Multivalent CXCR4-targeting nanobody formats differently affect affinity, receptor clustering, and antagonism.

The chemokine receptor CXCR4 is involved in the development and migration of stem and immune cells but is also implicated in tumor progression and metastasis for a variety of cancers. Antagonizing ligand (CXCL12)-induced CXCR4 signaling is, therefore, of therapeutic interest. Currently, there are two small-molecule CXCR4 antagonists on the market for the mobilization of hematopoietic stem cells. Other molecules with improved potencies and safety profiles are being developed for different indications, including cancer. Moreover, multiple antagonistic nanobodies targeting CXCR4 displayed similar or better potencies as compared to the CXCR4-targeting molecule AMD3100 (Plerixafor), which was further enhanced through avid binding of bivalent derivatives. In this study, we aimed to compare the affinities of various multivalent nanobody formats which might be differently impacted by avidity. By fusion to a flexible GS-linker, Fc-region of human IgG1, different C4bp/CLR multimerization domains, or via site-directed conjugation to a trivalent linker scaffold, we generated different types of multivalent nanobodies with varying valencies ranging from bivalent to decavalent. Of these, C-terminal fusion, especially to human Fc, was most advantageous with a 2-log-fold and 3-log-fold increased potency in inhibiting CXCL12-mediated Gαi- or ß-arrestin recruitment, respectively. Overall, we describe strategies for generating multivalent and high-potency CXCR4 antagonistic nanobodies able to induce receptor clustering and conclude that fusion to an Fc-tail results in the highest avidity effect irrespective of the hinge linker.

Novel Small-Molecule Atypical Chemokine Receptor 3 Agonists: Design, Synthesis, and Pharmacological Evaluation for Antiplatelet Therapy.

ACKR3, an atypical chemokine receptor, has been associated with prothrombotic events and the development of cardiovascular events. We designed, synthesized, and evaluated a series of novel small molecule ACKR3 agonists. Extensive structure-activity relationship studies resulted in several promising agonists with potencies ranging from the low micromolar to nanomolar range, for example, 23 (EC50 = 111 nM, Emax = 95%) and 27 (EC50 = 69 nM, Emax = 82%) in the ß-arrestin-recruitment assay. These compounds are selective for ACKR3 versus ACKR2, CXCR3, and CXCR4. Several agonists were subjected to investigations of their P-selectin expression reduction in the flow cytometry experiments. In particular, compounds 23 and 27 showed the highest potency for platelet aggregation inhibition, up to 80% and 97%, respectively. The most promising compounds, especially 27, exhibited good solubility, metabolic stability, and no cytotoxicity, suggesting a potential tool compound for the treatment of platelet-mediated thrombosis.

Fluorophore-Labeled Pyrrolones Targeting the Intracellular Allosteric Binding Site of the Chemokine Receptor CCR1.

In this study, we describe the structure-based development of the first fluorescent ligands targeting the intracellular allosteric binding site (IABS) of the CC chemokine receptor type 1 (CCR1), a G protein-coupled receptor (GPCR) that has been pursued as a drug target in inflammation and immune diseases. Starting from previously reported intracellular allosteric modulators of CCR1, tetramethylrhodamine (TAMRA)-labeled ligands were designed, synthesized, and tested for their suitability as fluorescent tracers to probe binding to the IABS of CCR1. In the course of these studies, we developed LT166 (12) as a highly versatile fluorescent CCR1 ligand, enabling cell-free as well as cellular NanoBRET-based binding studies in a nonradioactive and high-throughput manner. Besides the detection of intracellular allosteric ligands by direct competition with 12, we were also able to monitor the binding of extracellular antagonists due to their positive cooperative binding with 12. Thereby, we provide a straightforward and nonradioactive method to easily distinguish between ligands binding to the IABS of CCR1 and extracellular negative modulators. Further, we applied 12 for the identification of novel chemotypes for intracellular CCR1 inhibition that feature high binding selectivity for CCR1 over CCR2. For one of the newly identified intracellular CCR1 ligands (i.e., 23), we were able to show CCR1 over CCR2 selectivity also on a functional level and demonstrated that this compound inhibits basal ß-arrestin recruitment to CCR1, thereby acting as an inverse agonist. Thus, our fluorescent CCR1 ligand 12 represents a highly promising tool for future studies of CCR1-targeted pharmacology and drug discovery.

