Palmitoylation of the 5-hydroxytryptamine4a receptor regulates receptor phosphorylation, desensitization, and beta-arrestin-mediated endocytosis.
The mouse 5-hydroxytryptamine4a (5-HT4a) 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-HT4a receptor variants with distinct functional characteristics. In this study, we showed that upon 5-HT stimulation, the 5-HT4a receptor undergoes rapid (t(1/2) approximately 2 min) and dose-dependent (EC50 approximately 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 beta-arrestin-2 mediated pathway compared with the wild-type 5-HT4a. 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-HT4a receptor functions and that G protein activation, phosphorylation, desensitization, and internalization depend on the different receptor conformations.
Ponimaskin
Evgeni
E
Abteilung Neuro- und Sinnesphysiologie, Physiologisches Institut, Universität Göttingen, Germany. eponima@gwdg.de
Dumuis
Aline
A
Gaven
Florence
F
Barthet
Gaël
G
Heine
Martin
M
Glebov
Konstantin
K
Richter
Diethelm W
DW
Oppermann
Martin
M
D000818:Animals; D019390:Arrestins / Q000494:pharmacology*; D019556:COS Cells; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D004305:Dose-Response Relationship, Drug; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology*; D009154:Mutation / Q000187:drug effects / Q000502:physiology; D010168:Palmitates / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects; D044422:Receptors, Serotonin, 5-HT4 / Q000378:metabolism*; D012701:Serotonin / Q000494:pharmacology; D058828:Serotonin 5-HT4 Receptor Agonists; D018411:Spodoptera; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins
C000605258:Arrb2 protein, mouse; D019390:Arrestins; D010168:Palmitates; D058828:Serotonin 5-HT4 Receptor Agonists; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins; D044422:Receptors, Serotonin, 5-HT4; D012701:Serotonin