APLNR Regulates IFN-γ signaling via ß-arrestin 1 mediated JAK-STAT1 pathway in melanoma cells.

The apelin receptor (APLNR) regulates many biological processes including metabolism, angiogenesis, circulating blood volume and cardiovascular function. Additionally, APLNR is overexpressed in various types of cancer and influences cancer progression. APLNR is reported to regulate tumor recognition during immune surveillance by modulating the IFN-γ response. However, the mechanism of APLNR cross-talk with intratumoral IFN-γ signaling remains unknown. Here, we show that activation of APLNR up-regulates IFN-γ signaling in melanoma cells through APLNR mediated ß-arrestin 1 but not ß-arrestin 2 recruitment. Our data suggests that ß-arrestin 1 directly interacts with STAT1 to inhibit STAT1 phosphorylation to attenuate IFN-γ signaling. The APLNR mutant receptor, I109A, which is deficient in ß-arrestins recruitment, is unable to enhance intratumoral IFN-γ signaling. While APLNR N112G, a constitutively active mutant receptor, increases intratumoral sensitivity to IFN-γ signaling by enhancing STAT1 phosphorylation upon IFN-γ exposure. We also demonstrate in a co-culture system that APLNR regulates tumor survival rate. Taken together, our findings reveal that APLNR modulates IFN-γ signaling in melanoma cells and suggest that APLNR may be a potential target to enhance the efficacy of immunotherapy.

A general Fc engineering platform for the next generation of antibody therapeutics.

Rationale: Fc engineering has become the focus of antibody drug development. The current mutagenesis and in silico protein design methods are confined by the limited throughput and high cost, while the high-throughput phage display and yeast display technologies are not suitable for screening glycosylated Fc variants. Here we developed a mammalian cell display-based Fc engineering platform. Methods: By using mammalian cell display and next generation sequencing, we screened millions of Fc variants for optimized affinity and specificity for FcγRIIIa or FcγRIIb. The identified Fc variants with improved binding to FcγRIIIa were substituted into trastuzumab and rituximab and the effector function of antibodies were examined in the PBMC-based assay. On the other hand, the identified Fc variants with selectively enhanced FcγRIIb binding were applied to CD40 agonist antibody and the activities of the antibodies were measured on different cell assays. The immunostimulatory activity of CD40 antibodies was also evaluated by OVA-specific CD8+ T cell response model in FcγR/CD40-humanized mice. Results: Using this approach, we screened millions of Fc variant and successfully identified several novel Fc variants with enhanced FcγRIIIa or FcγRIIb binding. These identified Fc variants displayed a dramatic increase in antibody-dependent cellular cytotoxicity in PBMC-based assay. Novel variants with selectively enhanced FcγRIIb binding were also identified. CD40 agonist antibodies substituted with these Fc variants displayed activity more potent than the parental antibody in the in vitro and in vivo models.Conclusions: This approach increased the throughput of Fc variant screening from thousands to millions magnitude, enabled screening variants containing multiple mutations and could be integrated with glycoengineering technology, represents an ideal platform for Fc engineering. The initial efforts demonstrated the capability of the platform and the novel Fc variants could be substituted into nearly any antibody for the next generation of antibody therapeutics.

Loss of APJ mediated ß-arrestin signalling improves high-fat diet induced metabolic dysfunction but does not alter cardiac function in mice.

