Conformation- and activation-based BRET sensors differentially report on GPCR-G protein coupling.

G protein-coupled receptors (GPCRs) regulate cellular signaling processes by coupling to diverse combinations of heterotrimeric G proteins composed of Gα, Gß, and Gγ subunits. Biosensors based on bioluminescence resonance energy transfer (BRET) have advanced our understanding of GPCR functional selectivity. Some BRET biosensors monitor ligand-induced conformational changes in the receptor or G proteins, whereas others monitor the recruitment of downstream effectors to sites of G protein activation. Here, we compared the ability of conformation-and activation-based BRET biosensors to assess the coupling of various class A and B GPCRs to specific Gα proteins in cultured cells. These GPCRs included serotonin 5-HT2A and 5-HT7 receptors, the GLP-1 receptor (GLP-1R), and the M3 muscarinic receptor. We observed different signaling profiles between the two types of sensors, highlighting how data interpretation could be affected by the nature of the biosensor. We also found that the identity of the Gßγ subunits used in the assay could differentially influence the selectivity of a receptor toward Gα subtypes, emphasizing the importance of the receptor-Gßγ pairing in determining Gα coupling specificity. Last, the addition of epitope tags to the receptor could affect stoichiometry and coupling selectivity and yield artifactual findings. These results highlight the need for careful sensor selection and experimental design when probing GPCR-G protein coupling.

Molecular Mechanisms of Desensitization Underlying the Differential Effects of Formyl Peptide Receptor 2 Agonists on Cardiac Structure-Function Post Myocardial Infarction.

Formyl peptide receptor 2 (FPR2) plays an integral role in the transition of macrophages from a pro-inflammatory program to one that is pro-resolving. FPR2-mediated stimulation of resolution post myocardial infarction has demonstrated efficacy in rodent models and is hypothesized to reduce progression into heart failure. FPR2 agonists that promote long-lasting receptor internalization can lead to persistent desensitization and diminished therapeutic benefits. In vitro signaling profiles and propensities for receptor desensitization of two clinically studied FPR2 agonists, namely, BMS-986235 and ACT-389949, were evaluated. In contrast to BMS-986235, pre-stimulation with ACT-389949 led to a decrease in its potency to inhibit cAMP production. Moreover, ACT-389949 displayed greater efficacy for ß-arrestin recruitment, while efficacy of Gi activation was similar for both agonists. Following agonist-promoted FPR2 internalization, effective recycling to the plasma membrane was observed only with BMS-986235. Use of G protein-coupled receptor kinase (GRK) knock-out cells revealed a differential impact of GRK2 versus GRK5/6 on ß-arrestin recruitment and Gi activation promoted by the two FPR2 agonists. In vivo, decreases of granulocytes in circulation were greatly diminished in mice treated with ACT-389949 but not for BMS-986235. With short-term dosing, both compounds induced a pro-resolution polarization state in cardiac monocyte/macrophages post myocardial infarction. By contrast, with long-term dosing, only BMS-986235 preserved the infarct wall thickness and increased left ventricular ejection fraction in a rat model of myocardial infarction. Altogether, the study shows that differences in the desensitization profiles induced by ACT-389949 and BMS-986235 at the molecular level may explain their distinct inflammatory/pro-resolving activities in vivo.

Effector membrane translocation biosensors reveal G protein and ßarrestin coupling profiles of 100 therapeutically relevant GPCRs.

The recognition that individual GPCRs can activate multiple signaling pathways has raised the possibility of developing drugs selectively targeting therapeutically relevant ones. This requires tools to determine which G proteins and ßarrestins are activated by a given receptor. Here, we present a set of BRET sensors monitoring the activation of the 12 G protein subtypes based on the translocation of their effectors to the plasma membrane (EMTA). Unlike most of the existing detection systems, EMTA does not require modification of receptors or G proteins (except for Gs). EMTA was found to be suitable for the detection of constitutive activity, inverse agonism, biased signaling and polypharmacology. Profiling of 100 therapeutically relevant human GPCRs resulted in 1500 pathway-specific concentration-response curves and revealed a great diversity of coupling profiles ranging from exquisite selectivity to broad promiscuity. Overall, this work describes unique resources for studying the complexities underlying GPCR signaling and pharmacology.

