New Insights Into Modes of GPCR Activation

Review
New Insights into Modes of GPCR Activation

Wenjing Wang,1,2,3,4,5,y Yuhui Qiao,1,2,3,4,5,y and Zijian Li1,2,3,4,*
In classical G-protein-coupled receptor (GPCR) activation, GPCRs couple to a Highlights

variety of heterotrimeric G proteins on the membrane and then activate down- GPCRs are the largest family of cell
surface receptors and are significant
stream signaling pathways. More recently, GPCRs have been found to couple
drug targets.
to different effector proteins, including different G protein subtypes and regu-
latory proteins, such as arrestins. Some novel modes of GPCR activation have In recent years, many advanced bio-
chemical and biophysical methods,
been proposed to explain their complex behaviors. In this review, we summa- such as single molecule methods,
rize the main novel modes of GPCR activation, including biased activation, and single particle electron cryomicro-
intracellular activation, dimerization activation, transactivation, and biphasic scopy, have been used to study
GPCRs.
activation. In addition, we also discuss the relationship among the five modes
to show the complex picture of GPCR activation. The complex activation With advanced methods and in-depth
research, several novel modes of
modes regulate precisely GPCR downstream signaling, including physiological
GPCR activation have been discov-
and pathological signaling. Thus, there is the potential to develop GPCR ered in addition to the classical activa-
precision drugs that target precise GPCR activation modes to accurately tion (GPCR–G protein signaling
pathways) mode. These diverse
strengthen their beneficial functions and block specific pathological processes. modes of GPCR activation help explain
their complex behaviors.
The Activation of GPCR
Varied modes of GPCR activation
GPCRs, also called 7TM receptors because they contain seven transmembrane helices, are
allow the concept of precision drug
the largest family of cell-surface receptors. They have important roles in various physiological development and provide a theoretical
and pathological processes, including proliferation, differentiation, chemotaxis, and communi- basis for the research and develop-
cation [1]. As membrane proteins, GPCRs can be activated by a diverse range of extracellular ment of GPCR-targeted functionally
selective drugs.
ligands. In the classical GPCR signaling pathway, ligand-activated GPCRs function through
their interaction with intracellular G proteins [2]. The activated G proteins can then transduce
and amplify GPCR signals via second messengers to produce a variety of cell responses [1].
In contrast to the classic ‘GPCR–G protein’ activation mode, several novel modes of GPCR 1
Department of Cardiology and
activation have been discovered to explain their complex behavior. Here, we summarize five Institute of Vascular Medicine, Peking
modes of GPCR activation. Approximately 10 years ago, the multifunctional adaptor b-arrest- University Third Hospital, Beijing,
100191, China
ins were found to be able to initiate parallel G protein-independent signaling pathways. The 2
Key Laboratory of Cardiovascular
distinct b-arrestin-mediated signaling defined a new GPCR activation mode called ‘biased Molecular Biology and Regulatory
activation’ [1]. That is, GPCRs may bias towards either G protein-dependent pathways or Peptides, Ministry of Health, Peking
University Third Hospital, Beijing,
b-arrestin-dependent pathways. GPCRs were once thought to be located and activated on the 100191, China
cell surface. However, some GPCRs have also been found and can be activated inside cells 3
Key Laboratory of Molecular
[3,4]. Here, we call this activation mode ‘intracellular activation’. In addition, GPCRs do not Cardiovascular Sciences, Ministry of
Education, Peking University Third
always exist and mediate signals as monomers. The dimerization of GPCRs is important for Hospital, Beijing, 100191, China
certain signaling pathways, as well as for the function of receptors. The receptors assume 4
Beijing Key Laboratory of
different states as monomers or dimers, and their signals and functions are also different. Cardiovascular Receptors Research,
Peking University Third Hospital,
Therefore, we call activation that depends on dimerization ‘dimerization activation’ [5]. It is also Beijing, 100191, China
established that ligands that target GPCRs are able to transactivate other types of receptor, 5
These authors contributed equally to
such as tyrosine kinase receptors (RTKs), which expands the repertoire of GPCR signaling and this work
y
Co-first authors.
functions. We call this activation ‘transactivation’ of the receptor [6]. ‘Biphasic activation’ is
another unique activation pattern of GPCRs. Some GPCRs can be activated at two different *Correspondence:
phases, early phase (5–10 min) and late phase (90–120 min), and the two phases of signals lizijian@bjmu.edu.cn (Z. Li).
Trends in Pharmacological Sciences, April 2018, Vol. 39, No. 4 https://doi.org/10.1016/j.tips.2018.01.001 367
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may have different functions. Thus, the concept and significance of ‘biphasic activation’ are
also gradually being elucidated [7]. Here, we summarize these five novel modes of GPCR
activation and their mechanisms (Figure 1). Understanding these novel modes of GPCR
activation will not only deepen our knowledge of receptor activation, but also facilitate the
development of more-precise and effective GPCR-targeted drugs.
Biased Activation
Initially, GPCR signaling was considered to be simply mediated by intracellular G proteins.
However, this concept was completely revised after b-arrestin was also found to have the
capability to mediate diverse GPCR signaling independently [1]. Namely, when GPCRs are
activated, they can activate either the G protein or b-arrestin pathway. It is now appreciated that
several GPCR ligands can selectively activate either the G protein pathway or the b-arrestin
pathway, which is called biased activation [8].
The G protein pathway and the b-arrestin pathway are often spatially and temporally distinct,
and mediate unique physiological or pathophysiological effects [1,9]. Taking the angiotensin II
(A) Classical acvaon (B) Biased acvaon (C) Intracellular acvaon
G-protein G-protein β-arresn Clathrin-