Apelin receptor (APJ) is a G protein-coupled receptor that contributes to many physiological processes and is emerging as a therapeutic target to treat a variety of diseases. For most disease indications the role of G protein vs ß-arrestin signalling in mitigating disease pathophysiology remains poorly understood. This hinders the development of G protein biased APJ agonists, which have been proposed to have several advantages over balanced APJ signalling agonists. To elucidate the contribution of APJ ß-arrestin signalling, we generated a transgenic mouse harbouring a point mutation (APJ I107A) that maintains full G protein activity but fails to recruit ß-arrestin following receptor activation. APJ I107A mutant mice did not alter cardiac function at rest, following exercise challenge or in response to pressure overload induced cardiac hypertrophy. Additionally, APJ I107A mice have comparable body weights, plasma glucose and lipid levels relative to WT mice when fed a chow diet. However, APJ I107A mice showed significantly lower body weight, blood insulin levels, improved glucose tolerance and greater insulin sensitivity when fed a high-fat diet. Furthermore, loss of APJ ß-arrestin signalling also affected fat composition and the expression of lipid metabolism related genes in adipose tissue from high-fat fed mice. Taken together, our results suggest that G protein biased APJ activation may be more effective for certain disease indications given that loss of APJ mediated ß-arrestin signalling appears to mitigate several aspects of diet induced metabolic dysfunction.

Cardiovascular response to small-molecule APJ activation.

Heart failure (HF) remains a grievous illness with poor prognosis even with optimal care. The apelin receptor (APJ) counteracts the pressor effect of angiotensin II, attenuates ischemic injury, and has the potential to be a novel target to treat HF. Intravenous administration of apelin improves cardiac function acutely in patients with HF. However, its short half-life restricts its use to infusion therapy. To identify a longer acting APJ agonist, we conducted a medicinal chemistry campaign, leading to the discovery of potent small-molecule APJ agonists with comparable activity to apelin by mimicking the C-terminal portion of apelin-13. Acute infusion increased systolic function and reduced systemic vascular resistance in 2 rat models of impaired cardiac function. Similar results were obtained in an anesthetized but not a conscious canine HF model. Chronic oral dosing in a rat myocardial infarction model reduced myocardial collagen content and improved diastolic function to a similar extent as losartan, a RAS antagonist standard-of-care therapy, but lacked additivity with coadministration. Collectively, this work demonstrates the feasibility of developing clinical, viable, potent small-molecule agonists that mimic the endogenous APJ ligand with more favorable drug-like properties and highlights potential limitations for APJ agonism for this indication.

Author Correction: Cryo-EM structures of PAC1 receptor reveal ligand binding mechanism.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Function-based high-throughput screening for antibody antagonists and agonists against G protein-coupled receptors.

Hybridoma and phage display are two powerful technologies for isolating target-specific monoclonal antibodies based on the binding. However, for complex membrane proteins, such as G protein-coupled receptors (GPCRs), binding-based screening rarely results in functional antibodies. Here we describe a function-based high-throughput screening method for quickly identifying antibody antagonists and agonists against GPCRs by combining glycosylphosphatidylinositol-anchored antibody cell display with ß-arrestin recruitment-based cell sorting and screening. This method links antibody genotype with phenotype and is applicable to all GPCR targets. We validated this method by identifying a panel of antibody antagonists and an antibody agonist to the human apelin receptor from an immune antibody repertoire. In contrast, we obtained only neutral binders and antibody antagonists from the same repertoire by phage display, suggesting that the new approach described here is more efficient than traditional methods in isolating functional antibodies. This new method may create a new paradigm in antibody drug discovery.

Cryo-EM structures of PAC1 receptor reveal ligand binding mechanism.

The pituitary adenylate cyclase-activating polypeptide type I receptor (PAC1R) belongs to the secretin receptor family and is widely distributed in the central neural system and peripheral organs. Abnormal activation of the receptor mediates trigeminovascular activation and sensitization, which is highly related to migraine, making PAC1R a potential therapeutic target. Elucidation of PAC1R activation mechanism would benefit discovery of therapeutic drugs for neuronal disorders. PAC1R activity is governed by pituitary adenylate cyclase-activating polypeptide (PACAP), known as a major vasodilator neuropeptide, and maxadilan, a native peptide from the sand fly, which is also capable of activating the receptor with similar potency. These peptide ligands have divergent sequences yet initiate convergent PAC1R activity. It is of interest to understand the mechanism of PAC1R ligand recognition and receptor activity regulation through structural biology. Here we report two near-atomic resolution cryo-EM structures of PAC1R activated by PACAP38 or maxadilan, providing structural insights into two distinct ligand binding modes. The structures illustrate flexibility of the extracellular domain (ECD) for ligands with distinct conformations, where ECD accommodates ligands in different orientations while extracellular loop 1 (ECL1) protrudes to further anchor the ligand bound in the orthosteric site. By structure-guided molecular modeling and mutagenesis, we tested residues in the ligand-binding pockets and identified clusters of residues that are critical for receptor activity. The structures reported here for the first time elucidate the mechanism of specificity and flexibility of ligand recognition and binding for PAC1R, and provide insights toward the design of therapeutic molecules targeting PAC1R.

