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Originally published In Press as doi:10.1074/jbc.M708118200 on December 17, 2007

J. Biol. Chem., Vol. 283, Issue 9, 5669-5676, February 29, 2008
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β-Arrestin-biased Agonism at the β2-Adrenergic Receptor*

Matthew T. Drake{ddagger}1, Jonathan D. Violin{ddagger}2, Erin J. Whalen{ddagger}, James W. Wisler{ddagger}3, Sudha K. Shenoy{ddagger}, and Robert J. Lefkowitz, An investigator of the Howard Hughes Medical Institute{ddagger}§4

From the Departments of {ddagger}Medicine and §Biochemistry and the Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710

Received for publication, September 28, 2007 , and in revised form, December 5, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 {alpha}-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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
7TMRs,5 also known as G protein-coupled receptors, are the largest family of proteins involved in the transduction of signals from the extracellular milieu to intracellular effectors (1) and account for nearly 2% of all human genes (2). Clinically, 7TMR ligands are the single largest class of pharmacologic agents (3). According to a well established and evolutionarily conserved paradigm, 7TMRs signal through direct activation of heterotrimeric G proteins to promote the activation or inhibition of second messenger-generating enzymes and changes in second messenger-dependent effector activities (4). Thus, for example, agonist stimulation of β2AR promotes Gs-mediated activation of adenylyl cyclase, with subsequent cAMP production and cAMP-associated signaling events. Signal termination and 7TMR desensitization result from recruitment of β-arrestin proteins to the cytoplasmic surface of 7TMR following agonist-stimulated receptor phosphorylation by the G protein-coupled receptor kinase (GRK) family of proteins (5, 6).

When originally identified, the β-arrestin proteins were believed to play a role only in limiting 7TMR signaling by physically interceding between the receptor and G protein. Recent work has demonstrated, however, that for a variety of 7TMRs, β-arrestin proteins can mediate G protein-independent 7TMR signaling by selectively scaffolding signaling cascade components, including small GTP-binding proteins and members of the MAPK cascade (5, 7). These findings have had a profound impact on our understanding of 7TMR ligand pharmacology.

Historically, 7TMR ligands have been classified according to their ability to promote receptor activation (agonists) or inhibit agonist-mediated receptor activation (antagonists). This binary categorization underestimates, however, the true complexity of 7TMR ligand behavior. The identification of inverse agonists, which inhibit ligand-independent (i.e. constitutive) activity of 7TMRs for G protein activation (8), broadened the spectrum of 7TMR ligands to at least three fundamentally distinct classes. In this pharmacologic paradigm, 7TMR ligands were thought to exhibit "correlated efficacies," stimulating or inhibiting all functions of a receptor to the same extent. Consistent with this were studies performed 2 decades ago in which purified β2AR was reconstituted in a phospholipid vesicle system either with GRK2 or with both Gs and adenylyl cyclase (9). Analysis of the intrinsic abilities of a series of βAR ligands defined as partial agonists for G protein activation demonstrated a near-perfect correlation (coefficient of 0.996) in the ability of each ligand to stimulate G protein-mediated (adenylyl cyclase activation) or β-arrestin-associated (βAR phosphorylation) activities (9). More recent results, however, suggest that some β2AR antagonists are actually agonists for some signaling pathways, thus, for example, stimulating MAPK activation while blocking G protein (10–12). We refer to such selective signal activation as "ligand bias" (13), but the same phenomenon has also been described as "ligand-directed trafficking" (14), "protean agonism" (15), "pleuridimensional efficacy" (12), and "collateral efficacy" (16).

Recent evidence demonstrates that biased ligands can selectively activate β-arrestin signaling without activating G protein signaling. For example, angiotensin II-induced signaling via angiotensin II receptor type 1a (AT1aR) leads to G{alpha}q/11-mediated activation of phospholipase C- and β-arrestin-dependent functions, whereas AT1aR activation by a peptide analogue of angiotensin II ([Sar1,Ile4,Ile4]angiotensin II, denoted SII) leads to β-arrestin-dependent MAPK activation alone (17–20). The physiologic relevance of this AT1aR/G protein-independent signaling has recently been demonstrated in mice with cardiac-specific overexpression of a mutant AT1aR capable of activating only G protein-independent pathways in response to angiotensin II (21), as well as in isolated cardiomyocytes derived from wild-type mice treated with SII (19). Additional studies of β2AR showed that a mutant β2AR incapable of agonist-induced signaling via Gs promoted agonist-induced activation of the MAPK cascade via β-arrestins (22). Other studies have also demonstrated ligand-induced, G protein-independent, β-arrestin-dependent signaling by 7TMRs believed previously to signal only via conventional G protein-dependent mechanisms, including the V2 vasopressin receptor (23) and parathyroid hormone receptor (24), among others (see Ref. 25).

