β-arrestin plays a key role in G protein-coupled receptor (GPCR) signaling and desensitization. Despite recent structural advances, the mechanisms that govern receptor-β-arrestin interactions at the plasma membrane of living cells remain elusive. Here, we combine single-molecule microscopy with molecular dynamics simulations to dissect the complex sequence of events involved in β-arrestin interactions with both receptors and the lipid bilayer. Unexpectedly, our results reveal that β-arrestin spontaneously inserts into the lipid bilayer and transiently interacts with receptors via lateral diffusion on the plasma membrane. Moreover, they indicate that, following receptor interaction, the plasma membrane stabilizes β-arrestin in a longer-lived, membrane-bound state, allowing it to diffuse to clathrin-coated pits separately from the activating receptor. These results expand our current understanding of β-arrestin function at the plasma membrane, revealing a critical role for β-arrestin preassociation with the lipid bilayer in facilitating its interactions with receptors and subsequent activation.
G protein-coupled receptors (GPCRs) are implicated in virtually every physiological process and are major drug targets.1,2 Following agonist-mediated GPCR activation and phosphorylation by G protein-coupled-receptor kinases (GRKs), β-arrestins translocate from the cytosol to bind agonist-occupied, phosphorylated receptors on the plasma membrane. There are four arrestins—two visual arrestins (also known as arrestin 1 and 4), β-arrestin 1 (βArr1) (arrestin 2), and β-arrestin 2 (βArr2) (arrestin 3).
By interacting with the receptor core, β-arrestins mediate rapid signal desensitization.3 In addition, β-arrestins trigger receptor internalization via interaction with the adaptor protein 2 (AP2) and clathrin heavy chain.4 Moreover, β-arrestins have been proposed to mediate “non-classical” G protein-independent effects,5 providing a mechanism for “biased” signaling.6 This diversity of functions has been linked to multiple conformations in receptor-arrestin complexes revealed by structural and biophysical studies with purified proteins.7,8,9,10,11,12,13,14,15,16,17 Furthermore, recent findings of prolonged β-arrestin activation18,19,20 suggest that β-arrestin signaling might be more complex than previously thought. However, how this complexity operates on the plasma membrane of living cells remains largely unknown.
Here, we combined an innovative multicolor single-molecule microscopy approach21,22 with molecular dynamics (MD) simulations to dissect the sequence of events in receptor-β-arrestin interactions at the plasma membrane of living cells with ∼20 nm spatial and 30 ms temporal resolution.21,22 Our results reveal that β-arrestin binds directly to the lipid bilayer, allowing it to transiently interact with receptors via lateral diffusion, and reaches clathrin-coated pits (CCPs) separately from the activating receptor.
Single-molecule imaging reveals spontaneous membrane translocation and lateral diffusion of βArr2
As a main model, we chose βArr2 and the β2-adrenergic receptor (β2AR), a prototypical family-A GPCR that regulates numerous physiological processes.23,24 To investigate the behavior of individual βArr2 and β2AR molecules on the plasma membrane of living cells, we labeled them with two distinct organic fluorophores via fusion of Halo25 and SNAP26 tags to their C and N termini, respectively (Figure 1A). Both constructs were transiently expressed at low physiological levels in Chinese hamster ovary (CHO) cells, which have no detectable β1AR/β2AR.27 Both βArr2-Halo and SNAP-β2AR constructs are functional; βArr2-Halo binds receptors and mediates receptor internalization to a similar extent as wild-type (WT) βArr2, and co-localizes with internalized receptors in endosomes (Figures S1A–S1D; see STAR Methods for details). CHO cells were then labeled with saturating concentrations of both organic fluorophores (Figure S1E) and were simultaneously imaged by fast multicolor total internal reflection fluorescence (TIRF) microscopy combined with single-particle tracking22 (Figure 1B; Videos S1 and S2); unspecific labeling was <1% (Figure S1F). We additionally visualized CCPs by co-transfection of GFP-labeled clathrin light chain. Data were acquired both under basal conditions and after early (2–7 min) and late (8–15 min) stimulation with the β-adrenergic full agonist isoproterenol (Iso). An excess of 5.8 million individual molecular trajectories were acquired and analyzed in this study. Numbers of trajectories and particle densities in the analyzed groups are given in Table S1.