Blocker Action: Carvedilol Stimulates ฿-Arrestin Signaling
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A unique mechanism of -blocker action: Carvedilol stimulates -arrestin signaling James W. Wisler*, Scott M. DeWire†, Erin J. Whalen*, Jonathan D. Violin‡, Matthew T. Drake†§, Seungkirl Ahn†, Sudha K. Shenoy*, and Robert J. Lefkowitz*†‡¶ Departments of *Medicine and ‡Biochemistry and †Howard Hughes Medical Institute, Duke University Medical Center, Durham, NC 27710; and §Division of Endocrinology, Mayo Clinic College of Medicine, 200 First Street SW, Rochester, MN 55905 Contributed by Robert J. Lefkowitz, August 22, 2007 (sent for review July 11, 2007) For many years, -adrenergic receptor antagonists (-blockers or though the exact mechanisms behind this regulation are largely AR antagonists) have provided significant morbidity and mortal- unknown. ity benefits in patients who have sustained acute myocardial Ligands targeting 7TMRs such as AR antagonists are used infarction. More recently, -adrenergic receptor antagonists have therapeutically in numerous conditions. By virtue of their ability been found to provide survival benefits in patients suffering from to block the deleterious G protein-mediated effects of excess heart failure, although the efficacy of different -blockers varies catecholamine stimulation in the heart and other organs, AR widely in this condition. One drug, carvedilol, a nonsubtype- antagonists have become important therapeutic tools for a selective AR antagonist, has proven particularly effective in the variety of cardiovascular conditions including hypertension (18), treatment of heart failure, although the mechanism(s) responsible angina (19), post-acute myocardial infarction (AMI) (20) and for this are controversial. Here, we report that among 16 clinically heart failure (21). The AR antagonist carvedilol recently was relevant AR antagonists, carvedilol displays a unique profile of in demonstrated to significantly reduce morbidity and mortality in vitro signaling characteristics. We observed that in 2 adrenergic heart failure and in post-AMI patients (22–25). Moreover, receptor (2AR)-expressing HEK-293 cells, carvedilol has inverse although controversial, some evidence suggests carvedilol may efficacy for stimulating Gs-dependent adenylyl cyclase but, none- possess unique survival advantages in heart failure over other theless, stimulates (i) phosphorylation of the receptor’s cytoplas- AR antagonists (23). The mechanisms of such advantages are mic tail on previously documented G protein-coupled receptor currently unknown but have been variously ascribed to ancillary kinase sites; (ii) recruitment of -arrestin to the 2AR; (iii) receptor properties of carvedilol including antioxidant (26), antiinflam- internalization; and (iv) activation of extracellular regulated kinase matory (27), antiproliferative (28), and antiarrhythmic abilities 1/2 (ERK 1/2), which is maintained in the G protein-uncoupled (29) as well as its capacity to serve as an ␣1 adrenergic receptor mutant 2ART68F,Y132G,Y219A (2ARTYY) and abolished by - antagonist (30). arrestin2 siRNA. Taken together, these data indicate that carvedilol Given recent advances in understanding 7TMR signaling and is able to stabilize a receptor conformation which, although un- regulation, we examined a subset of 16 known AR antagonists coupled from Gs, is nonetheless able to stimulate -arrestin- for both AC and ERK 1/2 efficacy at the 2AR in simple cellular mediated signaling. We hypothesize that such signaling may systems. This subset encompassed a wide range of clinically used contribute to the special efficacy of carvedilol in the treatment of agents including both nonselective and subtype-selective ligands. heart failure and may serve as a prototype for a new generation of The results suggest a unique profile of activities for carvedilol therapeutic 2AR ligands. that might explain, in part, its positive therapeutic attributes and promote development of 2AR ligands that antagonize Gs- -adrenergic receptor ͉ antagonist ͉ ERK 1/2 ͉ scaffold ͉ internalization mediated signaling while stimulating -arrestin-mediated signaling. even transmembrane receptors (7TMRs), are one of the Results Smost important target classes of therapeutic agents, account- ing for Ͼ30% of all prescription medications (1). Traditionally, cAMP Accumulation. Typically, receptor ligands have been classi- ligands for such receptors have been classified as agonists, which fied as either agonist (full or partial) or antagonist with respect promote signaling through activation of heterotrimeric G pro- to G protein-coupling efficiency. Work over the last 20 years has teins and generation of second messengers such as cyclic aden- expanded this classification to include the concept of inverse