Future Directions in GPCR Biased Signaling and Ligand Pharmacology.

Biased signaling in GPCRs: Structural insights and implications for drug development.

G protein‐coupled receptors (GPCRs) are the therapeutic target of nearly half of the current drugs in the market. It has been now well established that GPCRs can signal through multiple transducers, including G proteins and beta‐arrestins. These signalling pathways can be activated or blocked by “balanced” agonists or antagonists, but also, they can also be selectively activated in a “biased” response. Until now, biased signalling has been induced by biased ligands and biased receptors, any of which can result in preferential signalling through G proteins or beta‐arrestins. However, the discovery and development of GPCR biased agonists has been quite challenging, since in most GPCRs there is no structure activity relationship studies available, moreover, different active conformations for each pathway are likely very similar. Traditionally, biased agonists have been developed to target only the orthosteric site which is the binding site of the endogenous ligands. Development of biased agonists targeting the orthosteric site might be extremely difficult, especially in the absence of structure‐activity relationship information. Biased agonism holds great promise as a mechanism to significantly reduce the side effects that current drugs in used in the clinic, as well as develop drugs for use on their own. Also, ligands that produce biased signalling will serve as valuable tools for elucidation of the molecular mechanisms underlying GPCR‐signalling. Allosteric modulators are ligands which bind to a receptor at a site distinct from that of the endogenous agonist and they do not activate the receptor rather than stabilize or induce intermediate active or inactive structural conformations modulating signalling of the orthosteric ligands. We tested the hypothesis that biased signalling of the receptor could be achieved by targeting allosteric domains which show high diversity in structure and amino acid sequence among the GPCRs. The rationale is that the development of a novel series of allosteric modulators will regulate GPCR signalling in such way that the endogenous ligand would produce biased signalling only when the target receptor is bound to an allosteric modulator. In this study, we targeted the intracellular domains of the beta 2 adrenergic receptor (b2‐AR) to produce beta‐arrestin biased signalling when activated by traditional orthosteric ligands. A highly selective b2‐AR biased allosteric modulator would be highly desirable to treat a number of cardiovascular diseases. Thus, we used a combination of state of art drug discovery platforms, in silico calculations and peptidomimetics to develop a beta‐arrestin biased allosteric modulator AR1981. This allosteric modulator increased the beta‐arrestin recruitment efficacy of isoproterenol by two‐fold. Also, AR1981 was able to increase the potency of isoproterenol in beta‐arrestin recruitment by more than ten‐fold. Thus, AR1981 is a promising tool to access a novel pharmacological profile stimulating cardioprotective signalling through the b2‐AR and can serve as a model for the next generation of cardiovascular drug development. The outcome of the proposed work will facilitate the development of a new generation of allosteric modulators for GPCRs.

G protein-coupled receptors (GPCRs) represent promising therapeutic targets due to their involvement in numerous physiological processes mediated by downstream G protein- and β-arrestin-mediated signal transduction cascades. Although the precise control of GPCR signaling pathways is therapeutically valuable, the molecular details for governing biased GPCR signaling remain elusive. The Angiotensin II type 1 receptor (AT1R), a prototypical class A GPCR with profound implications for cardiovascular functions, has become a focal point for biased ligand-based clinical interventions. Herein, we used single-molecule live-cell imaging techniques to evaluate the changes in stoichiometry and dynamics of AT1R with distinct biased ligand stimulations in real time. It was revealed that AT1R existed predominantly in monomers and dimers and underwent oligomerization upon ligand stimulation. Notably, β-arrestin-biased ligands induced the formation of higher-order aggregates, resulting in a slower diffusion profile for AT1R compared to G protein-biased ligands. Furthermore, we demonstrated that the augmented aggregation of AT1R, triggered by activation from each biased ligand, was completely abrogated in β-arrestin knockout cells. These findings furnish novel insights into the intricate relationship between GPCR aggregation states and biased signaling, underscoring the pivotal role of molecular behaviors in guiding the development of selective therapeutic agents.