GRK specificity and Gßγ dependency determines the potential of a GPCR for arrestin-biased agonism.

G protein-coupled receptors (GPCRs) are mainly regulated by GPCR kinase (GRK) phosphorylation and subsequent ß-arrestin recruitment. The ubiquitously expressed GRKs are classified into cytosolic GRK2/3 and membrane-tethered GRK5/6 subfamilies. GRK2/3 interact with activated G protein ßγ-subunits to translocate to the membrane. Yet, this need was not linked as a factor for bias, influencing the effectiveness of ß-arrestin-biased agonist creation. Using multiple approaches such as GRK2/3 mutants unable to interact with Gßγ, membrane-tethered GRKs and G protein inhibitors in GRK2/3/5/6 knockout cells, we show that G protein activation will precede GRK2/3-mediated ß-arrestin2 recruitment to activated receptors. This was independent of the source of free Gßγ and observable for Gs-, Gi- and Gq-coupled GPCRs. Thus, ß-arrestin interaction for GRK2/3-regulated receptors is inseparably connected with G protein activation. We outline a theoretical framework of how GRK dependence on free Gßγ can determine a GPCR's potential for biased agonism. Due to this inherent cellular mechanism for GRK2/3 recruitment and receptor phosphorylation, we anticipate generation of ß-arrestin-biased ligands to be mechanistically challenging for the subgroup of GPCRs exclusively regulated by GRK2/3, but achievable for GRK5/6-regulated receptors, that do not demand liberated Gßγ. Accordingly, GRK specificity of any GPCR is foundational for developing arrestin-biased ligands.

Development of a NanoBRET Assay Platform to Detect Intracellular Ligands for the Chemokine Receptors CCR6 and CXCR1.

A conserved intracellular allosteric binding site (IABS) was recently identified at several G protein-coupled receptors (GPCRs). This target site allows the binding of allosteric modulators and enables a new mode of GPCR inhibition. Herein, we report the development of a NanoBRET-based assay platform based on the fluorescent ligand LT221 (5), to detect intracellular binding to CCR6 and CXCR1, two chemokine receptors that have been pursued as promising drug targets in inflammation and immuno-oncology. Our assay platform enables cell-free as well as cellular NanoBRET-based binding studies in a nonisotopic and straightforward manner. By combining this screening platform with a previously reported CXCR2 assay, we investigated CXCR1/CXCR2/CCR6 selectivity profiles for both known and novel squaramide analogues derived from navarixin, a known intracellular CXCR1/CXCR2 antagonist and phase II clinical candidate for the treatment of pulmonary diseases. By means of these studies we identified compound 10, a previously reported tert-butyl analogue of navarixin, as a low nanomolar intracellular CCR6 antagonist. Further, our assay platform clearly indicated intracellular binding of the CCR6 antagonist PF-07054894, currently evaluated in phase I clinical trials for the treatment of ulcerative colitis, thereby providing profound evidence for the existence and the pharmacological relevance of a druggable IABS at CCR6.

Systematic Assessment of Human CCR7 Signalling Using NanoBRET Biosensors Points towards the Importance of the Cellular Context.

