Abstract
In addition to G protein-coupled receptor (GPCR) desensitization and endocytosis, β-arrestin recruitment to ligand-stimulated GPCRs promotes non-canonical signalling cascades. Distinguishing the respective contributions of β-arrestin recruitment to the receptor and β-arrestin-promoted endocytosis in propagating receptor signalling has been limited by the lack of selective analytical tools. Here, using a combination of virtual screening and cell-based assays, we have identified a small molecule that selectively inhibits the interaction between β-arrestin and the β2-adaptin subunit of the clathrin adaptor protein AP2 without interfering with the formation of receptor/β-arrestin complexes. This selective β-arrestin/β2-adaptin inhibitor (Barbadin) blocks agonist-promoted endocytosis of the prototypical β2-adrenergic (β2AR), V2-vasopressin (V2R) and angiotensin-II type-1 (AT1R) receptors, but does not affect β-arrestin-independent (transferrin) or AP2-independent (endothelin-A) receptor internalization. Interestingly, Barbadin fully blocks V2R-stimulated ERK1/2 activation and blunts cAMP accumulation promoted by both V2R and β2AR, supporting the concept of β-arrestin/AP2-dependent signalling for both G protein-dependent and -independent pathways.
Highlights
In addition to G protein-coupled receptor (GPCR) desensitization and endocytosis, b-arrestin recruitment to ligand-stimulated GPCRs promotes non-canonical signalling cascades
To gain insight on the inhibitory properties of Barbadin on clathrin-mediated internalization of GPCRs, we examined the sub-cellular distribution of b-arrestin, AP2 and clathrin by confocal fluorescence microscopy in V2R-expressing cells transfected with b-arrestin2-mCherry, b2-adaptin-CFP and clathrin-light-chain-YFP
The present study led to the identification of the first chemical probe that inhibits the interaction between b-arrestin and the b2-adaptin subunit of AP2
Summary
In addition to G protein-coupled receptor (GPCR) desensitization and endocytosis, b-arrestin recruitment to ligand-stimulated GPCRs promotes non-canonical signalling cascades. Using a combination of virtual screening and cell-based assays, we have identified a small molecule that selectively inhibits the interaction between b-arrestin and the b2-adaptin subunit of the clathrin adaptor protein AP2 without interfering with the formation of receptor/b-arrestin complexes. This selective b-arrestin/b2-adaptin inhibitor (Barbadin) blocks agonist-promoted endocytosis of the prototypical b2-adrenergic (b2AR), V2-vasopressin (V2R) and angiotensin-II type-1 (AT1R) receptors, but does not affect b-arrestin-independent (transferrin) or AP2-independent (endothelin-A) receptor internalization. From five compounds found to modulate the b-arrestin/b2-adaptin interaction, two were inhibitors and one of them, which we named b-arrestin/b2-adaptin interaction inhibitor (Barbadin), was further characterized and used as a tool to study the interplay between receptor GPCR internalization and signalling
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