G protein-coupled receptors (GPCRs) constitute the largest membrane protein family in human body. Over 800 kinds of GPCRs have been characterized in human and are divided into Rhodopsin, Adhesion, Secretin, Glutamate and Frizzled/Taste 2 families. GPCRs can be stimulated by a variety of cell signal molecules, including hormones, neurotransmitters, ions, light etc. As the most widely distributed membrane proteins, GPCRs play important roles in almost all of the physiological activities and serve as drug targets for many diseases, such as cardiovascular diseases, central nervous disorders, inflammation, metabolic diseases and cancer. Despite their pivotal roles in the physiological and pharmaceutical fields, structure determination of GPCRs remains to be extremely challenging due to low protein yield when expressed in vitro , poor protein stability and multiple conformational states. To date, structures of 48 GPCRs have been determined, accounting for ~5% of characterized GPCRs. The most important developments of technique include fusion partner insertion and lipidic cubic phase (LCP) crystallization. Fusion partners, such as T4 lysozyme, thermo-stabilized apocytochrome b562RIL and flavodoxin etc, are inserted into the N terminus or intracellular loops to replace the unstable regions and provide hydrophilic contact for crystal packing. LCP is essential for crystallization as it mimics native environment of membrane proteins and is used to solve most of GPCR structures. Besides, stabilization mutations and disulfide-bond engineering are also widely used for GPCR structure determination. The X-ray free-electron laser (XFEL) and cryo-electron microscopy (EM) pave the way for obtaining high-resolution protein structure information from small size crystals or without the need for crystallization. According to the solved structures, GPCRs share a canonical seven transmembrane architecture despite their sequence diversity. However, the ligand binding pockets of different GPCRs vary in shape, size, location and electrostatics. The binding sites of orthostreric and allosteric ligands locate at extracellular, middle, intracellular and even outside of the transmembrane region. The diversity of the binding pockets provides structural basis for recognizing various ligands. Upon activation, GPCRs undergo conformational changes including a large outward movement of TM6 on the intracellular side, which exposes a pocket and engages downstream signal proteins. Three classes of downstream signal proteins have been reported, namely G proteins, G protein-coupled receptor kinases (GRKs) and arrestins. Approximate 34% of the US Food and Drug Administration approved drugs act at GPCRs. During 2011−2015, drugs targeting GPCRs accounted for about 27% of the global therapeutic drugs market share. Biologics, allosteric modulators and biased ligands are increasing in clinical trials targeting GPCRs, while major disease indications for GPCRs-targeted drugs show a shift from traditional popular areas such as allergy and hypertension toward diabetes, oncology and central nerves system disorders etc. Recent breakthroughs on GPCR structural determination provide insights into the mechanisms of ligand recognition and signal transduction, and facilitate structure-based drug design. However, more structural information is needed, including the interaction patterns of GPCR with other G proteins and basis of biased ligand signaling, to fully understand the superfamily membrane proteins. Here, we summarize the recent progresses on GPCR structural studies and drug discovery, and give suggestions for future research directions.

G protein-coupled receptors (GPCRs) are omnipresent in the regulation of physiological processes and therefore account for the most prominent drug target class. However, nearly all drugs targeting GPCRs have been developed by the concept of receptors as simple on–off switches. This is surprising, because specifically addressing a distinct intracellular signaling pathway holds the potential to develop safer and more efficient drugs. In recent years, more and more ligands have been reported that shift the naturally imprinted preference of a receptor’s signaling profile, so-called biased ligands. It has been demonstrated for several aminergic GPCRs that an extension of their molecular structure towards extracellular receptor regions results in biased signaling. The underlying mechanism is a specific interference with the allosteric coupling mechanism by which extra- and intracellular sides of the receptor are conformationally linked. We propose a potential blueprint for the design of biased ligands based on the concept of specific interference with the extracellular receptor region and a restriction of its conformational space by extended ligand structures. While this design concept will likely identify new biased ligands, it remains a challenge to specifically design ligands with a desired signaling profile.

