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 β-arrestins, which are key proteins in the internalization of most of the G protein-coupled receptors (GPCRs), and the agonist-induced GABABR internalization and the interaction of GABABR with β-arrestin1 and β-arrestin2 were investigated in real time using GABABR 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 co-internalize with β-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 non-tagged GABABR.
In cells coexpressing GB1aR, GB2R-V, and β-arrestin1-Cerulean (βarr1-C) or β-arrestin2-Cerulean (βarr2-C), real-time imaging studies showed that baclofen treatment neither internalized GB2R-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 GABABR and causes GABABR 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 (FRET) assay also indicated scarce association of GB2R-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 GABABR's failure to undergo agonist-induced internalization results in part from its failure to interact with β-arrestins.
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