Generation of comprehensive GPCR-transducer-deficient cell lines to dissect complexity of GPCR signaling.

G-protein-coupled receptors (GPCRs) compose the largest family of transmembrane receptors and are targets of approximately one-third of FDA approved drugs owing to their involvement in almost all physiological processes. GPCR signaling occurs through the activation of heterotrimeric G-protein complexes and ß-arrestins, both of which serve as transducers resulting in distinct cellular responses. Despite seeming simple at first glance, accumulating evidence indicates that activation of either transducer is not a straightforward process, as a stimulation of a single molecule has the potential to activate multiple signaling branches. The complexity of GPCR signaling arises from the aspects of G-protein-coupling selectivity, biased signaling, interpathway crosstalk, and variable molecular modifications generating these diverse signaling patterns. Numerous questions relative to these aspects of signaling remained unanswered until the recent development of CRISPR-Cas9 genome editing. Such genome editing technology presents opportunities to chronically eliminate the expression of G-protein subunits, ß-arrestins, GRKs, and many other signaling nodes in the GPCR pathways at one's convenience. Here, we review the practicality of using CRISPR-Cas9-derived knockout (KO) cells in the experimental contexts of unravelling the molecular details of GPCR signaling mechanisms. To mention a few, KO cells have revealed the contribution of ß-arrestins in ERK activation, Gα protein selectivity, GRK-based regulation of GPCRs, and many more, hence validating its broad applicability in GPCR studies. Significance Statement This review emphasizes the practical application of GPCR-effector KO cells in dissecting the intricate regulatory mechanisms of the GPCR signaling network. Currently available cell lines, along with accumulating KO cell lines in diverse cell types, offer valuable resources for systematically elucidating GPCR signaling regulation. Given the association of GPCR signaling with numerous diseases, uncovering the system-based signaling map is crucial for advancing the development of novel drugs targeting specific diseases.

Therapeutic potentials of nonpeptidic V2R agonists for partial cNDI-causing V2R mutants.

Loss-of-function mutations in the type 2 vasopressin receptor (V2R) are a major cause of congenital nephrogenic diabetes insipidus (cNDI). In the context of partial cNDI, the response to desmopressin (dDAVP) is partially, but not entirely, diminished. For those with the partial cNDI, restoration of V2R function would offer a prospective therapeutic approach. In this study, we revealed that OPC-51803 (OPC5) and its structurally related V2R agonists could functionally restore V2R mutants causing partial cNDI by inducing prolonged signal activation. The OPC5-related agonists exhibited functional selectivity by inducing signaling through the Gs-cAMP pathway while not recruiting ß-arrestin1/2. We found that six cNDI-related V2R partial mutants (V882.53M, Y1283.41S, L1614.47P, T2736.37M, S3298.47R and S3338.51del) displayed varying degrees of plasma membrane expression levels and exhibited moderately impaired signaling function. Several OPC5-related agonists induced higher cAMP responses than AVP at V2R mutants after prolonged agonist stimulation, suggesting their potential effectiveness in compensating impaired V2R-mediated function. Furthermore, docking analysis revealed that the differential interaction of agonists with L3127.40 caused altered coordination of TM7, potentially contributing to the functional selectivity of signaling. These findings suggest that nonpeptide V2R agonists could hold promise as potential drug candidates for addressing partial cNDI.

GPCR signaling bias: an emerging framework for opioid drug development.

Biased signaling, also known as functional selectivity, has emerged as an important concept in drug development targeting G-protein-coupled receptors (GPCRs). Drugs that provoke biased signaling are expected to offer an opportunity for enhanced therapeutic effectiveness with minimized side effects. Opioid analgesics, whilst exerting potent pain-relieving effects, have become a social problem owing to their serious side effects. For the development of safer pain medications, there has been extensive exploration of agonists with a distinct balance of G-protein and ß-arrestin (ßarr) signaling. Recently, several approaches based on protein-protein interactions have been developed to precisely evaluate individual signal pathways, paving the way for the comprehensive analysis of biased signals. In this review, we describe an overview of bias signaling in opioid receptors, especially the µ-opioid receptor (MOR), and how to evaluate signaling bias in the GPCR field. We also discuss future directions for rational drug development through the integration of diverse signal datasets.

