G protein-coupled receptors (GPCRs) are a family of cell-surface proteins that play critical roles in regulating a variety of pathophysiological processes and thus are targeted by almost a third of currently available therapeutics. It was originally thought that GPCRs convert extracellular stimuli into intracellular signals through activating G proteins, whereas β-arrestins have important roles in internalization and desensitization of the receptor. Over the past decade, several novel functional aspects of β-arrestins in regulating GPCR signaling have been discovered. These previously unanticipated roles of β-arrestins to act as signal transducers and mediators of G protein-independent signaling have led to the concept of biased agonism. Biased GPCR ligands are able to engage with their target receptors in a manner that preferentially activates only G protein- or β-arrestin-mediated downstream signaling. This offers the potential for next generation drugs with high selectivity to therapeutically relevant GPCR signaling pathways. In this review, we provide a summary of the recent studies highlighting G protein- or β-arrestin-biased GPCR signaling and the effects of biased ligands on disease pathogenesis and regulation.
G protein-coupled receptors (GPCRs) represent the largest family of cell surface molecules involved in signal transduction. More than 1000 receptors for sensory (e.g., odor and light) and chemical stimuli (e.g., catecholamines, amino acids, peptides, and ions) have been identified based on their common structural and biochemical properties (Muller, 2000). Because GPCRs represent 1–5% of the total cell surface proteins in mammals, it is not surprising that nearly 30% of United States Food and Drug Administration (FDA)-approved drugs target GPCRs (Overington
Historically, GPCRs were assumed to exist in equilibrium between active and inactive states, and thus activation of GPCRs would equally affect all downstream signaling pathways. However, accumulating evidences indicate that GPCRs exist in multiple conformational states where each conformation confers different downstream effects. In this context, some ligands are able to induce a differential receptor conformation which activates a different subset of signaling events, causing bias receptor signaling (Liu
GPCRs are often referred to as seven-transmembrane receptors (7TMRs) because their structures are characterized by the presence of seven α-helices crossing the plasma membrane. GPCRs are consisted of intracellular and extracellular loops. The NH2 terminus is exposed to the extracellular environment and the COOH terminus is located in the intracellular part. The intracellular domains and loops mediate the interaction between the receptor and intracellular signaling partners such as G proteins (Gether, 2000; Hermans, 2003). The binding of exogenous ligands alters the conformation of critical domains of the seven-transmembrane helix pocket, which in turn causes the conformation changes of intracellular domains of the receptor. These changes promote the association of the receptor with a variety of heterotrimeric G proteins. They are composed of an α-subunit interacting with a βγ complex. Activation of the receptor promotes the exchange of a molecule of GDP by a molecule of GTP within the active site of the α-subunit. The binding of GTP to α-subunit causes the dissociation of the heterotrimeric complex, and both the GTP-bound α-subunit and the released βγ complex are then able to interact with intracellular or membrane effectors (e.g., enzymes or ion channels). The intrinsic GTPase activity of the α-subunit hydrolyses GTP into GDP, restoring its initial inactive conformation and its affinity for the βγ complex [for detailed reviews, see (Wess, 1997; Bockaert and Pin, 1999; Gether, 2000; Hermans, 2003)]. Up to now, at least 23 α-subunits derived from 17 different genes have been identified and are classified into four families (Gαi/o, Gαs, Gαq/11, and Gα12). At least 6 different β-subunits and 12 γ-subunits have been also discovered (Gautam
In addition to signaling through G proteins, GPCRs can also activate G protein-independent signaling pathways mainly through multi-functional adaptor proteins called arrestins. The arrestins are a small family of proteins originally discovered in the visual system. Arrestins, which include arrestin-1 and -4 (expressed in retinal rods and cones) and ubiquitously expressed arrestin-2 (β-arrestin1) and arrestin-3 (β-arrestin2), were initially characterized for their roles in GPCR desensitization (uncoupling of the G protein from the cognate receptor) (Shukla
As our understanding on
Although the detailed molecular mechanism of biased signaling is not yet well understood, it has been reported that biased GPCR ligands induce a unique receptor conformation, activating a particular signaling pathway. It is understood that the GPCR conformation stabilized by a G protein-biased ligand is distinct from the conformation stabilized by a β-arrestin-biased ligand. For example, a fluorescence-based study on activation of the arginine-vasopressin type 2 receptor by biased and unbiased ligands provided an interesting experimental notion, suggesting that the transmembrane helix 6 (TM6) and third intracellular loop at the receptor are associated with selective G protein signaling, whereas the TM7 and helix 8 (H8) regions at the receptor are required for selective β-arrestin recruitment (Rahmeh
Another recent study used site-specific fluorine-19 nuclear magnetic resonance (19F-NMR) labels in the β2-AR to unveil conformational changes of the receptor upon activation by biased and unbiased ligands. It was shown that unbiased ligand’s binding to the receptor primarily shifts the equilibrium toward the G protein-specific active state of helix 6, while β-arrestin-biased ligands predominantly regulate the conformational states of helix 7 (Liu
It is well appreciated that the G protein-independent effector on GPCRs, β-arrestin, is recruited upon phosphorylation of GPCRs by GRKs specifically on their C terminal and intracellular loops. An interesting “barcode” hypothesis suggests that different GRKs phosphorylate distinct sites on the C terminus and internal loops of the receptor, thereby establishing a “barcode” that would instruct or determine the conformation for different β-arrestin functions. This would in turn determine the differential roles of GRKs and β-arrestins (Butcher
Another interesting phenomenon in biased signaling observed for multiple GPCRs
Altogether, recent advances on GPCR biased signaling field suggest that biased GPCR ligands may have an important therapeutic potential in various diseases including cardiovascular diseases, neurological diseases, and cancers. As summarized in the following chapter and Table 1, we seek to provide recent scientific progress on identifying novel biased ligands on selected highly-profiled GPCRs.