Because the standard approach to 7TMR ligand discovery and biochemical characterization is based solely on G protein-dependent activities such as second messenger accumulation, we set out to develop a systematic approach that would allow us to unequivocally distinguish the ability of a ligand to modulate classic G protein-dependent activities from distinct signals. Accordingly, we chose to use the prototypical 7TMR β2AR as a model system both because we have shown previously that a mutant β2AR was capable of G protein-independent/β-arrestin-dependent signaling and because a wealth of well characterized ligands that have been demonstrated previously to modulate G protein-dependent activities at β2AR are readily available commercially. Using a set of FRET-based live-cell biosensors, we interrogated a series of ligands in search of β-arrestin-biased agonists in hopes of establishing structure-activity relationships for β-arrestin ligand bias at β2AR.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Deoxyepinephrine, epinephrine, formoterol, ICI-118,551, isoproterenol, norepinephrine, propranol, salbutamol, and salmeterol were obtained from Sigma. N-Cyclopentylbutanephrine (CPB) was supplied previously by Sterling-Winthrop. Additional CPB was obtained by the Duke University Small Molecule Synthesis Facility and determined to be equivalent to that provided by Sterling-Winthrop for all assays tested. Anti-phospho-β2AR (Ser355/Ser356) was obtained from Santa Cruz Biotechnology. Ligands were used at receptor-saturating concentrations, calculated as 100 x kd as follows: isoproterenol (1 µM), epinephrine (10 µM), norepinephrine (300 µM), isoetharine (100 µM), cyclopentylbutanephrine (1 µM), ethylnorepinephrine (300 µM), methylnorepinephrine (300 µM), protokylol (30 µM), deoxyepinephrine (200 µM), zinterol (1 µM), metaproterenol (200 µM), terbutaline (200 µM), fenoterol (200 µM), procaterol (1 µM), formoterol (1 µM), albuterol (30 µM), salbutamol (30 µM), salmeterol (3 µM), soterenol (30 µM), ritodrine (200 µM), and phenylephrine (300 µM).

Plasmids—pcDNA3.1 expressing β2AR-mCFP and β-arrestin2-mYFP, as well as cell lines stably expressing these plasmids, are described elsewhere (26). pcDNA3 expressing the cAMP biosensor ICUE2 (indicator of cAMP using Epac) was obtained from Jin Zhang (Johns Hopkins Medical Institutions) and is described elsewhere (27, 28). MyrPalm-mYFP was a gift of Roger Tsien (University of California, San Diego) (29).

Cell Culture—HEK-293 cells were maintained in minimal essential Eagle's medium (M2279; Sigma) plus 10% fetal bovine serum and penicillin/streptomycin. For stable cell line generation, G418 was used at 400 µg/ml for selection and 100 µg/ml for maintenance, and hygromycin was used at 250 µg/ml for selection and 150 µg/ml for maintenance. Stably transfected cell lines were generated as described elsewhere, with β2AR cell surface expression determined by 125I-cyanopindolol binding of 1.0 pmol/mg of protein (26).

Imaging—For all imaging experiments, cells were seeded onto fibronectin-coated imaging dishes 24 h prior to assay. Cells were washed once with 1x phosphate-buffered saline, placed in imaging buffer (125 mM NaCl, 5 mM KCl, 1.5 mM MgCl2, 1.5 mM CaCl2, 10 mM glucose, 0.2% bovine serum albumin, 10 mM HEPES, pH 7.4), and imaged in the dark on a stage heated to 37 °C. Images were acquired on a Zeiss Axiovert 200M microscope (Carl Zeiss MicroImaging Inc.) with a Roper Micromax cooled charge-coupled device camera (Photometrics) controlled by SlideBook 4.0 (Intelligent Imaging Innovations). CFP and FRET images were obtained through a 436/20 excitation filter (20-nm bandpass centered at 436 nm), a 455DCLP dichroic longpass mirror, and separate emission filters (480/30 for CFP and 535/30 for FRET). YFP intensity was imaged from a 500/20 excitation filter, a 515LP dichroic mirror, and a 535/30 emission filter. All optical filters were obtained from Chroma Technologies. Excitation and emission filters were switched in filter wheels (Lambda 10-2; Sutter Instruments). Integration times were varied between 100 and 300 ms to optimize signal and minimize photobleaching.