Structure-guided discovery of a single-domain antibody agonist against human apelin receptor.

Developing antibody agonists targeting the human apelin receptor (APJ) is a promising therapeutic approach for the treatment of chronic heart failure. Here, we report the structure-guided discovery of a single-domain antibody (sdAb) agonist JN241-9, based on the cocrystal structure of APJ with an sdAb antagonist JN241, the first cocrystal structure of a class A G protein-coupled receptor (GPCR) with a functional antibody. As revealed by the structure, JN241 binds to the extracellular side of APJ, makes critical contacts with the second extracellular loop, and inserts the CDR3 into the ligand-binding pocket. We converted JN241 into a full agonist JN241-9 by inserting a tyrosine into the CDR3. Modeling and molecular dynamics simulation shed light on JN241-9-stimulated receptor activation, providing structural insights for finding agonistic antibodies against class A GPCRs.

Fragment-Based Computational Method for Designing GPCR Ligands.

G protein-coupled receptors (GPCRs) are the largest family of cell surface receptors, which is arguably the most important family of drug target. With the technology breakthroughs in X-ray crystallography and cryo-electron microscopy, more than 300 GPCR-ligand complex structures have been publicly reported since 2007, covering about 60 unique GPCRs. Such abundant structural information certainly will facilitate the structure-based drug design by targeting GPCRs. In this study, we have developed a fragment-based computational method for designing novel GPCR ligands. We first extracted the characteristic interaction patterns (CIPs) on the binding interfaces between GPCRs and their ligands. The CIPs were used as queries to search the chemical fragments derived from GPCR ligands, which were required to form similar interaction patterns with GPCR. Then, the selected chemical fragments were assembled into complete molecules by using the AutoT&T2 software. In this work, we chose ß-adrenergic receptor (ß-AR) and muscarinic acetylcholine receptor (mAChR) as the targets to validate this method. Based on the designs suggested by our method, samples of 63 compounds were purchased and tested in a cell-based functional assay. A total of 15 and 22 compounds were identified as active antagonists for ß2-AR and mAChR M1, respectively. Molecular dynamics simulations and binding free energy analysis were performed to explore the key interactions (e.g., hydrogen bonds and π-π interactions) between those active compounds and their target GPCRs. In summary, our work presents a useful approach to the de novo design of GPCR ligands based on the relevant 3D structural information.

GPCR structure and function relationship: identification of a biased apelin receptor mutant.

