SPECIAL ARTICLE Circ J 2008; 72: 1725–1729

Beta-Arrestin-Mediated Signaling in the Heart

Priyesh A. Patel, BS; Douglas G. Tilley, PhD*; Howard A. Rockman, MD*,**

Beta-arrestin is a multifunctional adapter protein well known for its role in G-protein-coupled receptor (GPCR) desensitization. Exciting new evidence indicates thatβ-arrestin is also a signaling molecule capable of initiating its own G-protein-independent signaling at GPCRs. One of the best-studiedβ-arrestin signaling pathways is the one involvingβ-arrestin-dependent activation of a mitogen-activated protein kinase cascade, the extracellular regulated kinase (ERK). ERK signaling, which is classically activated by agonist stimulation of the epidermal growth factor receptor (EGFR), can be activated by a number of GPCRs in aβ-arrestin-dependent manner. Recent work in animal models of heart failure suggests thatβ-arrestin-dependent activation of EGFR/ERK signaling by theβ-1-adrenergic receptor, and possibly the angiotensin II Type 1A receptor, are cardioprotective. Hence, a new model of signaling at cardiac GPCRs has emerged and implicates classical G-protein-mediated signaling with promoting harmful remodeling in heart failure, while concurrently linkingβ-arrestin-dependent, G-protein-inde- pendent signaling with cardioprotective effects. Based on this paradigm, a new class of drugs could be identified, termed “biased ligands”, which simultaneously block harmful G-protein signaling, while also promoting cardio- protectiveβ-arrestin-dependent signaling, leading to a potential breakthrough in the treatment of chronic cardiac disease. (Circ J 2008; 72: 1725–1729) Key Words: Beta-arrestin; Beta-1-adrenergic receptor; Extracellular regulated kinase (ERK); G protein- coupled receptor

-protein-coupled receptors (GPCR) are ubiquitously Beta-Arrestin as a Desensitizing expressed, 7-transmembrane spanning receptors and Signaling Molecule G that regulate a vast multitude of physiologic proc- esses.1 The classic paradigm is that agonist stimulation of a Desensitization GPCR activates a receptor-associated G protein to promote Beta-arrestin exists as 2 isoforms, β-arrestin1 and β- downstream signaling via generation of second messengers.1 arrestin2, both of which are important in the regulation of Termination of GPCR signaling occurs by a process known GPCR signaling. It has long been known that β-arrestins as desensitization,2 which involves phosphorylation of the prevent G protein signaling by physically uncoupling the activated receptor by a GPCR kinase and subsequent recruit- interaction between a GPCR and its associated G protein.1,3 ment of the multifunctional adapter proteinβ-arrestin. The Moreover, recent studies of the β-2-adrenergic receptor β-arrestin in turn physically uncouples the G protein from (β2AR) and the muscarinic M1 receptor show thatβ-arres- the receptor, effectively terminating signaling.1,3 Although tins directly promote degradation of second messengers via the role ofβ-arrestin in desensitization is well established, recruitment of phosphodiesterase and diacylglycerol kinase recent evidence indicates thatβ-arrestin is far more func- respectively, hinting at a more elegant and versatile role for tionally versatile than previously appreciated.1,3 β-arrestins in desensitization than previously understood.5,6 Perhaps the most important discovery in the past decade In addition to their role in desensitization, bothβ-arrestin1 is the ability ofβ-arrestin to facilitate trafficking of the re- andβ-arrestin2 are intricately involved in GPCR trafficking, ceptor and initiate cell signaling events in its own right, recycling, and degradation following agonist stimulation independent of G protein activation. This new paradigm (reviewed by Moore et al3). establishes that, while agonist stimulation of a GPCR leads to immediate G-protein-mediated signaling,β-arrestin itself Signaling promotes a second wave of β-arrestin-dependent, G-pro- Although β-arrestins indirectly alter signaling through tein-independent signaling.1,3,4 Here we present evidence their roles in receptor desensitization and trafficking, new highlighting the importance of distinguishing the discrete data show that they can initiate signaling independent of G physiologic outcomes ofβ-arrestin-dependent vs G-protein- protein activation. The best studied example of aβ-arrestin- dependent signaling at GPCRs. activated signaling pathway is the extracellular regulated kinase (ERK) cascade. ERK signaling is prototypically activated by growth factor stimulation of receptor tyrosine (Received August 1, 2008; accepted August 1, 2008; released online 7 October 7, 2008) kinases (RTKs). However, recent studies have shown that School of Medicine, *Department of Medicine and **Department of several GPCRs can themselves initiate ERK signaling by Cell Biology and Genetics, Duke University, Durham, NC, USA both G-protein- andβ-arrestin-dependent processes. Exam- Mailing address: Howard A. Rockman, MD, Department of Medicine, ples of such receptors include the angiotensin II Type 1A Duke University Medical Center, DUMC 3104, 226 CARL Building, receptor (AT1AR),8β-1-adrenergic receptor (β1AR),9β2AR,10 Research Drive, Durham, NC 27710, USA. E-mail: h.rockman@ vasopressin V2 receptor11 and parathyroid hormone recep- duke.edu Supported by grants from the National Institutes of Health 12 to HAR (HL56687 and HL75443) tor. Interestingly, the time course and molecular conse- All rights are reserved to the Japanese Circulation Society. For per- quences of activating ERK signaling through G-protein- missions, please e-mail: [email protected] mediated pathways vs β-arrestin-mediated pathways are

Circulation Journal Vol.72, November 2008 1726 PATEL PA et al.

