title: Characterization of the interaction between the dopamine D4 receptor, KLHL12 and β-arrestins.
abstract: Dopamine receptors are G protein-coupled receptors involved in regulation of cognition, learning, movement and endocrine signaling. The action of G protein-coupled receptors is highly regulated by multifunctional proteins, such as β-arrestins which can control receptor desensitization, ubiquitination and signaling. Previously, we have reported that β-arrestin 2 interacts with KLHL12, a BTB-Kelch protein which functions as an adaptor in a Cullin3-based E3 ligase complex and promotes ubiquitination of the dopamine D4 receptor. Here, we have investigated the molecular basis of the interaction between KLHL12 and β-arrestins and questioned its functional relevance. Our data demonstrate that β-arrestin 1 and β-arrestin 2 bind constitutively to the most common dopamine D4 receptor polymorphic variants and to KLHL12 and that all three proteins can interact within a single macromolecular complex. Surprisingly, stimulation of the receptor has no influence on the association between these proteins or their cellular distribution. We found that Cullin3 also interacts with both β-arrestins but has no influence on their ubiquitination. Knockout of one of the two β-arrestins hampers neither interaction between the dopamine D4 receptor and KLHL12, nor ubiquitination of the receptor. Finally, our results indicate that p44/42 MAPK phosphorylation, the signaling pathway which is often regulated by β-arrestins is not influenced by KLHL12, but seems to be exclusively mediated by Gαi protein upon dopamine D4 receptor stimulation.
authors.affiliation: Laboratory of GPCR Expression and Signal Transduction (L-GEST), Ghent University, Proeftuinstraat 86, 9000 Ghent, Belgium. Electronic address: kamila.skieterska@ugent.be.
authors.forename: Kamila
authors.initials: K
authors.lastname: Skieterska
authors.affiliation: Department of Pharmacology, University of California at Davis (UCDavis), 2426 Tupper Hall, One Shields Ave, CA 95616, USA. Electronic address: aoshen@ucdavis.edu.
authors.forename: Ao
authors.initials: A
authors.lastname: Shen
authors.affiliation: Laboratory of GPCR Expression and Signal Transduction (L-GEST), Ghent University, Proeftuinstraat 86, 9000 Ghent, Belgium. Electronic address: dorien.clarisse@ugent.be.
authors.forename: Dorien
authors.initials: D
authors.lastname: Clarisse
authors.affiliation: Laboratory of GPCR Expression and Signal Transduction (L-GEST), Ghent University, Proeftuinstraat 86, 9000 Ghent, Belgium. Electronic address: pieter.rondou@ugent.be.
authors.forename: Pieter
authors.initials: P
authors.lastname: Rondou
authors.affiliation: Department of Neuroscience, Karolinska Institutet, Retzius väg 8, 17177 Stockholm, Sweden. Electronic address: dasiel.borroto.escuela@ki.se.
authors.forename: Dasiel Oscar
authors.initials: DO
authors.lastname: Borroto-Escuela
authors.affiliation: Laboratory of GPCR Expression and Signal Transduction (L-GEST), Ghent University, Proeftuinstraat 86, 9000 Ghent, Belgium. Electronic address: beatrice.lintermans@ugent.be.
authors.forename: Béatrice
authors.initials: B
authors.lastname: Lintermans
authors.affiliation: Department of Neuroscience, Karolinska Institutet, Retzius väg 8, 17177 Stockholm, Sweden. Electronic address: kjell.fuxe@ki.se.
authors.forename: Kjell
authors.initials: K
authors.lastname: Fuxe
authors.affiliation: Department of Pharmacology, University of California at Davis (UCDavis), 2426 Tupper Hall, One Shields Ave, CA 95616, USA. Electronic address: ykxiang@ucdavis.edu.
authors.forename: Yang Kevin
authors.initials: YK
authors.lastname: Xiang
authors.affiliation: Laboratory of GPCR Expression and Signal Transduction (L-GEST), Ghent University, Proeftuinstraat 86, 9000 Ghent, Belgium. Electronic address: kathleen.vancraenenbroeck@ugent.be.
authors.forename: Kathleen
authors.initials: K
authors.lastname: Van Craenenbroeck
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D044842:Cullin Proteins / Q000378:metabolism; D004298:Dopamine / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D000071776:Kelch Repeat; D051379:Mice; D018345:Mice, Knockout; D008840:Microfilament Proteins / Q000737:chemistry / Q000378:metabolism*; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D008954:Models, Biological; D050505:Mutant Proteins / Q000378:metabolism; D037342:Pertussis Toxin / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D000072417:Protein Domains; D055503:Protein Multimerization / Q000187:drug effects; D050638:Receptors, Dopamine D4 / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism; D054875:Ubiquitination / Q000187:drug effects; D000071557:beta-Arrestins / Q000378:metabolism*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D044842:Cullin Proteins; C493279:KLHL12 protein, human; D008840:Microfilament Proteins; D050505:Mutant Proteins; D000071557:beta-Arrestins; D050638:Receptors, Dopamine D4; D044767:Ubiquitin-Protein Ligases; D037342:Pertussis Toxin; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D019204:GTP-Binding Proteins; D004298:Dopamine
keywords: GPCR; KLHL12; dopamine D(4) receptor; ubiquitination; β-arrestin