192 Current Pharmaceutical Design, 2012, 18, 192-198

Beta-arrestin Biased Agonism/Antagonism at Cardiovascular Seven Transmem-

brane-spanning Receptors

Anastasios Lymperopoulos*

Department of Pharmaceutical Sciences, Nova Southeastern University College of Pharmacy, Fort Lauderdale, FL 33328, USA

Abstract: Heptahelical, G protein-coupled or seven transmembrane-spanning receptors, such as the -adrenergic and the angiotensin II
type 1 receptors, are the most diverse and therapeutically important family of receptors in the human genome, playing major roles in the
physiology of various organs/tissues including the heart and blood vessels. Ligand binding activates heterotrimeric G proteins that trans-
mit intracellular signals by regulating effector enzymes or ion channels. G protein signaling is terminated, in large part, by phosphoryla-
tion of the agonist-bound receptor by the G-protein coupled receptor kinases (GRKs), followed by arrestin binding, which uncouples the
phosphorylated receptor from the G protein and subsequently targets the receptor for internalization. As the receptor-arrestin complex
enters the cell, arrestin-1 and -2, the two mammalian arrestin isoforms, serve as ligand-regulated scaffolds that recruit a host of intra-
cellular proteins and signal transducers, thus promoting their own wave of signal transduction independently of G-proteins. A constantly
increasing number of studies over the past several years have begun to uncover specific roles played by these ubiquitously expressed re-
ceptor adapter proteins in signal transduction of several important heptahelical receptors regulating the physiology of various or-
gans/systems, including the cardiovascular (CV) system. Thus, arrestin-dependent signaling has increasingly been implicated in CV
physiology and pathology, presenting several exciting opportunities for therapeutic intervention in the treatment of CV disorders. Addi-
tionally, the discovery of this novel mode of heptahelical receptor signaling via arrestins has prompted a revision of classical pharma-
cological concepts such as receptor agonism/antagonism, as well as introduction of new terms such as biased signaling, which refers to
ligand-specific activation of selective signal transduction pathways by the very same receptor. The present review gives an overview of
the current knowledge in the field of arrestin-dependent signaling, with a specific focus on CV heptahelical receptor arrestin-mediated
signaling and on biased CV heptahelical receptor ligands that promote or inhibit it. Exciting new possibilities for cardiovascular thera-
peutics arising from the delineation of this arrestin-dependent signaling are also discussed.

Keywords: Heptahelical receptor (7TMR); cardiovascular; arrestin-dependent signalling; -adrenergic receptor; angiotensin II type 1 recep-
tor; arrestin biased agonist or antagonist.

INTRODUCTION with a well-conserved, central catalytic domain (~270 aa), similar

The heptahelical or seven-transmembrane spanning receptors
(7TMRs) or G protein-coupled receptors (GPCRs) are the largest
and most diverse superfamily of cell surface receptors. Approxi-
mately 600 distinct genes encoding non-olfactory 7TMRs make up
greater than 1% of the human genome [1,2]. Such evolutionary
diversity enables 7TMRs to detect an extraordinary array of ex-
tracellular stimuli. 7TMRs function in neurotransmission, neuro-
endocrine control of physiologic homeostasis and reproduction, and
regulation of hemodynamics and intermediary metabolism, and
they control the growth, proliferation, differentiation, and death of
multiple cell types. It is estimated that more than half of all drugs in
clinical use target 7TMRs, acting either to mimic endogenous
7TMR ligands, to block ligand access to the receptor, or to modu-
late ligand production [3]. Agonist binding promotes interaction of
the receptor with heterotrimeric G proteins, which initiates the clas-
sical intracellular signaling of these receptors that ultimately leads
to a variety of cellular responses/physiological effects.
At the same time, agonist binding promotes the phenomenon of
homologous or agonist-dependent receptor desensitization, which is
the molecular basis of the waning of the cellular responsiveness to
persistent receptor stimulation and constitutes a major classical
homeostatic mechanism of cellular physiology [4]. This agonist-
dependent desensitization is conferred, at the molecular level, by
phosphorylation of the receptor by the family of kinases known as
G-protein-coupled receptor kinases (GRKs). GRKs are serine-
threonine protein kinases comprising a family of seven mem-
bers(GRK1-7), which all share a common structural architecture
*Address correspondence to this author at the Department of Pharma-
ceutical Sciences, Nova Southeastern University College of Pharmacy, 3200
S. University Dr., HPD (Terry) Bldg/Room 1338, Fort Lauderdale, FL
33328, USA; Tel: 954-262-1338; FAX: 954-262-2278;
E-mail: al806@nova.edu
to that of other serine-threonine kinases, flanked by an amino-
terminal (NT) domain (~ 185 aa) and a variable-length carboxyl-
terminal (CT) domain (~ 105-230 aa) that contains specific regula-
tory sites dictating subcellular localization (4). GRK2 is the most
prominent member of this family, binding to and phosphorylating a
vast majority of agonist-occupied 7TMRs, amongst them - and
2-
adrenergic receptors (ARs &
2ARs) and angiotensin II type 1
receptors (AT1Rs) [5]. It is also the most abundant GRK isoform in
the heart and in the cardiovascular (CV) system in general [6].
When the receptor is activated by agonist, agonist binding induces
conformational changes in the receptor that persist for a brief pe-
riod, during which heterotrimeric G-proteins are activated and dis-
sociate into G
and G subunits. The CT of GRK2 (GRK2ct or
ARKct) interacts with the free G subunits resulting in transloca-
tion of GRK2 (which is cytosolic at rest) to the plasma membrane
where it can phosphorylate intracellular domains (i.e. the third in-
tracellular loop or the CT tail) of agonist-occupied 7TMRs [4-7].
This phosphorylation enhances the affinity of the receptor for
binding to the adapter proteins arrestins (arrs), which comprise
two ubiquitously expressed isoforms in mammals, arrestin-1 and -
2 (arr1 and -2), also known as the two non-visual arrestins, ar-
restin-2 and -3, respectively [4]. arrs are also abundantly ex-
pressed in the heart and in the vasculature, as well as in several
other tissues [4]. Similarly to the two other mammalian arrestins,
the visual arrestins (arrestin-1 and -4), arrs bind directly to GRK-
phosphorylated 7TMRs, forming a stoichiometric complex that is
stereochemically incapable of further G protein coupling. arr acti-
vation occurs when the polar core located in the hinge region be-
tween the two globular domains of the arr molecule interacts with
GRK-phosphorylated residues on the receptor, displacing the arr
CT and exposing the concave surface of the globular domains to
interact with the receptor [8]. Receptor binding produces significant
conformational changes in the arr molecule [9], whereas, con-

