title: Characterization of C-terminal tail determinants involved in CRTH2 receptor trafficking: identification of a recycling motif.
abstract: The molecular mechanisms regulating the trafficking of the CRTH2 receptor are poorly understood. In the present study, we characterize C-terminal tail determinants involved in the agonist-induced trafficking of the CRTH2 receptor for prostaglandin D(2). Our results showed that progressive deletion of C-terminal tail residues from amino acid 395 up to 337 gradually impaired CRTH2 internalization by approximately 50% as measured by ELISA in HEK293 cells. Surprisingly, further deletion of the C-tail to amino acid 328 or 317 resulted in receptor mutants displaying internalization similar to the wild-type receptor. Individual mutations of Asp(330), Ser(331), Glu(332), and Leu(333) to Ala in the C-tail of the full length receptor resulted in a 45% increase in internalization of the receptor mutants relative to the wild-type receptor. Pretreatment with the recycling inhibitor monensin increased internalization of the wild-type receptor but did not affect that of the D330A, S331A, E332A and L333A mutants, indicating that these residues are part of a recycling motif. Further experiments revealed that Asp(330), Ser(331) and Glu(332) are not only involved in receptor recycling, but are also required for promotion of CRTH2 internalization by GRK2 and GRK5. Site-directed mutagenesis identified Thr(347) as a major site for PKC-induced internalization of the receptor. Confocal microscopy revealed that arrestin-3 dissociated from the receptor after agonist stimulation and internalization, suggesting that CRTH2 is a class A G protein-coupled receptor. Our study identified specific amino acids in the CRTH2 receptor C-tail implicated in the agonist-induced internalization and the recycling of the receptor.
authors.affiliation: Service de Rhumatologie, Département de Médecine, Centre de Recherche Clinique-Etienne Lebel, Canada.
authors.forename: Sébastien J
authors.initials: SJ
authors.lastname: Roy
authors.forename: Audrey
authors.initials: A
authors.lastname: Parent
authors.forename: Maxime A
authors.initials: MA
authors.lastname: Gallant
authors.forename: Artur J
authors.initials: AJ
authors.lastname: de Brum-Fernandes
authors.forename: Jana
authors.initials: J
authors.lastname: Stanková
authors.forename: Jean-Luc
authors.initials: JL
authors.lastname: Parent
mesh_terms: D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D019390:Arrestins / Q000378:metabolism; D002460:Cell Line; D004705:Endocytosis / Q000235:genetics; D054769:G-Protein-Coupled Receptor Kinase 2 / Q000378:metabolism; D054774:G-Protein-Coupled Receptor Kinase 5 / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000166:cytology; D016297:Mutagenesis, Site-Directed; D015230:Prostaglandin D2 / Q000819:agonists; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D017434:Protein Structure, Tertiary / Q000235:genetics; D021381:Protein Transport / Q000235:genetics; D011971:Receptors, Immunologic / Q000235:genetics / Q000378:metabolism*; D011982:Receptors, Prostaglandin / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion; D014162:Transfection
chemical_list: D019390:Arrestins; D011971:Receptors, Immunologic; D011982:Receptors, Prostaglandin; C082333:arrestin3; D011493:Protein Kinase C; D054769:G-Protein-Coupled Receptor Kinase 2; D054774:G-Protein-Coupled Receptor Kinase 5; D015230:Prostaglandin D2; C036293:prostaglandin D2 receptor