coated pit
Cell signaling Cell signaling Cell signaling
(D) Dimerizaon acvaon (E) Transacvaon (F) Biphasic acvaon
src P P
y
wa
Sig
th
pa
nal
g
ing
lin
Downstream
na
pat
β-arresn
effector
Sig
G-protein
hw
ay
Cell signaling
Cell signaling
Time
Figure 1. Classical and Novel Modes of G-Protein-Coupled Receptor (GPCR) Activation. (A) Classical mode, (B) biased activation, (C) intracellular activation,
(D) dimerization activation, (E) transactivation, and (F) biphasic activation.
368 Trends in Pharmacological Sciences, April 2018, Vol. 39, No. 4
type 1 receptor (AT1R) as an example, the G protein pathway mediates potent vasoconstriction
and increases blood pressure, whereas the b-arrestin pathway mediates potentially beneficial
effects, including cytoprotection and antiapoptotic effects [10,11]. Thus, the discovery and
development of ‘biased ligands’, which selectively activate either G protein-biased or
b-arrestin-biased signaling, could selectively promote beneficial effects and block detrimental
or unwanted effects induced by GPCR activation (Figure 2A,B). Thus, the discovery of biased
ligands completely changed the classical concept of GPCR-targeted drugs [12–15].
So far, many biased ligands, including b-arrestin-biased or G protein-biased ligands, have been
identified and developed (Table 1), some of which have been applied to clinical practice
[15–18]. Most research on b-arrestin-biased ligands has been targeted at b-adrenergic
receptors (b-ARs) and AT1R. Carvedilol, a b-arrestin-biased agonist for b-AR, has been
demonstrated to inhibit the G protein-mediated toxic effect of catecholamines and stimulate
b-arrestin-mediated cell survival signaling pathways. Large-scale clinical trials indicated that
carvedilol may significantly improve survival in patients with heart failure compared with other
nonbiased b-blockers [19–21]. In addition, the activation of angiotensin receptor subtype 1a
(AT1aR) can be biased towards b-arrestin by the modified peptide [Sar1I-le4-Ile8] angiotensin II
(SII) [22]. SII binds to AT1R and activates the b-arrestin-dependent mitogen-activated protein
kinase (MAPK) pathway without coupling to G proteins. However, SII has low affinity for AT1R
and, thus, it has been difficult to study the pharmacological effects of the in vivo administration
of SII [23]. TRV120027, another b-arrestin-biased ligand of AT1R, has overcome this limitation
and shows high affinity for AT1R. TRV120027 inhibits AngII-stimulated vascular contraction,
but increases b-arrestin-mediated cardiomyocyte contractility and promotes cardiac output in
vivo. Thus, TRV120027 has the potential to treat acute heart failure [24]. By contrast, in some
GPCRs, the G protein pathway mediates beneficial effects, while the b-arrestin pathway
mediates undesirable adverse effects. Therefore, G protein-biased ligands have been devel-
oped to treat relevant diseases [1]. For example, the m-opioid receptor (MOR) is the target of the
prototypical m-opioid receptor agonist, morphine. Similar to most opioid narcotics, morphine
has adverse effects, such as pain tolerance, constipation, and respiratory suppression, thereby
(A) Ligand (B)
G protein β-arres n G protein β-arres n
Downstream Downstream
effector effector
Figure 2. Biased Activation. Two major patterns of based activation: (A) b-arrestin-mediated biased signaling and (B) G
protein-mediated biased signaling.
Trends in Pharmacological Sciences, April 2018, Vol. 39, No. 4 369
Table 1. Activation Modes of GPCRsa
Activation Mode GPCR Ligand/effector Biological effects Refs
Biased activation b-AR Carvedilol Extends survival in patients with chronic heart failure [19]
b2-AR ICL1-9 Stimulates cardioprotective signaling and inotropic effects [150]
AT1R SII Mediates inotropic effects [151]
TRV120027 Reduces blood pressure and increases cardiac performance [24]
TRV120023 Supports cardiac function and reduces cellular injury [152]
PTHR (D-Trp12,Tyr34)-PTH Supports anabolic bone formation [153]
MOR TRV130 Reduces gastrointestinal and respiratory adverse effects [28]
PZM21 Efficacious reduction of adverse effects [31]
GPR109A MK0354 Reduces cutaneous flushing [32]
pyrazole Reduces cutaneous flushing [154]
UTR Urantide Modulates cell survival [155]
Intracellular activation b1-AR Clathrin-coated pit ERK1/2 activation [45]
b2-AR Clathrin-coated pit ERK1/2 activation [3]
Endosome Activates cAMP signaling [54]
AT1aR Endosome Determines spatial distribution and activity of JNK3 [156]
PAR2 Clathrin-coated pit Intracellular targeting of activated ERK1/2 [157]
NTR1 Endosome Regulates proinflammatory signaling [158]
NK1R Endosome Facilitates proliferative and antiapoptotic effects [159]
THSR Golgi Induces local Gs protein signaling and gene transcription [55]
F2rl1 Nuclear Governs angiogenesis [160]
b-AR Golgi Contributes significantly to overall cellular cAMP response [61]
mGluR5 Nuclear Regulates nucleoplasmic Ca2+ in situ [60]
OA1 Melanosome Regulates organelle biogenesis and maturation [4]
CB1R Mitochondria Regulates neuronal energy metabolism [56]
Lysosomal Interacts with G proteins and mediates signaling [161]
GPR30 Endoplasmic reticulum Results in intracellular calcium mobilization [57]
Dimerization activation b1-AR b2-AR Optimizes b-adrenergic modulation of cardiac contractility [162]
b2-AR DOR Modulates receptor trafficking and signal transduction [163]
KOR Modulates receptor trafficking and signal transduction. [163]
a1A-AR a1BAR Correlates with characters of receptor internalization [164]
a2A-AR MOR Modulates receptor function [165]
b1-AR Crosstalk between, and mutual regulation of, these receptors [166]
a2C-AR AT1R Results in atypical Gs-PKA signaling [167]
AT1R b2-AR Influences response to drugs that antagonize these receptors [168]
Apelin receptor Increases NO production and inhibiting AT1R signaling [169]
MOR DOR Influences ethanol consumption [170]
DOR KOR Influences nociception [170]
CB1R Might be relevant to issue of multiple drug abuse [171]
P2YlR A1R Increases diversity of purine signaling [172]
NPY1R NPY5R Selectively modifies pharmacology [173]
Table 1. (continued)
CB1R CB2R Shows bidirectional cross-antagonism phenomenon [174]
SSTR1 SSTR5 Crosstalk between GPCRs for greater functional diversity [175]
SSTR2 D2R Increases D2R signaling and SSTR2 internalization [176]
SSTR5 D2R Creates novel receptor with enhanced functional activity [177]
D1R D2R Increases intracellular calcium levels [86]
D2R A2aR Modulates neuronal excitability and transmitter release [178]
V1AR V2R Regulates endocytotic processing of receptors [179]
T1R3 T1R1, T1R2 Combine to function as a sweet receptor [180]
AChRs M1 mAChRs M1,2,3 GPCR long-term regulation [181]
CCK-A CCK-B Promotes cell growth [182]
GABABR1 GABABR2 Inwardly rectifying potassium channels [84]
Transactivation b1-AR EGFR Cardioprotective role in chronic catecholamine stimulation [97]
b2-AR EGFR Activates ERK1/2 [98]
a1D-AR EGFR Maintains neuron-associated functions [183]
a1B-AR EGFR Adrenergic vasoconstriction [99]
a2-AR EGFR Mediates ERK activation [184]
AT1R EGFR Promotes cardiac myocyte growth [103]
D2R EGFR Stimulates MAPK [185]
MOR EGFR Stimulates ERK1/2 [102]
NMBR EGFR Human pancreatic cancer growth and metastatic spread [186]
GnRHR EGFR Induces transcription factors c-Jun and c-Fos [105]
LHRH EGFR Promotes ERK signaling and steroidogenesis [187]
AChRs EGFR Cancer cell migration and proliferation [188]
c-Met Oncogenic potential and tumor invasion [106]
GABABR IGF-1R Induces dynamic assembly and disassembly of a protein complex [101]
5-HT2B EGFR Maintains neuron-associated functions [183]
5-HTR PDGFR-b Activates serotonergic and neurotrophic signaling [100]
TrkB Activates serotonergic and neurotrophic signaling [100]
H2R EGFR Regulates gastric exocrine and epithelial cell function [189]
CXCR1 EGFR Cell proliferation and migration [104]
CXCR2 EGFR Cell proliferation and migration [104]
P2Y2R EGFR ERK1/2 phosphorylation [190]
LPAR EGFR MAPK activation, induction of cell proliferation [6]
LPAR c-Met Production of ROS, nuclear translocation of b-catenin [106]
FPR EGFR Activates STAT3 pathway, promotes cell growth [191]
FPR c-Met Activates STAT3, PLC-g1/PKCa, and PI3K/Akt pathways [192]
FPR TrkA ROS production, CD11b membrane integrin upregulation [193]
BDKRB1-2 c-Met Production of ROS, nuclear translocation of b-catenin [106]
PARs EGFR MMP-13 production, cartilage destruction during arthritis [194]
c-Met Production of ROS, nuclear translocation of b-catenin [106]
Table 1. (continued)
ETR c-Met Production of ROS, nuclear translocation of b-catenin [106]
NTRs EGFR Proliferation of prostate cancer cells [195]
V2R IGFR ERK1/2 activation, kidney cell proliferation [196]
AR TrkA Trk activation, neuronal survival [197]
ETR EGFR MAPK activation, induction of cell proliferation [6]
SSTR1 EGFR Activates MAPK signaling, regulates tumor growth [123]
SSTR5 EGFR Activates MAPK signaling, regulates tumor growth [123]
CXCR4 IGF-1 Activates migrational signaling pathways [125]
b2-AR insulin Leads to supersensitization of b2-AR [126]
S1P PDGF Cell motility and migration [127]
NGF Neurite outgrowth [198]
LPA NGF Neurite outgrowth [199]
CCR5 IGF-1 Cell migration [200]
PAC1 IGF-1 Protects against apoptosis [131]
S1P IGF-1 Cell migration [129]
Biphasic activation AT1R Ins-1,4,5-P3, Ca2+ Calcium mobilization [201]
MAPK Activates MAPK and induces DNA synthesis [7]
JAK/STAT Unclear [202]
b2-AR P38 Unclear [139]
b3-AR ERK1/2 Induces primary smooth muscle cell proliferation [140]
PTH1R ERK1/2 Unclear [137]
a
Abreviations: 5-HT2B, serotonin receptor type 2B; 5-HTR, serotonin receptor; a1A-AR, a1A-adrenergic receptor; a1B-AR, a1B-adrenergic receptor; a1D-AR, a1D-
adrenergic receptor; a2-AR, a2-adrenergic receptor; a2A-AR, a2A-adrenergic receptor; a2C-AR, a2C-adrenergic receptor; AChRs M1/2/3, acetylcholine receptor
type 1/2/3; AR, adenosine receptor; BDKRB1–2, bradykinin receptor B1–2; c-Met, hepatocyte growth factor receptor; CB1R, cannabinoid B1 receptor; CB2R,
cannabinoid B2 receptor; CCK-A/B, cholecystokinin receptor type A/B; CCR5, chemokine receptor 5; CXCR1/2/4, CXC chemokine receptor 1/2/4; D1R, Dopamine
D1 receptor; D2R, Dopamine D2 receptor; DOR, d-opioid receptor; EGFR, epidermal growth factor receptor; ETaR, endothelin type A receptor; ETR, endothelin
receptor; F2rl1, factor II receptor-like 1; FPR, N-formyl peptide receptor; GABABR1/2, g-aminobutyric acid B receptor 1/2; GnRHR, gonadotropin-releasing hormone
receptor; H2R, histamine receptor; IGF, insulin-like growth factors; IGF-1R, insulin-like growth factor type I receptor; KOR, k-opioid receptor; LHRH, luteinizing-
hormone receptor; LPAR, lysophosphatidic acid receptor; mGluR5, metabotropic glutamate receptor 5; MOR, m-opioid receptor; NGF, nerve growth factor; NK1R,
neurokinin-1 receptor; NMBR, neuromedin B receptor; NPY1R, neuropeptide Y1 receptor; NPY5R, neuropeptide Y5 receptor; NTR1, neurotensin receptor 1; OA1,
ocular albinism type 1; P2Y2R, G-protein-coupled P2Y2 receptor; P2YlR, P2Y-like adenosine receptor; PAC1, PACAP type I receptor; PARs, protease-activated
receptors; PDGF, platelet-derived growth factor; PDGFR-b, platelet-derived growth factor receptor-b; PTHR, parathyroid hormone receptor; SSTR1/2/5, Soma-
tostatin receptors 1/2/5; T1R1/2/3, taste receptor type 1/2/3; TRHR, thyrotropin-releasing hormone receptor; TrkA/B, Neurotrophic tyrosine kinase receptor type 1/2;
UTR, urotensin receptor; V1AR, V1A vasopressin receptor; V2R, V2 vasopressin receptor.
limiting its clinical utility. Furthermore, the b-arrestin pathway was found to mediate those
adverse effects [25–27]. TRV130, a G protein-biased ligand of MOR, showed lower rates of
gastrointestinal dysfunction and respiratory depression in rodent models compared with
morphine [28]. However, its analgesic effect is equivalent to that of morphine. Thus,
TRV130 is currently in clinical studies for the treatment of acute pain in humans [29,30].
Furthermore, PZM21, another G protein-biased ligand of MOR, was identified by computa-
tionally docking over three million molecules against the MOR structure, followed by structure-
based optimization. PZM21 proved to be structurally distinct, with novel properties compared
with TRV130. PZM21 not only shows considerable substantial signaling bias, analgesic effects,
and efficacious reduction of adverse effects, but also has higher opioid receptor selectivity than
TRV130. Thus, PZM21 is positioned to become a therapeutic lead in the field of analgesia
[18,31]. Another example is the niacin receptor GPR109A. Nicotinic acid, the classical ligand of
GPR109A, decreases serum-free fatty acids through the G protein-signaling pathway and also
induces b-arrestin-mediated adverse effects, such as cutaneous flushing. MK-0354, as a G
protein-biased ligand of GPR109A, appears to decrease serum-free fatty acids without causing
flushing. Thus, MK-0354 is a promising agent for hyperlipidemia treatment [32,33].
Initiation of biased GPCR signaling is imparted by GPCR conformational changes. Different