Cyclic AMP FRET Assays—G protein efficacy was measured by detection of the second messenger cAMP. cAMP was measured using the biosensor ICUE2 (28), an improved version of ICUE (27). ICUE2 is an Epac1 (exchange protein directly activated by cAMP)-based sensor fused to both CFP and YFP; cAMP binds to Epac, causing conformational changes that induce a change in intramolecular FRET. G protein efficacy was quantified as the integrated change in the FRET ratio (CFP intensity over YFP intensity) over the 5 min following ligand addition. This measure captures both the kinetics and amplitude of G protein efficacy.

β-Arrestin Translocation FRET Assays—GRK/β-arrestin efficacy was measured as the rate of β-arrestin recruitment to β2AR. We measured this rate by detecting formation of the receptor-arrestin complex, reported by FRET, between β2AR-mCFP and β-arrestin2-mYFP (13).

Endocytosis FRET Assays—β2AR internalization was measured by co-expressing β2AR-mCFP and a membrane-targeted mYFP bearing a sequence coding for myristoylation and palmitoylation, MyrPalm-mYFP (29). In the basal state, these two proteins colocalize in the plasma membrane, where their effective concentration is sufficiently high to result in intermolecular FRET, even in the absence of any intermolecular affinity. This "packing" effect can lead to artifactual results when studying protein-protein interactions by FRET (30), but is used here simply to generate a marker of membrane colocalization. When β2AR-mCFP internalizes from the cell surface after stimulation, MyrPalm-mYFP remains behind; this loss of colocalization leads to a reduction in basal FRET in proportion to the amount of β2AR-mCFP that is sequestered. Thus, this assay provides a real-time kinetic measurement of receptor internalization and can in principle be extended to the study of any protein that traffics to or from the plasma membrane.

Small Interfering RNA (siRNA) Silencing of Gene Expression—Chemically synthesized, double-stranded siRNAs with 19-nucleotide duplex RNA and 2-nucleotide 3'-dTdT overhangs were purchased from Xeragon (Germantown, MD) in a deprotected and desalted form. The siRNA sequence targeting human β-arrestin2 was 5'-AAGGACCGCAAAGUGUUUGUG-3', corresponding to positions 148–168 relative to the start codon (31). A nonsilencing RNA duplex, 5'-AAUUCUCCGAACGUGUCACGU-3', was used, as per the manufacturer's instructions, as the control (31). For siRNA experiments, early passage HEK-293 cells that were ~40–50% confluent on 100-mm dishes were transfected with 20 µg of siRNA using the GeneSilencer transfection reagent (Gene Therapy Systems). Forty-eight hours later, the cells were split and seeded into 6- or 12-well dishes for pERK assays. Only experiments with validated silencing were analyzed; the average silencing of β-arrestin2 was 69.9 ± 1.6%.

pERK Assays—HEK-293 cells in 6- or 12-well plates were starved for 4 h in serum-free medium prior to stimulation. After stimulation with the appropriate ligand for 5 min, cells were solubilized directly by adding 2x SDS sample buffer, followed by boiling at 100 °C for 5 min. For each transfection, an equal aliquot of cells was set aside for protein determination (Bradford).

Immunoblotting—Equal microgram quantities of cellular extracts were separated on 4–20% (for pERK1/2 detection) or 10% (for β-arrestin detection) Tris/glycine-polyacrylamide gels (Invitrogen) and transferred to nitrocellulose membrane for immunoblotting, according to standard protocols. Phosphorylated ERK1/2 and total ERK1/2 were detected by immunoblotting with rabbit polyclonal anti-phospho-p44/42 MAPK (1:2000; Cell Signaling) and anti-MAPK1/2 (1:10,000; Millipore) antibodies, respectively. To determine β-arrestin2 protein silencing, endogenous β-arrestin1/2 were detected with anti-β-arrestin antibody (A1CT; 1:3000) as described previously (32). Chemiluminescence detection was performed with horseradish peroxidase-coupled secondary antibody (Amersham Biosciences) and SuperSignal West Pico reagent (Pierce). Chemiluminescence was quantified by a charge-coupled device camera (Syngene ChemiGenius2) according to the manufacturer's instructions. GraphPad PRISM software was used for data analyses.


Figure 1
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  		FIGURE 1.
Ligand stimulation reveals dynamic changes in β2AR cellular responses demonstrated by FRET. A, changes in cellular cAMP levels measured by ICUE2. HEK-293 cells with stable expression of the FRET-based cAMP reporter ICUE2 (27, 28) were incubated with the appropriate vehicle or with one of seven ligands at concentrations 100-fold above the reported EC50. Time course data are representative of three to five independent experiments. The FRET emission ratio, corresponding to cAMP concentration, was sampled every 2 s. NE, norepinephrine; Salb, salbutamol; Salm, salmeterol; Prop, propranol; ICI, ICI-118,551. B, changes in β2AR and β-arrestin2 association determined by FRET. HEK-293 cells with stable co-expression of human β2AR-mCFP (1.0 pmol/mg) and rat β-arrestin2-mYFP were incubated with the same ligands described in A. FRET is quantified as a percentage of whole-cell total CFP-excited fluorescence (%F). Data shown are representative of three to five independent experiments.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Given the recent recognition that 7TMRs are capable of G protein-independent/β-arrestin-dependent signaling, we tested a broad panel of previously described full and partial agonists of G protein activity at β2AR for β-arrestin-biased activities using HEK-293 cells as the basis for our β2AR model systems.