Biased ligands of G protein-coupled receptors (GPCRs) may have improved therapeutic benefits and safety profiles. However, the molecular mechanism of GPCR biased signaling remains largely unknown. Using apelin receptor (APJ) as a model, we systematically investigated the potential effects of amino acid residues around the orthosteric binding site on biased signaling. We discovered that a single residue mutation I109A (I1093.32) in the transmembrane domain 3 (TM3) located in the deep ligand-binding pocket was sufficient to convert a balanced APJ into a G protein signaling biased receptor. APJ I109A mutant receptor retained full capabilities in ligand binding and G protein activation, but was defective in GRK recruitment, ß-arrestin recruitment, and downstream receptor-mediated ERK activation. Based on molecular dynamics simulations, we proposed a molecular mechanism for biased signaling of I109A mutant receptor. We postulate that due to the extra space created by I109A mutation, the phenyl group of the last residue (Phe-13) of apelin rotates down and initiates a cascade of conformational changes in TM3. Phe-13 formed a new cluster of hydrophobic interactions with the sidechains of residues in TM3, including F1103.33 and M1133.36, which stabilizes the mutant receptor in a conformation favoring biased signaling. Interruption of these stabilizing interactions by double mutation F110A/I109A or M113A/I109A largely restored the ß-arrestin-mediated signaling. Taken together, we describe herein the discovery of a biased APJ mutant receptor and provide detailed molecular insights into APJ signaling selectivity, facilitating the discovery of novel therapeutics targeting APJ.

Structural Basis for Apelin Control of the Human Apelin Receptor.

Apelin receptor (APJR) is a key regulator of human cardiovascular function and is activated by two different endogenous peptide ligands, apelin and Elabela, each with different isoforms diversified by length and amino acid sequence. Here we report the 2.6-Å resolution crystal structure of human APJR in complex with a designed 17-amino-acid apelin mimetic peptide agonist. The structure reveals that the peptide agonist adopts a lactam constrained curved two-site ligand binding mode. Combined with mutation analysis and molecular dynamics simulations with apelin-13 binding to the wild-type APJR, this structure provides a mechanistic understanding of apelin recognition and binding specificity. Comparison of this structure with that of other peptide receptors suggests that endogenous peptide ligands with a high degree of conformational flexibility may bind and modulate the receptors via a similar two-site binding mechanism.

Human and mouse trace amine-associated receptor 1 have distinct pharmacology towards endogenous monoamines and imidazoline receptor ligands.

TAARs (trace amine-associated receptors) are G-protein-coupled receptors that respond to low abundance, endogenous amines such as tyramine and tryptamine, and represent potential targets for neuropsychiatric diseases. However, some members of this receptor subfamily either have no ligand identified or remain difficult to express and characterize using recombinant systems. In the present paper we report the successful expression of human and mouse TAAR1, and the characterization of their responses to various natural and synthetic agonists. In HEK (human embryonic kidney)-293/CRE-bla cells, mouse TAAR1 showed a robust response to trace amines as measured using either a cAMP assay or a beta-lactamase reporter assay, whereas human TAAR1 showed a weaker, but still measurable, response. When certain fragments of human TAAR1 were replaced with the corresponding regions of mouse TAAR1, the chimaeric receptor showed a much stronger response in cAMP production. Examination of a series of agonists on these receptors revealed that the human and the chimaeric receptor are almost identical in pharmacology, but distinct from the mouse receptor. We also screened small libraries of pharmacologically active agents on TAAR1 and identified a series of synthetic agonists, some of which are also ligands of the enigmatic imidazoline receptor. The findings of the present study not only shed light on the pharmacological species difference of TAAR1, but also raise new possibilities about the mechanism of some of the imidazoline-related agents.

Identification of surrogate agonists and antagonists for orphan G-protein-coupled receptor GPR139.

GPR139 is an orphan G-protein-coupled receptor (GPCR) that is expressed nearly exclusively in the central nervous system and may play a role in the control of locomotor activity. The signal transduction pathway and pharmacological function of GPR139, however, are still controversial due to the lack of natural or synthetic ligands. The authors report the characterization of human GPR139 signaling pathway and identification of surrogate agonists and antagonists. In both transient and stable transfections of HEK293F cells, overexpression of GPR139 increased basal intracellular cAMP concentrations compared to control cells. Furthermore, forskolin and isoproterenol-stimulated cAMP responses were enhanced in GPR139-expressing cells, suggesting that GPR139 is predominantly coupled to Galpha(s). The authors screened a large library of small molecules for compounds that increase cAMP levels in GPR139-expressing cells and identified a compound with GPR139 agonist activity. This compound increased cAMP production specifically in cells expressing GPR139 but not in cells expressing its highly homologous receptor GPR142. Furthermore, this compound did not induce calcium mobilization in GPR139 cells, indicating no Galpha(q)-mediated response. In addition, antagonist screening with the identified agonist yielded 2 classes of compounds as antagonists. The identification of surrogate agonists and antagonists of human GPR139 provides important tools for further study of this orphan GPCR.