β-adrenergic receptors (β-ARs), prototypical members of GPCR superfamily, are known for their regulation of contractile function in the heart. The stimulation of cardiac β1-and β2-AR by catecholamines such as adrenaline and noradrenaline activates the canonical Gs-AC-cAMP-PKA signaling cascade, which increases calcium mobilization across different cellular compartments and sensitizes contractile proteins to cytosolic calcium. The overall physiological effect of cardiac β-AR stimulation is an increase in heart contractility (inotropic effect) and heart rate (chronotropic effect) (Rodefeld
More importantly, carvedilol has been identified as a biased ligand on β1-AR and β2-AR, which selectively stimulates GRK5/6-and β-arrestin-dependent cardioprotective signaling without activating G proteins (Wisler
In both diabetes and heart failure, circulating insulin levels are chronically elevated, leading to persistent stimulation of IRs. Despite the insulin resistance of adipocytes and skeletal muscle cells, the heart retains its insulin sensitivity to activate IR signaling cascades in type 2 diabetes (Wright
Angiotensin II (AngII) type I receptor (AT1R), a primary regulator of blood pressure, is a prototype GPCR in the study of biased agonism. Upon binding of its natural ligand AngII to the receptor, Gαq proteins are activated, resulting in intracellular inositol triphosphate (IP3) production, calcium mobilization, protein kinase C (PKC) activation, which altogether mediate the physiological effects of AT1R such as vasoconstriction and fluid retention. Moreover, the conformational rearrangement of 7 transmembrane α helices of the receptor also leads to the recruitment of β-arrestins, which mediates G protein-independent signaling, leading to overall positive inotropic and cardioprotective effects (Ikeda
The apelin receptor (also known as APJ, APLNR, AGTRL1) is a class A GPCR discovered in 1993 based on its sequence similarity with the AT1R (O’Dowd
Apelin receptor system represents an attractive target in pathologies such as pulmonary hypertension and heart failure (Chong
The human histamine H4 receptor (H4R) belongs to the GPCR family and is considered as an important receptor in immune and inflammatory processes (Leurs
Currently, one of the most clinically relevant therapies for histamine receptors is achieved through the regulation of H2R, which is pathologically implicated in gastric acid-related diseases but widely expressed in most tissues. The H2R stimulation increases adenylate cyclase activity and induces cAMP accumulation. The most commonly used H2R blocker, famotidine was shown to act as an “inverse agonist” by diminishing G protein-mediated increase of cAMP. Interestingly, famotidine also mimicked the effect of histamine, and induced receptor desensitization and internalization along with increased ERK phosphorylation in gastric epithelial cells (Alonso
Dopamine receptors are another well-studied family of GPCRs largely because dopamine neurotransmission is important in multiple neuropsychiatric disorders. Among the dopamine receptors, dopamine receptor D2 (D2R) is one of the most validated drug targets in neurology and psychiatry. However, most drugs targeting the D2R are problematic, either being less efficacious than desired or possessing adverse side effects due to the activation or blockade of a subset of downstream signaling pathways.
