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.

The mechanism of agonist-induced GABAB receptor (GABABR) internalization is not well understood. To investigate this process, we focused on the interaction of GABABR with beta-arrestins, which are key proteins in the internalization of most of the G protein-coupled receptors, and the agonist-induced GABABR internalization and the interaction of GABABR with beta-arrestin1 and beta-arrestin2 were investigated in real time using GABABR and beta-arrestins both of which were fluorescent protein-tagged. We then compared these profiles with those of mu-opioid receptors (mu OR), well-studied receptors that associate and cointernalize with beta-arrestins. When stimulated by the specific GABABR agonist baclofen, GABABR composed of GABAB1aR (GB1aR) and fluorescent protein-tagged GABAB2R-Venus (GB2R-V) formed functional GABABR; they elicited G protein-activated inwardly rectifying potassium channels as well as nontagged GABABR. In cells coexpressing GB1aR, GB2R-V, and beta-arrestin1-Cerulean (beta arr1-C) or beta-arrestin2-Cerulean (beta arr2-C), real-time imaging studies showed that baclofen treatment neither internalized GB2R-V nor mobilized beta arr1-C or beta arr2-C to the cell surface. This happened regardless of the presence of G protein-coupled receptor kinase 4 (GRK4), which forms a complex with GABABR and causes GABABR desensitization. On the other hand, in cells coexpressing mu OR-Venus, GRK2, and beta arr1-C or beta arr2-C, the mu OR molecule formed mu OR/beta arr1 or mu OR/beta arr2 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 GB2R-V and beta-arrestins-C with or without the stimulation of baclofen, while robust association of mu OR-V with beta-arrestins-C was detected after mu OR activation. These findings suggest that GABABRs failure to undergo agonist-induced internalization results in part from its failure to interact with beta-arrestins. Synapse 66:759769, 2012.(c) 2012 Wiley Periodicals, Inc.