Sequestration and phosphorylation of the prostaglandin E2 EP4 receptor: dependence on the C-terminal tail.
The prostaglandin E2 (PGE2) EP4 subtype is one of four prostanoid receptors that use PGE2 as the preferred ligand. We have investigated the agonist-mediated regulation of EP4 using a multifaceted approach. Short-term (30 min) agonist challenge of recombinant EP4 expressed in human embryonic kidney 293 cells (EP4-HEK293 cells) with PGE2 (1 microM) resulted in the desensitization of intracellular cyclic AMP (cAMP) accumulation and a reduction in cell surface [3H]PGE2 specific binding sites. These events correlated with sequestration of EP4, as visualized by immunofluorescence confocal microscopy and phosphorylation, as shown by [32P]orthophosphate labeling of the receptor. Stimulation of protein kinase A activity in EP4-HEK293 cells (10 microM forskolin or 1 mM 8-bromo-cAMP) did not induce EP4 desensitization, sequestration, or phosphorylation. In contrast, stimulation of protein kinase C activity (100 nM phorbol 12-myristate 13-acetate) attenuated PGE2-induced adenylyl cyclase activity and increased EP4 phosphorylation, but did not induce sequestration or a reduction in [3H]PGE2 specific binding sites. EP4 receptors containing a third intracellular loop deletion [EP4 (del. 215-263)] or a carboxyl-terminal tail truncation [EP4 (del. 355)] of EP4 were used to demonstrate that the C-terminal tail governs sequestration as well as phosphorylation of the receptor.
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Department of Biochemistry and Molecular Biology, Merck Frosst Centre for Therapeutic Research, 16711 Transcanada Hwy, H9H-3L1, Kirkland, Quebec, Canada. deborah_slipetz@merck.com
Buchanan
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Mackereth
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Brewer
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Pellow
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Hao
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Adam
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Abramovitz
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Metters
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D000595:Amino Acid Sequence; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D015232:Dinoprostone / Q000378:metabolism*; D004789:Enzyme Activation; D005455:Fluorescent Antibody Technique; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D017434:Protein Structure, Tertiary; D018078:Receptors, Prostaglandin E / Q000235:genetics / Q000378:metabolism*; D058309:Receptors, Prostaglandin E, EP4 Subtype; D015290:Second Messenger Systems / Q000502:physiology; D014162:Transfection
D008565:Membrane Proteins; C545650:PTGER4 protein, human; D018078:Receptors, Prostaglandin E; D058309:Receptors, Prostaglandin E, EP4 Subtype; D000242:Cyclic AMP; D011493:Protein Kinase C; D015232:Dinoprostone