Fig1. Theβ-arrestin-dependent and G-protein-de- pendent pathways differentially activate extracellular regulated kinase (ERK) signaling. The angiotensin II Type 1A receptor (AT1AR) is a Gq coupled receptor that can activate ERK signaling by both G-protein- andβ-arrestin-dependent processes. Upon angioten- sin II stimulation, inhibition ofβ-arrestin signaling by the AT1AR usingβ-arrestin2 siRNA reveals that Gq promotes rapid, short-lived activation of ERK (blue line). In contrast,β-arrestin mediates a late, extended phase of ERK activation as seen by PKC inhibition of Gq signaling (orange line). Inhibiting both G protein and β-arrestin-signaling prevents ERK activation (green line), suggesting that the AT1AR cannot acti- vate ERK in a manner independent of G protein orβ- arrestin. PKC, protein kinase C; CTL, control siRNA. Printed with permission from Science 2005; 308: 512–517 and J Biol Chem 2004; 279: 35518–35525.

Fig2. Theβ-1-adrenergic receptor (β1AR)-mediated transactivation of epidermal growth factor receptor (EGFR) isβ- arrestin dependent. HEK293 cells stably overexpressing wild-typeβ1AR were transiently transfected with EGFR-GFP and eitherβ-arrestin si-RNA or control si-RNA. After treatment with theβ1AR agonist dobutamine (Dob), EGFR redis- tributed into cellular aggregates, thereby indicating activation of the EGFR (panel 2, arrowheads). Redistribution of the EGFR into aggregates was prevented in the presence ofβ-arrestin siRNA, in which case EGFR remained localized to the plasma membrane (panel 5, arrow). Taken together, these findings indicate thatβ-arrestin is necessary forβ1AR-mediated transactivation of the EGFR. ICI, ICI118551, a selectiveβ-2-adrenergic receptor blocker; IB, immunoblot; EGF, epidermal growth factor; GFP, green fluorescent protein. Adapted with permission from J Clin Invest 2007; 117: 2445–2458. quite different. For example, in HEK293 cells overexpress- creasingly been investigated in order to uncover the cross- ing the AT1AR, G protein activation leads to peak activity talk between the GPCR and RTK signaling pathways. ERK within 2min and promotes both nuclear and cytoplasmic signaling is classically activated by agonist stimulation of localization of activated ERK. In contrast,β-arrestin-medi- RTKs, an example being the epidermal growth factor recep- ated signaling following angiotensin II stimulation of tor (EGFR). EGFR initiates a signaling cascade consisting AT1AR has a slower and more prolonged pattern and of Raf, MEK, and ERK, as well as recruitment of several promotes only cytoplasmic ERK localization (Fig1).13 In adapter and scaffolding proteins, including Src, Grb2 and addition, G-protein-mediated nuclear localization of ERK Ras, to promote mitogenic and anti-apoptotic effects.7,15 promotes increased transcription of early growth response 1, Anti-apoptotic signaling can be initiated through ERK phos- indicating that only G-protein-dependent signaling promotes phorylation of Bad, thereby allowing homodimerization of transcriptional events.8,13,14 These results suggest that G- Bcl-2 to promote pro-survival pathways.16,17 ERK has also protein- vsβ-arrestin-dependent signaling promote distinct been shown to phosphorylate and inactivate pro-apoptotic ERK signaling events downstream of the AT1AR. proteins such as caspase 918 and Bim,19 while also enhancing cellular proliferation by activation of proteins involved in Beta-Arrestin-Mediated Activation of nucleic acid synthesis,20 transcription,21,22 and translation.23 The β1AR can activate the EGFR to signal through EGFR Initiates ERK Signaling ERK pathways in aβ-arrestin-dependent manner through a Mitogenic ERK signaling activated by GPCRs has in- process known as “transactivation” (Fig2).9 Transactivation

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Fig3. Theβ-arrestin-mediated transactivation of the epidermal growth factor receptor is cardioprotective. Comparisons of (A) left ventricular function by echocardiography, (B) fractional shortening, and (C) TUNEL staining in non-transgenic (NTG) vs transgenic mice stably expressing the wild-typeβ-1-adrenergic receptor (WTβ1AR TG) or G-protein coupled receptor kinase-β-1-adrenergic receptor (GRK-β1AR TG, aβ1AR mutant that is unable to signal throughβ-arrestin-medi- ated pathways). GRK-β1AR mice have (A) greatly enlarged ventricle (enlarged black space), (B) decreased fractional shortening, and (C) increased apoptosis (arrowheads) vs NTG and WTβ1AR TG mice after chronic exposure to isopro- terenol (ISO). Adapted with permission from Clin Invest 2007; 117: 2445–2458.