ligands stabilize distinct active GPCR conformations, which is considered to result in varying
degrees of biased signaling towards G proteins or b-arrestins [34–36]. However, the mecha-
nisms underlying how the conformational differences in GPCRs are connected to different
signaling behaviors remain elusive. The current theory is that a ‘phospho-barcode’ on the
GPCR has an important role in GPCR-biased signaling. In this hypothesis, the activation of
receptors can mediate different signaling pathways via distinct barcodes constituted by
receptor phosphorylation sites. For instance, PKA-mediated phosphorylation of the b-AR
serves to switch coupling of this receptor from Gs to Gi [37]. GRK2 or GRK5/6-mediated
phosphorylation is considered to represent barcodes of selective b-arrestin-mediated signaling
for b2-AR [38–40]. Furthermore, a ‘flute’ model suggests that the ‘barcodes’ determine the
functional differences after b-arrestin is recruited. The arrangement of the phosphate-binding
pockets in the N-terminal of b-arrestin-1 is similar to the hole alignment of a flute. These
phosphate-binding sites function as sensors of the GPCR phospho-barcodes, which function
as ‘fingers’ on the flute and could instruct the specific arrestin conformation and function
[38,41,42]. The phospho-barcode theory is one proposed mechanism to account for GPCR-
biased signaling.
Intracellular Activation
As plasma membrane receptors, GPCRs are thought to be located and activated on the cell
surface. However, accumulating data indicate that some GPCRs can be activated inside the
cell and trigger specific downstream effects, suggesting that the activation of GPCRs does not
arise solely at the cell surface, but may result from intracellular signaling events [3,4]. There are
at least two possibilities to explain intracellular GPCR activation. First, GPCRs can continue
signaling after internalization together with their agonists. Second, intracellular GPCRs located
at different organelles can also be activated intracellularly.
The reports of GPCR internalization date from initial studies on the b2-adrenergic receptor
(b2-AR) in frog erythrocytes in 1979 [43] and in human astrocytoma cells in 1980 [44]. At first, it
was thought that GPCR internalization served to desensitize the receptors and terminate
signaling. However, in 1999, b2-AR internalization was found to mediate ERK1/2 activation [3].
Furthermore, b-arrestin was demonstrated to mediate b2-AR on clathrin-coated pits, which
have critical roles in the process of signal transduction. Similar phenomena were then found for
many other GPCRs (Table 1). It is now well established that the internalization and trafficking of
receptors are also one of the most important mechanisms mediating GPCR signaling; that is,
the signal can be activated in the cytoplasm in an internalization-dependent manner. Here, we
call this internalization-dependent activation. b-arrestin has a critical role in internalization-
dependent activation through the promotion of clathrin-mediated endocytosis of GPCRs and
mediation of the MAP kinase signaling pathway by scaffolding and interacting with many
components of the clathrin-mediated endocytosis machinery [45–50]. In addition, different
characteristics of the interaction between GPCRs and arrestins are connected to different
patterns of GPCR internalization. Class A and class B are two major classes of GPCR. Class A
receptors, which bind both biogenic amines and peptide ligands, bind to b-arrestin2 with
higher affinity than to b-arrestin1 and do not interact with visual arrestin. Class B receptors,
which bind peptide ligands, bind to b-arrestin1 and b-arrestin2 with similar high affinities and
also interact with visual arrestin. Class A GPCRs and b-arrestins form unstable endocytic
complexes, whereas class B GPCRs and b-arrestins form stable endocytic complexes during
GPCR internalization [47,51,52]. These findings provide the foundation for predicting the
patterns of internalization-dependent activation.
Here, we summarize four different patterns of internalization-dependent activation (Figure 3).