osine monophosphate (cAMP), or antagonists that block such agonism, and it has been observed that numerous classical stimulation (2). Recent work, however, has suggested that this neutral antagonists actually act as either partial agonists or simple classification is inadequate and that receptors may exist inverse agonists (2). Here, we used the live-cell biosensor ICUE2  in multiple active conformations, each of which could display a to assess Gs-dependent cAMP efficacy at the 2AR (31). distinct signaling profile (3–6). Acebutolol, alprenolol, atenolol, labetalol, oxprenolol, pindolol, Moreover, it has recently been appreciated that, in addition to and practolol displayed weak partial agonism for Gs-dependent signaling through G proteins, 7TMRs can also use the multi- functional adaptor proteins -arrestins 1 and 2 to activate Author contributions: J.W.W., S.M.D., E.J.W., J.D.V., M.T.D., S.A., S.K.S., and R.J.L. designed cellular pathways (7, 8). These proteins were originally discov- research; J.W.W., S.M.D., E.J.W., J.D.V., and M.T.D. performed research; J.W.W., S.M.D., ered to bind to activated GRK-phosphorylated receptors and E.J.W., J.D.V., and S.K.S. analyzed data; and J.W.W. and R.J.L. wrote the paper. thus block or ‘‘desensitize’’ further receptor-stimulated G pro- The authors declare no conflict of interest. MEDICAL SCIENCES  tein activation (9). Consequently, -arrestins exist as bifunc- Freely available online through the PNAS open access option. tional cellular mediators: even as they terminate G protein Abbreviations: AR, -adrenergic receptor; 7TMR, 7 transmembrane receptor; GRK, G signaling, they can function as scaffolds for signaling networks protein-coupled receptor kinase; 2ARTYY, 2ART68F,Y132G,Y219A; AC, adenylyl cyclase; ERK such as mitogen-activated protein kinases (MAPK) including 1/2, extracellular-regulated kinase 1/2; NS, nonstimulated. ERK 1/2 (10, 11), cJun N-terminal kinase (JNK3) (12), and p38 ¶To whom correspondence should be addressed. E-mail: [email protected]. kinase (13) as well as phosphatidylinositol 3 kinase (PI3K) and This article contains supporting information online at www.pnas.org/cgi/content/full/ Akt (14–16). Recent work also suggests that -arrestin may have 0707936104/DC1. significant antiapoptotic roles within the cell (10, 14, 17), al- © 2007 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0707936104 PNAS ͉ October 16, 2007 ͉ vol. 104 ͉ no. 42 ͉ 16657–16662 Downloaded by guest on September 23, 2021 Fig. 1. cAMP responses monitored by ICUE2. HEK-293 cells stably expressing 2AR and the cAMP biosensor ICUE2 were treated for 2 min with a panel of Fig. 2. ERK activation in 2AR and 2ARTYY stable cells. HEK-293 cells stably ligands described as 2AR antagonists. (A) cAMP agonism was measured as expressing 2AR (A)or2ARTYY (B) were stimulated with the panel of 2AR the rate of change of the ICUE2 FRET ratio corresponding to the rate of cAMP ligands used in Fig. 1 at 10 M for 5 min, and cell lysates were analyzed for accumulation. Ligands that did not induce cAMP generation were tested for pERK and ERK by Western blot. pERK was normalized to total ERK protein. inverse agonism in the same cells. (B) These cells exhibit constitutive 2AR Data represent mean Ϯ SE of at least three independent experiments done in activity that, although too weak to generate high basal cAMP, causes a rapid duplicate. Quantification of pERK bands is as a percentage of maximal activity increase in cAMP when phosphodiesterases are inhibited with isoxybutylm- observed for isoproterenol. , P Ͻ 0.05 vs. NS, , P Ͻ 0.001 vs. NS. ethylxanthine (IBMX). This effect is receptor-specific, because there is no * ** IBMX-induced cAMP increase in cells lacking overexpressed receptor (data not shown). We measured inverse agonism by pretreating cells with ligand for 5 min and quantifying the rate of cAMP increase for 30 sec after IBMX treat- carvedilol-stimulated pERK was insensitive to pertussis toxin ment. Inverse agonists are those ligands that do not stimulate cAMP accumu- pretreatment, excluding a role for Gi coupling in carvedilol- lation on their own and decrease the rate of IBMX-induced cAMP accumula- stimulated pERK [supporting information (SI) Fig. 6]. Further- tion caused by constitutive receptor activity. Data represent mean Ϯ SE from more, carvedilol-stimulated pERK was completely blocked by five independent experiments. **, P Ͻ 0.001 vs. nonstimulated cells (NS). pretreatment with the 2AR selective antagonist ICI 118,551 (data not shown), demonstrating 2AR specificity. AC activation (Fig. 1A). Ligands that did not stimulate signifi- 2AR Phosphorylation. We then focused our screen on carvedilol cant cAMP generation were further analyzed for inverse ago- and propranolol, which were the only AR antagonists that nism.