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Importance of Constitutive Activity and Arrestin-Independent Mechanisms for Intracellular Trafficking of the Ghrelin Receptor

Institute of Cell Signalling (N.D.H.), Queen’s Medical Centre, Nottingham NG7 2UH, United Kingdom; Wolfson Centre for Age-Related Diseases (E.A.R., H.M.C.), King’s College London, Guy’s Campus, London SE1 1UL, United Kingdom; and Laboratory for Molecular Pharmacology (B.H., T.W.S.), Panum Institute, University of Copenhagen, DK-2200 Copenhagen, Denmark

Address all correspondence and requests for reprints to: Dr. Nick Holliday, Institute of Cell Signalling, Queen’s Medical Centre, Nottingham NG7 2UH, United Kingdom. E-mail: nicholas.holliday{at}nottingham.ac.uk.

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 β-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-Arg¹, D-Phe⁵, D-Trp^7,9, Leu¹¹] 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 β-arrestin1(319–418) fragment or by expression in β-arrestin1/2 double-knockout mouse embryonic fibroblasts. In contrast, agonist-stimulated GhrelinRs recruited the clathrin adaptor green fluorescent protein-tagged β-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 β-arrestins.

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