1873-4286/12 $58.00+.00 2012 Bentham Science Publishers

Beta-arrestin Biased Agonism/Antagonism
versely, arr binding stabilizes a high-agonist affinity state of the
receptor, prompting some authors to characterize the receptor-arr
complex as an alternative ternary complex analogous to the ter-
nary complex existing between agonist-receptor-G protein in the
absence of GTP [10]. arrs (but, interestingly enough, not the visual
arrestins) further dampen G protein signaling by linking receptors
to the clathrin-dependent endocytic machinery [4,11]. The arr CT
that is displaced upon engagement of the receptor directly binds
clathrin heavy chain and the 2 adaptin subunit of the AP-2 com-
plex, both important components of the endocytic machinery [12-
15]. Clathrin/AP-2 binding causes arr-bound receptors to cluster in
clathrin-coated pits, which are pinched off the plasma membrane by
the motor protein dynamin. This arr-dependent endocytosis (re-
ceptor internalization or sequestration) removes receptors from the
cell surface, rendering them less responsive to subsequent stimuli.
From that point on, most 7TMRs fall into one of two classes based
on their affinity for the two arr isoforms and the longevity of the
receptor-arr interaction [16]. One class exhibits higher affinity for
arr2 than arr1 and forms transient receptor-arr complexes that
dissociate soon after the receptor internalizes. These receptors (e.g.
the 2AR) rapidly recycle back to the plasma membrane ready to
signal again upon the next encounter with agonist (receptor resensi-
tization). The other class exhibits equivalent affinities for arr1 or -
2 and forms more stable receptor-arr complexes that remain intact
as the receptor undergoes endosomal sorting. These receptors (e.g.
AT1R & vasopressin V2 receptor) are sequestered in endosomes and
tend to recycle slowly or undergo lysosomal targeting for degrada-
tion (receptor downregulation, i.e. total cellular receptor number
reduced).
Unlike the catalytic 7TMR-G protein interaction, arr-bound
receptors form relatively stable complexes that persist on a time
scale of minutes to hours [17]. It was the discovery that arrs serve
as adapters not only in the context of 7TMR sequestration but also
in linking activated receptors to other enzymatic effectors that ush-
ered in a new paradigm shift in 7TMR signal transduction [18-21].
It is now clear that arrs bind a number of catalytically active pro-
teins and recruit them to agonist-occupied 7TMRs, among them Src
family tyrosine kinases [22], components of the ERK1/2 and c-Jun
N-terminal kinase 3 (JNK3) mitogen-activated protein (MAP)
kinase cascades [23], the E3 ubiquitin ligase Mdm2 [24], the cAMP
phosphodiesterases (PDE) PDE4D3/5 [25], diacylglycerol kinase
(DGK) [26], the inhibitor of nuclear factor (NF)-B IB
[27], and
the Ser/Thr protein phosphatase (PP) PP2A [28]. It is via these
interactions that arr binding to the receptor initiates secondary
waves of 7TMR signal transduction independently of G proteins,
which persist long after receptor signaling via the latter proteins has
been terminated by the very same binding of arr. Of course, arr-
mediated signaling displays several important qualitative differ-
ences compared to G protein-mediated signal transduction; for in-
stance, arr signaling does not result in signal amplification, as it
usually proceeds through a 1:1 stoichiometry. Additionally, it ex-
hibits specific subcellular localization, dictated by the location of
the arr-based molecular signaling scaffold, which significantly
affects the ultimate signaling events produced, i.e. arr signaling is
more compartmentalized compared to the more diffuse, and their inhibition should theoretically be beneficial in acute heart
throughout the cell, G protein signaling [8,29].
Herein, we will review the current literature regarding
arrestin-dependent signaling, with a specific focus on cardiovascu-
lar 7TMRs and their biased ligands that selectively promote or
inhibit this signaling. Exciting new therapeutic possibilities emerg-
ing from uncovering the physiological roles in vivo in the cardio-
vascular system (and the pathological implications in various CV
diseases) of this heptahelical receptor arrestin-dependent signaling
will also be discussed. For more extensive accounts of the physio-
logical roles of arr signaling in vitro and in vivo in additional tis-
sues and organ systems, the reader is referred to several excellent
recent reviews [29-31].
Current Pharmaceutical Design, 2012, Vol. 18, No. 2 193
ARR-DEPENDENT BIASED AGONISM/ANTAGONISM:
GENERAL CONSIDERATIONS
Almost all agonists for 7TMRs have been characterized through
measurement of G protein signals mediating cell second messen-
gers or calcium. However, as the measurement of arr signaling
effects is becoming routine, agonists are now being classified as
actually being biased toward the arr system. For example, angio-
tensin II (AngII) produces activation of G proteins and arrs
through binding to AT1Rs. However, the AngII agonist analog SII
(Sar1,Ile4,Ile8-AngII) (Fig. 1) [32] is perfectly biased toward pro-
ducing activation of only the arr pathway through this receptor
[33,34] (Fig. 2). Other examples of perfect bias for arr activation
can be found in standard -blockers. Thus, propranolol (Fig. 1), the
prototypic non-subtype selective -blocker and a known inverse
agonist for G protein interaction with 2ARs [35], produces activa-
tion of arrs [36,37]. Similar profiles are observed for carvedilol
[38] and bucindolol [37] (Fig. 1). One of the major mechanisms of
coding for functional selectivity of 7TMRs is the phosphorylation
of the ligand-bound receptor by the GRKs. This barcoding of the
receptor by GRK phosphorylation forms the link between the ago-
nist (as it stabilizes unique 7TMR conformations) and the rest of the
cytosolic machinery [39].
The ultimate result of biased agonism/antagonism is a pheno-
typic response that can be unique to a given cell. The first step in
the prosecution of this mechanism for new drug discovery is to
have the ability to detect the effect. To this end, there has been vast
technological progress in pharmacological assay systems over the
past few years. One of the most facile approaches has been whole-
system real-time response reading of pharmacologic response from
human cells in culture rather than from isolated tissues. Recent
technological advances have expanded possible modes of detection
of receptor function. A great deal of information is obtained from
high-content assays based on imaging techniques that use fluores-
cent signals to yield information about receptor interaction with
arr and subsequent movement of the receptor/arr complex within
the cytoplasm. These responses can be monitored directly through
observation of receptor/arr green fluorescent protein (GFP) com-
plexes, with bioluminescence resonance energy transfer (BRET),
with enzyme fragment complementation, with protease-activated
transcriptional reporter genes, or with whole-cell response meas-
urements using optical biosensors that can measure dynamic mass
redistribution signals from whole cells and measurement of the
electrical impedance of layers of cells in culture caused by receptor-
mediated changes in cell mass redistribution (see Ref. 30 for an
excellent review of all the experimental approaches used in dissect-
ing arr biased signaling, and references therein). Below, several
specific examples of cardiovascular 7TMR arr biased signaling
(and its agonism/antagonism) in specific organs/tissues of the car-
diovascular system are discussed.
CARDIAC
AR ARR-DEPENDENT BIASED SIGNALING
arrs are abundantly expressed in cardiac muscle. As co-factors
of GRKs in
AR desensitization/downregulation, they contribute to
the diminished inotropic and adrenergic reserve of the failing heart
failure, as it would enhance the G
s-adenylyl cyclase-PKA-
mediated pro-contractile signaling of cardiac
ARs which increases
cardiac contractility [6,40,41]. However, and exactly because arrs
do a lot more than merely ceasing G protein-mediated signaling
(i.e. they actually promote signaling in their own right), a number
of recent studies point to a beneficial role played by arr signaling
in the heart, especially when the cardiac
1AR is engaged. In heart
failure, chronic catecholamine stimulation of the
1AR promotes
cardiac hypertrophy, decreased contractility, and increased myocyte
apoptosis [42]. Especially this latter effect is, quite intriguingly,
postulated to be opposed by cardiac
2AR signaling [42,43]. As a
result, administration of
-blockers is currently part of standard care
194 Current Pharmaceutical Design, 2012, Vol. 18, No. 2 Anastasios Lymperopoulos