First, for Class A GPCRs, b-arrestin translocates to the plasma membrane and interacts with
the GPCR following GPCR activation. The receptor–b-arrestin complex is internalized and
ERK1/2 is then activated near the membrane. The interaction between b-arrestin and the class
A receptor is transient, and dissociates rapidly after internalization. Then, the receptor rapidly
recycles back to the plasma membrane (Figure 3A). Second, in contrast to class A receptors,
class B receptors interact robustly with b-arrestin until targeted to endosomes for subsequent
GPCR degradation and ERK1/2 activation (Figure 3B) [45,47,51,52]. Third, b1-AR was
(A) (B) (C)

Ligand
β-arres n Clathrin-
coated pit
β-arres n β-arres n
Kis
s
Endosome
β-arres n runand β-arres n
Clathrin-
coated pit Clathrin-
coated pit
β-arres n
β-arres n ERK1/2 P
β-arres n
ERK1/2 P
ERK1/2 P
(D) (E) (F)
Transloca on
Endosome
ca on
β-arrea n
or golgi
hesis
Sy
nt
Translo
Organelle
Synt
Downstream he
Gs sis Downstream
Clathrin- effector effector
coated pit
cAMP
GPCR mRNA
Nuclear
β-arrea n
ERK1/2 P GPCR mRNA
Nuclear
Figure 3. Intracellular Activation. Four major patterns of internalization-dependent intracellular activation: (A) Class A receptors interact with b-arrestin and the
complexes are targeted to clathrin-coated pits for subsequent ERK1/2 activation near the membrane. The process is transient and is followed by rapid recycling of the
receptors back to the plasma membrane. (B) Class B receptors interact robustly with b-arrestin and the complexes are targeted to endosomes for subsequent G-
protein-coupled receptor (GPCR) degradation and ERK1/2 activation. (C) b-arrestin is internalized without forming a complex with the b1-adrenergic receptor (b1-AR).
b-arrestin briefly ‘kisses’ b1-AR, locates to clathrin-coated structures at the plasma membrane, and then activates ERK1/2. (D) Internalization-dependent activation is
mediated by G protein in the cytoplasm. After internalization, GPCRs target the endosome or Golgi. The receptors then recruit the Gs protein, resulting in cAMP
accumulation that induces ERK1/2 activation in the endosome or Golgi. Two patterns of internalization-independent intracellular activation are proposed: GPCRs can
reside on either the organelle (E) or nuclear membrane (F) and then initiate internal signaling in situ, which is independent of receptor internalization from the plasma
membrane.
reported to exhibit a novel pattern. In this pattern, b-arrestin is internalized without the formation
of a complex with b1-AR. b-arrestin briefly ‘kisses’ b1-AR, locates in clathrin-coated structures
at the plasma membrane, and then activates ERK1/2 (Figure 3C). In the above-three patterns,
the activated signals are all mediated by b-arrestin [45]. In the fourth pattern, the activated
signals are mediated by a G protein in the cytoplasm. After receptor internalization, some
GPCRs (such as b2-AR) target endosomes, and some GPCRs (such as thyroid-stimulating
hormone receptor; THSR) target the Golgi. The receptors, in the endosome or Golgi, then
recruit the Gs protein, resulting in cAMP accumulation that induces ERK1/2 activation
(Figure 3D) [53–55].
In contrast to internalization-dependent activation, internalization-independent intracellular