Selective FPR2 Agonism Promotes a Proresolution Macrophage Phenotype and Improves Cardiac Structure-Function Post Myocardial Infarction.

Dysregulated inflammation following myocardial infarction (MI) leads to maladaptive healing and remodeling. The study characterized and evaluated a selective formyl peptide receptor 2 (FPR2) agonist BMS-986235 in cellular assays and in rodents undergoing MI. BMS-986235 activated G proteins and promoted ß-arrestin recruitment, enhanced phagocytosis and neutrophil apoptosis, regulated chemotaxis, and stimulated interleukin-10 and monocyte chemoattractant protein-1 gene expression. Treatment with BMS-986235 improved mouse survival, reduced left ventricular area, reduced scar area, and preserved wall thickness. Treatment increased macrophage arginase-1 messenger RNA and CD206 receptor levels indicating a proresolution phenotype. In rats following MI, BMS-986235 preserved viable myocardium, attenuated left ventricular remodeling, and increased ejection fraction relative to control animals. Therefore, FPR2 agonism improves post-MI healing, limits remodeling and preserves function, and may offer an innovative therapeutic option to improve outcomes.

BRET-based effector membrane translocation assay monitors GPCR-promoted and endocytosis-mediated Gq activation at early endosomes.

G protein-coupled receptors (GPCRs) are gatekeepers of cellular homeostasis and the targets of a large proportion of drugs. In addition to their signaling activity at the plasma membrane, it has been proposed that their actions may result from translocation and activation of G proteins at endomembranes-namely endosomes. This could have a significant impact on our understanding of how signals from GPCR-targeting drugs are propagated within the cell. However, little is known about the mechanisms that drive G protein movement and activation in subcellular compartments. Using bioluminescence resonance energy transfer (BRET)-based effector membrane translocation assays, we dissected the mechanisms underlying endosomal Gq trafficking and activity following activation of Gq-coupled receptors, including the angiotensin II type 1, bradykinin B2, oxytocin, thromboxane A2 alpha isoform, and muscarinic acetylcholine M3 receptors. Our data reveal that GPCR-promoted activation of Gq at the plasma membrane induces its translocation to endosomes independently of ß-arrestin engagement and receptor endocytosis. In contrast, Gq activity at endosomes was found to rely on both receptor endocytosis-dependent and -independent mechanisms. In addition to shedding light on the molecular processes controlling subcellular Gq signaling, our study provides a set of tools that will be generally applicable to the study of G protein translocation and activation at endosomes and other subcellular organelles, as well as the contribution of signal propagation to drug action.

In Vitro and In Vivo Evaluation of a Small-Molecule APJ (Apelin Receptor) Agonist, BMS-986224, as a Potential Treatment for Heart Failure.