Fig. (1). Structures of some important 7TMR ligands that display bias towards
arr-dependent signaling. Structures of various AT1R arr biased ligands (ago-
nists & antagonists) are shown on the left, while the structures of some adrenergic receptor (and other 7TMR) arr biased ligands can be seen on the right.

in the clinical management of congestive heart failure [44,45]. arrs
have been shown in vitro to mediate the mitogenic signaling of
EGF (Epidermal Growth Factor) receptor transactivation by the

1AR. As inhibition of EGF receptors contributes to dilated car-
diomyopathy,
1AR signaling via arr2 appears to be protective
rather than deleterious for the heart [46] and arr2-dependent EGF
receptor transactivation might exert a cardioprotective effect.
Therefore, arr2-mediated
1AR signaling might be of therapeutic
benefit in heart failure (Table 1).
With regards to arr biased agonism/antagonism at the
AR, a
considerable bias for association of 2ARs with arr was observed
for selective compounds containing an ethyl substitution of the

carbon within a series of phenylethylamines [47]. Stereoisomers
of fenoterol, a 2AR-selective agonist (Fig. 1), differentially acti-
vate Gs and Gi proteins in rat cardiomyocytes [48]. It has been
shown that, in addition to blockade of AR-induced G protein acti-
vation, some -blockers produce activation of ERK through arr
[36]. In view of the fact that a number of -blockers have been
tested in clinical trials for therapy of congestive heart failure and
only a few have shown beneficial effect, this ERK-stimulating ef-
fect may be relevant. In particular, carvedilol has been identified as
producing beneficial effects in congestive heart failure [45], and it
also has been shown to produce arr-mediated activation of ERK
[38].
Beta-arrestin Biased Agonism/Antagonism
Table 1. Pharmacological Interventions and Potential Therapeutic Effects of Targeting 7TMR
arr Signaling in the Cardiovascu-
lar System
Tissue/Receptor Targeted Isoform
Cardiac AT1R arr2
Cardiac 1AR arr2
Adrenal AT1R arr1
Adrenal
2AR arr1 (GRK2)
VSM AT1 R arr1/arr2
VSM GPR109A arr1
Abbreviations: 7TMR, seven transmembrane-spanning receptor; arr, beta-arrestin; AT1R, angiotensin II type 1 receptor; 1AR, beta1-adrenergic receptor;
2AR, alpha2-adrenergic
receptor; arr1, beta-arrestin1; arr2, beta-arrestin2; CA, catecholamine; GPR109A, G protein-coupled receptor 109A (nicotinic acid receptor); GRK2, G protein-coupled receptor
kinase-2; HF, heart failure; VSM, vascular smooth muscle.
SII
AT1R
arr
Fig. (2). The concept of arr-selective biased agonism exemplified by the
AngII analog SII (Sar1,Ile 4,Ile 8-AngII) at the AT1R. SII selectively activates
arr signaling, while simultaneously prohibiting heterotrimeric G protein
activation from the receptor. See text for details.
CARDIAC AT1R ARR-DEPENDENT BIASED SIGNALING
The AT1R represents another very important heptahelical recep-
tor in CV physiology and pathology. An artificially constructed
AT1AR mutant (AT1-i2m), which fails to activate G proteins but
nonetheless interacts with arrs, when expressed in cardiomyocytes
of transgenic mice in vivo, leads to greater cardiomyocyte hypertro-
phy, bradycardia, and fetal cardiac gene expression than compara-
ble overexpression of the wild type receptor [49]. In primary car-
diomyocytes, the arr biased agonist at the AT1R SII stimulates
cardiomyocyte proliferation independently of G proteins, but not
hypertrophy, which requires Gq/11 protein signaling [50]. In addi-
tion, SII produces positive inotropic and lusitropic effects on iso-
lated murine cardiomyocytes [51]. These effects require GRK6 and
arr2, whereas GRK2 seems to oppose them, consistent with the
specialized role of GRK isoforms described in a transfected system
[52] and with the concept of GRK-induced receptor barcoding
discussed earlier [39]. On the other hand, SII does not produce
inotropic or chronotropic effects in isolated Langendorff-perfused
cardiac preparations despite its ability to activate ERK1/2 [50].
Thus, it seems that the AT1AR promotes cardiac hypertrophy and
Current Pharmaceutical Design, 2012, Vol. 18, No. 2 195
Pharmacological Intervention Desired Therapeutic Effect (Disease Context)
stimulation  contractility (HF)
stimulation  survival-antiapoptosis (HF)
inhibition  aldosterone (HF)
inhibition  CA (HF)
stimulation/inhibition  VSM hyperplasia, hypertrophy (atherosclerosis)
inhibition vasodilation-flushing (niacin drug treatment)
cardiomyocyte proliferation via its classical Gq/11 protein-PKC (pro-
tein kinase C) signaling on one hand, and on the other hand in-
creases cardiac contractility via arr2 signaling. Since arr2 is
bound to stop the G protein-mediated signaling of the receptor and
GRK2 also seems to oppose this pro-contractile signaling of arr2
[51], stimulation of arr2 activity at the cardiac AT1AR would ap-
pear therapeutically desirable for the treatment of heart failure and
cardiac hypertrophy (Table 1).