G protein-coupled receptors (GPCRs) are seven-transmembrane proteins that mediate most cellular responses to hormones and neurotransmitters, representing the largest group of therapeutic targets. Recent studies show that some GPCRs signal through both G protein and arrestin pathways in a ligand-specific manner. Ligands that direct signaling through a specific pathway are known as biased ligands. The arginine-vasopressin type 2 receptor (V2R), a prototypical peptide-activated GPCR, is an ideal model system to investigate the structural basis of biased signaling. Although the native hormone arginine-vasopressin leads to activation of both the stimulatory G protein (Gs) for the adenylyl cyclase and arrestin pathways, synthetic ligands exhibit highly biased signaling through either Gs alone or arrestin alone. We used purified V2R stabilized in neutral amphipols and developed fluorescence-based assays to investigate the structural basis of biased signaling for the V2R. Our studies demonstrate that the Gs-biased agonist stabilizes a conformation that is distinct from that stabilized by the arrestin-biased agonists. This study provides unique insights into the structural mechanisms of GPCR activation by biased ligands that may be relevant to the design of pathway-biased drugs.

Cannabinoid receptor 1 ligands: Biased signaling mechanisms driving functionally selective drug discovery.

Signaling bias is a promising characteristic of G protein-coupled receptors (GPCRs) as it provides the opportunity to develop more efficacious and safer drugs. This is because biased ligands can avoid the activation of pathways linked to side effects whilst still producing the desired therapeutic effect. In this respect, a deeper understanding of receptor dynamics and implicated allosteric communication networks in signaling bias can accelerate the research on novel biased drug candidates. In this review, we aim to provide an overview of computational methods and techniques for studying allosteric communication and signaling bias in GPCRs. This includes (i) the detection of allosteric communication networks and (ii) the application of network theory for extracting relevant information pipelines and highly communicated sites in GPCRs. We focus on the most recent research and highlight structural insights obtained based on the framework of allosteric communication networks and network theory for GPCR signaling bias.

Biased signaling of G protein coupled receptors (GPCRs): Molecular determinants of GPCR/transducer selectivity and therapeutic potential.

G protein-coupled receptors (GPCRs) interact with multiple intracellular effector proteins such that different ligands may preferentially activate one signal pathway over others, a phenomenon known as signaling bias. Signaling bias can be quantified to optimize drug selection for preclinical research. Here, we describe moderate-throughput methods to quantify signaling bias of known and novel compounds. In the example provided, we describe a method to define cannabinoid-signaling bias in a cell culture model of Huntington's disease (HD). Decreasing type 1 cannabinoid receptor (CB1) levels is correlated with chorea and cognitive deficits in HD. There is evidence that elevating CB1 levels and/or signaling may be beneficial for HD patients while decreasing CB1 levels and/or signaling may be detrimental. Recent studies have found that Gαi/o-biased CB1 agonists activate extracellular signal-regulated kinase (ERK), increase CB1 protein levels, and improve viability of cells expressing mutant huntingtin. In contrast, CB1 agonists that are β-arrestin1-biased were found to reduce CB1 protein levels and cell viability. Measuring agonist bias of known and novel CB1 agonists will provide important data that predict CB1-specific agonists that might be beneficial in animal models of HD and, following animal testing, in HD patients. This method can also be applied to study signaling bias for other GPCRs.