Rules and mechanisms governing G protein coupling selectivity of GPCRs.

G protein-coupled receptors (GPCRs) convert extracellular stimuli into intracellular signaling by coupling to heterotrimeric G proteins of four classes: Gi/o, Gq, Gs, and G12/13. However, our understanding of the G protein selectivity of GPCRs is incomplete. Here, we quantitatively measure the enzymatic activity of GPCRs in living cells and reveal the G protein selectivity of 124 GPCRs with the exact rank order of their G protein preference. Using this information, we establish a classification of GPCRs by functional selectivity, discover the existence of a G12/13-coupled receptor, G15-coupled receptors, and a variety of subclasses for Gi/o-, Gq-, and Gs-coupled receptors, culminating in development of the predictive algorithm of G protein selectivity. We further identify the structural determinants of G protein selectivity, allowing us to synthesize non-existent GPCRs with de novo G protein selectivity and efficiently identify putative pathogenic variants.

Membrane phosphoinositides regulate GPCR-ß-arrestin complex assembly and dynamics.

Binding of arrestin to phosphorylated G protein-coupled receptors (GPCRs) is crucial for modulating signaling. Once internalized, some GPCRs remain complexed with ß-arrestins, while others interact only transiently; this difference affects GPCR signaling and recycling. Cell-based and in vitro biophysical assays reveal the role of membrane phosphoinositides (PIPs) in ß-arrestin recruitment and GPCR-ß-arrestin complex dynamics. We find that GPCRs broadly stratify into two groups, one that requires PIP binding for ß-arrestin recruitment and one that does not. Plasma membrane PIPs potentiate an active conformation of ß-arrestin and stabilize GPCR-ß-arrestin complexes by promoting a fully engaged state of the complex. As allosteric modulators of GPCR-ß-arrestin complex dynamics, membrane PIPs allow for additional conformational diversity beyond that imposed by GPCR phosphorylation alone. For GPCRs that require membrane PIP binding for ß-arrestin recruitment, this provides a mechanism for ß-arrestin release upon translocation of the GPCR to endosomes, allowing for its rapid recycling.

Structural basis of sphingosine-1-phosphate receptor 1 activation and biased agonism.

Sphingosine-1-phosphate receptor 1 (S1PR1) is a master regulator of lymphocyte egress from the lymph node and an established drug target for multiple sclerosis (MS). Mechanistically, therapeutic S1PR1 modulators activate the receptor yet induce sustained internalization through a potent association with ß-arrestin. However, a structural basis of biased agonism remains elusive. Here, we report the cryo-electron microscopy (cryo-EM) structures of Gi-bound S1PR1 in complex with S1P, fingolimod-phosphate (FTY720-P) and siponimod (BAF312). In combination with functional assays and molecular dynamics (MD) studies, we reveal that the ß-arrestin-biased ligands direct a distinct activation path in S1PR1 through the extensive interplay between the PIF and the NPxxY motifs. Specifically, the intermediate flipping of W2696.48 and the retained interaction between F2656.44 and N3077.49 are the key features of the ß-arrestin bias. We further identify ligand-receptor interactions accounting for the S1PR subtype specificity of BAF312. These structural insights provide a rational basis for designing novel signaling-biased S1PR modulators.

Synthesis of unnatural morphinan compounds to induce itch-like behaviors in mice: Towards the development of MRGPRX2 selective ligands.

