Sequestration and phosphorylation of the prostaglandin E2EP4 receptor:: dependence on the C-terminal tail

The prostaglandin E-2 (PGE(2)) EP4 subtype is one of four prostanoid receptors that use PGE(2) 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 PGE(2) (1 muM) resulted in the desensitization of intracellular cyclic AMP (cAMP) accumulation and a reduction in cell surface [H-3]PGE(2) specific binding sites. These events correlated with sequestration of EP4, as visualized by immunofluorescence confocal microscopy and phosphorylation, as shown by [P-32]orthophosphate labeling of the receptor. Stimulation of protein kinase A activity in EP4-HEK293 cells (10 muM 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 PGE(2)-induced adenylyl cyclase activity and increased EP4 phosphorylation, but did not induce sequestration or a reduction in [H-3]PGE(2) 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. (C) 2001 Elsevier Science Inc. All rights reserved.