The human CC chemokine receptor 7 (CCR7) is activated by two natural ligands, CC chemokine ligand 19 (CCL19) and 21 (CCL21). The CCL19-CCL21-CCR7 axis has been extensively studied in vitro, but there is still debate over whether CCL21 is an overall weaker agonist or if the axis displays biased signalling. In this study, we performed a systematic analysis at the transducer level using NanoBRET-based methodologies in three commonly used cellular backgrounds to evaluate pathway and ligand preferences, as well as ligand bias and the influence of the cellular system thereon. We found that both CCL19 and CCL21 activated all cognate G proteins and some non-cognate couplings in a cell-type-dependent manner. Both ligands recruited ß-arrestin1 and 2, but the potency was strongly dependent on the cellular system. Overall, CCL19 and CCL21 showed largely conserved pathway preferences, but small differences were detected. However, these differences only consolidated in a weak ligand bias. Together, these data suggest that CCL19 and CCL21 share mostly overlapping, weakly biased, transducer profiles, which can be influenced by the cellular context.

Pharmacological Characterization and Radiolabeling of VUF15485, a High-Affinity Small-Molecule Agonist for the Atypical Chemokine Receptor ACKR3.

Atypical chemokine receptor 3 (ACKR3), formerly referred to as CXCR7, is considered to be an interesting drug target. In this study, we report on the synthesis, pharmacological characterization and radiolabeling of VUF15485, a new ACKR3 small-molecule agonist, that will serve as an important new tool to study this ß-arrestin-biased chemokine receptor. VUF15485 binds with nanomolar affinity (pIC50 = 8.3) to human ACKR3, as measured in [125I]CXCL12 competition binding experiments. Moreover, in a bioluminescence resonance energy transfer-based ß-arrestin2 recruitment assay VUF15485 acts as a potent ACKR3 agonist (pEC50 = 7.6) and shows a similar extent of receptor activation compared with CXCL12 when using a newly developed, fluorescence resonance energy transfer-based ACKR3 conformational sensor. Moreover, the ACKR3 agonist VUF15485, tested against a (atypical) chemokine receptor panel (agonist and antagonist mode), proves to be selective for ACKR3. VUF15485 labeled with tritium at one of its methoxy groups ([3H]VUF15485), binds ACKR3 saturably and with high affinity (K d = 8.2 nM). Additionally, [3H]VUF15485 shows rapid binding kinetics and consequently a short residence time (<2 minutes) for binding to ACKR3. The selectivity of [3H]VUF15485 for ACKR3, was confirmed by binding studies, whereupon CXCR3, CXCR4, and ACKR3 small-molecule ligands were competed for binding against the radiolabeled agonist. Interestingly, the chemokine ligands CXCL11 and CXCL12 are not able to displace the binding of [3H]VUF15485 to ACKR3. The radiolabeled VUF15485 was subsequently used to evaluate its binding pocket. Site-directed mutagenesis and docking studies using a recently solved cryo-EM structure propose that VUF15485 binds in the major and the minor binding pocket of ACKR3. SIGNIFICANCE STATEMENT: The atypical chemokine receptor atypical chemokine receptor 3 (ACKR3) is considered an interesting drug target in relation to cancer and multiple sclerosis. The study reports on new chemical biology tools for ACKR3, i.e., a new agonist that can also be radiolabeled and a new ACKR3 conformational sensor, that both can be used to directly study the interaction of ACKR3 ligands with the G protein-coupled receptor.

Systematic assessment of chemokine ligand bias at the human chemokine receptor CXCR2 indicates G protein bias over ß-arrestin recruitment and receptor internalization.