Mas-related G protein-coupled receptor X2 (MRGPRX2) mediates the itch response in neurons and is involved in atopic dermatitis (AD)-associated inflammation and itch. Potent and MRGPRX2-selective ligands are essential to an understanding of the detailed function of the receptor and to develop new therapeutic agents for its related diseases. (+)-TAN-67 (1), the enantiomer of the δ-opioid receptor (DOR) selective ligand (-)-TAN-67 (1), has been reported to activate MRGPRX2, although (+)-1 also interacts with DOR, which prevents investigators from interrogating the function of MRGPRX2. Here, we have succeeded in developing a novel unnatural morphinan compound (+)-2a by a transformation based on the structure of (+)-1, which removes the DOR binding affinity. (+)-2a activated both human MRGPRX2 and the mouse orthologue Mrgprb2 in in vitro experiments and induced itch-like behaviors in mice to the same extent as (+)-1. The (+)-2a-induced itch response in mice was suppressed by administration of the tripeptide QWF, an MRGPRX2/Mrgprb2 antagonist, or the antipruritic drug nalfurafine. Together, (+)-2a serves as a useful tool to elucidate the itch-related function/action of MRGPRX2 and its mouse orthologue Mrgprb2.

Vibrational spectroscopy analysis of ligand efficacy in human M2 muscarinic acetylcholine receptor (M2R).

The intrinsic efficacy of ligand binding to G protein-coupled receptors (GPCRs) reflects the ability of the ligand to differentially activate its receptor to cause a physiological effect. Here we use attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy to examine the ligand-dependent conformational changes in the human M2 muscarinic acetylcholine receptor (M2R). We show that different ligands affect conformational alteration appearing at the C=O stretch of amide-I band in M2R. Notably, ATR-FTIR signals strongly correlated with G-protein activation levels in cells. Together, we propose that amide-I band serves as an infrared probe to distinguish the ligand efficacy in M2R and paves the path to rationally design ligands with varied efficacy towards the target GPCR.

An ATX-LPA6-Gα13-ROCK axis shapes and maintains caudal vein plexus in zebrafish.

Lysophosphatidic acid (LPA) is a potential regulator of vascular formation derived from blood. In this study, we utilized zebrafish as a model organism to monitor the blood vessel formation in detail. Zebrafish mutant of ATX, an LPA-producing enzyme, had a defect in the caudal vein plexus (CVP). Pharmacological inhibition of ATX resulted in a fusion of the delicate vessels in the CVP to form large sac-like vessels. Mutant embryos of LPA6 receptor and downstream Gα13 showed the same phenotype. Administration of OMPT, a stable LPA-analog, induced rapid CVP constriction, which was attenuated significantly in the LPA6 mutant. We also found that blood flow-induced CVP formation was dependent on ATX. The present study demonstrated that the ATX-LPA6 axis acts cooperatively with blood flow and contributes to the formation and maintenance of the CVP by generating contractive force in endothelial cells.

Intrinsic bias at non-canonical, ß-arrestin-coupled seven transmembrane receptors.

G-protein-coupled receptors (GPCRs), also known as seven transmembrane receptors (7TMRs), typically interact with two distinct signal-transducers, i.e., G proteins and ß-arrestins (ßarrs). Interestingly, there are some non-canonical 7TMRs that lack G protein coupling but interact with ßarrs, although an understanding of their transducer coupling preference, downstream signaling, and structural mechanism remains elusive. Here, we characterize two such non-canonical 7TMRs, namely, the decoy D6 receptor (D6R) and the complement C5a receptor subtype 2 (C5aR2), in parallel with their canonical GPCR counterparts. We discover that D6R and C5aR2 efficiently couple to ßarrs, exhibit distinct engagement of GPCR kinases (GRKs), and activate non-canonical downstream signaling pathways. We also observe that ßarrs adopt distinct conformations for D6R and C5aR2, compared to their canonical GPCR counterparts, in response to common natural agonists. Our study establishes D6R and C5aR2 as ßarr-coupled 7TMRs and provides key insights into their regulation and signaling with direct implication for biased agonism.

Identification and biochemical characterization of a second zebrafish autotaxin gene.

