Comparison of agonist-induced internalization of the human EP2 and EP4 prostaglandin receptors: role of the carboxyl terminus in EP4 receptor sequestration.
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 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.
Desai
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S
Department of Pharmacology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.
April
H
H
Nwaneshiudu
C
C
Ashby
B
B
D000595:Amino Acid Sequence; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay; D005455:Fluorescent Antibody Technique; D006801:Humans; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary / Q000502:physiology; D018078:Receptors, Prostaglandin E / Q000235:genetics / Q000502:physiology*; D058307:Receptors, Prostaglandin E, EP2 Subtype; D058309:Receptors, Prostaglandin E, EP4 Subtype; D012694:Serine / Q000502:physiology; D013912:Threonine / Q000502:physiology
C545644:PTGER2 protein, human; C545650:PTGER4 protein, human; D018078:Receptors, Prostaglandin E; D058307:Receptors, Prostaglandin E, EP2 Subtype; D058309:Receptors, Prostaglandin E, EP4 Subtype; D013912:Threonine; D012694:Serine