Accordingly, the ability of each compound to induce cAMP production via the endogenously expressed β2AR was assessed in HEK-293 cells stably expressing ICUE2, an improved version of the FRET-based cAMP reporter ICUE (27). This highly sensitive technique allows for the quantitative comparison of both the relative amount of cAMP generated in response to ligand stimulation and the rigorous quantification of the rate at which cAMP production occurs. As shown in Fig. 1A, the classic βAR agonist isoproterenol (Iso) resulted in a rapid but transient cAMP response, as did a series of βAR partial agonists. The ICUE2 response was completely abrogated by pretreatment of cells with the βAR antagonist propranolol or ICI-118,551 and exhibited a standard dose-response relationship for varying concentrations of isoproterenol, with an EC50 similar to previous findings (28). The ICUE2 dose-response relationship was the same regardless of whether the ICUE2 response was measured as the initial linear rate, the maximum response, or the integrated response over time (data not shown). Because the integrated response captures both the kinetics and amplitude of cAMP dynamics, we chose that measure as the simplest representation of G protein efficacy.

The proximal event in β-arrestin-dependent signaling is thought to be ligand-stimulated 7TMR phosphorylation by GRKs, followed by β-arrestin translocation from the cytosol to the phosphorylated 7TMR (5). We thus used a FRET-based measurement of receptor/β-arrestin interaction as an indicator of β-arrestin-dependent signaling. We have found previously that the rate of β-arrestin recruitment, and not the amount of β-arrestin recruited, is sensitive to changes in GRK concentrations (26); thus, differences in GRK/β-arrestin efficacy will manifest most acutely as changes in the kinetics of β-arrestin recruitment. Accordingly, we used the rate of β-arrestin recruitment to measure GRK/β-arrestin efficacy. HEK-293 cells with stable co-expression of β2AR-mCFP and β-arrestin2-mYFP were incubated with the same expanded panel of βAR ligands utilized in the initial screen described for Fig. 1A. As seen in Fig. 1B, these ligands demonstrated a range of efficacies. Interestingly, we found one ligand, CPB, that exhibits a marked bias: robust β-arrestin efficacy despite only moderate G protein efficacy.

We investigated this bias more fully by characterizing a larger set of βAR ligands, including a number that are used clinically. The addition of each ligand at a receptor-saturating concentration (100-fold higher than published kd values) revealed a range of efficacies for G protein activation and β-arrestin recruitment. As seen in Fig. 2A, a correlation plot clearly shows that although most ligands were essentially Iso-efficacious for G protein-dependent and β-arrestin-dependent activities, several ligands were more efficacious for β-arrestin translocation to β2AR than for cAMP production. CPB exhibits G protein efficacy nearly identical to that of the partial agonist norepinephrine (Fig. 1A), but β-arrestin efficacy greater than that of the full agonist isoproterenol. Indeed, CPB engendered β-arrestin recruitment 3.2-fold more rapidly than did norepinephrine. Furthermore, relative to the full G protein agonist Iso, which did not demonstrate any bias in its ability to promote cAMP production or β-arrestin translocation, CPB was only 58% as efficient for G protein activation, but 1.3-fold more efficient for β-arrestin recruitment. Several additional ligands (Fig. 2A, shown in red) were also found to have significant β-arrestin bias. These were ethylnorepinephrine and isoetharine, structural derivatives of norepinephrine and Iso, respectively. Specificity of these ligands' activities was demonstrated by blocking all effects with the β2AR-specific antagonist ICI-118,551 and by the absence of any effect of blocking endogenous {alpha}-adrenergic receptors with the antagonist yohimbine (data not shown).


