Diversity in arrestin function

Abstract

The termination of heptahelical receptor signaling is a multilevel process coordinated, in large part, by members of the arrestin family of proteins. Arrestin binding to agonist-occupied receptors promotes desensitization by interrupting receptor-G protein coupling, while simultaneously recruiting machinery for receptor endocytosis, vesicular trafficking, and receptor fate determination. By simultaneously binding other proteins, arrestins also act as ligand-regulated scaffolds that recruit protein and lipid kinase, phosphatase, phosphodiesterase, and ubiquitin ligase activity into receptor-based multiprotein 'signalsome' complexes. Arrestin-binding thus 'switches' receptors from a transient G protein-coupled state to a persistent arrestin-coupled state that continues to signal as the receptor transits intracellular compartments. While it is clear that signalsome assembly has profound effects on the duration and spatial characteristics of heptahelical receptor signals, the physiologic functions of this novel signaling mechanism are poorly understood. Growing evidence suggests that signalsomes regulate such diverse processes as endocytosis and exocytosis, cell migration, survival, and contractility.

Fig. 1

Role of arrestins in GPCR desensitization and sequestration. a Line diagram comparing visual arrestin (arrestin 1) with the non-visual arrestins 2 and 3. The location of the crystallographically defined globular N and C domains are shown in relation to the functionally defined N-terminal (A) domain primarily responsible for receptor recognition, the C-terminal (B) domain responsible for secondary receptor recognition, the phosphate sensor domain (P), and the N- (R1) and C- (R2) terminal regulatory domains. Also indicated are the locations of binding sites for inositol 6-phosphate (IP6), clathrin and AP2, as well as sites of phosphorylation by casein kinase 2 (CK2) and extracellular signal regulated kinases (ERK). b Stability of the receptor–arrestin interaction defines two GPCR classes. Homologous desensitization of GPCRs results from arrestin (Arr) binding to agonist (H)-occupied receptors that have been phosphorylated by GRKs. The two non-visual arrestins direct GPCR sequestration by linking the receptor to clathrin and β2-adaptin (AP-2). Sequestration reflects the dynamin (Dyn)-dependent endocytosis of GPCRs via clathrin-coated pits. Once internalized, GPCRs exhibit two distinct patterns of arrestin interaction. Class A receptors dissociate from β-arrestin and are rapidly recycled to the plasma membrane. Class B receptors form stable receptor–arrestin complexes. These accumulate in endocytic vesicles and are either targeted for degradation or slowly recycled

Fig. 2

GPCRs adopt both G protein-coupled and arrestin-coupled signaling states. Upon agonist (H) binding, GPCRs engage heterotrimeric G proteins, rapidly activating G protein-regulated effectors (E1) at the plasma membrane. Concurrently, GRK phosphorylation of the receptor creates high affinity arrestin binding sites. Arrestin binding uncouples the receptor from heterotrimeric G proteins while targeting it for endocytosis. As arrestins translocate from the cytosol to the receptor on the membrane, they recruit additional catalytically-active proteins (E2), that transmit a distinct set of signals as the receptor internalizes and transits the intracellular compartment