activation is also found in some GPCRs at their subcellular locations, where they may couple
to different signaling systems. For example, GPCRs have been found on mitochondria [56],
melanosomes [4], endoplasmic reticulum (ER) membranes [57], lysosomes [58,59], and
nuclear membranes [60]. Ocular albinism type 1 gene (OA1) represents the first example of
intracellular GPCR-mediated signal transduction systems in mammalian cells. OA1 was not
found at the plasma membrane, being instead targeted to specialized intracellular organelles,
the melanosomes, in which it regulates organelle biogenesis and maturation through activation
of G proteins on the cytoplasmic side of the melanosomal membrane (Figure 3E) [4]. b1-AR can
also steadily reside on the Golgi and initiate an internal Gs–cAMP signal from the Golgi
apparatus, which is independent of receptor internalization from the plasma membrane.
(Figure 3E) [61]. In addition, the metabotropic glutamate receptor 5 (mGluR5) is located at
the inner nuclear membrane via interactions with chromatin, where it is activated and mediates
Ca2+ changes in the nucleoplasm by coupling with Gq/11. (Figure 3F) [60]. Although it remains
unclear whether those residential GPCRs exhibit functions distinct from their cell-surface
counterparts, some clues indicate that they could induce location-specific effects [62]. For
example, by coupling with G proteins in Golgi, GPCRs are able to regulate Golgi transport
activities and induce changes in Golgi structural organization [61,63,64]. Nuclear localization of
GPCRs may favor in situ interactions with chromatin to regulate gene transcription, chromo-
some remodeling, and genomic integrity [60]. Collectively, the present findings reinforce the
notion that GPCRs are also residential and localized inside the cell, where they may couple to
different signaling systems, and exhibit distinct subcellular distribution patterns and functions.
Dimerization Activation
GPCRs were once thought to exist and function solely as monomers on the cell membrane, but
the existence of GPCR dimerization was first reported in 1975 as a result of the development of
new biological techniques. A direct kinetic binding method revealed that treatment with
unlabeled alprenolol increased the rate of labeled alprenolol dissociation with b-ARs, suggest-
ing there were negatively cooperative interactions among the b-AR-binding sites [65] During
the 1980s, it was shown by photoaffinity labeling, target size analysis, or cross-linking experi-
ments that some GPCRs could form dimers, such as homodimers of b2-ARs and heterodimers
between cholecystokinin-8 (CCK-8) and dopamine (DA) receptors [66–72]. However, there was
no direct evidence to confirm the existence of GPCR dimers at that time. When differential
epitope-tagging and immunoprecipitation became available for the study of GPCRs, solid
biochemical evidence for the presence of GPCR dimers and/or oligomers was provided [73–
75]. Later work using either fluorescence or bioluminescence resonance energy transfer (FRET
or BRET) methods further confirmed the existence of GPCR dimers [76–78]. Recently, powerful
advances in single-molecule microscopy have facilitated the measurements of the dynamic
process of GPCR dimerization in real time [79–81]. Compared with monomers, the dimers
show different agonist affinity, efficacy, and trafficking properties, as well as the mediation of
different signals and biological functions (Table 1) [5,82,83]. Given that GPCR dimers exhibit a
unique signaling activation mode that differs from that of GPCR monomers, here we call it
‘dimerization activation’.
GPCRs that show dimerization activation can be classified into three categories. First, some
GPCRs can mediate signaling only in the condition of dimerization (Figure 4A). For example,
g-aminobutyric acid type B receptor (GABABR) comprises GABABR1 and GABABR2. Ligands
can only bind to GABABR1 because the binding site is only located in GABABR1. However,
GABABR1 has no ability to transduce subsequent signaling, which is the role of GABABR2.
Thus, neither GABABR1 nor GABABR2 can activate GIRK-type potassium channels individu-
ally. Only when GABABR1 and GABABR2 comprise the complex GABABR, can channel activity
be robustly stimulated [75,84,85]. Second, compared with monomeric GPCRs, dimeric
GPCRs couple with different G proteins and downstream signaling pathways (Figure 4B).
For example, the dopamine D1 monomer transduces signals through Gas, while the dopamine
D2 monomer transduces signals through Gai. However, the dopamine D1/D2 heterodimer
transduces signals through Gaq [86]. Third, dimeric GPCRs induce a ‘G protein/b-arrestin
coupling switch’ (from G protein to b-arrestin; Figure 4C). For instance, MOR mediates
signaling through G proteins when it exists as monomer. However, when MOR heterodimerizes
with the d opioid receptor (DOR), the signal is transduced through b-arrestin [87].
GPCR dimerization has important biological and pharmacological significance. Pre-eclampsia

was the first disorder identified to be associated with altered GPCR heterodimerization. A
significant increase in AT1R/bradykinin B2 receptor (B2R) heterodimers was found in pre-
eclamptic hypertensive women. Increased AT1R/B2R heterodimers contribute to promoting
(A) (B) (C)

Ligand
Gαs/i Gαq G protein β-arres n
Downstream Downstream Downstream Downstream

Cell signaling effector effector effector effector
Figure 4. Dimerization Activation. Two major patterns of dimerization activation: (A) G-protein-coupled receptors (GPCRs) can mediate signaling only in the
condition of dimerization; and (B,C) dimers couple with different effector proteins from monomers.
the AngII-stimulated activation of Gaq/11 on membranes and upregulating AngII hypersensitivity
in patients with pre-eclampsia [88,89]. Another example is functional antagonism between
adenosine A1 receptors (A1R) and dopamine D1 receptors (D1R) in the brain. A1R agonists
promote A1R/D1R heteromerization, which induces the uncoupling of D1R with the Gs protein
and reduces D1R-mediated cAMP. Thus, compared with D1R activation alone, the coactivation
of A1R and D1R increases the level of A1R/D1R heteromerization and reduces the accumula-
tion of cAMP significantly. This antagonistic mechanism offers a basis for the development of
novel agents to treat D1R-involved diseases, such as Parkinson's disease [90,91].
The frequency and relevance of GPCR dimerization in vivo remains under debate. Some of the
published results are from receptor overexpression experiments, which could lead to experi-
mental artifacts [92]. In addition, there are concerns that the methods currently used for
assessment of GPCR dimerization have some limitations. For example, FRET and BRET report
close proximity rather than association per se between membrane molecules. Thus, the
random and nonspecific proximity between proteins on the membrane can generate false
positive signals [93,94]. However, improved RET methods, such as time-resolved FRET (TR-
FRET) [76] and the proximity ligation in situ assay (P-LISA) [95] partially overcome these
disadvantages. In addition, more advanced technologies are currently under development.
For example, total internal reflection fluorescence microscope (TIRFM), a powerful tool for
imaging membrane proteins, can be used to clearly observe receptor dimers on the plasma
membrane [80]. Super-resolution microscopy technologies have overcome the limit of resolu-
tion and can provide quantitative information of molecular numbers in oligomers [96]. These
technologies have their own unique advantages and, thus, it would be promising to combine
the advantages of these techniques to observe the formation and dissociation of GPCR dimers
accurately and dynamically in living cells.
Transactivation
The initial report of GPCR transactivation was published in 1996. Daub et al. demonstrated that
several GPCR agonists resulted in rapid tyrosine phosphorylation and activation of epidermal
growth factor receptor (EGFR), which could be inhibited by selective antagonists of GPCRs.
These results indicated that GPCRs could transactivate another kind of membrane receptor,
EGFR [6]. Later, the transactivation was found for many GPCRs, including neurotransmitter
receptors (such as catecholamine, GABABR, and serotonin receptor) [97–101], peptide
receptors (such as MOR, AngII, and chemokine receptors) [102–104], and gonadotropin-
releasing hormone receptor [105] (Table 1). Moreover, the membrane receptors that can be
transactivated by GPCRs include not only EGFR, but also other kinase-linked receptors,
including tyrosine kinase receptors (RTKs) [6,101,106], members of the transforming growth
factor (TGF)-b receptor superfamily [107–109], and pattern recognition receptors (PRR) (i.e.,
Toll-like receptors, TLRs) [110].
A typical example of transactivation is the transactivation of EGFR by GPCRs, which could be