2AR ARR-DEPENDENT BIASED SIGNALING & HY-
PERTENSION
The activation of
2ARs results in a beneficial hypotensive
response in hypertension but also in unwanted sedation [52]. This
latter effect can be gauged through observation of reduced coordi-
nation and balance in the rotorod test in mice. The resistance to
impairment of rotorod coordination to the
2AR agonist 5-bromo-
N-(4,5-dihydro-1Himidazol-2-yl)-6-quinoxalinamine (brimonidine
or UK14304) (Fig. 1) in arr2 knockout mice suggests that sedation
may be related to the arr signaling pathway [53]. Thus, a
2AR-
biased agonist devoid of arr2 stimulation properties might be of
therapeutic value in the pharmacotherapy of hypertension [54].
ADRENAL AT1R &
2AR ARR-DEPENDENT SIGNALING
Aldosterone is one of a number of hormones whose levels are
elevated in heart failure and produces a multitude of negative ef-
fects on the failing heart, including promotion of post-myocardial
infarction adverse cardiac remodeling and heart failure progression
[55-57]. It is produced and secreted by the adrenocortical zona
glomerulosa cells in response to AT1R activation by AngII [58].
Until recently, the general consensus for AT1R signaling to aldos-
terone production was that it proceeded via activation of Gq/11 pro-
teins, to which the AT1R normally couples [59]. Recently however,
we described a crucial role for arr1 in mediating the signaling
from AT1R to aldosterone synthesis and secretion in vitro and in
vivo [60]. Specifically, arr1 was found to stimulate sustained
ERK1/2 activation and subsequent upregulation of steroidogenic
acute regulatory protein (StAR), a steroid transport protein that
catalyzes the rate-limting step in adrenal steroid biosynthesis.
Moreover, arr1 was shown to promote aldosterone production
independently of G-proteins, since SII could recapitulate the effects
of AngII on aldosterone turnover [60]. Importantly, in vivo, adrenal
arr1 was shown to be a major regulator of circulating aldosterone
levels under normal healthy conditions, since its upregulation, spe-
cifically in the adrenal gland, caused hyperaldosteronism [60]. Sub-
sequently, we investigated the effects of adrenal arr1 on aldoster-
one levels also in diseased animals, as they progressed to HF after a
vast experimental myocardial infarction [61]. We found that adrenal
196 Current Pharmaceutical Design, 2012, Vol. 18, No. 2
arr1 overexpression promoted aldosterone elevation post-
myocardial infarction, resulting in accelerated cardiac adverse re-
modeling and deterioration of left ventricular function. Importantly,
these detrimental effects of aldosterone were prevented when adre-
nal arr1 was inhibited in vivo via gene therapy with a arr1 CT
domain-derived fragment-encoding mini-gene which we designed
and developed. Adrenal arr1 inhibition with this mini-gene mark-
edly reduced circulating aldosterone levels and improved cardiac
structure and function in post-myocardial infarction animals,
largely preventing (and/or even reversing) the ensuing cardiac ad-
verse remodeling [61]. Thus, it appears that in the adrenal gland,
arr1 is a major driving force behind elevation of the cardiotoxic
aldosterone in HF, and its inhibition might be of therapeutic value
in treatment of hyperaldosteronism of post-myocardial infarction
heart failure (Table 1).
In addition, in that very same study, we found that losartan, the
protoypic AT1R-selective antagonist (angiotensin receptor blocker,
ARB or sartan), virtually fails to suppress adrenal arr1-dependent
post-myocardial infarction hyperaldosteronism [61]. This prompted
us to test various other sartans, structurally similar to losartan (i.e.
biphenylmethyl derivatives, Fig. 1) and important drugs used in
current clinical practice, in terms of their efficacy at inhibiting the
AT1R-arr1 interaction and thereby suppressing AT1R-induced
aldosterone production. Our latest data indicate that losartan, as
well as irbesartan (Fig. 1), are very weak biased antagonists to-
wards arr inhibition at the AT1R, displaying very little (if any)
bias for arr inhibition vs. G protein inhibition and consequently
are ineffective at curbing aldosterone production [Ref. 62 & Lym-
peropoulos A et al., unpublished data]. In contrast, among the
AT1R antagonists tested, candesartan and valsartan (Fig. 1) were
found to be the most potent arr inhibitors at the AT1R and the
most biased arr antagonists, and this was reflected also on their
effects on aldosterone production, which they dramatically sup-
pressed in vitro and in vivo [Ref. 62 & Lymperopoulos A et al.,
unpublished data]. Based on these data and on a crude comparison
of the structures of these biphenylmethyl derivatives (Fig. 1), which
are all very potent antagonists of the AT1R in terms of G protein
inhibition, some very interesting conclusions about the structural
requirements for arr biased antagonism at the AT1R can be drawn.
Specifically, it appears that: a) the tetrazole ring substitution at the
R2 position of the biphenylmethyl backbone is necessary for arr
inhibition (Fig. 1), and b) the bulkier the substitution at the R 1
position, the more potent arr biased antagonist the compound is
(Fig. 1), since candesartan and valsartan have the bulkiest R 1
substitutions, whereas losartan and irbesartan the smallest. We also
tested a series of AngII analogs with various substitutions at amino-
acid position 5 at their potency as arr biased agonists. These ana-
logs had the sarcosine substitution at aminoacid position 1, so they
were all antagonists with regards to G protein activation. Based on
our data with these peptide analogs, we were able to conclude that,
when it comes to arr biased agonism at the AT1R, the bulkier
the aminoacid substitution at position 5, the more potent arr biased
agonist the resultant compound is [Ref. 62 & Lymperopoulos A et
al., unpublished data]. Of note, SII, the prototypic arr biased
agonist at the AT1R, has no substitution at this position (it contains
a substitution at aminoacid position 4 instead, Ile in the place of
Tyr, Fig. 1). Of course, the substitutions at positions 1 and 8 are
always indispensable in order for the compound to be devoid of any
G protein activating properties [Refs. 32, 62 & Lymperopoulos A et
al., unpublished data].
