Sudha K. Shenoy

β-Arrestin-dependent, G Protein-independent ERK1/2 Activation by the β2 Adrenergic Receptor

Physiological effects of β adrenergic receptor (β2AR) stimulation have been classically shown to result from Gs-dependent adenylyl cyclase activation. Here we demonstrate a novel signaling mechanism wherein β-arrestins mediate β2AR signaling to extracellular-signal regulated kinases 1/2 (ERK 1/2) independent of G protein activation. Activation of ERK1/2 by the β2AR expressed in HEK-293 cells was resolved into two components dependent, respectively, on Gs-Gi/protein kinase A (PKA) or β-arrestins. G protein-dependent activity was rapid, peaking within 2-5 min, was quite transient, was blocked by pertussis toxin (Gi inhibitor) and H-89 (PKA inhibitor), and was insensitive to depletion of endogenous β-arrestins by siRNA. β-Arrestin-dependent activation was slower in onset (peak 5-10 min), less robust, but more sustained and showed little decrement over 30 min. It was insensitive to pertussis toxin and H-89 and sensitive to depletion of either β-arrestin1 or -2 by small interfering RNA. In Gs knock-out mouse embryonic fibroblasts, wild-type β2AR recruited β-arrestin2-green fluorescent protein and activated pertussis toxin-insensitive ERK1/2. Furthermore, a novel β2AR mutant (β2ART68F,Y132G,Y219A or β2ARTYY), rationally designed based on Evolutionary Trace analysis, was incapable of G protein activation but could recruit β-arrestins, undergo β-arrestin-dependent internalization, and activate β-arrestin-dependent ERK. Interestingly, overexpression of GRK5 or -6 increased mutant receptor phosphorylation and β-arrestin recruitment, led to the formation of stable receptor-β-arrestin complexes on endosomes, and increased agonist-stimulated phospho-ERK1/2. In contrast, GRK2, membrane translocation of which requires Gβγ release upon G protein activation, was ineffective unless it was constitutively targeted to the plasma membrane by a prenylation signal (CAAX). These findings demonstrate that the β2AR can signal to ERK via a GRK5/6-β-arrestin-dependent pathway, which is independent of G protein coupling.

Independent β-arrestin 2 and G protein-mediated pathways for angiotensin II activation of extracellular signal-regulated kinases 1 and 2

Stimulation of a mutant angiotensin type 1A receptor (DRY/AAY) with angiotensin II (Ang II) or of a wild-type receptor with an Ang II analog ([sarcosine1,Ile4,Ile8]Ang II) fails to activate classical heterotrimeric G protein signaling but does lead to recruitment of β-arrestin 2-GFP and activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) (maximum stimulation ≈50% of wild type). This G protein-independent activation of mitogen-activated protein kinase is abolished by depletion of cellular β-arrestin 2 but is unaffected by the PKC inhibitor Ro-31-8425. In parallel, stimulation of the wild-type angiotensin type 1A receptor with Ang II robustly stimulates ERK1/2 activation with ≈60% of the response blocked by the PKC inhibitor (G protein dependent) and the rest of the response blocked by depletion of cellular β-arrestin 2 by small interfering RNA (β-arrestin dependent). These findings imply the existence of independent G protein- and β-arrestin 2-mediated pathways leading to ERK1/2 activation and the existence of distinct “active” conformations of a seven-membrane-spanning receptor coupled to each.

β-Arrestin-mediated receptor trafficking and signal transduction.

β-Arrestins function as endocytic adaptors and mediate trafficking of a variety of cell-surface receptors, including seven-transmembrane receptors (7TMRs). In the case of 7TMRs, β-arrestins carry out these tasks while simultaneously inhibiting upstream G-protein-dependent signaling and promoting alternate downstream signaling pathways. The mechanisms by which β-arrestins interact with a continuously expanding ensemble of protein partners and perform their multiple functions including trafficking and signaling are currently being uncovered. Molecular changes at the level of protein conformation as well as post-translational modifications of β-arrestins probably form the basis for their dynamic interactions during receptor trafficking and signaling. It is becoming increasingly evident that β-arrestins, originally discovered as 7TMR adaptor proteins, indeed have much broader and more versatile roles in maintaining cellular homeostasis. In this review paper, we assess the traditional and novel functions of β-arrestins and discuss the molecular attributes that might facilitate multiple interactions in regulating cell signaling and receptor trafficking.

