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.

Novel Mechanism of Voltage-Gated N-type (Cav2.2) Calcium Channel Inhibition Revealed through α-Conotoxin Vc1.1 Activation of the GABAB Receptor

“…Another potential mechanism of action is GABA B Ractivated internalization of the GABA B R-Ca v 2.2 complex. However, it was recently shown that b-arrestin, which is important for receptor internalization, is not bound to GABA B R (Sudo et al, 2012). Furthermore, surface biotinylation showed that GABA B R cell surface expression was not affected by exposure to Vc1.1 (Cuny et al, 2012).…”

Transduction of group I mGluR-mediated synaptic plasticity by β-arrestin2 signalling

“…First, signal transduction by arrestins has gradually gained wider recognition through pioneering studies with a number of family A and family B 7TMRs over the last 15 years, but analogous evidence linking mGluRs and other family C 7TMRs with arrestins is limited: calcium sensing receptor and mGlu7 receptor function are sensitive to β-arrestin knockdown or overexpression323334, whereas GABA B receptors do not appear to associate with or initiate β-arrestin-mediated trafficking or signal transduction35. Second, in previous studies G protein-independent signalling has been associated experimentally with mGlu1 receptors rather than mGluR5 (ref.…”

Reversal of inhibition of putative dopaminergic neurons of the ventral tegmental area: Interaction of GABAB and D2 receptors

“…In contrast, it has been shown that desensitization of GABA B receptors can occur through a phosphorylation-independent mechanism via GRK4 in HEK293 cells and cerebellar granule cells (Perroy, et al, 2003). Phosphorylation of D2 receptors by PKC results in desensitization and internalization of those receptors through β-arrestins (Namkung and Sibley, 2004; Thibault, et al, 2011), whereas it has been shown that baclofen stimulation of GABA B receptors does not result in internalization of that receptor, nor did it result in interaction with β-arrestins even in the presence of GRK4 (Sudo, et al, 2012). Despite the cross-desensitization between dopamine and baclofen in the present study, the mechanisms for desensitization for each receptor may be different.…”