mediated by two different pathways: a ligand-dependent pathway and a ligand-independent
pathway (Figure 5). The ligand-dependent pathway is characterized by the production of
endogenous ligand induced by GPCR-activated signal molecules (Figure 5A). Several endog-
enous ligands have been reported to mediate EGFR transactivation, including heparin-binding
EGF-like growth factor (HB-EGF), amphiregulin (ARG), epigen (EGN), and TGF-a. Among
these, HB-EGF, a member of the EGF family, is the most classical endogenous ligand
[111–113]. Soluble mature HB-EGF is proteolytically processed from membrane-anchored
precursor (pro-HB-EGF) and binds to EGFR with high affinity, leading to cell growth and
differentiation [44,114,115]. In the ligand-dependent pathway, GPCRs activate Src kinases.
(A) Ligand HB-EGF
GPCR MMP
EGFR
P P
src
ERK1/2 P
(B) (C)
P P P P
src
ERK1/2 P ERK1/2 P
Figure 5. Transactivation. Three major patterns are proposed for G-protein-coupled receptor (GPCR)-mediated
epidermal growth factor receptor (EGFR) transactivation. (A) In the ligand-dependent pathway, the Src activated by
GPCRs enhances matrix metalloproteinases (MMP) expression, which leads to cleavage and shedding of heparin-binding
EGF-like growth factor (HB-EGF) from the membrane. The fallen HB-EGF binds to EGFR and induces its activation. The
ligand-independent pathway includes (B) direct phosphorylation of EGFR in the cytoplasm by Src and (C) activation of
EGFR through formation of GPCR–EGFR signaling complexes. Direct phosphorylation of EGFR by Src means that
activated Src could directly phosphorylate the cytosolic tyrosine residues of EGFR. In addition, some GPCRs can form
GPCR–EGFR signaling complexes and trigger EGFR transactivation.
The activated Src enhances matrix metalloproteinases (MMP) expression, which leads to
cleavage and shedding of HB-EGF from the membrane. The fallen HB-EGF binds to the EGFR
and induces its activation. Given that the cellular signal needs to cross the membrane three
times during the process, the ligand-dependent pathway is also designated the triple mem-
brane passing signal (TMPS) pathway [52,97,111,116–118]. By contrast, the ligand-indepen-
dent pathway does not need a ligand. Two main ligand-independent patterns have been
identified so far: direct phosphorylation of EGFR in cytoplasm by Src (Figure 5B) and activation
of EGFR through formation of GPCR–EGFR signaling complexes (Figure 5C) [119]. Direct
phosphorylation of EGFR by Src was reported in bovine sperm acrosome reactions. Stimula-
tion of GPCR leads to Src activation, which could directly phosphorylate the cytosolic tyrosine
residues of EGFR [120,121]. In addition, some GPCRs, such as b2-AR, chemokine receptor
CXCR7, and somatostatin receptors, can form GPCR–EGFR signaling complexes and trigger
EGFR transactivation [98,122,123]. Compared with the ligand-dependent pathway, ligand-
independent EGFR transactivation may result in incomplete downstream signaling [111]. For
example, lysophosphatidic acid receptor (LPAR) transactivates the EGFR ligand dependently
by releasing HB-EGF, resulting in a full transactivation pattern with simultaneous activation of
the Grb/Sos-Ras-ERK, PLCg-PKC, and PI-3 kinase-Akt pathways. However, in COS-7 cells,
b2-AR, which transactivates EGFR ligand independently without releasing EGF-like peptides, is
capable of inducing the activation of the Grb/Sos-Ras-ERK pathway, but fails to mediate
activation of the PLCg-PKC and PI-3 kinase-Akt pathways [124].
A large body of evidence indicates that GPCRs transactivate RTKs. Reciprocally, an increasing
body of evidence has revealed that GPCRs can be transactivated by other cytokines and RTK
ligands (Table 1). For example, growth factor insulin-like growth factor receptor (IGF-1R) can
transactivate the chemokine receptor CXCR4 [125] and insulin can transactivate the b2-
adrenoceptor [126]. Platelet-derived growth factor (PDGF) can transactivate S1P1 [127].
Two distinct mechanisms of GPCR transactivation by RTKs have been identified so far. In
the first, RTK stimulation induces the synthesis and secretion of the transactivated GPCR
ligands, which bind and activate the GPCR [128–130]. The second mode of GPCR trans-
activation by RTK occurs in a ligand-independent manner, in which the GPCR is not activated
by an extracellular ligand but by the formation of GPCR–RTK complexes [125,131]. GPCR
transactivation by RTKs might contribute to the signaling and functions of these growth factors.
Transactivation has a key role in various physiological and pathological processes. For exam-
ple, it promotes the proliferation, migration, and invasion of cancer cells [132–134]. In the
cardiovascular system, it may exacerbate cardiac pathological remodeling and endothelial
dysfunction [113]. Thus, transactivation provides new insights and targets for the treatment of
diseases, including cancer and cardiovascular disorders [111,116].
Biphasic Activation
Biphasic activation of GPCRs was first found in the AT1R. AT1R-mediated ERK1/2 phos-
phorylation reached a first transient peak at 2 min, followed by a second sustained peak at 120–
150 min. Both phases of ERK1/2 activation contributed to c-jun transcriptional activity and cell
proliferation. However, only the second phase of ERK1/2 activation had an effect on DNA
synthesis and cellular growth, induced by AngII [7]. Later, biphasic activation was further found
in different GPCRs (Figure 1E and Table 1). Thus, as a new mode of activation, biphasic
activation has been gradually recognized and has triggered many studies into its characters
and potential mechanisms.
The different intensity and duration of GPCR downstream effectors often lead to different
biological effects, suggesting that the two phases have distinct functions. Taking ERK1/2 as an
example, it was reported that the higher intensity and longer duration of activation of ERK1/2
led to stronger PC12 cell differentiation [135]. After treatment of neonatal rat myocytes with
phenylephrine, the early and immediate peaks of ERK1/2 activity were insufficient to lead to a
hypertrophic phenotype, but were necessary to start this process. However, the delayed and
sustained peak of ERK1/2 activity may contribute to the hypertrophic response by regulating
gene expression [136]. Further study of the mechanisms of biphasic activation will provide more
powerful approaches to reveal the distinct functions of the different time phases involved.
We summarize three activation patterns based on the mechanisms of biphasic activation. First,
the early phase is mediated by the G protein pathway, while the late phase is mediated by the
b-arrestin pathway. For example, type I PTH/PTH-related peptide receptor (PTH1R) exhibits
biphasic activation of ERK1/2 after stimulation by PTH. The first phase occurs at 10 min, and
depends on G protein-meditated PKA and PKC activation. The second phase is from 30 to
60 min, and is mediated by b-arrestin [137]. In addition, a similar phenomenon occurs with the
k-opioid receptor (KOR). ERK1/2 is phosphorylated, with an early period of activity between 5
and 15 min mediated by the G protein pathway, and a late phase at 2 h mediated by the
b-arrestin2 pathway [138]. Second, the early phase is mediated by the b-arrestin pathway,
whereas the late phase is mediated by the G protein pathway. b2-AR activation can lead to the
biphasic activation of p38 MAPK. Acute early activation peaked at 10 min and returned to a
basic level at 60 min, while the weak delayed activation occurred at 90 min and was sustained
for 6 h. Furthermore, early activation and late activation of MAPK p38 were mediated by the
b-arrestin-1/Rac1/NADPH oxidase pathway and classical Gs/AC/cAMP/PKA pathway,
respectively [139]. Third, biphasic activation can be mediated by different G proteins. It was
found that specific b3-AR stimulation could also lead to biphasic activation of ERK1/2. The first
phase of ERK1/2 activation at 3 min is Gs dependent, and the second phase of ERK1/2
activation, which occurs at 8 h, is Gi dependent; Src is considered to have a potential role in
triggering the switch of coupling during this second phase [140]. When both Gs and Gi
pathways were pharmacologically inhibited, b3-AR-induced primary smooth muscle cell pro-
liferation was abolished.
The observation of biphasic activation is limited to a few GPCRs. It is unclear whether this
activation mode is universal among other GPCRs. In addition, the function and mechanisms of
biphasic activation are also unclear.
Interrelations of the Five Modes of GPCR Activation