Autotaxin (ATX) is a secreted enzyme that produces a bioactive lysophospholipid, lysophosphatidic acid (LPA). ATX plays a role in vascular and neural development in embryos but its mechanisms remain unclear. At the beginning of this study, only one zebrafish atx gene (atxa) was known and had been investigated. In this study, we generated ATX knockout (KO) fish by TALEN targeting atxa. Unexpectedly, atxa KO fish showed neither vascular defects nor reduction of ATX activity, implying the existence of one or more other ATXs in the genome. By a BLAST search using ATXa protein fragments as a query, we found a genomic sequence that closely resembled atxa exons 13, 14 and 15. Consequently, we cloned a cDNA encoding a second zebrafish autotaxin (ATXb), and found that it was transcribed in various tissues. The atxb gene encoded a protein of 832 amino acids (compared to 850 amino acids in ATXa) with 60% amino acid identity to ATXa and clustered with ATXs from other species. A recombinant ATXb protein showed lysophospholipase D (lysoPLD) activities with substrate specificities similar to those of ATXa and mammalian ATXs. These results indicate that ATXb is a second zebrafish ATX, which possibly shares redundant roles with ATXa in embryonic development.

ATX-LPA1 axis contributes to proliferation of chondrocytes by regulating fibronectin assembly leading to proper cartilage formation.

The lipid mediator lysophosphatidic acid (LPA) signals via six distinct G protein-coupled receptors to mediate both unique and overlapping biological effects, including cell migration, proliferation and survival. LPA is produced extracellularly by autotaxin (ATX), a secreted lysophospholipase D, from lysophosphatidylcholine. ATX-LPA receptor signaling is essential for normal development and implicated in various (patho)physiological processes, but underlying mechanisms remain incompletely understood. Through gene targeting approaches in zebrafish and mice, we show here that loss of ATX-LPA1 signaling leads to disorganization of chondrocytes, causing severe defects in cartilage formation. Mechanistically, ATX-LPA1 signaling acts by promoting S-phase entry and cell proliferation of chondrocytes both in vitro and in vivo, at least in part through ß1-integrin translocation leading to fibronectin assembly and further extracellular matrix deposition; this in turn promotes chondrocyte-matrix adhesion and cell proliferation. Thus, the ATX-LPA1 axis is a key regulator of cartilage formation.

LPP3 localizes LPA6 signalling to non-contact sites in endothelial cells.

Lysophosphatidic acid (LPA) is emerging as an angiogenic factor, because knockdown of the enzyme that produces it (autotaxin, also known as ENPP2) and its receptors cause severe developmental vascular defects in both mice and fish. In addition, overexpression of autotaxin in mice causes similar vascular defects, indicating that the extracellular amount of LPA must be tightly regulated. Here, we focused on an LPA-degrading enzyme, lipid phosphate phosphatase 3 (LPP3, also known as PPAP2B), and showed that LPP3 was localized in specific cell-cell contact sites of endothelial cells and suppresses LPA signalling through the LPA6 receptor (also known as LPAR6). In HEK293 cells, overexpression of LPP3 dramatically suppressed activation of LPA6. In human umbilical vein endothelial cells (HUVECs), LPA induced actin stress fibre formation through LPA6, which was substantially upregulated by LPP3 knockdown. LPP3 was localized to cell-cell contact sites and was missing in non-contact sites to which LPA-induced actin stress fibre formation mediated by LPA6 was restricted. Interestingly, the expression of LPP3 in HUVECs was dramatically increased after forskolin treatment in a process involving Notch signalling. These results indicate that LPP3 regulates and localizes LPA signalling in endothelial cells, thereby stabilizing vessels through Notch signalling for proper vasculature.

Autotaxin overexpression causes embryonic lethality and vascular defects.

Autotaxin (ATX) is a secretory protein, which converts lysophospholipids to lysophosphatidic acid (LPA), and is essential for embryonic vascular formation. ATX is abundantly detected in various biological fluids and its level is elevated in some pathophysiological conditions. However, the roles of elevated ATX levels remain to be elucidated. In this study, we generated conditional transgenic (Tg) mice overexpressing ATX and examined the effects of excess LPA signalling. We found that ATX overexpression in the embryonic period caused severe vascular defects and was lethal around E9.5. ATX was conditionally overexpressed in the neonatal period using the Cre/loxP system, which resulted in a marked increase in the plasma LPA level. This resulted in retinal vascular defects including abnormal vascular plexus and increased vascular regression. Our findings indicate that the ATX level must be carefully regulated to ensure coordinated vascular formation.