BACKGROUND: The human CXC chemokine receptor 2 (CXCR2) is a G protein-coupled receptor (GPCR) interacting with multiple chemokines (i.e., CXC chemokine ligands CXCL1-3 and CXCL5-8). It is involved in inflammatory diseases as well as cancer. Consequently, much effort is put into the identification of CXCR2 targeting drugs. Fundamental research regarding CXCR2 signaling is mainly focused on CXCL8 (IL-8), which is the first and best described high-affinity ligand for CXCR2. Much less is known about CXCR2 activation induced by other chemokines and it remains to be determined to what extent potential ligand bias exists within this signaling system. This insight might be important to unlock new opportunities in therapeutic targeting of CXCR2. METHODS: Ligand binding was determined in a competition binding assay using labeled CXCL8. Activation of the ELR + chemokine-induced CXCR2 signaling pathways, including G protein activation, ß-arrestin1/2 recruitment, and receptor internalization, were quantified using NanoBRET-based techniques. Ligand bias within and between these pathways was subsequently investigated by ligand bias calculations, with CXCL8 as the reference CXCR2 ligand. Statistical significance was tested through a one-way ANOVA followed by Dunnett's multiple comparisons test. RESULTS: All chemokines (CXCL1-3 and CXCL5-8) were able to displace CXCL8 from CXCR2 with high affinity and activated the same panel of G protein subtypes (Gαi1, Gαi2, Gαi3, GαoA, GαoB, and Gα15) without any statistically significant ligand bias towards any one type of G protein. Compared to CXCL8, all other chemokines were less potent in ß-arrestin1 and -2 recruitment and receptor internalization while equivalently activating G proteins, indicating a G protein activation bias for CXCL1,-2,-3,-5,-6 and CXCL7. Lastly, with CXCL8 used as reference ligand, CXCL2 and CXCL6 showed ligand bias towards ß-arrestin1/2 recruitment compared to receptor internalization. CONCLUSION: This study presents an in-depth analysis of signaling bias upon CXCR2 stimulation by its chemokine ligands. Using CXCL8 as a reference ligand for bias index calculations, no ligand bias was observed between chemokines with respect to activation of separate G proteins subtypes or recruitment of ß-arrestin1/2 subtypes, respectively. However, compared to ß-arrestin recruitment and receptor internalization, CXCL1-3 and CXCL5-7 were biased towards G protein activation when CXCL8 was used as reference ligand.

G protein-receptor kinases 5/6 are the key regulators of G protein-coupled receptor 35-arrestin interactions.

Human G protein-coupled receptor 35 is regulated by agonist-mediated phosphorylation of a set of five phospho-acceptor amino acids within its C-terminal tail. Alteration of both Ser300 and Ser303 to alanine in the GPR35a isoform greatly reduces the ability of receptor agonists to promote interactions with arrestin adapter proteins. Here, we have integrated the use of cell lines genome edited to lack expression of combinations of G protein receptor kinases (GRKs), selective small molecule inhibitors of subsets of these kinases, and antisera able to specifically identify either human GPR35a or mouse GPR35 only when Ser300 and Ser303 (orce; the equivalent residues in mouse GPR35) have become phosphorylated to demonstrate that GRK5 and GRK6 cause agonist-dependent phosphorylation of these residues. Extensions of these studies demonstrated the importance of the GRK5/6-mediated phosphorylation of these amino acids for agonist-induced internalization of the receptor. Homology and predictive modeling of the interaction of human GPR35 with GRKs showed that the N terminus of GRK5 is likely to dock in the same methionine pocket on the intracellular face of GPR35 as the C terminus of the α5 helix of Gα13 and, that while this is also the case for GRK6, GRK2 and GRK3 are unable to do so effectively. These studies provide unique and wide-ranging insights into modes of regulation of GPR35, a receptor that is currently attracting considerable interest as a novel therapeutic target in diseases including ulcerative colitis.

GPR182 is a broadly scavenging atypical chemokine receptor influencing T-independent immunity.