In this review, we have summarized five novel modes of GPCR activation to highlight the
complexity of this process. However, the five modes are interconnected. In certain conditions,
intracellular activation may be a part of transactivation. For example, during EGFR trans-
activation by b2-AR, a b2-AR–EGFR complex is formed that is internalized in a clathrin-
dependent manner, followed by activation of ERK1/2 [98]. Sometimes, transactivation is a
special kind of b-arrestin-biased activation. The signaling pathways activated by some
b-arrestin-biased ligands, such as carvedilol, are reported to be mediated by EGFR trans-
activation, which can be blocked by inhibitors of Src and EGFR [141]. In addition, under certain
conditions, dimerization is essential for biased activation. b2-AR commonly signals as a
monomer in physiological states. However, when stimulated by the biased ligand carvedilol,
b2-AR dimerizes and then drives b-arrestin-biased signaling [142]. In addition, the biphasic
activation is often due to the switch between different biased signaling pathways. For example,
the early phasic activation of PTH1R is mediated by G protein-biased activation, whereas the
late phasic activation is mediated by b-arrestin-biased activation [137].
Moreover, a few GPCRs have been shown to involve in all five modes, such as b2-AR. The
activation modes of b2-AR include b-arrestin-biased activation [21], intracellular activation of
ERK1/2 [55], homologous dimerization activation [142], EGFR transactivation [143], and
biphasic activation of p38 MAPK [139]. However, how a GPCR selectively switches from
one activation mode to another remains an open question. Here, we propose five potential
solutions to this puzzle. First, the conformation of the GPCR determines the different activation
modes. For example, a specific conformation of b2-AR that can recruit GRK2/6 will initiate
b-arrestin-mediated biased activation rather than the classical G protein-mediated activation
mode [38–40]. Second, the GPCR ligand determines the different activation modes. For
instance, specific biased ligands of a receptor can lead to biased signaling [31]. Third, the
dosage of the GPCR ligand determines the different activation modes. At low concentrations of
an agonist, b2-AR signals activate the MAPK pathway through the Gs protein in mouse
embryonic fibroblast cells. At high agonist concentrations, b2-AR signals are transduced
through an additional pathway that is G protein independent but tyrosine kinase Src dependent
[144]. Thus, the receptor signals are transduced through two mechanisms, with the agonist
dosage acting as the switch. Fourth, the location of GPCRs determines the different activation
modes. As mentioned above, residential GPCRs on the plasma membrane or different
subcellular structures may couple to different signaling systems, and exhibit distinct subcellular Outstanding Questions
distribution patterns and functions. Fifth, the time duration determines the different activation What are the temporal and spatial rela-
modes. Biphasic activation is a typical example, in which the first phase is quick and transient tions among the different modes of
GPCR activation?
and the second phase usually occurs 1 h after the first phase. This long-term duration
determines the biphasic activation mode but not other single activation modes. To determine Is it possible to develop a real-time
the relationships between these different activation modes will open a new vista for exploring method to dynamically monitor the
the complex functions of GPCRs in physiological and pathological conditions and novel process of specific modes of GPCR
activation?
interventions for GPCR-related diseases.
Are there agonists or antagonists that
Concluding Remarks interfere specifically with each activa-
GPCRs constitute a superfamily of 7TM receptors, which represent the largest and most tion mode?
ubiquitous family of membrane receptors. GPCRs sense and transduce extracellular signals
into cytoplasm by activating diverse and complex signaling pathways. In this review, we have Can we find other proteins that medi-
ate new modes of GPCR activation
summarized five novel modes to show the complex and diverse features of GPCR activation
independent of G protein and
and signaling. This raises an important question about how cells integrate and interpret multiple b-arrestin?
complex signals. In addition to the relationship among the five modes of GPCR activation
described here, we further propose an integrated activation model that different modes may What is the specific mechanism of
coexist in the same cell simultaneously. mGluR5 is thought to be located on the cell surface, dimerization activation?
where it transmits extracellular signals to the cytoplasm. However, recent studies indicate that
What are the mechanisms and func-
only approximately 10–40% of mGluR5s are located on plasma membrane, where they
tions of the second phases in biphasic
respond to signals to undergo endocytosis and recycling [145]. Most (approximately 60– activation? What is the functional dif-
90%) mGluR5s are located at the inner nuclear membrane, where they are activated to regulate ference between the two phases?
nucleoplasmic Ca2+ in situ [60]. Thus, mGluR5 can be activated on plasma membranes and
nuclear membranes simultaneously, suggesting that the GPCR-mediated biological responses GPCRs can mediate signaling both in
the cytoplasm and on the membrane.
result from the integration of extracellular and intracellular activation events. For those con- What is the difference between these
comitant activation modes, which one is dominant depends on the GPCR population distribu- different signaling locations?
tion and location. mGluR5 is predominately located at the inner nuclear membrane, thus most
(50–90%) mGluR5 function occurs at the nuclear membrane [146]. However, in certain
conditions, a low abundance of receptors can also bear crucial functional significance in
specific locations. Although representing only approximately 15% of the receptors located
on the mitochondrial membrane, type 1 cannabinoid receptors (CB1) exert many functions. For
instance, CB1 directly controls cellular respiration and energy production via the intracellular
activation of mitochondrial membrane CB1 [56]. Although some simple integration models
have been identified, the integration of complex activation modes is still an important challenge
for future investigations.
This complex GPCR activation not only ensures normal physiological functions, but is also
involved in the generation and development of many major diseases, including cardiac dis-
eases, cancer, and neurodegeneration. For instance, EGFR transactivation by GPCRs is
involved in the development of cancer, while intracellular activation occurs in the development
of many cardiovascular disorders [147].
Thus, understanding of these novel modes of GPCR activation will provide not only new insights
into GPCR functions, but also more drug targets. The relatively new concept of ‘functional
selectivity’ suggests that certain ligands are able to activate selective signaling pathways and
downstream responses. Based on the related studies, functional targeting could enable drug
effects to be refined to improve their beneficial effects and avoid any adverse effects. However,
to achieve these advances, an in-depth understanding of receptor dynamics and their signaling
transduction mechanisms is required [148,149] (see Outstanding Questions). Therefore, iden-
tifying these novel modes of GPCR activation and their complex interconnections will provide a
new perspective for the development of functionally selective drugs. First, each activation mode
may be a potential specific target, for which we could develop specific drugs to treat the
relevant activation mode-associated diseases. Second, based on the deeper understanding of
the interrelations among the five activation modes described herein, we may screen and
develop multitarget drugs that target different activation modes that mediate the same or
different diseases.
In our opinion, further studies are required to identify more novel modes of GPCR activation.
Understanding of these novel activation modes will deepen our understanding of functionally
selective drugs to facilitate the development of more-precise and effective GPCR-targeted
drugs.
Acknowledgments
The authors acknowledge funding support from the National Basic Research Program of China (Grant no.
2014CBA02000), the National Natural Science Foundation of China (81471893, 81270157, 91539123, 81070078),
and Beijing Municipal Natural Science Foundation (7172235).
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New Insights into Modes of GPCR Activation

