A new inhibitor of the b-arrestin/AP2 endocytic complex reveals interplay between GPCR internalization and signalling
In addition to G protein-coupled receptor (GPCR) desensitization and endocytosis, b-arrestin recruitment to ligand-stimulated GPCRs promotes non-canonical signalling cascades. Distinguishing the respective contributions of b-arrestin recruitment to the receptor and b-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 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. Interestingly, Barbadin fully blocks V2R-stimulated ERK1/2 activation and blunts cAMP accumulation promoted by both V2R and b2AR, supporting the concept of b-arrestin/AP2-dependent signalling for both G protein-dependent and -independent pathways.
Beta-arrestins (b-arrestin1 and b-arrestin2) play central roles in the mechanisms regulating G protein-coupled receptor (GPCR) signalling and trafficking1,2. The recruitment of b-arrestin to phosphorylated activated GPCRs at the plasma membrane, following sustained agonist stimulation, promotes desensitization by functionally uncoupling the activated receptors from their heterotrimeric G proteins3. Complexes formed between ligand-occupied GPCRs and b-arrestin are then directed to the endocytic machinery, leading to their interaction with clathrin and its adaptor protein AP2, followed by the internalization of the receptors4–6. Once internalized and targeted to early endosomes, receptors can either be recycled back to the plasma membrane or targeted toward lysosomal degradation. The fate of the endocytosed receptors has been inferred to depend largely on the stability of the interaction between the internalized receptor and b-arrestin7; where the more labile complexes lead to rapid recycling of receptor, whereas more stable interactions prevent recycling and favour receptor degradation. Although endocytosis was first linked to receptor desensitization and resensitization, more recent evidence suggest that internalized receptors can engage in various signalling activities8,9. For instance, endocytosis has been linked to the b-arrestindependent activation of mitogen-activated protein kinases (MAPK)10 and the sustained activation of adenylyl-cyclase by some receptors11–13. Despite the well-established contribution of b-arrestins in various steps of receptor trafficking and signalling, spatial and temporal causative links between these responses have remained difficult to probe, mainly because of the lack of selective pharmacological tools.
Several endocytic paths have been described for GPCRs, including both clathrin-dependent and -independent events14,15, with the vast majority of GPCRs undergoing b-arrestindependent, clathrin-mediated endocytosis. Upon binding to agonist-bound receptor, b-arrestin undergoes a major conformational change, adopting an active conformation that is characterized by the release of its C-terminus, which is buried in its polar core16. The exposed b-arrestin C-tail constitutes an accessible site (DxxFxxFxxxR motif) for the binding of the b2-adaptin subunit of the AP2 complex, which directs the receptor/b-arrestin complexes into maturing clathrin-coated pits (CCPs)17,18. A tripartite interaction exists between b-arrestin, AP2 and clathrin in CCPs (that is, each protein involved in a direct interaction with the other two proteins) and the interaction between b-arrestin and AP2 is an important initial step in localizing GPCRs into the CCP, following agonist stimulation17. The structural determinants for b-arrestin and AP2 interaction have been well-defined17,19, and more recently validated by a co-crystal between the b2-adaptin subunit of AP2 and a peptide fragment of b-arrestin1 C-terminus20. Most of our knowledge concerning GPCR endocytosis and b-arrestin-dependent signalling has been gained through mutagenesis and protein depletion studies in particular for b-arrestins, which would indiscriminately affect recruitment of b-arrestin to receptors, desensitization, endocytosis and b-arrestin-mediated signalling21–23. Specific pharmacological probes targeting b-arrestin/AP2 complexes would be invaluable for determining the role of this complex and clathrin-mediated endocytosis in GPCR signalling.
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