Figure 2
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  		FIGURE 2.
Quantification of ligand-induced β-arrestin recruitment and cAMP formation rates demonstrates β-arrestin bias. A, a correlation plot of the intrinsic activity rates determined for the ligands shown in Fig. 1 to promote β-arrestin recruitment (ordinate) and cAMP formation (abscissa) shows that the majority of ligands do not demonstrate any bias. Three ligands (shown in red) demonstrate enhanced ability to promote β-arrestin recruitment to β2AR relative to their ability to induce cAMP production. Efficacy for G protein activation is reported as the integrated FRET ratio change of ICUE2, indicative of cumulative cAMP production. Efficacy for β-arrestin is reported as the rate of recruitment (min-1), which corresponds to the amount of elicited GRK activity (26). B, the ratio of β-arrestin efficacy to G protein efficacy is expressed as the bias factor for each ligand. A bias factor of 1.0 indicates no bias. As a benchmark for inferring bias, a line is shown demarcating the mean + 2 S.D. of the bias factor of isoproterenol. CPB, shown in red, exhibits a bias factor unlikely to be randomly detected by assay variability and leads to the hypothesis that CPB is significantly biased. Epi, epinephrine; Norepi, norepinephrine; EtNE, ethylnorepinephrine; MeNE, methylnorepinephrine; deoxyepi, deoxyepinephrine.

 
We also measured the bias present among the tested ligands by calculating a "bias factor," the ratio of β-arrestin efficacy to G protein (cAMP) efficacy (Fig. 2B). This analysis allowed us to rapidly discern which ligands appear biased and are of interest for further study. As a benchmark, we chose a guideline of twice the S.D. of isoproterenol's bias factor: although isoetharine and ethylnorepinephrine exhibit only modest bias factors, CPB exhibits a bias factor appreciably larger than this benchmark. Thus, we hypothesize that isoetharine, ethylnorepinephrine, and especially CPB are β-arrestin-biased ligands and as such will differ in downstream effects when compared with unbiased agonists.


Figure 3
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  		FIGURE 3.
β-Arrestin-biased agonists induce more rapid β2AR phosphorylation. A, HEK-293 cells with stable overexpression of FLAG-β2AR/pcDNA3 as described previously (22) were stimulated for the indicated times (30 s or 1, 2, 4, 8, or 20 min) with the ligand and concentrations as described in the Fig. 1 legend. Following SDS-PAGE and transfer to nitrocellulose, β2AR phosphorylated by GRKs on serines 355 and 356 (p355/p356) in the C-terminal tail of the receptor was detected with a phospho-specific antibody (33). Each data point represents the mean ± S.E. from three independent experiments. NE, norepinephrine; Salb, salbutamol; Salm, salmeterol. B, for visual comparison, the initial rates of phosphorylation derived from A are shown graphically; the rate of phosphorylation stimulated by CPB is indistinguishable from that elicited by Iso.

 
β-Arrestin translocation to 7TMRs in response to ligand stimulation follows receptor phosphorylation at cytoplasmic serines and threonines by the GRK family of kinases (6). As another measure of efficacy for GRK/β-arrestin functions, we assayed the rate of agonist-stimulated, GRK-mediated phosphorylation of β2AR. HEK-293 cells stably expressing β2AR (22) were stimulated at times ranging from 30 s to 20 min and were analyzed for receptor phosphorylation with an antibody specific for β2AR phosphorylated on serines 355 and 356, sites shown previously to undergo phosphorylation by GRKs (33, 34). The initial rate of CPB-stimulated β2AR phosphorylation closely mirrored that stimulated by Iso, whereas ligands with partial agonist activity for both G protein and β-arrestin activities (norepinephrine, salbutamol, and salmeterol) stimulated rates much slower than that stimulated by Iso (Fig. 3A). The initial rates of GRK-mediated phosphorylation of β2AR are shown graphically in Fig. 3B. These results show that, consistent with its effects on β-arrestin recruitment, CPB is a full agonist for receptor phosphorylation by GRKs despite its modest partial agonism for G protein activity.

As CPB leads to both rapid β-arrestin recruitment (Fig. 2) and GRK-mediated β2AR phosphorylation (Fig. 3), we hypothesized that CPB may also promote more efficient β2AR internalization. Indeed, an enhanced rate or amount of β2AR internalization would be a functional readout for CPB bias. To examine this directly, we developed a FRET-based receptor internalization assay in which β2AR-CFP was co-expressed in HEK-293 cells with YFP modified by the covalent linkage of both myristoyl and palmitoyl moieties (29). With these lipid modifications, YFP is localized nearly exclusively to the plasma membrane, where it exhibits density-dependent FRET with β2AR-CFP (data not shown), arising from the high effective concentration of fluorophores confined to a two-dimensional plane, even in the absence of intermolecular affinity (29). This constitutive colocalization is disrupted by internalization of the receptor, but not the lipid-modified YFP, into endocytic vesicles after agonist stimulation (Fig. 4A). This corresponds to a loss of FRET after agonist stimulation, with a time course matching the change in spatial separation (Fig. 4B). Importantly, preincubation with 450 mM sucrose, a well established technique for the inhibition of internalization (35), completely abrogated β2AR endocytosis in response to isoproterenol (Fig. 4B). As anticipated, ligands demonstrated to be less efficacious for β-arrestin recruitment (Fig. 1B), such as salbutamol and norepinephrine, resulted in both less rapid and less robust β2AR internalization than the reference compound, Iso (Fig. 4C). Importantly and concordant with its high efficacy for β-arrestin recruitment (Fig. 1B), stimulation with CPB resulted in rapid β2AR internalization (Fig. 4, C and D).