Fig. 3

G proteins and arrestins regulate spatially, temporally and functionally distinct pools of ERK1/2. a G protein-dependent and arrestin-dependent mechanisms of ERK1/2 activation. Transactivation of EGF receptors (HER1/2) results from GPCR-stimulated shedding of preformed EGF-family ligands, such as heparin-binding (HB)-EGF, by membrane-anchored ADAM family matrix metalloproteases. Activated HER1/2 recruits a Ras activation complex containing the adapter proteins Shc and Grb2, and the Ras-GEF, mSos, leading to Ras-dependent activation of the Raf-1-MEK-ERK1/2 cascade. By acting as scaffolds, arrestins (Arr) promote the assembly and membrane targeting of a GPCR-based Raf-1-MEK-ERK1/2 signalsome on the plasma membrane. This process does not require prior G protein activation [50]. In the case of GPCRs that form stable receptor–arrestin complexes, the signalsome complex traffics to early endosomes. b ERK1/2 activated by G protein-dependent mechanisms is distributed diffusely throughout the cytosol and accumulates in the nucleus (green shading) to elicit a transcriptional response. Active ERK1/2 in GPCR-arrestin signalsomes (red shading) is spatially constrained, accumulating at the leading edge of cells in a chemoattractant gradient (arrow) or in endosomal vesicles off the plasma membrane. c Arrestins limit the duration of G protein-dependent ERK1/2 while conferring long-lasting arrestin-dependent ERK1/2 activity. EGF receptor transactivation is the dominant mechanism of LPA receptor-mediated ERK1/2 activation in arrestin 2/3 null MEFs [63]. In the absence of arrestins, LPA stimulates prolonged EGF receptor-dependent ERK1/2 activation (green stripes). When arrestin 3 is reintroduced, the duration of transactivation-dependent signaling is markedly shortened (solid green), reflecting arrestin-dependent desensitization of receptor-G protein coupling. Conversely, EGF receptor-independent ERK1/2 activation (red stripes) is a minor component of ERK1/2 activity in the null background, while restoration of arrestin expression confers long-lasting G protein-independent ERK1/2 activity (solid red). Figure adapted from [63]

Fig. 4

Diverse functions of GPCR-arrestin signalsomes. a Negative regulation of β-catenin signaling by dopamine D2 receptors. PP2A activity in arrestin3-PP2A-Akt-GSK3β complexes promotes dephosphorylation and inactivation of co-scaffolded Akt. This releases Akt-mediated negative regulation of GSK3β activity. GSK3β phosphorylates β-catenin, accelerating its degradation [83]. Without arrestin scaffolding, free Akt phosphorylates and inactivates GSK3β resulting in nuclear accumulation of β-catenin. b Determination of receptor fate by arrestin and GPCR ubiquitination. Arrestin3 binds the E3 ligase, mdm2. Upon recruitment to the receptor, mdm2 ubiquitinates arrestin 3, stabilizing the receptor–arrestin complex [22]. After the receptor internalizes, de-ubiquitination of arrestin 3 destabilizes the complex producing a Class A pattern of arrestin binding, e.g., β2 adrenergic receptor. Stable arrestin ubiquitination produces a Class B pattern leading to accumulation of internalized receptor–arrestin signalsomes, e.g., V2 vasopressin receptor. In some cases, GPCR ubiquitination by other E3 ligases, e.g., Nedd4, promotes receptor degradation over recycling [25]. c Actin filament assembly during chemotaxis. In a chemotactic gradient (arrow), arrestin binding promotes the assembly of a complex containing, cofilin, chronophin, and LIMK on PAR-2 receptors. Activation of chronophin and inhibition of LIMK within the complex increases cofilin activity, forming free actin barbed ends for filament elongation [138]

Fig. 5

Models of GPCR agonist efficacy. a In a two-state model, the receptor exists in equilibrium between inactive (R) and active (R*) conformations. Ligands exhibit varying degrees of preferential binding to R and R*, such that full agonists preferentially stabilize R* while inverse agonists stabilize R. True neutral antagonists bind indiscriminately to both conformations with no net effect on the equilibrium. According to the model, any given ligand should have similar properties, e.g., agonist, antagonist or inverse agonist, regardless of the assay used to detect receptor activation state. b Examples of biased agonism in a multi-state model. Cartesian plot depicting the relationship between three different readouts of PTH1 receptor activity; cAMP production, PI hydrolysis, and ERK1/2 activation. The line of unity predicted by a two-state model, from full agonist (efficacy 1:1:1), through neutral antagonist (efficacy 0:0:0), to full inverse agonist (efficacy −1:−1:−1) is shown, as are the efficacy profiles produced by three PTH1 receptor ligands (stars); the full agonist PTH(1-34), a cAMP pathway-selective, agonist Trp1-PTHrP(1-36), and an arrestin pathway-selective agonist, (d-Trp12, Tyr34)PTH(7-34), that acts as an inverse agonist for receptor-Gs coupling. Such ‘reversal of efficacy’ can only be modeled assuming the existence of more than one signaling conformation. Figure based on [64]