Differential Kinetic and Spatial Patterns of β-Arrestin and G Protein-mediated ERK Activation by the Angiotensin II Receptor

The seven-membrane-spanning angiotensin II type 1A receptor activates the mitogen-activated protein kinases extracellular signal-regulated kinases 1 and 2 (ERK1/2) by distinct pathways dependent on either G protein (likely Gq/G11) or β-arrestin2. Here we sought to distinguish the kinetic and spatial patterns that characterize ERK1/2 activated by these two mechanisms. We utilized β-arrestin RNA interference, the protein kinase C inhibitor Ro-31-8425, a mutant angiotensin II receptor (DRY/AAY), and a mutant angiotensin II peptide (SII-angiotensin), which are incapable of activating G proteins, to isolate the two pathways in HEK-293 cells. G protein-dependent activation was rapid (peak <2 min), quite transient (t½ ∼2 min), and led to nuclear translocation of the activated ERK1/2 as assessed by confocal microscopy. In contrast, β-arrestin2-dependent activation was slower (peak 5–10 min), quite persistent with little decrement noted out to 90 min, and entirely confined to the cytoplasm. Moreover, ERK1/2 activated via β-arrestin2 accumulated in a pool of cytoplasmic endosomal vesicles that also contained the internalized receptors and β-arrestin. Such differential regulation of the temporal and spatial patterns of ERK1/2 activation via these two pathways strongly implies the existence of distinct physiological endpoints.

A unique mechanism of β-blocker action: Carvedilol stimulates β-arrestin signaling

For many years, β-adrenergic receptor antagonists (β-blockers or βAR antagonists) have provided significant morbidity and mortality benefits in patients who have sustained acute myocardial infarction. More recently, β-adrenergic receptor antagonists have been found to provide survival benefits in patients suffering from heart failure, although the efficacy of different β-blockers varies widely in this condition. One drug, carvedilol, a nonsubtype-selective βAR antagonist, has proven particularly effective in the treatment of heart failure, although the mechanism(s) responsible for this are controversial. Here, we report that among 16 clinically relevant βAR antagonists, carvedilol displays a unique profile of in vitro signaling characteristics. We observed that in β2 adrenergic receptor (β2AR)-expressing HEK-293 cells, carvedilol has inverse efficacy for stimulating Gs-dependent adenylyl cyclase but, nonetheless, stimulates (i) phosphorylation of the receptors cytoplasmic tail on previously documented G protein-coupled receptor kinase sites; (ii) recruitment of β-arrestin to the β2AR; (iii) receptor internalization; and (iv) activation of extracellular regulated kinase 1/2 (ERK 1/2), which is maintained in the G protein-uncoupled mutant β2ART68F,Y132G,Y219A (β2ARTYY) and abolished by β-arrestin2 siRNA. Taken together, these data indicate that carvedilol is able to stabilize a receptor conformation which, although uncoupled from Gs, is nonetheless able to stimulate β-arrestin-mediated signaling. We hypothesize that such signaling may contribute to the special efficacy of carvedilol in the treatment of heart failure and may serve as a prototype for a new generation of therapeutic β2AR ligands.

Multifaceted roles of β-arrestins in the regulation of seven-membrane-spanning receptor trafficking and signalling

Beta-arrestins are cytosolic proteins that bind to activated and phosphorylated G-protein-coupled receptors [7MSRs (seven-membrane-spanning receptors)] and uncouple them from G-protein-mediated second messenger signalling pathways. The binding of beta-arrestins to 7MSRs also leads to new signals via activation of MAPKs (mitogen-activated protein kinases) such as JNK3 (c-Jun N-terminal kinase 3), ERK1/2 (extracellular-signal-regulated kinase 1/2) and p38 MAPKs. By binding to endocytic proteins [clathrin, AP2 (adapter protein 2), NSF (N -ethylmaleimide-sensitive fusion protein) and ARF6 (ADP-ribosylation factor 6)], beta-arrestins also serve as adapters to link the receptors to the cellular trafficking machinery. Agonist-promoted ubiquitination of beta-arrestins is a prerequisite for their role in receptor internalization, as well as a determinant of the differing trafficking patterns of distinct classes of receptors. Recently, beta-arrestins have also been implicated as playing novel roles in cellular chemotaxis and apoptosis. By virtue of their ability to bind, in a stimulus-dependent fashion, to 7MSRs as well as to different classes of cellular proteins, beta-arrestins serve as versatile adapter proteins that regulate the signalling and trafficking of the receptors.

Distinct Phosphorylation Sites on the β2-Adrenergic Receptor Establish a Barcode That Encodes Differential Functions of β-Arrestin