In classical G-protein-coupled receptor (GPCR) activation, GPCRs couple to a Highlights

(A) Classical acvaon (B) Biased acvaon (C) Intracellular acvaon

G-protein G-protein β-arresn Clathrin-

Cell signaling Cell signaling Cell signaling

(D) Dimerizaon acvaon (E) Transacvaon (F) Biphasic acvaon

368 Trends in Pharmacological Sciences, April 2018, Vol. 39, No. 4

(A) Ligand (B)

G protein β-arres n G protein β-arres n

Trends in Pharmacological Sciences, April 2018, Vol. 39, No. 4 369

b2-AR ICL1-9 Stimulates cardioprotective signaling and inotropic effects [150]

AT1R SII Mediates inotropic effects [151]

TRV120027 Reduces blood pressure and increases cardiac performance [24]

TRV120023 Supports cardiac function and reduces cellular injury [152]

PTHR (D-Trp12,Tyr34)-PTH Supports anabolic bone formation [153]

MOR TRV130 Reduces gastrointestinal and respiratory adverse effects [28]

PZM21 Efﬁcacious reduction of adverse effects [31]

GPR109A MK0354 Reduces cutaneous ﬂushing [32]

pyrazole Reduces cutaneous ﬂushing [154]

UTR Urantide Modulates cell survival [155]

Intracellular activation b1-AR Clathrin-coated pit ERK1/2 activation [45]

b2-AR Clathrin-coated pit ERK1/2 activation [3]

Endosome Activates cAMP signaling [54]

AT1aR Endosome Determines spatial distribution and activity of JNK3 [156]

PAR2 Clathrin-coated pit Intracellular targeting of activated ERK1/2 [157]

NTR1 Endosome Regulates proinﬂammatory signaling [158]

NK1R Endosome Facilitates proliferative and antiapoptotic effects [159]

F2rl1 Nuclear Governs angiogenesis [160]

b-AR Golgi Contributes signiﬁcantly to overall cellular cAMP response [61]

mGluR5 Nuclear Regulates nucleoplasmic Ca2+ in situ [60]

OA1 Melanosome Regulates organelle biogenesis and maturation [4]

CB1R Mitochondria Regulates neuronal energy metabolism [56]

Lysosomal Interacts with G proteins and mediates signaling [161]

GPR30 Endoplasmic reticulum Results in intracellular calcium mobilization [57]

b2-AR DOR Modulates receptor trafﬁcking and signal transduction [163]

KOR Modulates receptor trafﬁcking and signal transduction. [163]

a1A-AR a1BAR Correlates with characters of receptor internalization [164]

a2A-AR MOR Modulates receptor function [165]

a2C-AR AT1R Results in atypical Gs-PKA signaling [167]

Apelin receptor Increases NO production and inhibiting AT1R signaling [169]

MOR DOR Inﬂuences ethanol consumption [170]

DOR KOR Inﬂuences nociception [170]

CB1R Might be relevant to issue of multiple drug abuse [171]

P2YlR A1R Increases diversity of purine signaling [172]

NPY1R NPY5R Selectively modiﬁes pharmacology [173]

370 Trends in Pharmacological Sciences, April 2018, Vol. 39, No. 4

CB1R CB2R Shows bidirectional cross-antagonism phenomenon [174]

SSTR2 D2R Increases D2R signaling and SSTR2 internalization [176]

D1R D2R Increases intracellular calcium levels [86]

D2R A2aR Modulates neuronal excitability and transmitter release [178]

V1AR V2R Regulates endocytotic processing of receptors [179]

T1R3 T1R1, T1R2 Combine to function as a sweet receptor [180]

AChRs M1 mAChRs M1,2,3 GPCR long-term regulation [181]

CCK-A CCK-B Promotes cell growth [182]

GABABR1 GABABR2 Inwardly rectifying potassium channels [84]

Transactivation b1-AR EGFR Cardioprotective role in chronic catecholamine stimulation [97]

b2-AR EGFR Activates ERK1/2 [98]