BACKGROUND: New heart failure therapies that safely augment cardiac contractility and output are needed. Previous apelin peptide studies have highlighted the potential for APJ (apelin receptor) agonism to enhance cardiac function in heart failure. However, apelin's short half-life limits its therapeutic utility. Here, we describe the preclinical characterization of a novel, orally bioavailable APJ agonist, BMS-986224. METHODS: BMS-986224 pharmacology was compared with (Pyr1) apelin-13 using radio ligand binding and signaling pathway assays downstream of APJ (cAMP, phosphorylated ERK [extracellular signal-regulated kinase], bioluminescence resonance energy transfer-based G-protein assays, ß-arrestin recruitment, and receptor internalization). Acute effects on cardiac function were studied in anesthetized instrumented rats. Chronic effects of BMS-986224 were assessed echocardiographically in the RHR (renal hypertensive rat) model of cardiac hypertrophy and decreased cardiac output. RESULTS: BMS-986224 was a potent (Kd=0.3 nmol/L) and selective APJ agonist, exhibiting similar receptor binding and signaling profile to (Pyr1) apelin-13. G-protein signaling assays in human embryonic kidney 293 cells and human cardiomyocytes confirmed this and demonstrated a lack of signaling bias relative to (Pyr1) apelin-13. In anesthetized instrumented rats, short-term BMS-986224 infusion increased cardiac output (10%-15%) without affecting heart rate, which was similar to (Pyr1) apelin-13 but differentiated from dobutamine. Subcutaneous and oral BMS-986224 administration in the RHR model increased stroke volume and cardiac output to levels seen in healthy animals but without preventing cardiac hypertrophy and fibrosis, effects differentiated from enalapril. CONCLUSIONS: We identify a novel, potent, and orally bioavailable nonpeptidic APJ agonist that closely recapitulates the signaling properties of (Pyr1) apelin-13. We show that oral APJ agonist administration induces a sustained increase in cardiac output in the cardiac disease setting and exhibits a differentiated profile from the renin-angiotensin system inhibitor enalapril, supporting further clinical evaluation of BMS-986224 in heart failure.

Tetrahydroisoquinoline CXCR4 Antagonists Adopt a Hybrid Binding Mode within the Peptide Subpocket of the CXCR4 Receptor.

The rationale for the structural and mechanistic basis of a tetrahydroisoquinoline (THIQ) based series of CXCR4 antagonists is presented. Using the previously reported crystal structures which reveal two distinct binding sites of CXCR4 defined as the small molecule (IT1t or minor) binding pocket and peptide (CVX15 or major) binding pocket, we hypothesized our THIQ small molecule series could bind like either molecule in these respective receptor configurations (IT1t versus CVX15 based poses). To this end, a thorough investigation was performed through a combination of receptor mutation studies, medicinal chemistry, biological testing, conformational analysis, and flexible docking. Our findings showed that the CVX15 peptide-based CXCR4 receptor complexes (red pose) were consistently favored over the small molecule IT1t based CXCR4 receptor configurations (blue pose) to correctly explain the computational and mutational studies as well as key structural components of activity for these small molecules.

Preservation of Post-Infarction Cardiac Structure and Function via Long-Term Oral Formyl Peptide Receptor Agonist Treatment.

Dysregulated inflammation following myocardial infarction (MI) promotes left ventricular (LV) remodeling and loss of function. Targeting inflammation resolution by activating formyl peptide receptors (FPRs) may limit adverse remodeling and progression towards heart failure. This study characterized the cellular and signaling properties of Compound 43 (Cmpd43), a dual FPR1/FPR2 agonist, and examined whether Cmpd43 treatment improves LV and infarct remodeling in rodent MI models. Cmpd43 stimulated FPR1/2-mediated signaling, enhanced proresolution cellular function, and modulated cytokines. Cmpd43 increased LV function and reduced chamber remodeling while increasing proresolution macrophage markers. The findings demonstrate that FPR agonism improves cardiac structure and function post-MI.

Allosteric modulation of metabotropic glutamate receptor 4 activates IDO1-dependent, immunoregulatory signaling in dendritic cells.

Metabotropic glutamate receptor 4 (mGluR4) possesses immune modulatory properties in vivo, such that a positive allosteric modulator (PAM) of the receptor confers protection on mice with relapsing-remitting experimental autoimmune encephalomyelitis (RR-EAE). ADX88178 is a newly-developed, one such mGluR4 modulator with high selectivity, potency, and optimized pharmacokinetics. Here we found that application of ADX88178 in the RR-EAE model system converted disease into a form of mild-yet chronic-neuroinflammation that remained stable for over two months after discontinuing drug treatment. In vitro, ADX88178 modulated the cytokine secretion profile of dendritic cells (DCs), increasing production of tolerogenic IL-10 and TGF-ß. The in vitro effects required activation of a Gi-independent, alternative signaling pathway that involved phosphatidylinositol-3-kinase (PI3K), Src kinase, and the signaling activity of indoleamine 2,3-dioxygenase 1 (IDO1). A PI3K inhibitor as well as small interfering RNA targeting Ido1-but not pertussis toxin, which affects Gi protein-dependent responses-abrogated the tolerogenic effects of ADX88178-conditioned DCs in vivo. Thus our data indicate that, in DCs, highly selective and potent mGluR4 PAMs such as ADX88178 may activate a Gi-independent, long-lived regulatory pathway that could be therapeutically exploited in chronic autoimmune diseases such as multiple sclerosis.