Different patterns of GPCR phosphorylation produce distinct conformations of β-arrestin and specific downstream responses. Cracking a Phosphorylation Code Not only can ligands for G protein–coupled receptors (GPCRs) trigger signaling through two completely different pathways—G protein–mediated and β-arrestin–mediated—Nobles et al. report that phosphorylation of one of these receptors, the β2-adrenergic receptor, by isoform-specific GPCR kinases (GRKs) produces distinct phosphorylation patterns that influence β-arrestin conformation and induce distinct downstream responses. As noted in the Perspective by Liggett, GPCRs are the largest class of signaling proteins in the human genome and are common targets of clinically used therapeutic agents. Drugs that bias signaling down G protein–coupled pathways or the β-arrestin pathways already exist. That the β-arrestin pathways depend on the specific GRK-induced “barcode” triggered by receptor activation has implications for understanding the effects of existing drugs and the development of selective therapies targeting specific β-arrestin–mediated pathways. Phosphorylation of G protein–coupled receptors (GPCRs, which are also known as seven-transmembrane spanning receptors) by GPCR kinases (GRKs) plays essential roles in the regulation of receptor function by promoting interactions of the receptors with β-arrestins. These multifunctional adaptor proteins desensitize GPCRs, by reducing receptor coupling to G proteins and facilitating receptor internalization, and mediate GPCR signaling through β-arrestin–specific pathways. Detailed mapping of the phosphorylation sites on GPCRs targeted by individual GRKs and an understanding of how these sites regulate the specific functional consequences of β-arrestin engagement may aid in the discovery of therapeutic agents targeting individual β-arrestin functions. The β2-adrenergic receptor (β2AR) has many serine and threonine residues in the carboxyl-terminal tail and the intracellular loops, which are potential sites of phosphorylation. We monitored the phosphorylation of the β2AR at specific sites upon stimulation with an agonist that promotes signaling by both G protein–mediated and β-arrestin–mediated pathways or with a biased ligand that promotes signaling only through β-arrestin–mediated events in the presence of the full complement of GRKs or when either GRK2 or GRK6 was depleted. We correlated the specific and distinct patterns of receptor phosphorylation by individual GRKs with the functions of β-arrestins and propose that the distinct phosphorylation patterns established by different GRKs establish a “barcode” that imparts distinct conformations to the recruited β-arrestin, thus regulating its functional activities.

Functional specialization of β-arrestin interactions revealed by proteomic analysis

β-arrestins are cytosolic proteins that form complexes with seven-transmembrane receptors after agonist stimulation and phosphorylation by the G protein-coupled receptor kinases. They play an essential role in receptor desensitization and endocytosis, and they also serve as receptor-regulated signaling scaffolds and adaptors. Moreover, in the past decade, a growing list of protein–protein interactions of β-arrestins pertinent to these functions has been documented. The discovery of several novel functions of β-arrestins stimulated us to perform a global proteomics analysis of β-arrestin-interacting proteins (interactome) as modulated by a model seven-transmembrane receptor, the angiotensin II type 1a receptor, in an attempt to assess the full range of functions of these versatile molecules. As determined by LC tandem MS, 71 proteins interacted with β-arrestin 1, 164 interacted with β-arrestin 2, and 102 interacted with both β-arrestins. Some proteins bound only after agonist stimulation, whereas others dissociated. Bioinformatics analysis of the data indicates that proteins involved in cellular signaling, organization, and nucleic acid binding are the most highly represented in the β-arrestin interactome. Surprisingly, both S-arrestin (visual arrestin) and X-arrestin (cone arrestin) were also found in heteromeric complex with β-arrestins. The β-arrestin interactors distribute not only in the cytoplasm, but also in the nucleus as well as other subcellular compartments. The binding of 16 randomly selected newly identified β-arrestin partners was validated by coimmunoprecipitation assays in HEK293 cells. This study provides a comprehensive analysis of proteins that bind β-arrestin isoforms and underscores their potentially broad regulatory roles in mammalian cellular physiology.

Trafficking of G Protein–Coupled Receptors

G protein–coupled receptors (GPCRs) play an integral role in the signal transduction of an enormous array of biological phenomena, thereby serving to modulate at a molecular level almost all components of human biology. This role is nowhere more evident than in cardiovascular biology, where GPCRs regulate such core measures of cardiovascular function as heart rate, contractility, and vascular tone. GPCR/ligand interaction initiates signal transduction cascades, and requires the presence of the receptor at the plasma membrane. Plasma membrane localization is in turn a function of the delivery of a receptor to and removal from the cell surface, a concept defined most broadly as receptor trafficking. This review illuminates our current view of GPCR trafficking, particularly within the cardiovascular system, as well as highlights the recent and provocative finding that components of the GPCR trafficking machinery can facilitate GPCR signaling independent of G protein activation.

β-Arrestin-biased Agonism at the β2-Adrenergic Receptor

Classically, the β2-adrenergic receptor (β2AR) and other members of the seven-transmembrane receptor (7TMR) superfamily activate G protein-dependent signaling pathways in response to ligand stimulus. It has recently been discovered, however, that a number of 7TMRs, including β2AR, can signal via β-arrestin-dependent pathways independent of G protein activation. It is currently unclear if among β2AR agonists there exist ligands that disproportionately signal via G proteins or β-arrestins and are hence “biased.” Using a variety of approaches that include highly sensitive fluorescence resonance energy transfer-based methodologies, including a novel assay for receptor internalization, we show that the majority of known β2AR agonists exhibit relative efficacies for β-arrestin-associated activities (β-arrestin membrane translocation and β2AR internalization) identical to the irrelative efficacies for G protein-dependent signaling (cyclic AMP generation). However, for three βAR ligands there is a marked bias toward β-arrestin signaling; these ligands stimulate β-arrestin-dependent receptor activities to a much greater extent than would be expected given their efficacy for G protein-dependent activity. Structural comparison of these biased ligands reveals that all three are catecholamines containing an ethyl substitution on the α-carbon, a motif absent on all of the other, unbiased ligands tested. Thus, these studies demonstrate the potential for developing a novel class of 7TMR ligands with a distinct bias for β-arrestin-mediated signaling.