To highlight the extent to which β2AR internalization reflects the ability of a ligand to promote either G protein- or β-arrestin-dependent actions, we compared the rate of β2AR internalization for several ligands (data from Fig. 4C) with cAMP formation and with the rate of β-arrestin recruitment (from Fig. 1). As shown in Fig. 5A, the correlation of the β2AR internalization rate and β-arrestin recruitment rate for a range of responses matched a hyperbolic model of ligand efficacy, consistent with a single "activated" receptor state (33). This model assumes that every ligand stabilizes "active" and "inactive" receptor conformations and that efficacy results from the proportion of receptors shifted to the active state. Different assays of efficacy measure different saturable cellular responses, resulting in a hyperbolic efficacy function. However, correlation of the β2AR internalization rate and cAMP response is inconsistent with this hyperbolic model: the biased ligand CPB is disproportionately efficacious for β2AR internalization relative to its efficacy for cAMP generation (Fig. 5B). This finding is inconsistent with a single active state of the receptor and implies that multiple functionally distinct receptor conformations can be stabilized by agonists.

One potential concern regarding our measurement of ligand bias is that we used both kinetic and integrated responses to characterize ligand efficacy. Signal kinetics and signal magnitude are difficult to cleanly differentiate, so we sought a single downstream measurement to verify that CPB is indeed biased toward β-arrestin. In our previous studies of β-arrestin-biased signaling, we elucidated both G protein-dependent and β-arrestin-dependent contributions to β2AR-stimulated MAPK activation (22). Thus, we used activation of the MAPK ERK1/2 to verify the β-arrestin bias of CPB. Serum-starved HEK-293 cells were treated with Iso and CPB in the presence of either control siRNA or siRNA directed against β-arrestin2. We observed that in the presence of siRNA targeting β-arrestin2, Iso-stimulated pERK was reduced by 43.8 ± 1.9%, whereas for CPB, pERK stimulation was reduced by 60.3 ± 5.3% (p < 0.05; n = 4) (Fig. 6). Accordingly, these results demonstrate that although Iso and CPB induce similar levels of pERK stimulation, their utilization of G protein- versus β-arrestin-dependent signaling pathways to activate ERK differs significantly (i.e. there is a relatively greater contribution of β-arrestin signaling to the CPB ERK response). As such, these results further verify the β-arrestin bias of CPB relative to Iso and demonstrate that the observed bias leads to alterations in a downstream signaling kinase cascade. Furthermore, these results validate the use of proximal signal transduction signals, in this case the rate of β-arrestin recruitment and the integrated cAMP response, to discern β-arrestin bias among a set of ligands.


Figure 4
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  		FIGURE 4.
β-Arrestin-biased ligands induce more rapid β2AR internalization. A, HEK-293 cells stably expressing 1.0 pmol ofβ2AR-mCFP/mg of protein (shown in green) were transfected with MyrPalm-mYFP (shown in red) and treated for 1 h with vehicle alone (No Rx; left panel) or 1 µM isoproterenol (right panel). Exposure to isoproterenol results in β2AR translocation from the plasma membrane into punctate endocytic vesicles. Images were deconvolved by the no-neighbors algorithm in SlideBook software to sharpen the definition of membrane and vesicle fluorescence. B, FRET time lapse reveals that after the addition of 1 µM Iso, FRET is lost between β2AR-mCFP and MyrPalm-mYFP as β2AR is endocytosed. This signal was completely abrogated by the addition of 450 mM sucrose (Suc) to the cell imaging medium. C, the rate and extent of β2AR internalization vary greatly among different β2AR agonists. CPB stimulates β2AR internalization with a rate and extent indistinguishable from that caused by isoproterenol. Internalization is not found after treatment with either vehicle (MeOH) or the β2AR antagonist ICI-118,551 (ICI). Salm, salmeterol; NE, norepinephrine; Salb, salbutamol. D, a comparison of the initial β2AR internalization rates determined from Fig. 3C is shown; internalization stimulated by CPB is indistinguishable from that elicited by Iso.