Finally, adrenal arr1, in conjunction with its co-factor GRK2,
also contributes to adrenal
2AR desensitization and downregula-
tion which results in chronically elevated catecholamine secretion,
another enormous heurohormonal burden on the failing heart
[63,64]. Therefore, adrenal arr1 (and/or GRK2) inhibition poses as
an attractive therapeutic strategy for lowering the neurohormonal
burden of the failing heart, since it should lead to lower catechola-
Anastasios Lymperopoulos
mines (sympatholysis) as well as to lower aldosterone, both of
which hormones contribute significantly to the morbidity and mor-
tality of heart failure (Table 1).
VASCULAR AT1R & NICOTINIC ACID RECEPTOR ARR-
DEPENDENT SIGNALING
In vascular smooth muscle (VSM), arr-dependent signaling
appears to promote development and progression of atherosclerotic
vascular disease. Neointimal hyperplasia after carotid endothelial
injury is enhanced in arr1-knockout mice but diminished in arr2-
knockout mice. Loss of arr2 appears to decrease 7TMR-stimulated
VSM cell proliferation and ERK1/2 activation/migration, consistent
with a role for arr2 signaling in the injury response. When the
low-density lipoprotein receptor-knockout mouse, a genetic model
of enhanced atherogenesis, is crossed onto a arr2-knockout back-
ground, atheromas are significantly reduced [65]. In vitro, both G
protein- and arr-dependent pathways elicited by the AT1AR con-
verge on the EGF receptor in primary VSM cells to stimulate pro-
liferation. Specifically, arr2 seems to enhance Src-dependent EGF
receptor transactivation by the AT1AR [66]. Moreover, arr2-
dependent ERK1/2 activation downregulates the pro-apoptotic
phospho-BAD protein, thus inducing anti-apoptotic cytoprotective
effects in rat VSM [67]. Thus, it appears that arr2 can promote
AT1AR-dependent VSM proliferation and hypertrophy, thus con-
tributing to the development and progression of atheromas, whereas
arr1 again (similarly to cardiac AT1AR pro-contractile signaling)
opposes these actions of arr2. Therefore, in VSM and in athero-
sclerotic disease, arr2 inhibition and/or arr1 stimulation might be
therapeutically desirable (Table 1).
Another vascular-related physiological effect of arr-dependent
signaling appears to be facilitation of niacin (nicotinic acid)-
induced flushing, a major adverse effect of pharmacotherapy with
this very useful lipid-lowering drug that very often poses several
limitations on its use in patients. Through its actions on GPR109A,
a 7TMR, nicotinic acid has been shown to decrease serum free fatty
acids by activating Gi/o signaling and to increase cutaneous blood
flow/flushing in humans and in mice by stimulating cytosolic phos-
pholipase A2, leading to the production and secretion of prosta-
glandin D2 [68,69]. It was recently demonstrated that arr1, al-
though dispensable for the beneficial effects of niacin on free fatty
acid levels, actually promotes the niacin-related flushing, as meas-
ured by perfusion of the ventral ear. The underlying mechanism
appears to be niacin-induced arr1-mediated activation of phos-
pholipase A2, which stimulates arachidonic acid release, the pre-
cursor of vasodilatory prostaglandin D2 responsible for the flushing
response [70]. Thus, arr1 inhibition at the GPR109A receptor or a
biased GPR109A ligand capable of activating G protein signaling
without arr1 activation would be therapeutically desirable, as it
would confer the same therapeutic effect of niacin on lipid metabo-
lism without the adverse effect of flushing (Table 1). Notably, a
series of pyrazole derivatives with GPR109A receptor agonist
properties have been synthesized that are devoid of the ability to
internalize GPR109A and activate ERK, and thus they do not cause
flushing [71]. One of these pyrazole derivatives, 3-(1H-tetrazol-5-
yl)-1,4,5,6-tetrahydrocyclopentapyrazole (MK-0354) (Fig. 1), de-
creases serum free fatty acids without producing cutaneous flush-
ing, probably by causing selective G protein activation and simulta-
neous arr1 inhibition at the GPR109A receptor, and appears thus
superior to niacin for lipid lowering therapy [72].
CONCLUDING REMARKS/FUTURE DIRECTIONS
The emerging and increasingly expanding field of 7TMR arr-
dependent signaling offers several exciting new opprtunities for
therapeutic intervention in cardiovascular disease. Despite being
still in its infancy with respect to ascribing certain physiological
and pathophysiological effects to specific arr isoforms and in spe-
cific tissues/organs, the potential advantages of exploiting this area
Beta-arrestin Biased Agonism/Antagonism
for therapeutic purposes is enormous. Admittedly, the picture is still
hazy regarding physiological actions of
arrs, in particular with
relation to the cardiovascular system. For instance, cardiac
arr2
inhibits contractility by
ARs but appears to enhance it by the
AT1R. It also appears to have beneficial effects in the heart as it
increases survival and cardiomyocyte proliferation, as well as
AngII-induced contractility, but it also promotes atherosclerosis in
the vasculature. On the other hand,
arr1 seems to have negative
effects in most cardiovascular tissues/organs whose physiology it is
involved in: it opposes cardiac
AR (and AT1R) pro-contractile
signaling, promotes AngII-induced aldosterone produc-
tion/secretion by the adrenal glands and mediates niacin-induced
flushing. Further, since it theoretically competes with
arr2 for
receptor binding, inhibition of
arr1 would indirectly enhance
arr2
activity and its beneficial effects in the heart. Thus, it appears that
these two
arr isoforms by no means complement each other with
regard to their physiological actions, and, in fact, more often than
not, their signaling pathways result in diametrically opposing ulti-
mate cellular effects. Therefore, the situation in terms of their
therapeutic exploitation for cardiovascular disease treatment be-
comes even more complicated and it is quite plausible that isoform-
specific targeting might ultimately be warranted, if direct targeting
of
arr-dependent signaling (i.e. donwstream of the receptor) is
ever going to be seriously considered as a therapeutic strategy for
the treatment of cardiovascular disease. For instance, inhibitors of

