530627
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