Sequestration and phosphorylation of the prostaglandin E2 EP4 receptor: Dependence on the C-terminal tail

• Published in: Biochemical pharmacology Vol. 62; no. 8; pp. 997 - 1012
• Main Authors: SLIPETZ, Deborah, BUCHANAN, Stephanie, MACKERETH, Cameron, BREWER, Natalie, PELLOW, Vanessa, HAO, Chuan-Ming, ADAM, Mohammed, ABRAMOVITZ, Mark, METTERS, Kathleen M
• Format: Journal Article
• Language: English
• Published: New York, NY Elsevier Science 15.10.2001
• Subjects: Amino Acid Sequence; Biological and medical sciences; Cell receptors; Cell structures and functions; Cells, Cultured; Cyclic AMP - metabolism; Dinoprostone - metabolism; Enzyme Activation; Fluorescent Antibody Technique; Fundamental and applied biological sciences. Psychology; Hormone receptors. Growth factor receptors. Cytokine receptors. Prostaglandin receptors; Humans; Membrane Proteins - genetics; Membrane Proteins - metabolism; Molecular and cellular biology; Molecular Sequence Data; Mutation; Phosphorylation; Protein Kinase C - metabolism; Protein Structure, Tertiary; Receptors, Prostaglandin E - genetics; Receptors, Prostaglandin E - metabolism; Receptors, Prostaglandin E, EP4 Subtype; Second Messenger Systems - physiology; Transfection; Agonist; Phosphorylation; Desensitization; Enzyme; Transferases; Cyclic AMP; Prostaglandin E2; Activation; Binding site; 5'-End; Gene expression; Transfection; Protein kinase; Internalization; Established cell line; Prostanoid; Carboxyl group; Mutation; Mechanism of action; EP4 prostanoid receptor
• ISSN: 0006-2952; 1873-2968
• DOI: 10.1016/S0006-2952(01)00742-0

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.