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1000
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Abstract/Summary
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1. Two isoforms of the rat prostaglandin E2 receptor, rEP3α-R and rEP3β-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 rEP3α-R was mutated to Ala and both isoforms and the receptor mutant (rEP3α-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 rEP3α-R isoform to PGE2 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 rEP3β-R and the rEP3α-ST341-349A-R were not affected by prior agonist-stimulation.
4. Agonist-stimulation of HEK293-rEP3α-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 rEP3β-R was not phosphorylated and the rEP3α-ST341-349A-R was phosphorylated only weakly.
5. These results led to the hypothesis that agonist-induced desensitization of the rEP3α-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.
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