arr1 and enhancers of
arr2 activity (or vice versa in the case of
atherosclerosis) or inhibition of
arr1 only, which would also indi-
rectly increase
arr2 activity appear desirable for cardiovascular
signal transduction therapy. Future studies will hopefully help clar-
ify the picture with regards to the exact roles of
arr-dependent
signaling in vivo in the cardiovascular system. For the time being,
synthesis and development of new 7TMR biased ligands, which
selectively activate one pathway over the other from the same re-
ceptor or even activate one pathway (e.g. G protein signaling) while
inhibiting the other (e.g.
arr signaling), depending each time on
the specific 7TMR, cell type and desired cellular effect in question,
seems the most reasonable and pragmatic approach of exploiting
7TMR
arr signaling therapeutically. In any case, this
arr-
dependent 7TMR signal transduction represents a very exciting and
potentially very promising avenue of possibilities for novel thera-
peutic approaches in cardiovascular disease, a disease area with an
overwhelmingly high mortality burden compared to the therapeutic
arsenal available to clinicians for combating it.
REFERENCES
[1] Lander E S, Linton LM, Birren B, et al. Initial sequencing and
analysis of the human genome. Nature 2001; 409: 860-921.
[2] Venter JC, Adams MD, Myers, et al. The sequence of the human
genome. Science 2001; 291: 1304-51.
[3] Flower DR. Modeling G-protein-coupled receptors for drug design.
Biochem Biophys Acta 1999; 1422: 207-34.
[4] Ferguson SS. Evolving concepts in G protein-coupled receptor
endocytosis: the role in receptor desensitization and signaling.
Pharmacol Rev 2001; 53: 1-24.
[5] Hata JA, Koch WJ. Phosphorylation of G protein-coupled recep-
tors: GPCR kinases in heart disease. Mol Interv 2003; 3: 264-72.
[6] Rockman HA, Koch WJ, Lefkowitz RJ. Seven-transmembrane-
spanning receptors and heart function. Nature 2002; 415: 206-12.
[7] Reiter E, Lefkowitz RJ. GRKs and beta-arrestins: roles in receptor
silencing, trafficking and signaling. Tr Endocrinol Metab 2006; 17:
159-265.
[8] Gurevich VV, Gurevich EV. The structural basis of arrestin-
mediated regulation of G-protein-coupled receptors. Pharmacol
Ther 2006; 110: 465-502.
[9] Shukla AK, Violin JD, Whalen EJ, Gesty-Palmer D, Shenoy SK,
Lefkowitz RJ. Distinct conformational changes in beta-arrestin re-
port biased agonism at seven-transmembrane receptors. Proc Natl
Acad Sci USA 2008; 105: 9988-93.
Current Pharmaceutical Design, 2012, Vol. 18, No. 2 197
[10] Gurevich VV, Pals-Rylaarsdam R, Benovic JL, Hosey MM,
Onorato JJ. Agonist-receptor-arrestin, an alternative ternary com-
plex with high agonist affinity. J Biol Chem 1997; 272: 28849-52.
[11] Stoffel RH 3rd, Pitcher JA, Lefkowitz RJ. Targeting G protein-
coupled receptor kinases to their receptor substrates. J Membr Biol
1997; 157: 1-8.
[12] Goodman OB Jr, Krupnick JG, Santini F, et al. Beta-arrestin acts as
a clathrin adaptor in endocytosis of the beta2-adrenergic receptor.
Nature 1996; 383: 447-50.
[13] Krupnick JG, Goodman OB Jr, Keen JH, Benovic JL. Ar-
restin/clathrin interaction. Localization of the clathrin binding do-
main of nonvisual arrestins to the carboxy terminus. J Biol Chem
1997; 272: 15011-6.
[14] Laporte SA, Oakley RH, Holt JA, Barak LS, Caron MG. The inter-
action of beta-arrestin with the AP-2 adaptor is required for the
clustering of beta 2-adrenergic receptor into clathrin-coated pits. J
Biol Chem 2000; 275: 23120-6.
[15] Laporte SA, Oakley RH, Zhang J, Holt JA, Ferguson SS, Caron
MG, Barak LS. The beta2-adrenergic receptor/betaarrestin complex
recruits the clathrin adaptor AP-2 during endocytosis. Proc Natl
Acad Sci USA 1999; 96: 3712-7.
[16] Oakley RH, Laporte SA, Holt JA, Caron MG, Barak LS. Differen-
tial affinities of visual arrestin, beta arrestin1, and beta arrestin2 for
G protein-coupled receptors delineate two major classes of recep-
tors. J Biol Chem 2000; 275: 17201-10.
[17] Charest PG, Terrillon S, Bouvier M. Monitoring agonist-promoted
conformational changes of beta-arrestin in living cells by in-
tramolecular BRET. EMBO Rep 2005; 6: 334-40.
[18] Miller WE, Lefkowitz RJ. Expanding roles for beta-arrestins as
scaffolds and adapters in GPCR signaling and trafficking. Curr
Opin Cell Biol 2001; 13: 139-45.
[19] Perry SJ, Lefkowitz RJ. Arresting developments in heptahelical
receptor signaling and regulation. Trends Cell Biol 2002; 12: 130-
8.
[20] Maudsley S, Martin B, Luttrell LM. The origins of diversity and
specificity in G protein-coupled receptor signaling. J Pharmacol
Exp Ther 2005; 314: 485-94.
[21] Shenoy SK, Lefkowitz RJ. Seven-transmembrane receptor signal-
ing through beta-arrestin. Sci STKE 2005: cm10.
[22] Luttrell LM, Ferguson SS, Daaka Y, et al. Beta- arrestin-dependent
formation of beta2 adrenergic receptor-Src protein kinase com-
plexes. Science 1999; 283: 655-61.
[23] Luttrell LM, Roudabush FL, Choy EW, et al. Activation and target-
ing of extracellular signal-regulated kinases by beta-arrestin scaf-
folds. Proc Natl Acad Sci USA 2001; 98: 2449-54.
[24] Shenoy SK, McDonald PH, Kohout TA, Lefkowitz RJ. Regulation
of receptor fate by ubiquitination of activated beta 2-adrenergic re-
ceptor and beta-arrestin. Science 2001; 294: 1307-13.
[25] Perry SJ, Baillie GS, Kohout TA, et al. Targeting of cyclic AMP
degradation to beta 2-adrenergic receptors by beta-arrestins. Sci-
ence 2002; 298: 834-6.
[26] Nelson CD, Perry SJ, Regier DS, Prescott SM, Topham MK,
Lefkowitz RJ. Targeting of diacylglycerol degradation to M1 mus-
carinic receptors by beta-arrestins. Science 2007; 315: 663-6.
[27] Witherow DS, Garrison TR, Miller WE, Lefkowitz RJ. beta-
Arrestin inhibits NF-kappaB activity by means of its interaction
with the NF-kappaB inhibitor IkappaBalpha. Proc Natl Acad Sci
USA 2004; 101: 8603-7.
[28] Beaulieu JM, Sotnikova TD, Marion S, Lefkowitz RJ, Gainetdinov
RR, Caron MG. An Akt/beta-arrestin 2/PP2A signaling complex
mediates dopaminergic neurotransmission and behavior. Cell 2005;
122: 261-73.
[29] Luttrell LM, Gesty-Palmer D. Beyond desensitization: physiologi-
cal relevance of arrestin-dependent signaling. Pharmacol Rev 2010;
62: 305-30.
[30] Rajagopal S, Rajagopal K, Lefkowitz RJ. Teaching old receptors
new tricks: biasing seven-transmembrane receptors. Nat Rev Drug
Discov 2010; 9: 373-86.
[31] DeWire SM, Ahn S, Lefkowitz RJ, Shenoy SK. Beta-arrestins and
cell signaling. Annu Rev Physiol 2007; 69: 483-510.
[32] Holloway AC, Qian H, Pipolo L, et al. Side-chain substitutions
within angiotensin II reveal different requirements for signaling, in-
ternalization, and phosphorylation of type 1A angiotensin recep-
tors. Mol Pharmacol 2002; 61: 768-77.
[33] Ahn S, Nelson CD, Garrison TR, Miller WE, Lefkowitz RJ. Desen-
sitization, internalization, and signaling functions of beta-arrestins
198 Current Pharmaceutical Design, 2012, Vol. 18, No. 2
demonstrated by RNA interference. Proc Natl Acad Sci USA 2003;
100: 1740-4.
[34] Wei H, Ahn S, Shenoy SK, et al. Independent beta-arrestin 2 and G
protein-mediated pathways for angiotensin II activation of extracel-
lular signal-regulated kinases 1 and 2. Proc Natl Acad Sci USA
2003; 100: 10782-7.
[35] Baker JG, Hall IP, Hill SJ. Agonist and inverse agonist actions of