Immune responses highly depend on the effective trafficking of immune cells into and within secondary lymphoid organs (SLOs). Atypical chemokine receptors (ACKRs) scavenge chemokines to eliminate them from the extracellular space, thereby generating gradients that guide leukocytes. In contrast to canonical chemokine receptors, ACKRs do not induce classical intracellular signaling that results in cell migration. Recently, the closest relative of ACKR3, GPR182, has been partially deorphanized as a potential novel ACKR. We confirm and extend previous studies by identifying further ligands that classify GPR182 as a broadly scavenging chemokine receptor. We validate the "atypical" nature of the receptor, wherein canonical G-protein-dependent intracellular signaling is not activated following ligand stimulation. However, ß-arrestins are required for ligand-independent internalization and chemokine scavenging whereas the C-terminus is in part dispensable. In the absence of GPR182 in vivo, we observed elevated chemokine levels in the serum but also in SLO interstitium. We also reveal that CXCL13 and CCL28, which do not bind any other ACKR, are bound and efficiently scavenged by GPR182. Moreover, we found a cooperative relationship between GPR182 and ACKR3 in regulating serum CXCL12 levels, and between GPR182 and ACKR4 in controlling CCL20 levels. Furthermore, we unveil a new phenotype in GPR182-KO mice, in which we observed a reduced marginal zone (MZ), both in size and in cellularity, and thus in the T-independent antibody response. Taken together, we and others have unveiled a novel, broadly scavenging chemokine receptor, which we propose should be named ACKR5.

Cannabidiol negatively modulates adenosine A2A receptor functioning in living cells.

OBJECTIVES: Cannabidiol (CBD) is a phytocannabinoid with great potential in clinical applications. The mechanism(s) of action of CBD require further investigation. Previous studies suggested that adenosine A2A receptors (A2ARs) could play a role in CBD-induced effects. Here, we evaluated the ability of CBD to modify the function of A2AR. METHODS: We used HEK-293T cells transfected with the cDNA encoding the human A2AR and Gαs protein, both modified to perform bioluminescence-based assays. We first assessed the effect of CBD on A2AR ligand binding using an A2AR NanoLuciferase sensor. Next, we evaluated whether CBD modified A2AR coupling to mini-Gαs proteins using the NanoBiT™ assay. Finally, we further assessed CBD effects on A2AR intrinsic activity by recording agonist-induced cAMP accumulation. RESULTS: CBD did not bind orthosterically to A2AR but reduced the coupling of A2AR to Gαs protein and the subsequent generation of cAMP. CONCLUSION: CBD negatively modulates A2AR functioning.

Formate promotes invasion and metastasis in reliance on lipid metabolism.

Metabolic rewiring is essential for cancer onset and progression. We previously showed that one-carbon metabolism-dependent formate production often exceeds the anabolic demand of cancer cells, resulting in formate overflow. Furthermore, we showed that increased extracellular formate concentrations promote the in vitro invasiveness of glioblastoma cells. Here, we substantiate these initial observations with ex vivo and in vivo experiments. We also show that exposure to exogeneous formate can prime cancer cells toward a pro-invasive phenotype leading to increased metastasis formation in vivo. Our results suggest that the increased local formate concentration within the tumor microenvironment can be one factor to promote metastases. Additionally, we describe a mechanistic interplay between formate-dependent increased invasiveness and adaptations of lipid metabolism and matrix metalloproteinase activity. Our findings consolidate the role of formate as pro-invasive metabolite and warrant further research to better understand the interplay between formate and lipid metabolism.

New pairings and deorphanization among the atypical chemokine receptor family - physiological and clinical relevance.

Atypical chemokine receptors (ACKRs) form a small subfamily of receptors (ACKR1-4) unable to trigger G protein-dependent signaling in response to their ligands. They do, however, play a crucial regulatory role in chemokine biology by capturing, scavenging or transporting chemokines, thereby regulating their availability and signaling through classical chemokine receptors. ACKRs add thus another layer of complexity to the intricate chemokine-receptor interaction network. Recently, targeted approaches and screening programs aiming at reassessing chemokine activity towards ACKRs identified several new pairings such as the dimeric CXCL12 with ACKR1, CXCL2, CXCL10 and CCL26 with ACKR2, the viral broad-spectrum chemokine vCCL2/vMIP-II, a range of opioid peptides and PAMP-12 with ACKR3 as well as CCL20 and CCL22 with ACKR4. Moreover, GPR182 (ACKR5) has been lately proposed as a new promiscuous atypical chemokine receptor with scavenging activity notably towards CXCL9, CXCL10, CXCL12 and CXCL13. Altogether, these findings reveal new degrees of complexity of the chemokine network and expand the panel of ACKR ligands and regulatory functions. In this minireview, we present and discuss these new pairings, their physiological and clinical relevance as well as the opportunities they open for targeting ACKRs in innovative therapeutic strategies.

