title: The natural inverse agonist agouti-related protein induces arrestin-mediated endocytosis of melanocortin-3 and -4 receptors.
abstract: Agouti-related protein (Agrp), one of the two naturally occurring inverse agonists known to inhibit G protein-coupled receptor activity, regulates energy expenditure by decreasing basal and blocking agonist-promoted melanocortin receptor (MCR) signaling. Here we report that, in addition to its inverse agonistic activities, Agrp exhibits agonistic properties on the endocytosis pathway of melanocortin receptors. Sustained exposure of human embryonic kidney 293 cells to Agrp induced endocytosis of the MC3R or the MC4R. The extent and kinetics of Agrp-promoted MCR endocytosis were similar to the endocytosis induced by melanocortins. Using the bioluminescence resonance energy transfer technique, we further showed that after binding of Agrp both MCRs interacted with beta-arrestins. In line with this observation, in COS-7 cells co-expression of beta-arrestins enhanced Agrp-induced MCR endocytosis, whereas in human embryonic kidney 293 cells co-transfection of beta-arrestin-specific small interference RNAs diminished Agrp-promoted endocytosis. This new regulatory mechanism was likewise detectable in a cell line derived from murine hypothalamic neurons endogenously expressing MC4R, pointing to the physiological relevance of Agrp-promoted receptor endocytosis. In conclusion, we demonstrated that Agrp does not solely act by directly blocking MCR signaling but also by reducing the amount of MCR molecules accessible to melanocortins at the cell surface. This beta-arrestin-dependent mechanism reveals a new aspect of MCR signaling in particular and refines the concept of G protein-coupled receptor antagonism in general.
authors.affiliation: Institut für Pharmakologie und Toxikologie, Philipps-Universität Marburg, 35033 Marburg, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Breit
authors.forename: Katharina
authors.initials: K
authors.lastname: Wolff
authors.forename: Hermann
authors.initials: H
authors.lastname: Kalwa
authors.forename: Hubertus
authors.initials: H
authors.lastname: Jarry
authors.forename: Thomas
authors.initials: T
authors.lastname: Büch
authors.forename: Thomas
authors.initials: T
authors.lastname: Gudermann
mesh_terms: D054369:Agouti-Related Protein; D000818:Animals; D019390:Arrestins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D000242:Cyclic AMP / Q000096:biosynthesis; D004705:Endocytosis / Q000502:physiology*; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D011485:Protein Binding; D034741:RNA, Small Interfering / Q000235:genetics; D044104:Receptor, Melanocortin, Type 3 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D044105:Receptor, Melanocortin, Type 4 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection; D000071557:beta-Arrestins
chemical_list: C515198:AGRP protein, human; D054369:Agouti-Related Protein; C515199:Agrp protein, mouse; D019390:Arrestins; D036341:Intercellular Signaling Peptides and Proteins; D034741:RNA, Small Interfering; D044104:Receptor, Melanocortin, Type 3; D044105:Receptor, Melanocortin, Type 4; D011993:Recombinant Fusion Proteins; D000071557:beta-Arrestins; D000242:Cyclic AMP