-blockers at the human
2-adrenoceptor provide evidence for
agonist-directed signaling. Mol Pharmacol 2003; 64: 1357-69.
[36] Azzi M, Charest PG, Angers S, Rousseau G, Kohout T, Bouvier M,
Pineyro G.
- Arrestin-mediated activation of MAPK by inverse
agonists reveals distinct active conformations for G protein-
coupled receptors. Proc Natl Acad Sci USA 2003; 100: 11406-11.
[37] Galandrin S, Oligny-Longpre G, Bonin H, Ogawa K, Gales C,
Bouvier M. Conformational rearrangements and signaling cascades
involved in ligand-biased mitogen-activated protein kinase signal-
ing through the
1-adrenergic receptor. Mol Pharmacol 2008; 74:
162-172.
[38] Wisler JW, DeWire SM, Whalen EJ, et al. A unique mechanism of

-blocker action: carvedilol stimulates
-arrestin signaling. Proc
Natl Acad Sci USA 2007; 104: 16657-62.
[39] Zidar DA, Violin JD, Whalen EJ, Lefkowitz RJ. Selective engage-
ment of G protein coupled receptor kinases (GRKs) encodes dis-
tinct functions of biased ligands. Proc Natl Acad Sci USA 2009;
106: 9649-54.
[40] Rengo G, Lymperopoulos A, Koch WJ. Future g protein-coupled
receptor targets for treatment of heart failure. Curr Treatment Op-
tions Cardiovasc Med 2009; 11: 328-38.
[41] Rengo G, Lymperopoulos A, Zincarelli C, et al. Myocardial adeno-
associated virus serotype 6-betaARKct gene therapy improves car-
diac function and normalizes the neurohormonal axis in chronic
heart failure. Circulation 2009; 119: 89-98.
[42] Xiang Y, Kobilka BK. Myocyte adrenoceptor signaling pathways.
Science 2003; 300: 1530-2.
[43] Communal, C, Singh K, Sawyer DB, Colucci WS. Opposing ef-
fects of beta(1)- and beta(2)-adrenergic receptors on cardiac myo-
cyte apoptosis: role of a pertussis toxin-sensitive G protein. Circu-
lation 1999; 100: 2210-2.
[44] Bristow MR.
-adrenergic receptor blockade in chronic heart fail-
ure. Circulation 2000; 101: 558-69.
[45] Packer M, Bristow MR, Cohn JN, Colucci WS, Fowler MB,
Gilbert EM, Shusterman NH. The effect of carvedilol on morbidity
and mortality in patients with chronic heart failure. N Engl J Med
1996; 334: 1349-55.
[46] Noma T, Lemaire A, Naga Prasad SV, et al. Beta-arrestin-mediated
beta1-adrenergic receptor transactivation of the EGFR confers car-
dioprotection. J Clin Invest 2007; 117: 2445-58.
[47] Drake MT, Violin JD, Whalen EJ, Wisler JW, Shenoy SK,
Lefkowitz RJ.
-arrestin-biased agonism at the
2-adrenergic re-
ceptor. J Biol Chem 2008; 283: 5669- 5676.
[48] Woo AY, Wang TB, Zeng X, et al. Stereochemistry of an agonist
determines coupling preference of beta2-adrenoceptor to different
G proteins in cardiomyocytes. Mol Pharmacol 2009; 75: 158-65.
[49] Zhai P, Yamamoto M, Galeotti J, et al. Cardiac-specific overex-
pression of AT1 receptor mutant lacking G alpha q/G alpha i cou-
pling causes hypertrophy and bradycardia in transgenic mice. J Clin
Invest 2005; 115: 3045-56.
[50] Aplin M, Christensen GL, Schneider M, et al. Differential extracel-
lular signal-regulated kinases 1 and 2 activation by the angiotensin
type 1 receptor supports distinct phenotypes of cardiac myocytes.
Basic Clin Pharmacol Toxicol 2007; 100: 296-301.
[51] Rajagopal K, Whalen EJ, Violin JD, et al. Beta-arrestin2-mediated
inotropic effects of the angiotensin II type 1A receptor in isolated
cardiac myocytes. Proc Natl Acad Sci USA 2006; 103: 16284-9.
[52] Kukkonen JP. Regulation of receptor-coupling to (multiple) G
proteins. A challenge for basic research and drug discovery. Recep-
tors and Channels 2004; 10: 167-83.
Received: September 4, 2011 Accepted: November 9, 2011
Anastasios Lymperopoulos
[53] Wang Q, Zhao J, Brady AE, Feng J, et al. Spinophilin blocks ar-