Inhibition of MYC translation through targeting of the newly identified PHB-eIF4F complex as a therapeutic strategy in CLL.

Dysregulation of messenger RNA (mRNA) translation, including preferential translation of mRNA with complex 5' untranslated regions such as the MYC oncogene, is recognized as an important mechanism in cancer. Here, we show that both human and murine chronic lymphocytic leukemia (CLL) cells display a high translation rate, which is inhibited by the synthetic flavagline FL3, a prohibitin (PHB)-binding drug. A multiomics analysis performed in samples from patients with CLL and cell lines treated with FL3 revealed the decreased translation of the MYC oncogene and of proteins involved in cell cycle and metabolism. Furthermore, inhibiting translation induced a proliferation arrest and a rewiring of MYC-driven metabolism. Interestingly, contrary to other models, the RAS-RAF-(PHBs)-MAPK pathway is neither impaired by FL3 nor implicated in translation regulation in CLL cells. Here, we rather show that PHBs are directly associated with the eukaryotic initiation factor (eIF)4F translation complex and are targeted by FL3. Knockdown of PHBs resembled FL3 treatment. Importantly, inhibition of translation controlled CLL development in vivo, either alone or combined with immunotherapy. Finally, high expression of translation initiation-related genes and PHBs genes correlated with poor survival and unfavorable clinical parameters in patients with CLL. Overall, we demonstrated that translation inhibition is a valuable strategy to control CLL development by blocking the translation of several oncogenic pathways including MYC. We also unraveled a new and direct role of PHBs in translation initiation, thus creating new therapeutic opportunities for patients with CLL.

Reduced G protein signaling despite impaired internalization and ß-arrestin recruitment in patients carrying a CXCR4Leu317fsX3 mutation causing WHIM syndrome.

WHIM syndrome is an inherited immune disorder caused by an autosomal dominant heterozygous mutation in CXCR4. The disease is characterized by neutropenia/leukopenia (secondary to retention of mature neutrophils in bone marrow), recurrent bacterial infections, treatment-refractory warts, and hypogammaglobulinemia. All mutations reported in WHIM patients lead to the truncations in the C-terminal domain of CXCR4, R334X being the most frequent. This defect prevents receptor internalization and enhances both calcium mobilization and ERK phosphorylation, resulting in increased chemotaxis in response to the unique ligand CXCL12. Here, we describe 3 patients presenting neutropenia and myelokathexis, but normal lymphocyte count and immunoglobulin levels, carrying what we believe to be a novel Leu317fsX3 mutation in CXCR4, leading to a complete truncation of its intracellular tail. The analysis of the L317fsX3 mutation in cells derived from patients and in vitro cellular models reveals unique signaling features in comparison with R334X mutation. The L317fsX3 mutation impairs CXCR4 downregulation and ß-arrestin recruitment in response to CXCL12 and reduces other signaling events - including ERK1/2 phosphorylation, calcium mobilization, and chemotaxis - all processes that are typically enhanced in cells carrying the R334X mutation. Our findings suggest that, overall, the L317fsX3 mutation may be causative of a form of WHIM syndrome not associated with an augmented CXCR4 response to CXCL12.

Nanoluciferase-based complementation assays to monitor activation, modulation and signaling of receptor tyrosine kinases (RTKs).