A homogeneous G protein-coupled receptor ligand binding assay based on time-resolved fluorescence resonance energy transfer.

Fluorescence resonance energy transfer (FRET) has emerged as a powerful tool to the study of protein-protein interactions, such as receptor-ligand binding. However, the application of FRET to the study of G protein-coupled receptors (GPCRs) has been limited by the method of labeling receptor with fluorescence probes. Here we described a novel time-resolved (TR)-FRET method to study GPCR-ligand binding by using human complement 5a (C5a) receptor (C5aR) as a model system. Human C5aR was expressed in human embryonic kidney 293 cells with a hemagglutinin (HA) epitope at the N-terminus. Purified human C5a was labeled with terbium chelate and used as the fluorescence donor. Monoclonal anti-HA antibody conjugated with Alexa Fluor 488 was used as the fluorescence acceptor. Robust FRET signal was observed when the labeled ligand and C5aR membrane were mixed in the presence of the conjugated anti-HA antibody. This FRET signal was specific and saturable. C5a binding affinity to C5aR measured by the FRET assay was consistent with the data as determined by competition binding analysis using radiolabeled C5a. The FRET assay was also used to determine affinity of C5aR antagonists by competition binding analysis, and the data are similar to those from radioligand binding studies. Compared to the commonly used radioligand binding assay, this TR-FRET-based assay provides a nonradioactive, faster, and sensitive homogeneous assay format that could be easily adapted to high-throughput screening. The principle of this assay should also be applicable to other GPCRs, especially to those receptors with peptide or protein ligands.

Induction of substrate specificity shifts by placement of alanine insertions within the consensus amphipathic region of the Escherichia coli GABA (gamma-aminobutyric acid) transporter encoded by gabP.

The Escherichia coli GABA (gamma-aminobutyric acid) permease GabP is a prototypical APC (amine/polyamine/choline) super-family transporter that has a CAR (consensus amphipathic region) containing multiple specificity determinants, ostensibly organized on two helical surfaces, one hydrophobic [SHS (sensitive hydrophobic surface)] and the other hydrophilic [SPS (sensitive polar surface)]. To gauge the functional effects of placing alanine insertions at close intervals across the entire GabP CAR, 64 insertion variants were constructed. Insertions, particularly those in the SHS and the SPS, were highly detrimental to steady-state [(3)H]GABA accumulation. TSR (transport specificity ratio) analysis, employing [(3)H]nipecotic acid and [(14)C]GABA, showed that certain alanine insertions were associated with a specificity shift (i.e. a change in k (cat)/ K (m)). An insertion (INS Ala-269) located N-terminal to the SHS increased specificity for [(3)H]nipecotic acid relative to [(14)C]GABA, whereas an insertion (INS Ala-321) located C-terminal to the SPS had the opposite effect. Overall, the results are consistent with a working hypothesis that the GabP CAR contains extensive functional surfaces that may be manipulated by insertion mutagenesis to alter the specificity ( k (cat)/ K (m)) phenotype. The thermodynamic basis of TSR analysis provides generality, suggesting that amino acid insertions could affect specificity in many other transporters, particularly those such as the E. coli phenylalanine permease PheP [Pi, Chow and Pittard (2002) J. Bacteriol. 184, 5842-5847] that have a functionally significant CAR-like domain.

Dishevelled 2 recruits beta-arrestin 2 to mediate Wnt5A-stimulated endocytosis of Frizzled 4.

