RT Journal Article
SR Electronic
T1 Comparison of Agonist-Induced Internalization of the Human EP2 and EP4 Prostaglandin Receptors: Role of the Carboxyl Terminus in EP4 Receptor Sequestration
JF Molecular Pharmacology
JO Mol Pharmacol
FD American Society for Pharmacology and Experimental Therapeutics
SP 1279
OP 1286
DO 10.1124/mol.58.6.1279
VO 58
IS 6
A1 Snehal Desai
A1 Heather April
A1 Chinwe Nwaneshiudu
A1 Barrie Ashby
YR 2000
UL http://molpharm.aspetjournals.org/content/58/6/1279.abstract
AB Prostaglandin E2 (PGE2) couples to stimulation of adenylyl cyclase through two distinct G protein-coupled receptors designated EP2 and EP4. Although they have similar affinities for PGE2, the EP2 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 PGE2-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 full-length 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.