Agonist-Induced Phosphorylation by G Protein-Coupled Receptor Kinases of the EP4 Receptor Carboxyl-Terminal Domain in an EP3/EP4 Prostaglandin E₂Receptor Hybrid

Abstract

Prostaglandin E 2 receptors (EP-Rs) belong to the family of heterotrimeric G protein-coupled ectoreceptors with seven transmembrane domains. They can be subdivided into four subtypes according to their ligand-binding and G protein-coupling specificity: EP1 couple to G q , EP2 and EP4 to G s , and EP3 to G i . The EP4-R, in contrast to the EP3␤-R, shows rapid agonist-induced desensitization. The agonist-induced desensitization depends on the presence of the EP4-R carboxyl-terminal domain, which also confers desensitization in a G i -coupled rEP3hEP4 carboxyl-terminal domain receptor hybrid (rEP3hEP4-Ct-R). To elucidate the possible mechanism of this desensitization, in vivo phosphorylation stimulated by activators of second messenger kinases, by prostaglandin E 2 , or by the EP3-R agonist M&B28767 was investigated in COS-7 cells expressing FLAGepitope-tagged rat EP3␤-R (rEP3␤-R), hEP4-R, or rEP3hEP4-Ct-R. Stimulation of protein kinase C with phorbol-12-myristate-13-acetate led to a slight phosphorylation of the FLAG-rEP3␤-R but to a strong phosphorylation of the FLAG-hEP4-R and the FLAG-rEP3hEP4-Ct-R, which was suppressed by the protein kinase A and protein kinase C inhibitor staurosporine. Prostaglandin E 2 stimulated phosphorylation of the FLAG-hEP4-R in its carboxyl-terminal receptor domain. The EP3-R agonist M&B28767 induced a time-and dose-dependent phosphorylation of the FLAG-rEP3hEP4-Ct-R but not of the FLAGrEP3␤-R. Agonist-induced phosphorylation of the FLAGhEP4-R and the FLAG-rEP3hEP4-Ct-R were not inhibited by staurosporine, which implies a role of G protein-coupled receptor kinases (GRKs) in agonist-induced receptor phosphorylation. Overexpression of GRKs in FLAG-rEP3hEP4-Ct-R-expressing COS-7 cells augmented the M&B28767-induced receptor phosphorylation and receptor sequestration. These findings indicate that phosphorylation of the carboxyl-terminal hEP4-R domain possibly by GRKs but not by second messenger kinases may be involved in rapid agonist-induced desensitization of the hEP4-R and the rEP3hEP4-Ct-R.Prostaglandin E 2 receptors (EP-Rs), like most prostanoid receptors, belong to the class of G protein-coupled ectoreceptors (GPCR) with seven transmembrane domains (Negishi, 1994). There are four subtypes of E-prostaglandin receptors (EP-Rs) that differ in their affinity to synthetic ligands and their G protein coupling specificity. EP1-Rs are linked to G q and increase inositol trisphosphate (InsP 3 ) and, hence, cytosolic Ca 2ϩ concentration. EP2-Rs and EP4-Rs are coupled to G s and increase intracellular cAMP. EP3-Rs are coupled to G i and decrease hormone-stimulated cyclic AMP (cAMP) formation ( Fig. 1) (Coleman, 1994). These receptors display an overall sequence homology of about 50%; the putative transmembrane domains are the most conserved (Coleman, 1994).

Arrestin Specificity for G Protein-coupled Receptors in Human Airway Smooth Muscle

“…In contrast, the difference in the amount of EP4 receptor co-precipitating with ARR2-GFP versus ARR2(R169E)-GFP was minimal. Whereas a greater amount of the EP4 receptor migrating at ϳ55 kDa was observed to coprecipitate with ARR2(R169E)-GFP, aggregates of the receptor (migrating at ϳ100 -150-kDa oligomers (49)) that co-precipitated with ARR2-GFP were more abundant, such that the difference in total co-precipitating EP4 receptor was insignificant (less than 20% in duplicate experiments). Thus, the relative degree of interaction between EP receptors and the arrestin chimeras suggested by co-precipitation parallels the effects of the two arrestins on agonist-promoted internalization and desensitization.…”

Low Nociceptor GRK2 Prolongs Prostaglandin E2Hyperalgesia via Biased cAMP Signaling to Epac/Rap1, Protein Kinase Cε, and MEK/ERK

“…This classical model of GRK2 action predicts that GRK2 reduction would enhance EP4-mediated signaling, leading to a stronger cAMP response and increased PKA activation (Ribas et al, 2007). There is indeed evidence that human EP4 receptors can be phosphorylated by GRK2 (Neuschäfer-Rube et al, 1999). Conversely, PGE 2 -induced cAMP production in smooth muscle cells is unaffected in conditions of low GRK2 (Kong et al, 2008).…”

Internalization and Sequestration of the Human Prostacyclin Receptor

“…These observations extend to the eicosanoid receptors. The importance of the C-terminal region of the EP-4 receptor for PGE 2 (57) in sequestration has been reported. Furthermore, ␣ and ␤ isoforms of the TP receptor that differ only in their C-terminal regions demonstrate strikingly different rates of agonist-induced internalization (50).…”