Wnt proteins, regulators of development in many organisms, bind to seven transmembrane-spanning (7TMS) receptors called frizzleds, thereby recruiting the cytoplasmic molecule dishevelled (Dvl) to the plasma membrane.Frizzled-mediated endocytosis of Wg (a Drosophila Wnt protein) and lysosomal degradation may regulate the formation of morphogen gradients. Endocytosis of Frizzled 4 (Fz4) in human embryonic kidney 293 cells was dependent on added Wnt5A protein and was accomplished by the multifunctional adaptor protein beta-arrestin 2 (betaarr2), which was recruited to Fz4 by binding to phosphorylated Dvl2. These findings provide a previously unrecognized mechanism for receptor recruitment of beta-arrestin and demonstrate that Dvl plays an important role in the endocytosis of frizzled, as well as in promoting signaling.

GIPC interacts with the beta1-adrenergic receptor and regulates beta1-adrenergic receptor-mediated ERK activation.

Beta1-adrenergic receptors, expressed at high levels in the human heart, have a carboxyl-terminal ESKV motif that can directly interact with PDZ domain-containing proteins. Using the beta1-adrenergic receptor carboxyl terminus as bait, we identified the novel beta1-adrenergic receptor-binding partner GIPC in a yeast two-hybrid screen of a human heart cDNA library. Here we demonstrate that the PDZ domain-containing protein, GIPC, co-immunoprecipitates with the beta1-adrenergic receptor in COS-7 cells. Essential for this interaction is the Ser residue of the beta1-adrenergic receptor carboxyl-terminal ESKV motif. Our data also demonstrate that beta1-adrenergic receptor stimulation activates the mitogen-activated protein kinase, ERK1/2. beta1-adrenergic receptor-mediated ERK1/2 activation was inhibited by pertussis toxin, implicating Gi, and was substantially decreased by the expression of GIPC. Expression of GIPC had no observable effect on beta1-adrenergic receptor sequestration or receptor-mediated cAMP accumulation. This GIPC effect was specific for the beta1-adrenergic receptor and was dependent on an intact PDZ binding motif. These data suggest that GIPC can regulate beta1-adrenergic receptor-stimulated, Gi-mediated, ERK activation while having no effect on receptor internalization or Gs-mediated cAMP signaling.

G protein-coupled receptor kinase 5 regulates beta 1-adrenergic receptor association with PSD-95.

We previously reported that the beta(1)-adrenergic receptor (beta(1)AR) associates with PSD-95 through a PDZ domain-mediated interaction, by which PSD-95 modulates beta(1)AR function and facilitates the physical association of beta(1)AR with other synaptic proteins such as N-methyl-d-aspartate receptors. Here we demonstrate that beta(1)AR association with PSD-95 is regulated by G protein-coupled receptor kinase 5 (GRK5). When beta(1)AR and PSD-95 were coexpressed with either GRK2 or GRK5 in COS-7 cells, GRK5 alone dramatically decreased the association of beta(1)AR with PSD-95, although GRK2 and GRK5 both could be co-immunoprecipitated with beta(1)AR and both could enhance receptor phosphorylation in vivo. Increasing expression of GRK5 in the cells led to further decreased beta(1)AR association with PSD-95. Stimulation with the beta(1)AR agonist isoproterenol further decreased PSD-95 binding to beta(1)AR. In addition, GRK5 protein kinase activity was required for this regulatory effect since a kinase-inactive GRK5 mutant had no effect on PSD-95 binding to beta(1)AR. Moreover, the regulatory effect of GRK5 on beta(1)AR association with PSD-95 was observed only when GRK5 was expressed together with the receptor, but not when GRK5 was coexpressed with PSD-95. Thus, we propose that GRK5 regulates beta(1)AR association with PSD-95 through phosphorylation of beta(1)AR. Regulation of protein association through receptor phosphorylation may be a general mechanism used by G protein-coupled receptors that associate via PDZ domain-mediated protein/protein interactions.