D2R couples Gαi/o to negatively regulate cAMP-PKA pathways and modulate intracellular Ca2+ levels by acting on ion channels or by triggering the release of Ca2+ from intracellular stores. In addition, more recent discoveries showed that dopamine receptors exert their
Interestingly, biased ligands with the opposing pharmacology for the D2R, that is, the stimulation of G protein signaling pathways without activation of β-arrestin recruitment have been recently identified. The first example is MLS1547, which was shown to robustly activate G proteins while antagonizing of β-arrestin recruitment to the D2R (Free
Opioid receptors are GPCRs, which are widely studied due to their crucial roles in pain management, drug abuse/addiction, and mood disorders. There are three major subtypes of opioid receptors: δ-receptor (DOR), κ-receptor (KOR), and μ-receptor (MOR). Majority of opioids exert their analgesic activities primarily via activating MOR. Upon activation, MOR predominately couples to Gαi/o, which orchestrates downstream signaling cascades including those contributing to antinociception. On the other hand, activation of β-arrestins, especially β-arrestin2 induces receptor internalization and desensitization, diminishing G protein-mediated signaling. Recent studies have shown that some MOR agonists such as fentanyl and [D-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO) have the high efficacy to recruit β-arrestin, whereas other opioids such as morphine are biased toward G protein signaling (McPherson
Interestingly, biased ligands that are selective agonists at one desired pathway could also act as biased competitive antagonists for the undesired pathway. In general, MOR ligands with low efficacy for G protein activation also have low efficacy for β-arrestin2 recruitment, and in fact partial agonists such as buprenorphine do not significantly recruit β-arrestin2 in cell models (McPherson
In contrast to the successful implementation of GPCR biased signaling concept for clinical benefit in the cardiovascular, neurological, behavior fields, there have been no reports demonstrating the utility of GPCR biased signaling for the treatment of cancer. However, a few recent studies reported the scientific progress in the potential use of biased signaling on endothelin receptors in cancer treatment.
Endothelin-1 (ET-1) is a peptide belonging to a family of the most potent vasoconstrictors. In addition to this function in the circulation, it has been implicated in various physiological and pathological conditions such as development, cell proliferation, differentiation, cardiac function and cancer (Schorlemmer
Other GPCRs, which are involved in the progression of cancer and have been suggested as potential targets for yet-to-be-identified biased ligands, are CXC chemokine receptor 4 (CXCR4) and protease activated receptor 2 (PAR2). The enhanced expression of CXCR4 and aberrant downstream signaling are implicated in several cancers, where it is involved in tumor growth, vascularization, and metastasis (Guleng
Over the past decade, there has been a surge in publications to describe the identification of biased ligands at a wide variety of GPCRs. Accordingly, quantifying ligand bias has been an active area of research. One way to quantify ligand bias is to plot β-arrestin activity against G protein activity. For biased ligands, there would be different levels of β-arrestin-and G protein-mediated efficacies. Such data can also be represented as a matrix that incorporates data from multiple assays, or ligand bias factors that compare β-arrestin activity against G protein activity in different assays (Rajagopal
In the past years, most groups have relied on comparing the maximal effects (Emax) and potencies (EC50) of ligands for different signaling pathways. However, they are prone to errors in the interpretation in the setting of receptor reserve. For example, these parameters failed to account for the differences in the receptor reserve and amplification of different assays (Rajagopal
The key requirements for measuring bias signaling are a common reference compound to overcome observational and systemic bias as well as a scale, which accounts for both potency and maximal response of ligands. They allow the relative activity of ligands to be compared across assays. The current ‘gold standard’ method is the operational model of agonism that allows for the systematically independent quantification of agonist activity via the relative transduction ratio coefficient Δlog (τ/KA). The term τ incorporates agonist efficacy, receptor density and coupling within the system. The dissociation constant (KA) is the reciprocal of the conditional affinity of the agonist in the functional system [for detailed reviews, see (Kenakin and Christopoulos, 2013)]. The alternative to this method is to use the bias factor βlig, which is calculated by the ratios of the efficacy of agonists for a given signaling pathway in a cell. This method was first used to quantify ligand bias and to identify weak biased compounds in β2-AR and AT1R. This method differs from the previous one by assuming that a single estimate of KA for the receptor (obtained from biochemical binding studies) should be used to fit the data with the operational model [for detailed reviews, see (Rajagopal
One of increasingly recognized techniques for identification of GPCR signaling bias is BRET, which is a sensitive and non-destructive method commonly used in live cells to investigate protein-protein interactions or changes. BRET is a naturally occurring phenomenon resulting from the nonradioactive transfer of energy between luminescent donor and fluorescent acceptor proteins. In the sea pansy
In addition to BRET, fluorescence resonance energy transfer (FRET) proximity and conformation assays as well as signaling assays such as MAPK activation have been widely used for the discovery of biased ligands (Rajagopal
The drug discovery strategy targeting only the primary ligand binding sites of GPCRs is becoming more and more difficult. As our understanding on
We thank the editors for inviting us to write this review. Due to space restrictions, the authors cannot cite many important literatures on this field. The authors apologize to all colleagues whose work contributed significantly. This work was supported by the American Heart Association Postdoctoral Fellowship 16POST26990020 to Zuzana Bologna, American Heart Association Predoctoral Fellowship 16PRE30210016 to Jianpeng Teoh, and American Physiological Society Shih-Chun Wang Young Investigator Award, American Heart Association Grant-in-Aid 12GRNT12100048 and Scientist Development Grant 14SDG18970040, and National Institutes of Health R01 HL124251 to Il-man Kim.