G protein‐coupled receptors (GPCRs) are the largest family of cell‐surface receptors and are known to canonically signal through two main effector proteins: G proteins and β‐arrestins. Biased agonism at GPCRs refers to the functional selectivity of a ligand towards certain signaling pathways over others. In addition to biased agonists, there are various other sources of bias that influence GPCR signaling, such as receptor and location bias, which affect the downstream pathways of receptor activation, leading to disproportionate levels of signaling between the G protein and β‐arrestin pathway. One of the mechanisms underlying this signaling bias is the phosphorylation barcode hypothesis which states that ligand induced receptor phosphorylation promotes differential engagement with downstream transducers. Across the entire GPCR superfamily, GPCR kinases (GRKs) are known to be primarily responsible for receptor phosphorylation. Of the seven known isoforms in the GRK family, GRK2, GRK3, GRK5, and GRK6 are expressed ubiquitously, whereas GRK1 and GRK7 are mainly found in the eye while GRK4 is predominantly located in the testes. Although there is supportive evidence demonstrating that differential phosphorylation promotes biased signaling, there is relatively little known about the role of GRKs in directing GPCR functional selectivity. To study the role of GRKs in biased signaling, we studied the GPCR CXCR3, a chemokine receptor primarily involved in leukocyte migration, which has three endogenous biased ligands, CXCL9, CXCL10, and CXCL11, and two synthetic biased ligands, VUF10661 and VUF11418. We found that, following ligand stimulation, GRK2 and GRK3 are recruited to CXCR3, with VUF10661 and CXCL11 being the most efficacious ligands. However, we also observed that GRK3 and GRK5 are able to traffic to endosomes following ligand stimulation with VUF10661, even though all ligands are able to promote receptor internalization. Given biased engagement of the GRKs observed at both the receptor and in endosomes, we next studied the functional consequences of ligand and location bias by creating GRK 2, 3, 5 and 6 constructs that are specifically targeted to the plasma membrane or the endosome. Using GRK2/3/5/6 KO cells, we determined that localization of the GRKs impacts receptor internalization, ERK activity, and transcription in a ligand specific manner. Together, we observed that the endogenous and synthetic ligands of CXCR3 demonstrate biased engagement of the GRKs at different subcellular locations and highlight the functional significance of these findings on downstream receptor signaling. These data provide evidence that the functional selectivity of biased signaling extends beyond β‐arrestins and G‐proteins to other effector proteins that interact with GPCRs.

G protein-coupled receptors (GPCRs) mediate the effects of various endogenous and extracellular stimuli through multiple transducers, including heterotrimeric G proteins, GPCR kinases (GRKs), and arrestins. Biased signaling, which preferentially activates certain G protein or GRK/arrestin signaling pathways, provides great opportunities for developing drugs with enhanced therapeutic efficacy and minimized side effects. In this Review, we review studies addressing the structural dynamics of GPCRs bound to balanced and biased ligands and current consensus on how ligand-receptor interactions determine signaling outcomes. We also examine the conformational changes in GPCRs when in complex with G proteins, arrestins, and GRKs, highlighting a more profound impact of signal transducers on receptor rearrangements compared with biased ligands. This evidence supports the idea that biased signaling can be achieved through the promotion of multiple conformational states by biased agonists and the stabilization of specific active conformations by individual signal transducers.

Clinically, no human diseases are currently diagnosed as being directly driven by β- arrestins. Nonetheless, there is growing interest in developing β-arrestin–biased drugs, due to the key regulatory role of β-arrestins in modulating the cellular signaling of most G protein–coupled receptors (GPCRs). In cell-based studies of rhodopsin-family GPCRs, mutations in a conserved proline–hydrophobic (ProH) motif within the second intracellular loop (ICL2) have been shown to alter β-arrestin signaling bias. The clinical relevance of such mutations in humans is unknown. However, if naturally occurring single nucleotide polymorphisms (SNPs) affecting this ProH motif exist across the GPCR family, then cross-referencing these genetic variants with large-scale population sequencing and epidemiologic data could reveal potential roles for β-arrestin signaling in human health. In this report, we identify SNPs in human GPCRs that correspond to ProH substitutions, and we show that, in the neurotensin receptor NTSR1, these variants can indeed shift signaling bias. We also estimate a lower-bound population frequency for such SNPs, suggesting that although rare for any given receptor, their cumulative prevalence across the GPCR superfamily may be large enough to impact phenotypic variation. Together with emerging data from biased ligands, our findings support the idea that genetic variations in β-arrestin signaling could represent a meaningful source of therapeutic relevance.Significance statementMutations altering G protein-coupled receptor (GPCR) signaling can affect health. We have identified a specific amino acid determinant in intracellular loop (ICL2) of human rhodopsin family GPCRs that influences β-arrestin signaling. Although the medical consequences of this determinant remain unclear, we show that single nucleotide polymorphisms (SNPs) affecting the determinant routinely occur. By studying neurotensin receptor NTSR1, we confirm that the SNP alters β-arrestin signaling bias. While individually rare per receptor, these SNPs may collectively contribute to β-arrestin-related phenotypic changes in the human population. Our findings, combined with research on biased drugs, support the idea that β-arrestin signaling might serve as a useful therapeutic target, opening new possibilities for precision medicine.