Fig4. Theβ-1-adrenergic receptor (β1AR) mediated transactivation of the epidermal growth factor receptor (EGFR). Stimulation of theβ1AR activates signaling through 2 independent pathways, Gs-mediated pathways and newly discovered β-arrestin-mediated pathways. Gs activates adenylyl cyclase (AC) to generate the second messenger cAMP to promote downstream signaling. Gs-signaling promotes inotropy in the short term, but with chronic catecholamine stimulation pro- motes harmful cardiac remodeling including myocyte apoptosis, increased ventricular dilatation, and decreased fractional shortening. Following recruitment ofβ-arrestin to GRK5/6-phosphorylated receptors, theβ1AR initiates a second wave of cardioprotectiveβ-arrestin- mediated signaling via “transactivation” of the EGFR. Transactivation involves Src-dependent matrix-metalloproteinase (MMP)-mediated shedding of Heparin-Binding epidermal growth factor (HB-EGF) into the extracellular space, where HB-EGF acts as a ligand for the EGFR. PKA, cAMP-dependent protein kinase A; GRK, G-pro- tein coupled receptor kinase; ERK, extracellular regulated kinase; MAPK, mitogen-activated protein kinase; P, phospho- rylation site. Adapted with permission from J Clin Invest 2007; 117: 2396–2398.

of the EGFR following catecholamine stimulation of the by activation and internalization of EGFR and phosphoryla- β1AR involves Src-dependent matrix-metalloproteinase- tion of ERK.9 Although molecular signaling events in the mediated shedding of heparin-binding epidermal growth ERK signaling pathway have been extensively investigated, factor (HB-EGF). HB-EGF in turn acts as a ligand for the exciting new studies reveal the in vivo importance ofβ-ar- EGFR to initiate mitogenic signaling pathways, as evidenced restin-dependent ERK signaling in the heart.9

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Beta-Arrestin Dependent Signaling gest that identification of β-arrestin-biased ARBs and β- Confers Cardioprotection blockers may provide new therapeutic opportunities in the treatment of cardiac disease. Recent data indicates that β-arrestin-dependent, G-pro- tein-independent activation of the EGFR by the β1AR confers cardioprotection in mice chronically stimulated To the Future with catecholamine.9 Theβ1AR is a Gs-coupled receptor that Although the discovery of distinctβ-arrestin-dependent, regulates cardiac inotropy and chronotropy via activation G-protein-independent signaling is indeed exciting, all the of cAMP-mediated protein kinase A (PKA).24,25 Long-term mechanisms underlyingβ-arrestin-mediated signaling have chronic catecholamine stimulation of theβ1AR, as seen in yet to be fully appreciated. For instance, the 2 isoforms of heart failure, promotes receptor downregulation and harm- β-arrestin, β-arrestin1 and β-arrestin2, appear to have di- ful cardiac remodeling.26,27 vergent and sometimes opposing roles in signaling at a Interestingly, transgenic mice overexpressing mutant number of receptors, including the protease-activated β1ARs that are unable to recruitβ-arrestins show marked receptor (PAR) 1,34 PAR 2,35 parathyroid hormone receptor myocyte apoptosis and left ventricular dilatation under con- type 1,36 and the RTK toll-like receptor 4.37 Also, studies ditions of chronic catecholamine stress (Fig3).9 Moreover, with the β2AR indicate that β-arrestin dimerization is wild-type mice chronically stimulated with catecholamines needed forβ-arrestin-dependent ERK signaling, suggesting show the same deterioration in cardiac function when pre- that interactions between individualβ-arrestins themselves treated with the selective EGFR inhibitor, erlotinib.9 Those are necessary to initiateβ-arrestin-dependent signaling.38,39 results indicate that β-arrestin-mediated signaling by the Interestingly, proteomic analysis of β-arrestin1 and β- β1AR is cardioprotective under conditions of chronic cate- arrestin2 show that they may directly interact with as many cholamine stress, and further, that the mechanism of this as 337 proteins from various protein families,40 which sug- cardioprotection involvesβ-arrestin-dependent transactiva- gests thatβ-arrestins are likely players in a complex web of tion of the EGFR by theβ1AR (Fig4). protein–protein interactions and cellular signaling events. 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