Comparison of Agonist-Induced Internalization of the Human EP2 and EP4 Prostaglandin Receptors: Role of the Carboxyl Terminus in EP4 Receptor Sequestration

Abstract

Prostaglandin E 2 (PGE 2 ) couples to stimulation of adenylyl cyclase through two distinct G protein-coupled receptors designated EP2 and EP4. Although they have similar affinities for PGE 2 , the EP 2 and EP4 receptors have distinct structural characteristics. EP2 is a 358-amino-acid protein with short third intracellular loop and C-terminal domains, whereas EP4 consists of 488 amino acids with a long third intracellular loop and a long cytoplasmic tail. The ability of the HA epitope-tagged receptors to undergo PGE 2 -induced internalization was examined by enzyme-linked immunosorbent assay and immunofluorescence microscopy after expression in human embryonic kidney 293 cells. The EP2 receptor did not internalize, whereas the EP4 receptor underwent rapid internalization. Truncation of the EP4 receptor after amino acid 350, which removes 138 residues, abolished internalization. Truncation after amino acid 369 markedly attenuated internalization, whereas truncation after amino acid 383 had little effect. Serine and threonine residues in the region 350 to 383 were mutated to determine their role in internalization. The mutants S370-382A, a fulllength receptor containing six serine-to-alanine mutations in the region 370 to 382, and S354-369A, containing four serine mutations and one threonine mutation in the region 350 to 370, both internalized to the same extent as the wild-type. A further mutant, designated S354-382A, containing amino acid substitutions S354A, S359A, S364A, S366G, T369A, S370A, S371A, S374A, S377A, S379A, and S382A, also internalized to the same extent as the wild-type. We conclude that the C terminus of the EP4 receptor is involved in internalization; however, serine and threonine residues do not seem to be involved. PGE 2 is an important autocrine mediator in the cardiovascular and other systems (Campbell and Halushka, 1996). PGE 2 is a potent vasodilator in the microvasculature, although it may also cause constriction at selected sites. It causes a fall in systemic blood pressure and an increase in blood flow to the heart; it increases blood flow to the kidneys, leading to increased diuresis, natriuresis, and kaluresis, and causes secretion of renin from the renal cortex. PGE 2 exerts its actions through four distinct G protein-coupled receptors, designated EP1, EP2, EP3, and EP4, that are encoded by different genes (for a review, see Negishi et al., 1995). The EP1 receptor couples to phospholipase C, the EP2 and EP4 receptors couple to stimulation of adenylyl cyclase, and the EP3 receptor couples to inhibition of adenylyl cyclase.EP2 and EP4 receptors are widely distributed; both receptors are found in uterus, spleen, and lung; the EP4 receptor is also present in small intestine, thymus, pancreas, leukocytes, and kidney (Regan et al., 1994;Katsuyama et al., 1995). There are no selective EP receptor antagonists that can be used in studies to functionally distinguish the two receptors. However, the selective EP2 receptor agonist butaprost is not active at the EP4 receptor (Regan et al.,...

Prostaglandin E2 promotes cell proliferation via protein kinase C/extracellular signal regulated kinase pathway‐dependent induction of c‐Myc expression in human esophageal squamous cell carcinoma cells

“…EP4 receptor is readily internalized when activated by PGE 2 whereas EP2 receptor is not. 38,39 Although PGE 2 has also been reported to transactivate EGFR to induce ERK phosphorylation and cell proliferation in other cell types, 22,23 our results indicate that PGE 2 exerts no effect on EGFR phosphorylation. To this end, PKC has been shown to activate ERK in an EGFR-independent manner by stimulation of Raf-1 or Ras.…”

Vasopressin-independent targeting of aquaporin-2 by selective E-prostanoid receptor agonists alleviates nephrogenic diabetes insipidus

“…This may be due to elimination properties of the EP4 agonist, but when administered via minipumps, the agonist's effects were also diminished after 24 h, suggesting otherwise. An alternative explanation may lie in the properties of the receptors themselves; that is, the EP4 receptor is internalized and desensitized following ligand binding, whereas the EP2 receptor is not (29,30). Another advantage of EP2 is that, in comparison with EP4 that is the most widely distributed and abundantly expressed PGE2 receptor, EP2 is the least abundant (31).…”

Gene and protein expression profiles of prostaglandin E2 receptor subtypes in the human corpus cavernosum

“…20 According to reactivity for metabolic product and a difference of desensitization, transient action of PGE2 is shown by EP4 receptor, and persistent action is estimated to be shown by EP2 receptor. 21 It may be reasonable that EP4 receptor subtype expresses at higher level than EP2 in the human cavernosum tissue, from these functional standpoints. According to this hypothesis, agonists working for both EP2 and EP4 may become more efficient and promising drug with aspect of prolonged erection, in addition to EP4 agonist as a new therapy.…”