restin actions in vitro and in vivo at G protein-coupled receptors.
Science 2004; 304: 1940-1944.
[54] Schmid CL, Bohn LM. Physiological and pharmacological implica-
tions of beta-arrestin regulation. Pharmacol Ther 2009; 121: 285-
93.
[55] Weber KT. Aldosterone in congestive heart failure. N Engl J Med
2001; 345: 1689-97.
[56] Connell JM, Davies E. The new biology of aldosterone. J Endocri-
nol 2005; 186: 1-20.
[57] Marney AM, Brown NJ. Aldosterone and end-organ damage. Clin
Sci (Lond) 2007; 113: 267-78.
[58] Ganguly A, Davis JS. Role of calcium and other mediators in al-
dosterone secretion from the adrenal glomerulosa cells. Pharmacol
Rev 1994; 46: 417-47.
[59] De Gasparo M, Catt KJ, Inagami T, Wright JW, Unger T. Interna-
tional union of pharmacology. XXIII. The angiotensin II receptors.
Pharmacol Rev 2000; 52: 415-72.
[60] Lymperopoulos A, Rengo G, Zincarelli C, Kim J, Soltys S, Koch
WJ. An adrenal beta-arrestin 1-mediated signaling pathway under-
lies angiotensin II-induced aldosterone production in vitro and in
vivo. Proc Natl Acad Sci USA 2009; 106: 5825-30.
[61] Lymperopoulos A, Rengo G, Zincarelli C, Kim J, Koch WJ. Adre-
nal Beta-arrestin 1 inhibition in vivo attenuates post-myocardial in-
farction progression to heart failure and adverse remodeling via re-
duction of circulating aldosterone levels. J Am Coll Cardiol 2011;
57: 356-65.
[62] Lymperopoulos A, Rengo G, Magafa V, Cordopatis P, Koch WJ.
Biased Agonism/Antagonism of
-Arrestin activation by the An-
giotensin II Type 1 Receptor: A Study of Sartans and Angiotensin
II Analogs Using Aldosterone Turnover as a Readout. J. Card. Fail.
Suppl. 1 2010; 16: S30-1.
[63] Lymperopoulos A, Rengo G, Funakoshi H, Eckhart AD, Koch WJ.
Adrenal GRK2 upregulation mediates sympathetic overdrive in
heart failure. Nat Med 2007; 13: 315-23.
[64] Lymperopoulos A, Rengo G, Koch WJ. Adrenal adrenoceptors in
heart failure: fine-tuning cardiac stimulation. Tr Mol Med 2007;
13: 503-11.
[65] Kim J, Zhang L, Peppel K, et al. Beta-arrestins regulate atheroscle-
rosis and neointimal hyperplasia by controlling smooth muscle cell
proliferation and migration. Circ Res 2008; 103: 70-9.
[66] Kim J, Ahn S, Rajagopal K, Lefkowitz RJ. Independent beta-
arrestin2 and Gq/protein kinase Czeta pathways for ERK stimu-
lated by angiotensin type 1A receptors in vascular smooth muscle
cells converge on transactivation of the epidermal growth factor re-
ceptor. J Biol Chem 2009; 284: 11953-62.
[67] Ahn S, Kim J, Hara MR, Ren XR, Lefkowitz RJ. Beta-arrestin-2
mediates anti-apoptotic signaling through regulation of BAD phos-
phorylation. J Biol Chem 2009; 284: 8855-65.
[68] Kather H, Aktories K, Schulz G, Jakobs KH. Islet-activating pro-
tein discriminates the antilipolytic mechanism of insulin from that
of other antilipolytic compounds. FEBS Lett 1983; 161: 149-52.
[69] Pike NB. Flushing out the role of GPR109A (HM74A) in the clini-
cal efficacy of nicotinic acid. J Clin Invest 2005; 115: 3400-3.
[70] Walters RW, Shukla AK, et al. Beta-Arrestin1 mediates nicotinic
acid-induced flushing, but not its antilipolytic effect, in mice. J Clin
Invest 2009; 119: 1312-21.
[71] Richman JG, Kanemitsu-Parks M, Gaidarov I, et al. Nicotinic acid
receptor agonists differentially activate downstream effectors. J
Biol Chem 2007; 282: 18028-36.
[72] Semple G, Skinner PJ, Gharbaoui T, et al. 3-(1H-tetrazol-5-yl)-
1,4,5,6-tetrahydro-cyclopentapyrazole (MK-0354): a partial agonist
of the nicotinic acid receptor, G-protein coupled receptor 109a,
with antilipolytic but no vasodilatory activity in mice. J Med Chem
2008; 51: 5101-8.