title: Downregulation of 5-HT7 Serotonin Receptors by the Atypical Antipsychotics Clozapine and Olanzapine. Role of Motifs in the C-Terminal Domain and Interaction with GASP-1.
abstract: The human 5-HT7 serotonin receptor, a G-protein-coupled receptor (GPCR), activates adenylyl cyclase constitutively and upon agonist activation. Biased ligands differentially activate 5-HT7 serotonin receptor desensitization, internalization and degradation in addition to G protein activation. We have previously found that the atypical antipsychotics clozapine and olanzapine inhibited G protein activation and, surprisingly, induced both internalization and lysosomal degradation of 5-HT7 receptors. Here, we aimed to determine the mechanism of clozapine- and olanzapine-mediated degradation of 5-HT7 receptors. In the C-terminus of the 5-HT7 receptor, we identified two YXXΦ motifs, LR residues, and a palmitoylated cysteine anchor as potential sites involved in receptor trafficking to lysosomes followed by receptor degradation. Mutating either of these sites inhibited clozapine- and olanzapine-mediated degradation of 5-HT7 receptors and also interfered with G protein activation. In addition, we tested whether receptor degradation was mediated by the GPCR-associated sorting protein-1 (GASP-1). We show that GASP-1 binds the 5-HT7 receptor and regulates the clozapine-mediated degradation. Mutations of the identified motifs and residues, located in or close to Helix-VIII of the 5-HT7 receptor, modified antipsychotic-stimulated binding of proteins (such as GASP-1), possibly by altering the flexibility of Helix-VIII, and also interfered with G protein activation. Taken together, our data demonstrate that binding of clozapine or olanzapine to the 5-HT7 receptor leads to antagonist-mediated lysosomal degradation by exposing key residues in the C-terminal tail that interact with GASP-1.
authors.affiliation: †Department of Pharmacology, Institute of Clinical Medicine, University of Oslo and Oslo University Hospital, P.O. Box 1057 Blindern, 0316 Oslo, Norway.|‡K.G. Jebsen Cardiac Research Centre and Center for Heart Failure Research, Institute of Clinical Medicine, University of Oslo, 0316 Oslo, Norway.
authors.forename: Ornella
authors.initials: O
authors.lastname: Manfra
authors.affiliation: §Laboratory of GPCR Expression and Signal Transduction (LEGEST), Ghent University-Ghent, 9000 Ghent, Belgium.
authors.forename: Kathleen
authors.initials: K
authors.lastname: Van Craenenbroeck
authors.affiliation: §Laboratory of GPCR Expression and Signal Transduction (LEGEST), Ghent University-Ghent, 9000 Ghent, Belgium.
authors.forename: Kamila
authors.initials: K
authors.lastname: Skieterska
authors.affiliation: ∥The Novo Nordisk Foundation Center for Basic Metabolic Research, Faculty of Health and Medical Sciences, University of Copenhagen, DK-1165 Copenhagen, Denmark.
authors.forename: Thomas
authors.initials: T
authors.lastname: Frimurer
authors.affiliation: ∥The Novo Nordisk Foundation Center for Basic Metabolic Research, Faculty of Health and Medical Sciences, University of Copenhagen, DK-1165 Copenhagen, Denmark.|⊥Laboratory for Molecular Pharmacology, Department of Pharmacology, Panum Institute, University of Copenhagen, DK-22 00 Copenhagen, Denmark.
authors.forename: Thue W
authors.initials: TW
authors.lastname: Schwartz
authors.affiliation: †Department of Pharmacology, Institute of Clinical Medicine, University of Oslo and Oslo University Hospital, P.O. Box 1057 Blindern, 0316 Oslo, Norway.|‡K.G. Jebsen Cardiac Research Centre and Center for Heart Failure Research, Institute of Clinical Medicine, University of Oslo, 0316 Oslo, Norway.
authors.forename: Finn Olav
authors.initials: FO
authors.lastname: Levy
authors.affiliation: †Department of Pharmacology, Institute of Clinical Medicine, University of Oslo and Oslo University Hospital, P.O. Box 1057 Blindern, 0316 Oslo, Norway.|‡K.G. Jebsen Cardiac Research Centre and Center for Heart Failure Research, Institute of Clinical Medicine, University of Oslo, 0316 Oslo, Norway.
authors.forename: Kjetil Wessel
authors.initials: KW
authors.lastname: Andressen
mesh_terms: D000262:Adenylyl Cyclases / Q000378:metabolism; D014150:Antipsychotic Agents / Q000494:pharmacology*; D001569:Benzodiazepines / Q000494:pharmacology*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D003024:Clozapine / Q000494:pharmacology*; D015536:Down-Regulation / Q000187:drug effects; D057809:HEK293 Cells; D006801:Humans; D047468:Immunoprecipitation; D036341:Intercellular Signaling Peptides and Proteins; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D000077152:Olanzapine; D011506:Proteins / Q000378:metabolism*; D011869:Radioligand Assay; D011985:Receptors, Serotonin / Q000235:genetics / Q000378:metabolism*; D018490:Serotonin Agents / Q000494:pharmacology*; D014162:Transfection
chemical_list: D014150:Antipsychotic Agents; D036341:Intercellular Signaling Peptides and Proteins; D011506:Proteins; D011985:Receptors, Serotonin; D018490:Serotonin Agents; C424444:WFIKKN2 protein, human; C104425:serotonin 7 receptor; D001569:Benzodiazepines; D000262:Adenylyl Cyclases; D003024:Clozapine; D000077152:Olanzapine
keywords: 5-HT7 receptor; GASP-1; Helix-VIII; clozapine; lysosome; olanzapine