Summary

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.

Introduction

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.

Results

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.

1- Introduction

2- Materials and methods

3- Results

4- Discussion

References

Abstract

The Drosophila male stem cell niche is a well characterized structure in which a small cluster of somatic cells send self-renewal signals to neighbouring germ cells. Although the molecular information involved in the stem cell fate have been identified, much less is understand on the mechanisms driving their short-range specific release. Our ultrastructural analysis reveals distinct protrusions of the stem cell plasma membrane that interdigitate with membrane protrusions of the facing hub cells. Some of these protrusions are very elongated and extend into the hub and could correspond to the Mt-Nanotubes. Therefore, the interface between the stem cells and the hub appears more complex than previously reported and the membrane protrusions of the stem cells might represent specialized surface areas involved in the niche-stem cell communication. We also noticed the presence of clathrin-coated vesicles in the germline plasma membrane that might be also involved in delivering information from the hub.

Introduction

One of the best-known stem cell niche systems is restricted to the apical tip of the Drosophila testis. Here, a crown of 8–10 germline stem cells (GSCs) surrounds a cluster of small post-mitotic somatic cells, the hub [1]. The GSCs divide asymmetrically to produce two daughters, one daughter remains attached to the hub and maintains stem cell fate, while the other daughter, the gonialblast, is displaced away and initiates the differentiation program. A pair of somatic cyst stem cells (CySCs) surrounds each GSC and divide asymmetrically to produce a pair of somatic stem cell daughters and two cyst cells that surround the gonialblast. The different fate of the stem cell progeny is ensured by upstream signals that travel within the niche microenvironment triggering stemness or differentiation. In Drosophila, the hub represents the source of two main self-renewal factors, the cytokine-like ligand Unpaired (Upd) and the Bone Morphogenetic Protein ligand Decapentaplegic (Dpp). Upd through its receptor Domeless (Dome) activates the Janus Kinase Transducer and Activator of Transcription (JAK/ STAT) [2] pathway that supports GSC maintenance by promoting the stem cell adhesion to the hub [3]. The Dpp signal that is supplied by both the hub and the CySCs [4] activates the R-Smad transcription factor Mother against Dpp (Mad) that promotes GSC fate by preventing the expression of the specific differentiation gene Bam-of-marbles (Bam) so that Bam is silenced in GSCs but active in gonialblasts [5].

The plasma membrane (PM) H+ -ATPase is an important ion pump in the plant cell membrane. By extruding protons from the cell and generating a membrane potential, this pump energizes the PM, which is a prerequisite for growth. Modification of the autoinhibitory terminal domains activates PM H+ -ATPase activity, and on this basis it has been hypothesized that these regulatory termini are targets for physiological factors that activate or inhibit proton pumping. In this review, we focus on the posttranslational regulation of the PM H+ -ATPase and place regulation of the pump in an evolutionary and physiological context. The emerging picture is that multiple signals regulating plant growth interfere with the posttranslational regulation of the PM H+ -ATPase.

INTRODUCTION

The plasma membrane (PM) H+ -ATPase is an electrogenic pump that exports cellular protons. In addition to generating a transmembrane chemical gradient of H+ (DpH; acidic on the outside), it also establishes an electrical gradient (the membrane potential; negative on the inside). As almost all other transport proteins in the plant PM are energized by this combined electrochemical gradient of protons, it has been a long-standing hypothesis in plant physiology that the PM H+ – ATPase is a powerhouse for plant growth (reviewed in Palmgren, 2001; Arango et al., 2003; Sondergaard et al., 2004; Duby and Boutry, 2009). However, although the biochemistry of the pump is known in great detail and its structure has been determined (reviewed in Morth et al., 2011), strong evidence for its role in plant growth has remained sparse until recently, when strong links between posttranslational regulation of PM H+ -ATPase and essential physiological functions of plants have emerged. The aim of this review is to provide an overview of this exciting and rapidly developing field.

Abstract

Plants are permanently situated in a fixed location and thus are well adapted to sense and respond to environmental stimuli and developmental cues. At the cellular level, several of these responses require delicate adjustments that affect the activity and steady-state levels of plasma membrane proteins. These adjustments involve both vesicular transport to the plasma membrane and protein internalization via endocytic sorting. A substantial part of our current knowledge of plant plasma membrane protein sorting is based on studies of PIN-FORMED (PIN) auxin transport proteins, which are found at distinct plasma membrane domains and have been implicated in directional efflux of the plant hormone auxin. Here, we discuss the mechanisms involved in establishing such polar protein distributions, focusing on PINs and other key plant plasma membrane proteins, and we highlight the pathways that allow for dynamic adjustments in protein distribution and turnover, which together constitute a versatile framework that underlies the remarkable capabilities of plants to adjust growth and development in their ever-changing environment.

Introduction

Plasma membrane proteins play key roles in mediating responses to the endogenous and environmental cues that regulate various developmental and metabolic events. Intrinsic membrane proteins have been implicated in various transport processes across membranes as well as in perception and further transmission of regulatory signals. Crosstalk at the plasma membrane interface is all the more important for sessile organisms such as higher plants, which are required to maintain a diverse spectrum of plasma membrane receptor and transport proteins in order to be able to properly sense and respond to fluctuations in their environment (Geldner and Robatzek, 2008; Otegui and Spitzer, 2008; Richter et al., 2009; Robinson et al., 2012; Korbei and Luschnig, 2013). In plants, as in any other eukaryote, this is achieved by a complex system of internal membranes that serves to transport proteins to their site of action and degradation. Exchange of material between these membranes is achieved by vesicles that bud off from one membrane, mature and fuse with another in a highly controlled fashion.