 


Figure 5
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  		FIGURE 5.
A comparison of G protein and β-arrestin efficacies for β2AR internalization. A, the hyperbolic correlation of the β2AR internalization rate (ordinate) and β-arrestin recruitment rate (abscissa) for a range of responses is consistent with a single activated receptor state. Salm, salmeterol; Salb, salbutamol; NE, norepinephrine; Form, formoterol. B, the correlation of the β2AR internalization rate (ordinate) and cAMP response (abscissa) is inconsistent with this model: the biased ligand CPB is disproportionately efficacious for β2AR internalization relative to its efficacy for cAMP generation. Data are means ± S.E. from Figs. 2 and 4.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Although the ability of 7TMRs to induce cellular responses through G protein activation in response to ligand has long been recognized, an accumulation of evidence now clearly demonstrates that 7TMRs can also impart G protein-dependent or -independent β-arrestin-dependent signals. The present study is consistent with these more recent observations (18, 20, 22–24, 31) and further provides a robust approach that can be readily adapted for the identification of 7TMR ligands that promote β-arrestin-selective signaling.


Figure 6
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  		FIGURE 6.
The β-arrestin-biased ligand cyclopentylbutanephrine has an increased efficacy for β-arrestin-dependent MAPK activation relative to isoproterenol. Serum-starved HEK-293 cells stably expressing β2AR were transfected with either control siRNA (CTL) or siRNA targeting β-arrestin2 (WEM2). These cells were then stimulated (Stim) with either CPB (1 µM) or Iso (1 µM) for 5 min. Cell lysates were analyzed for pERK, ERK, and β-arrestin1/2 by Western blotting (A). For Iso, β-arrestin2 depletion decreased ERK activation by 43.8 ± 1.9%; for CPB, β-arrestin2 depletion decreased ERK activation by 60.3 ± 5.3% (*, p < 0.05), consistent with selective activation of β-arrestin signaling pathways by CPB (B). NS, not stimulated.

 
As postulated by Kenakin (16), all 7TMRs are likely capable of adopting a range of distinct conformations, each of which can lead to the activation of distinct intracellular signaling pathways. The adoption of these distinct conformations can, in turn, be modulated by the presence of ligand, such that distinct receptor configurations are stabilized or induced in response to a particular ligand. Our evaluation of a broad range of ligands, characterized previously by their ability to stimulate G protein-dependent activities at β2AR, is in agreement with the hypothesis that biased 7TMR signaling can be induced by ligand. Thus, although the majority of ligands assayed demonstrated comparable efficacies for G protein- and β-arrestin-associated activities, at least three ligands showed a clear bias for β-arrestin-associated activity. Previous biophysical studies of purified β2ARs exposed to an array of ligands also support this concept (36, 37). In addition, recent cellular studies examining the capacity of a variety of βAR ligands to activate adenylyl cyclase and MAPK signaling pathways at β2AR showed clear ligand-dependent activation profile differences, although the extent to which MAPK signaling was independent of G protein activation and the relationship of this response to β-arrestin function were not determined (12).

A corollary to these findings is that to determine the true efficacy of a ligand for a specific 7TMR, one must define the entire range of effector signaling pathways modulated in response to the ligand. 7TMR ligands have been described classically solely according to their relative abilities to affect G protein-dependent pathways (i.e. as agonists, partial agonists, antagonists, or inverse agonists). However, our data, as well as the work of several other groups, suggest that the efficacy of each ligand for multiple G protein-mediated as well as non-G protein-mediated pathways must also be considered during classification. It is unknown whether important therapeutic differences that have been described among members of a 7TMR ligand class relate to these non-G protein effects, but this provides an attractive and testable hypothesis.

Interestingly, although we observed ligands that demonstrated a bias toward β-arrestin-dependent cellular processes, we did not detect any ligands that exhibited a G protein bias relative to β-arrestin activity. However, there is no theoretical reason to dismiss the possibility of ligands that promote 7TMR conformations with a selective bias toward G protein activation. Our studies included only ligands demonstrated previously to have βAR biological activity (i.e. agonists); careful characterization of a more comprehensive collection of ligands may in fact identify some with a G protein bias relative to β-arrestin. It is interesting to note, however, that in a recent study in which a collection of βAR ligands was used to assess biased signaling at both the β1- and β2ARs, no compounds were found that promoted more adenylyl cyclase activity relative to MAPK activation (12), a result that is consistent with our own findings.

