A series of FRET-based β-arrestin2 biosensors are used to study the dynamics and conformational changes that occur when β-arrestin2 binds to and dissociates from the β2-adrenergic receptor in living cells; results show that after β-arrestin2 dissociates from the β2-adrenergic receptor, it stays at the cell membrane in an active conformation for a while, indicating that β-arrestin is able to signal in a G-protein-coupled receptor (GPCR)-free state. Much has been learnt about the structure of G-protein-coupled receptors (GPCRs), ubiquitous mediators of the actions of many hormones, neurotransmitters and drugs, but little is known about how the conformations of these membrane proteins change when they interact with proteins that regulate their function. Susanne Nuber et al. used a series of FRET-based β-arrestin2 biosensors to study the dynamics of the conformational changes that take place in β-arrestin2 when it binds to and eventually dissociates from the β-adrenergic receptor in living cells. They find that after dissociation, β-arrestin2 remains at the cell membrane in an active conformation at least initially, indicating that β-arrestin is able to signal in a GPCR-free state. Mi-Hye Lee et al. used a series of intramolecular FlAsH BRET reporters to monitor conformational changes in β-arrestin2 in the presence of six different GPCRs. They found that different GPCRs and ligands produce β-arrestin2 conformational signatures that correlate with the stability of the receptor–arrestin complex; this explains how different GPCRs can use a common effector for different purposes. (β-)Arrestins are important regulators of G-protein-coupled receptors (GPCRs)1,2,3. They bind to active, phosphorylated GPCRs and thereby shut off ‘classical’ signalling to G proteins3,4, trigger internalization of GPCRs via interaction with the clathrin machinery5,6,7 and mediate signalling via ‘non-classical’ pathways1,2. In addition to two visual arrestins that bind to rod and cone photoreceptors (termed arrestin1 and arrestin4), there are only two (non-visual) β-arrestin proteins (β-arrestin1 and β-arrestin2, also termed arrestin2 and arrestin3), which regulate hundreds of different (non-visual) GPCRs. Binding of these proteins to GPCRs usually requires the active form of the receptors plus their phosphorylation by G-protein-coupled receptor kinases (GRKs)1,3,4. The binding of receptors or their carboxy terminus as well as certain truncations induce active conformations of (β-)arrestins that have recently been solved by X-ray crystallography8,9,10. Here we investigate both the interaction of β-arrestin with GPCRs, and the β-arrestin conformational changes in real time and in living human cells, using a series of fluorescence resonance energy transfer (FRET)-based β-arrestin2 biosensors. We observe receptor-specific patterns of conformational changes in β-arrestin2 that occur rapidly after the receptor–β-arrestin2 interaction. After agonist removal, these changes persist for longer than the direct receptor interaction. Our data indicate a rapid, receptor-type-specific, two-step binding and activation process between GPCRs and β-arrestins. They further indicate that β-arrestins remain active after dissociation from receptors, allowing them to remain at the cell surface and presumably signal independently. Thus, GPCRs trigger a rapid, receptor-specific activation/deactivation cycle of β-arrestins, which permits their active signalling.

Background and Purpose—: Lymphocytes are important players in the pathophysiology of acute ischemic stroke. The interaction of lymphocytes with endothelial cells and platelets, termed thrombo-inflammation, fosters microvascular dysfunction and secondary infarct growth. FTY720, a sphingosine-1-phosphate receptor modulator, blocks the egress of lymphocytes from lymphoid organs and has been shown to reduce ischemic neurodegeneration; however, the underlying mechanisms are unclear. We investigated the mode of FTY720 action in models of cerebral ischemia. Methods—: Transient middle cerebral artery occlusion (tMCAO) was induced in wild-type and lymphocyte-deficient Rag1 ^−/− mice treated with FTY720 (1 mg/kg) or vehicle immediately before reperfusion. Stroke outcome was assessed 24 hours later. Immune cells in the blood and brain were counted by flow cytometry. The integrity of the blood–brain barrier was analyzed using Evans Blue dye. Thrombus formation was determined by immunohistochemistry and Western blot, and was correlated with cerebral perfusion. Results—: FTY720 significantly reduced stroke size and improved functional outcome in wild-type mice on day 1 and day 3 after transient middle cerebral artery occlusion. This protective effect was lost in lymphocyte-deficient Rag1 ^−/− mice and in cultured neurons subjected to hypoxia. Less lymphocytes were present in the cerebral vasculature of FTY720-treated wild-type mice, which in turn reduced thrombosis and increased cerebral perfusion. In contrast, FTY720 was unable to prevent blood–brain barrier breakdown and transendothelial immune cell trafficking after transient middle cerebral artery occlusion. Conclusions—: Induction of lymphocytopenia and concomitant reduction of microvascular thrombosis are key modes of FTY720 action in stroke. In contrast, our findings in Rag1 ^−/− mice and cultured neurons argue against direct neuroprotective effects of FTY720.