Received for publication October 29, 2004.
Revised February 2, 2005.
Accepted for publication February 2, 2005.

Palmitoylation of the 5-Hydroxytryptamine(4a) Receptor Regulates Receptor Phosphorylation, Desensitization and Arrestin mediated Endocytosis

Abstract

The mouse 5-hydroxytryptamine(4a) (5-HT_4(a)) 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. (Ponimaskin et al., (2002) J. Biol. Chem. 277, 2534-2546). By targeted mutation of the proximal (Cys-328/329) or the distal (Cys-386) palmitoylation sites, or a combination of both, we generated 5-HT_4(a) receptor variants with distinct functional characteristics. Here we show that upon 5-HT stimulation the 5-HT_4(a) receptor undergoes rapid (t^1/2 ~ 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 as compared to the wild-type 5-HT4(a). 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_4(a) receptor functions and that G protein activation, phosphorylation, desensitization and internalization depend on the different receptor conformations.

Key words: Serotonin, Desensitization/uncoupling, Sequestration/Internalization, GRKs, barrestins, Phosphorylation/Dephosphorylation