title: GABA(B) receptors do not internalize after baclofen treatment, possibly due to a lack of β-arrestin association: study with a real-time visualizing assay.
abstract: The mechanism of agonist-induced GABA(B) receptor (GABA(B) R) internalization is not well understood. To investigate this process, we focused on the interaction of GABA(B) R with β-arrestins, which are key proteins in the internalization of most of the G protein-coupled receptors, and the agonist-induced GABA(B) R internalization and the interaction of GABA(B) R with β-arrestin1 and β-arrestin2 were investigated in real time using GABA(B) R and β-arrestins both of which were fluorescent protein-tagged. We then compared these profiles with those of μ-opioid receptors (μOR), well-studied receptors that associate and cointernalize with β-arrestins. When stimulated by the specific GABA(B) R agonist baclofen, GABA(B) R composed of GABA(B1a) R (GB(1a) R) and fluorescent protein-tagged GABA(B2) R-Venus (GB₂ R-V) formed functional GABA(B) R; they elicited G protein-activated inwardly rectifying potassium channels as well as nontagged GABA(B) R. In cells coexpressing GB(1a) R, GB₂ R-V, and β-arrestin1-Cerulean (βarr1-C) or β-arrestin2-Cerulean (βarr2-C), real-time imaging studies showed that baclofen treatment neither internalized GB₂ R-V nor mobilized βarr1-C or βarr2-C to the cell surface. This happened regardless of the presence of G protein-coupled receptor kinase 4 (GRK4), which forms a complex with GABA(B) R and causes GABA(B) R desensitization. On the other hand, in cells coexpressing μOR-Venus, GRK2, and βarr1-C or βarr2-C, the μOR molecule formed μOR/βarr1 or μOR/βarr2 complexes on the cell surface, which were then internalized into the cytoplasm in a time-dependent manner. Fluorescence resonance energy transfer assay also indicated scarce association of GB₂ R-V and β-arrestins-C with or without the stimulation of baclofen, while robust association of μOR-V with β-arrestins-C was detected after μOR activation. These findings suggest that GABA(B) Rs failure to undergo agonist-induced internalization results in part from its failure to interact with β-arrestins.
authors.affiliation: Department of Cellular and Molecular Biology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8523, Japan.
authors.forename: Yuka
authors.initials: Y
authors.lastname: Sudo
authors.forename: Minoru
authors.initials: M
authors.lastname: Hojo
authors.forename: Yuko
authors.initials: Y
authors.lastname: Ando
authors.forename: Masafumi
authors.initials: M
authors.lastname: Takada
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Murata
authors.forename: Shinji
authors.initials: S
authors.lastname: Kurata
authors.forename: Noriyuki
authors.initials: N
authors.lastname: Nishida
authors.forename: Yasuhito
authors.initials: Y
authors.lastname: Uezono
mesh_terms: D000818:Animals; D019390:Arrestins / Q000378:metabolism*; D001418:Baclofen / Q000494:pharmacology*; D002460:Cell Line; D006224:Cricetinae; D031541:Fluorescence Resonance Energy Transfer; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels / Q000378:metabolism; D058786:GABA-B Receptor Agonists / Q000494:pharmacology*; D006801:Humans; D051381:Rats; D018080:Receptors, GABA-B / Q000378:metabolism*; D000071557:beta-Arrestins
chemical_list: D019390:Arrestins; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; D058786:GABA-B Receptor Agonists; D018080:Receptors, GABA-B; D000071557:beta-Arrestins; D001418:Baclofen