One of the most intriguing findings revealed by our study is that the three β-arrestin-biased ligands identified share a common structural feature absent from all of the nonbiased ligands assayed. CPB, ethylnorepinephrine, and isoetharine all contain an ethyl substituent on the catecholamine {alpha}-carbon. Structurally, ethylnorepinephrine is identical to norepinephrine except for the presence of the {alpha}-carbon ethyl substituent, whereas isoetharine is structurally identical to isoproterenol except for the addition of an {alpha}-carbon ethyl moiety. As seen in Fig. 2A, the addition of an {alpha}-carbon ethyl group to isoproterenol (creating isoetharine) resulted in a substantial decrease in β2AR-mediated G protein activation, with no change in the β-arrestin translocation rate. Moreover, addition of an ethyl substituent to the {alpha}-carbon of norepinephrine (to generate ethylnorepinephrine) substantially improved the rate of β-arrestin recruitment while having no significant effect on adenylyl cyclase activity. Thus, although the inclusion of the same structural motif on two ligands resulted in β-arrestin bias at β2AR relative to the nonbiased "parent" compounds, the chemical basis for this bias is unclear. Accordingly, the importance of {alpha}-carbon alkyl substitutions in β-arrestin-biased β2AR ligands, although suggestive, remains to be determined. Further investigations into the structure-activity relationship of β-arrestin-biased ligands for β2AR, using newly synthesized derivatives of the ligands studied here, should greatly aid in understanding the possibilities for even more highly biased ligands for this receptor.

Importantly, all 7TMR ligands currently used as therapeutic agents in humans have been characterized based upon their ability to modulate 7TMR G protein activities. For example, βAR antagonists currently used in clinical practice for the management of hypertension and heart failure and following acute myocardial infarction were developed based upon their ability to block catecholamine-induced βAR activation of G proteins. It is reasonable to hypothesize, however, that βAR antagonists with a similar ability to inhibit βAR G protein activation, but with the simultaneous capacity to stimulate β-arrestin-dependent signaling pathways, may have additional salutary effects to those already recognized for beta-blockers. Indeed, recent data from our laboratory demonstrate that carvedilol, a nonselective βAR antagonist with particular efficacy in the management of heart failure, appears to be unique among beta-blockers in current clinical use in possessing the ability to promote β-arrestin-mediated signaling while concomitantly antagonizing G protein activity (38).

Whether 7TMR ligands that promote biased signaling activities, as demonstrated for carvedilol, have superior efficacy for the management and treatment of other pathologic human conditions is at present unknown. The potential for β-arrestin-biased ligands to have physiologic properties that differ from ligands without bias is suggested, however, by studies of the β-arrestin-biased angiotensin receptor ligand, SII. This modified angiotensin peptide promotes positive inotropic and lusitropic responses in adult mouse cardiomyocytes (19) in the complete absence of G protein signaling. Whether other β-arrestin-biased ligands, such as the [D-Trp12,Tyr34]parathyroid hormone-(7–34) analogue (24) or any of the three ligands identified here, have unique physiologic properties in vivo remains to be determined. Accordingly, similar arguments may be proffered for nearly any current 7TMR that is a target of modern pharmacotherapy.

The recent recognition that 7TMRs are capable of G protein-independent/β-arrestin-dependent signaling has altered longheld dogmas in our understanding of 7TMR signal transduction mechanisms. Thus, it is perhaps not surprising that with the development of careful screening methods for the detection of both β-arrestin- and G protein-dependent activities, we were able to identify ligands that induce β-arrestin-biased activities at β2AR. Although our previous classification of 7TMR ligands was based solely on the efficacy of the ligand for modulation of G protein activity, our new appreciation for the growing complexity of 7TMR signaling suggests that future ligand characterizations will necessitate the inclusion of other signaling pathway(s) or effector systems modulated in response to ligand.


   FOOTNOTES
 
* This work was supported in part by National Institutes of Health Grants RO1 HL16037 and RO1 HL70631 (to R. J. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Present address: Div. of Endocrinology, Mayo Clinic College of Medicine, 200 First St. S. W., Rochester, MN 55905. Back

2 Recipient of an American Heart Association post-doctoral fellowship. Back

3 Supported by a Sarnoff Cardiovascular Research Foundation fellowship. Back

4 To whom correspondence should be addressed: Howard Hughes Medical Inst., Duke University Medical Center, Box 3821, Durham, NC 27710. Tel.: 919-684-2974; Fax: 919-684-8875; E-mail: lefko001{at}receptor-biol.duke.edu.

5 The abbreviations used are: 7TMR, seven-transmembrane receptor; β2AR, β2-adrenergic receptor; AT1aR, angiotensin II receptor type 1a; CFP, cyan fluorescent protein; mCFP, monomeric CFP; ERK, extracellular signal-regulated kinase; pERK, phosphorylated ERK; FRET, fluorescence resonance energy transfer; GRK, G protein-coupled receptor kinase; MAPK, mitogen-activated protein kinase; CPB, N-cyclopentylbutanephrine; siRNA, small interfering RNA; YFP, yellow fluorescent protein; mYFP, monomeric YFP; Iso, isoproterenol. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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