D. Slipetz et al., Sequestration and phosphorylation of the prostaglandin E2EP4 receptor: dependence on the C-terminal tail, BIOCH PHARM, 62(8), 2001, pp. 997-1012
The prostaglandin E-2 (PGE(2)) EP4 subtype is one of four prostanoid recept
ors that use PGE(2) as the preferred ligand. We have investigated the agoni
st-mediated regulation of EP4 using a multifaceted approach. Short-term (30
min) agonist challenge of recombinant EP4 expressed in human embryonic kid
ney 293 cells (EP4-HEK293 cells) with PGE(2) (1 muM) resulted in the desens
itization of intracellular cyclic AMP (cAMP) accumulation and a reduction i
n cell surface [H-3]PGE(2) specific binding sites. These events correlated
with sequestration of EP4, as visualized by immunofluorescence confocal mic
roscopy and phosphorylation, as shown by [P-32]orthophosphate labeling of t
he 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 kina
se C activity (100 nM phorbol 12-myristate 13-acetate) attenuated PGE(2)-in
duced adenylyl cyclase activity and increased EP4 phosphorylation, but did
not induce sequestration or a reduction in [H-3]PGE(2) specific binding sit
es. EP4 receptors containing a third intracellular loop deletion [EP4 (del.
215-263)] or a carboxyl-terminal tail truncation [EP4 (del. 355)] of EP4 w
ere used to demonstrate that the C-terminal tail governs sequestration as w
ell as phosphorylation of the receptor. (C) 2001 Elsevier Science Inc. All
rights reserved.