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ORIGINAL ARTICLE

Palmitoylation of the 5-Hydroxytryptamine_4a Receptor Regulates Receptor Phosphorylation, Desensitization, and -Arrestin-Mediated Endocytosis

Abteilung Neuround Sinnesphysiologie, Physiologisches Institut (E.P, M.H., K.G., D.W.R.), and Abteilung Immunologie (M.O.), Universität Göttingen, Göttingen, Germany; and Institute of Functional Genomics, Montpellier, France (A.D., F.G., G.B.)

Abstract

The mouse 5-hydroxytryptamine_4a (5-HT_4a) receptor is an unusual member of the G protein-coupled receptor superfamily because it possesses two separate carboxyl-terminal palmitoylation sites, which may allow the receptor to adopt different conformations in an agonist-dependent manner (J Biol Chem 277:2534–2546, 2002). By targeted mutation of the proximal (Cys-328/329) or distal (Cys-386) palmitoylation sites, or a combination of both, we generated 5-HT_4a receptor variants with distinct functional characteristics. In this study, we showed that upon 5-HT stimulation, the 5-HT_4a receptor undergoes rapid (t 2 min) and dose-dependent (EC₅₀ 180 nM) phosphorylation on serine residues by a staurosporine-insensitive receptor kinase. Overexpression of GRK2 significantly reduced the receptor-promoted cAMP formation. The Cys328/329-Ser mutant, which is constitutively active in the absence of ligand, exhibited enhanced receptor phosphorylation under both basal and agonist-stimulated conditions and was more effectively desensitized and internalized via a -arrestin-2 mediated pathway compared with the wild-type 5-HT_4a. In contrast, G protein activation, phosphorylation, desensitization, and internalization of the other palmitoylation-deficient receptor mutants were affected differently. These findings suggest that palmitoylation plays an important role in modulating 5-HT_4a receptor functions and that G protein activation, phosphorylation, desensitization, and internalization depend on the different receptor conformations.

Address correspondence to: Evgeni Ponimaskin, Abteilung Neuround Sinnesphysiologie, Physiologisches Institut, Universität Göttingen, Humboldtallee 23, D-37073 Göttingen, Germany. E-mail: eponima{at}gwdg.de

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