This Article Full Text Full Text (PDF) Supplemental Data Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Holliday, N. D. Articles by Cox, H. M. Search for Related Content PubMed PubMed Citation Articles by Holliday, N. D. Articles by Cox, H. M.

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.