Comparing label-free biosensors for pharmacological screening with cell-based functional assays.

The diversity and impact of label-free technologies continues to expand in drug discovery. Two classes of label-free instruments, using either an electrical impedance-based or an optical-based biosensor, are now available for investigating the effects of ligands on cellular targets. Studies of GPCR function have been especially prominent with these instruments due to the importance of this target class in drug discovery. Although both classes of biosensors share similar high sensitivity to changes in cell shape and structure, it is unknown whether these biosensors yield similar results when comparing the same GPCR response. Furthermore, since cell morphology changes induced by GPCRs differ depending on which G-protein is activated, there is potential for these instruments to have differential sensitivities to G-protein signaling. Here 1 impedance (CellKey)- and 2 optical-based instruments (BIND and Epic) are compared using Gi-coupled (ACh M2), Gq-coupled (ACh M1), and Gs-coupled (CRF1) receptors. All 3 instruments were robust in agonist and antagonist modes yielding comparable potencies and assay variance. Both the impedance and optical biosensors showed similar high sensitivity for detecting an endogenous D1/D5 receptor response and a melanocortin-4 receptor inverse agonist (agouti-related protein). The impedance-based biosensor was uniquely able to qualitatively distinguish G-protein coupling and reveal dual signaling by CRF1. Finally, responses with a ligand-gated ion channel, TRPV1, were similarly detectable in each instrument. Thus, despite some differences, both impedance- and optical-based platforms offer robust live-cell, label-free assays well suited to drug discovery and typically yield similar pharmacological profiles for GPCR ligands.

Functional calcitonin gene-related peptide receptors are formed by the asymmetric assembly of a calcitonin receptor-like receptor homo-oligomer and a monomer of receptor activity-modifying protein-1.

In addition to their interactions with hetero-trimeric G proteins, seven-transmembrane domain receptors are now known to form multimeric complexes that can include receptor homo- or hetero-oligomers and/or accessory proteins that modulate their activity. The calcitonin gene-related peptide (CGRP) receptor requires the assembly of the seven-transmembrane domain calcitonin receptor-like receptor with the single-transmembrane domain receptor activity-modifying protein-1 to reach the cell surface and be active. However, the relative stoichiometric arrangement of these two proteins within a receptor complex remains unknown. Despite recent advances in the development of protein-protein interactions assays, determining the composition and stoichiometric arrangements of such signaling complexes in living cells remains a challenging task. In the present study, we combined bimolecular fluorescence complementation (BiFC) with bioluminescence resonance energy transfer (BRET) to probe the stoichiometric arrangement of the CGRP receptor complex. Together with BRET competition assays, co-immunoprecipitation experiments, and BiFC imaging, dual BRET/BiFC revealed that functional CGRP receptors result from the association of a homo-oligomer of the calcitonin receptor-like receptor with a monomer of the accessory protein receptor activity-modifying protein-1. In addition to revealing the existence of an unexpected asymmetric oligomeric organization for a G protein-coupled receptor, our study illustrates the usefulness of dual BRET/BiFC as a powerful tool for analyzing constitutive and dynamically regulated multiprotein complexes.

Assembly and signaling of CRLR and RAMP1 complexes assessed by BRET.