Some G protein-coupled receptors (GPCRs) demonstrate biased signaling such that ligands of the same receptor exclusively or preferentially activate certain downstream signaling pathways over others. This phenomenon may result from ligand-specific receptor phosphorylation by GPCR kinases (GRKs). GPCR signaling can also exhibit location bias because GPCRs traffic to and signal from subcellular compartments in addition to the plasma membrane. Here, we investigated whether GRKs contributed to location bias in GPCR signaling. GRKs translocated to endosomes after stimulation of the chemokine receptor CXCR3 or other GPCRs in cultured cells. GRK2, GRK3, GRK5, and GRK6 showed distinct patterns of recruitment to the plasma membrane and to endosomes depending on the identity of the biased ligand used to activate CXCR3. Analysis of engineered forms of GRKs that localized to either the plasma membrane or endosomes demonstrated that biased CXCR3 ligands elicited different signaling profiles that depended on the subcellular location of the GRK. Each GRK exerted a distinct effect on the regulation of CXCR3 engagement of β-arrestin, internalization, and activation of the downstream effector kinase ERK. Our work highlights a role for GRKs in location-biased GPCR signaling and demonstrates the complex interactions between ligands, GRKs, and cellular location that contribute to biased signaling.

β-Adrenoceptor agonists relieve airflow obstruction by activating β2 -adrenoceptors, which are G protein-coupled receptors (GPCRs) expressed on human airway smooth muscle (HASM) cells. The currently available β-adrenoceptor agonists are balanced agonists, however, and signal through both the stimulatory G protein (Gs )- and β-arrestin-mediated pathways. While Gs signalling is beneficial and promotes HASM relaxation, β-arrestin activation is associated with reduced Gs efficacy. In this context, biased ligands that selectively promote β2 -adrenoceptor coupling to Gs signalling represent a promising strategy to treat asthma. Here, we examined several β-adrenoceptor agonists to identify Gs -biased ligands devoid of β-arrestin-mediated effects. Gs -biased ligands for the β2 -adrenoceptor were identified by high-throughput screening and then evaluated for Gs interaction, Gi interaction, cAMP production, β-arrestin interaction, GPCR kinase (GRK) phosphorylation of the receptor, receptor trafficking, ERK activation, and functional desensitization of the β2 -adrenoceptor. We identified ractopamine, dobutamine, and higenamine as Gs -biased agonists that activate the Gs /cAMP pathway upon β2 -adrenoceptor stimulation while showing minimal Gi or β-arrestin interaction. Furthermore, these compounds did not induce any receptor trafficking and had reduced GRK5-mediated phosphorylation of the β2 -adrenoceptor. Finally, we observed minimal physiological desensitization of the β2 -adrenoceptor in primary HASM cells upon treatment with biased agonists. Our work demonstrates that Gs -biased signalling through the β2 -adrenoceptor may prove to be an effective strategy to promote HASM relaxation in the treatment of asthma. Such biased compounds may also be useful in identifying the molecular mechanisms that determine biased signalling and in design of safer drugs.