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.,...