TY  - JOUR
A1  - Neuschäfer-Rube, Frank
A1  - Hermosilla, Ricardo
A1  - Rehwald, Matthias
A1  - Ronnstrand, Lars
A1  - Schülein, Ralf
A1  - Wernstedt, Christer
A1  - Püschel, Gerhard Paul
T1  - Identification of a Ser/Thr cluster in the C-terminal domain of the human prostaglandin receptor EP4 that is essential for agonist-induced beta-arrestin1 recruitment but differs from the apparent principal phosphorylation site
N2  - hEP4-R (human prostaglandin E2 receptor, subtype EP4) is a G(s)-linked heterotrimeric GPCR (G-protein-coupled receptor). It undergoes agonist-induced desensitization and internalization that depend on the presence of its C- terminal domain. Desensitization and internalization of GPCRs are often linked to agonist-induced beta-arrestin complex formation, which is stabilized by phosphorylation. Subsequently beta-arrestin uncouples the receptor from its G-protein and links it to the endocytotic machinery. The C-terminal domain of hEP4-R contains 38 Ser/Thr residues that represent potential phosphorylation sites. The present study aimed to analyse the relevance of these Ser/Thr residues for agonist- induced phosphorylation, interaction with beta-arrestin and internalization. In response to agonist treatment, hEP4-R was phosphorylated. By analysis of proteolytic phosphopeptides of the wild-type receptor and mutants in which groups of Ser/Thr residues had been replaced by Ala, the principal phosphorylation site was mapped to a Ser/Thr-containing region comprising residues 370-382, the presence of which was necessary and sufficient to obtain full agonist-induced phosphorylation. A cluster of Ser/Thr residues (Ser-389-Ser-390-Thr-391-Ser-392) distal to this site, but not the principal phosphorylation site, was essential to allow agonist-induced recruitment of beta-arrestin1. However, phosphorylation greatly enhanced the stability of the beta-arrestin1-receptor complexes. For maximal agonist-induced internalization, phosphorylation of the principal phosphorylation site was not required, but both beta-arrestin1 recruitment and the presence of Ser/Thr residues in the distal half of the C-terminal domain were necessary.
Y1  - 2004
UR  - http://www.biochemj.org/bj/379/0573/bj3790573.htm
ER  - 
TY  - JOUR
A1  - Neuschäfer-Rube, Frank
A1  - Hermosilla, Ricardo
A1  - Kuna, Manuela
A1  - Pathe-Neuschäfer-Rube, Andrea
A1  - Schulein, R.
A1  - Püschel, Gerhard Paul
T1  - A Ser/Thr cluster within the C-terminal domain of the rat prostaglandin receptor EP3 alpha is essential for agonist-induced phosphorylation, desensitization and internalization
N2  - 1 Two isoforms of the rat prostaglandin E-2 receptor, rEP3 alpha-R and rEP3 beta-R, differ only in their C- terminal domain. To analyze the function of the rEP3-R C-terminal domain in agonist induced desensitization, a cluster of Ser/Thr residues in the C-terminal domain of the rEP3 alpha-R was mutated to Ala and both isoforms and the receptor mutant (rEP3 alpha-ST341-349A-R) were stably expressed in HEK293 cells. 2 All rEP3-R receptors showed a similar ligand- binding profile. They were functionally coupled to Gi and reduced forskolin-induced cAMP-formation. 3 Repeated exposure of cells expressing the rEP3 alpha-R isoform to PGE(2) reduced the agonist induced inhibition of forskolin-stimulated cAMP-formation by 50% and led to internalization of the receptor to intracellular endocytotic vesicles. By contrast, Gi- response as well as plasma membrane localization of the rEP3 beta-R and the rEP3 alpha-ST341-349A-R were not affected by prior agonist-stimulation. 4 Agonist-stimulation of HEK293-rEP3 alpha-R cells induced a time- and dose-dependent phosphorylation of the receptor most likely by G protein-coupled receptor kinases and not by protein kinase A or protein kinase C. By contrast, upon agonist-stimulation the rEP3 beta-R was not phosphorylated and the rEP3 alpha-ST341-349A-R was phosphorylated only weakly. 5 These results led to the hypothesis that agonist-induced desensitization of the rEP3 alpha-R isoform is mediated most likely by a GRK-dependent phosphorylation of Ser/Thr residues 341 - 349. Phosphorylation then initiates uncoupling of the receptor from Gi protein and receptor internalization
Y1  - 2005
SN  - 0007-1188
ER  - 
TY  - JOUR
A1  - Neuschäfer-Rube, Frank
A1  - Hermosilla, Ricardo
A1  - Kuna, Manuela
A1  - Pathe-Neuschaefer-Rube, Andrea
A1  - Püschel, Gerhard Paul
T1  - Agonist-induced desensitization of rat prostaglandin EP3 receptor isoforms
Y1  - 2004
SN  - 0028-1298
ER  -