Receptor tyrosine kinases (RTKs) are transmembrane receptors activated by a wide diversity of growth factors, cytokines or hormones. They ensure multiple roles in cellular processes, including proliferation, differentiation and survival. They are also crucial drivers of development and progression of multiple cancer types, and represent important drug targets. Generally, ligand binding induces dimerization of RTK monomers, which induces auto-/transphosphorylation of tyrosine residues on the intracellular tails leading to the recruitment of adaptor proteins and modifying enzymes to promote and modulate various downstream signaling pathways. This chapter details easy, rapid, sensitive and versatile methods based on split Nanoluciferase complementation technology (NanoBiT) to monitor activation and modulation of two models of RTKs (EGFR and AXL) through the measurement of their dimerization and the recruitment of the adaptor protein Grb2 (SH2 domain-containing growth factor receptor-bound protein 2) and the receptor-modifying enzyme, the ubiquitin ligase Cbl.

Chemokine CXCL4 interactions with extracellular matrix proteoglycans mediate widespread immune cell recruitment independent of chemokine receptors.

Leukocyte recruitment from the vasculature into tissues is a crucial component of the immune system but is also key to inflammatory disease. Chemokines are central to this process but have yet to be therapeutically targeted during inflammation due to a lack of mechanistic understanding. Specifically, CXCL4 (Platelet Factor 4, PF4) has no established receptor that explains its function. Here, we use biophysical, in vitro, and in vivo techniques to determine the mechanism underlying CXCL4-mediated leukocyte recruitment. We demonstrate that CXCL4 binds to glycosaminoglycan (GAG) sugars on proteoglycans within the endothelial extracellular matrix, resulting in increased adhesion of leukocytes to the vasculature, increased vascular permeability, and non-specific recruitment of a range of leukocytes. Furthermore, GAG sulfation confers selectivity onto chemokine localization. These findings present mechanistic insights into chemokine biology and provide future therapeutic targets.

Computationally designed GPCR quaternary structures bias signaling pathway activation.

Communication across membranes controls critical cellular processes and is achieved by receptors translating extracellular signals into selective cytoplasmic responses. While receptor tertiary structures can be readily characterized, receptor associations into quaternary structures are challenging to study and their implications in signal transduction remain poorly understood. Here, we report a computational approach for predicting receptor self-associations, and designing receptor oligomers with various quaternary structures and signaling properties. Using this approach, we designed chemokine receptor CXCR4 dimers with reprogrammed binding interactions, conformations, and abilities to activate distinct intracellular signaling proteins. In agreement with our predictions, the designed CXCR4s dimerized through distinct conformations and displayed different quaternary structural changes upon activation. Consistent with the active state models, all engineered CXCR4 oligomers activated the G protein Gi, but only specific dimer structures also recruited ß-arrestins. Overall, we demonstrate that quaternary structures represent an important unforeseen mechanism of receptor biased signaling and reveal the existence of a bias switch at the dimer interface of several G protein-coupled receptors including CXCR4, mu-Opioid and type-2 Vasopressin receptors that selectively control the activation of G proteins vs ß-arrestin-mediated pathways. The approach should prove useful for predicting and designing receptor associations to uncover and reprogram selective cellular signaling functions.

How Carvedilol activates ß2-adrenoceptors.

Carvedilol is among the most effective ß-blockers for improving survival after myocardial infarction. Yet the mechanisms by which carvedilol achieves this superior clinical profile are still unclear. Beyond blockade of ß1-adrenoceptors, arrestin-biased signalling via ß2-adrenoceptors is a molecular mechanism proposed to explain the survival benefits. Here, we offer an alternative mechanism to rationalize carvedilol's cellular signalling. Using primary and immortalized cells genome-edited by CRISPR/Cas9 to lack either G proteins or arrestins; and combining biological, biochemical, and signalling assays with molecular dynamics simulations, we demonstrate that G proteins drive all detectable carvedilol signalling through ß2ARs. Because a clear understanding of how drugs act is imperative to data interpretation in basic and clinical research, to the stratification of clinical trials or to the monitoring of drug effects on the target pathway, the mechanistic insight gained here provides a foundation for the rational development of signalling prototypes that target the ß-adrenoceptor system.