title: Importance of constitutive activity and arrestin-independent mechanisms for intracellular trafficking of the ghrelin receptor.
abstract: The ghrelin receptor (GhrelinR) and its related orphan GPR39 each display constitutive signaling, but only GhrelinRs undergo basal internalization. Here we investigate these differences by considering the roles of the C tail receptor domains for constitutive internalization and activity. Furthermore the interaction between phosphorylated receptors and beta-arrestin adaptor proteins has been examined. Replacement of the FLAG-tagged GhrelinR C tail with the equivalent GPR39 domain (GhR-39 chimera) preserved G(q) signaling. However in contrast to the GhrelinR, GhR-39 receptors exhibited no basal and substantially decreased agonist-induced internalization in transiently transfected HEK293 cells. Internalized GhrelinR and GhR-39 were predominantly localized to recycling compartments, identified with transferrin and the monomeric G proteins Rab5 and Rab11. Both the inverse agonist [d-Arg(1), d-Phe(5), d-Trp(7,9), Leu(11)] substance P and a naturally occurring mutant GhrelinR (A204E) with eliminated constitutive activity inhibited basal GhrelinR internalization. Surprisingly, we found that noninternalizing GPR39 was highly phosphorylated and that basal and agonist-induced phosphorylation of the GhR-39 chimera was elevated compared with GhrelinRs. Moreover, basal GhrelinR endocytosis occurred without significant phosphorylation, and it was not prevented by cotransfection of a dominant-negative beta-arrestin1(319-418) fragment or by expression in beta-arrestin1/2 double-knockout mouse embryonic fibroblasts. In contrast, agonist-stimulated GhrelinRs recruited the clathrin adaptor green fluorescent protein-tagged beta-arrestin2 to endosomes, coincident with increased receptor phosphorylation. Thus, GhrelinR internalization to recycling compartments depends on C-terminal motifs and constitutive activity, but the high levels of GPR39 phosphorylation, and of the GhR-39 chimera, are not sufficient to drive endocytosis. In addition, basal GhrelinR internalization occurs independently of beta-arrestins.
authors: Institute of Cell Signalling, Queen's Medical Centre, Nottingham NG7 2UH, United Kingdom. nicholas.holliday@nottingham.ac.uk
authors: Nicholas D
authors: ND
authors: Holliday
authors: Birgitte
authors: B
authors: Holst
authors: Elena A
authors: EA
authors: Rodionova
authors: Thue W
authors: TW
authors: Schwartz
authors: Helen M
authors: HM
authors: Cox
mesh_terms: D000595:Amino Acid Sequence; D019393:Arrestin / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010766:Phosphorylation; D021381:Protein Transport; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D054440:Receptors, Ghrelin / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D020696:rab5 GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D000099193:rab11 GTP-Binding Proteins
chemical_list: D019393:Arrestin; D007295:Inositol Phosphates; D043562:Receptors, G-Protein-Coupled; D054440:Receptors, Ghrelin; D011994:Recombinant Proteins; D020691:rab GTP-Binding Proteins; D020696:rab5 GTP-Binding Proteins; D000099193:rab11 GTP-Binding Proteins; C495797:GPR39 protein, human