Biochemical and functional evidence suggest that the calcitonin receptor-like receptor (CRLR) interacts with receptor activity-modifying protein-1 (RAMP1) to generate a calcitonin gene-related peptide (CGRP) receptor. Using bioluminescence resonance energy transfer (BRET), we investigated the oligomeric assembly of the CRLR-RAMP1 signaling complex in living cells. As for their wild-type counterparts, fusion proteins linking CRLR and RAMP1 to the energy donor Renilla luciferase (Rluc) and energy acceptor green fluorescent protein (GFP) reach the cell surface only upon coexpression of CRLR and RAMP1. Radioligand binding and cAMP production assays also confirmed that the fusion proteins retained normal functional properties. BRET titration experiments revealed that CRLR and RAMP1 associate selectively to form heterodimers. This association was preserved for a mutated RAMP1 that cannot reach the cell surface, even in the presence of CRLR, indicating that the deficient targeting resulted from the altered conformation of the complex rather than a lack of heterodimerization. BRET analysis also showed that, in addition to associate with one another, both CRLR and RAMP1 can form homodimers. The homodimerization of the coreceptor was further confirmed by the ability of RAMP1 to prevent cell surface targeting of a truncated RAMP1 that normally exhibits receptor-independent plasma membrane delivery. Although the role of such dimerization remains unknown, BRET experiments clearly demonstrated that CRLR can engage signaling partners, such as G proteins and beta-arrestin, following CGRP stimulation, only in the presence of RAMP1. In addition to shed new light on the CRLR-RAMP1 signaling complex, the BRET assays developed herein offer new biosensors for probing CGRP receptor activity.

Calcitonin gene-related peptide analogues with aza and indolizidinone amino acid residues reveal conformational requirements for antagonist activity at the human calcitonin gene-related peptide 1 receptor.

Calcitonin gene-related peptide antagonists have potential for the treatment and prevention of disease states such as non-insulin-dependent diabetes mellitus, migraine headache, pain, and inflammation. To gain insight into the spatial requirements for CGRP antagonism, three strategies were employed to restrict the conformation of the potent undecapeptide antagonist, [D31,P34,F35]CGRP27-37. First, aza-amino acid scanning was performed, and ten aza-peptide analogues were synthesized and examined for biological activity. Second, (3S,6S,9S)-2-oxo-3-amino-indolizidin-2-one amino acid (I2aa) and (2S,6S,8S)-9-oxo-8-amino-indolizidin-9-one amino acid (I9aa) both were introduced at positions 31-32, 32-33, 33-34, and 34-35, regions of the backbone expected to adopt turns. Finally, the conformation of the backbone and side-chain of the C-terminal residue, Phe35-Ala36-Phe37-NH2, was explored employing (2S,4R,6R,8S)-9-oxo-8-amino-4-phenyl-indolizidin-9-one amino acid (4-Ph-I9aa) as a constrained phenylalanine mimic. The structure-activity relationships exhibited by our 26 analogues illustrate conformational requirements important for designing CGRP antagonists and highlight the importance of beta-turns centered at Gly33-Pro34 for potency.

Mutant Frizzled 4 associated with vitreoretinopathy traps wild-type Frizzled in the endoplasmic reticulum by oligomerization.

nt signalling pathways regulate cell proliferation, cell fate and morphogenetic movements. Here, we demonstrate that the Frizzled (Fz) family of Wnt receptors, similarly to G-protein-coupled receptors (GPCRs), form specific homo- and hetero-oligomers. Two lines of evidence suggest that oligomerization occurs in the endoplasmic reticulum: first, a mutant allele of Fz4, encoding a truncated protein that is retained in the endoplasmic reticulum, is linked to the autosomal-dominant retinal degenerative disease, familial exudative vitreoretinopathy (FEVR). We show that this mutant form of Fz4 oligomerizes with wild-type Fz4, retains it in the endoplasmic reticulum and inhibits its signalling. Second, a derivative of Fz1 targeted to the endoplasmic reticulum traps wild-type Fz1 in the endoplasmic reticulum and blocks its signalling. These data support the hypothesis that oligomerization of mutant and wild-type Fz proteins occurs in the endoplasmic reticulum and may explain the genetic dominance of this FEVR allele.