Endocytosis and intracellular trafficking of receptors are pivotal to maintain physiological functions and drug action; however, robust quantitative approaches are lacking to study such processes in live cells. Here we present new bioluminescence resonance energy transfer (BRET) sensors to quantitatively monitor G protein-coupled receptors (GPCRs) and β-arrestin trafficking. These sensors are based on bystander BRET and use the naturally interacting chromophores luciferase (RLuc) and green fluorescent protein (rGFP) fromRenilla. The versatility and robustness of this approach are exemplified by anchoring rGFP at the plasma membrane or in endosomes to generate high dynamic spectrometric BRET signals on ligand-promoted recruitment or sequestration of RLuc-tagged proteins to, or from, specific cell compartments, as well as sensitive subcellular BRET imaging for protein translocation visualization. These sensors are scalable to high-throughput formats and allow quantitative pharmacological studies of GPCR trafficking in real time, in live cells, revealing ligand-dependent biased trafficking of receptor/β-arrestin complexes.

G protein–coupled receptors (GPCRs) use diverse mechanisms to regulate the mitogen-activated protein kinases ERK1/2. β-Arrestins (βArr1/2) are ubiquitous inhibitors of G protein signaling, promoting GPCR desensitization and internalization and serving as scaffolds for ERK1/2 activation. Studies using CRISPR/Cas9 to delete βArr1/2 and G proteins have cast doubt on the role of β-arrestins in activating specific pools of ERK1/2. We compared the effects of siRNA-mediated knockdown of βArr1/2 and reconstitution with βArr1/2 in three different parental and CRISPR-derived βArr1/2 knockout HEK293 cell pairs to assess the effect of βArr1/2 deletion on ERK1/2 activation by four G_s-coupled GPCRs. In all parental lines with all receptors, ERK1/2 stimulation was reduced by siRNAs specific for βArr2 or βArr1/2. In contrast, variable effects were observed with CRISPR-derived cell lines both between different lines and with activation of different receptors. For β₂ adrenergic receptors (β₂ARs) and β₁ARs, βArr1/2 deletion increased, decreased, or had no effect on isoproterenol-stimulated ERK1/2 activation in different CRISPR clones. ERK1/2 activation by the vasopressin V₂ and follicle-stimulating hormone receptors was reduced in these cells but was enhanced by reconstitution with βArr1/2. Loss of desensitization and receptor internalization in CRISPR βArr1/2 knockout cells caused β₂AR-mediated stimulation of ERK1/2 to become more dependent on G proteins, which was reversed by reintroducing βArr1/2. These data suggest that βArr1/2 function as a regulatory hub, determining the balance between mechanistically different pathways that result in activation of ERK1/2, and caution against extrapolating results obtained from βArr1/2- or G protein–deleted cells to GPCR behavior in native systems.

In addition to G protein-coupled receptor (GPCR) desensitization and endocytosis, β-arrestin recruitment to ligand-stimulated GPCRs promotes non-canonical signalling cascades. Distinguishing the respective contributions of β-arrestin recruitment to the receptor and β-arrestin-promoted endocytosis in propagating receptor signalling has been limited by the lack of selective analytical tools. Here, using a combination of virtual screening and cell-based assays, we have identified a small molecule that selectively inhibits the interaction between β-arrestin and the β2-adaptin subunit of the clathrin adaptor protein AP2 without interfering with the formation of receptor/β-arrestin complexes. This selective β-arrestin/β2-adaptin inhibitor (Barbadin) blocks agonist-promoted endocytosis of the prototypical β2-adrenergic (β2AR), V2-vasopressin (V2R) and angiotensin-II type-1 (AT1R) receptors, but does not affect β-arrestin-independent (transferrin) or AP2-independent (endothelin-A) receptor internalization. Interestingly, Barbadin fully blocks V2R-stimulated ERK1/2 activation and blunts cAMP accumulation promoted by both V2R and β2AR, supporting the concept of β-arrestin/AP2-dependent signalling for both G protein-dependent and -independent pathways.

G protein–coupled receptors (GPCRs) are important therapeutic targets that exhibit functional selectivity (biased signaling), in which different ligands or receptor variants elicit distinct downstream signaling. Understanding all the signaling events and biases that contribute to both the beneficial and adverse effects of GPCR stimulation by given ligands is important for drug discovery. Here, we report the design, validation, and use of pathway-selective bioluminescence resonance energy transfer (BRET) biosensors that monitor the engagement and activation of signaling effectors downstream of G proteins, including protein kinase C (PKC), phospholipase C (PLC), p63RhoGEF, and Rho. Combined with G protein and β-arrestin BRET biosensors, our sensors enabled real-time monitoring of GPCR signaling at different levels in downstream pathways in both native and engineered cells. Profiling of the responses to 14 angiotensin II (AngII) type 1 receptor (AT1R) ligands enabled the clustering of compounds into different subfamilies of biased ligands and showed that, in addition to the previously reported functional selectivity between Gα_q and β-arrestin, there are also biases among G protein subtypes. We also demonstrated that biases observed at the receptor and G protein levels propagated to downstream signaling pathways and that these biases could occur through the engagement of different G proteins to activate a common effector. We also used these tools to determine how naturally occurring AT1R variants affected signaling bias. This suite of BRET biosensors provides a useful resource for fingerprinting biased ligands and mutant receptors and for dissecting functional selectivity at various levels of GPCR signaling.

The prostaglandin F2alpha (PGF2alpha) receptor (FP) is a key regulator of parturition and a target for pharmacological management of preterm labor. However, an incomplete understanding of signaling pathways regulating myometrial contraction hinders the development of improved therapeutics. Here we used a peptidomimetic inhibitor of parturition in mice, PDC113.824, whose structure was based on the NH(2)-terminal region of the second extracellular loop of FP receptor, to gain mechanistic insight underlying FP receptor-mediated cell responses in the context of parturition. We show that PDC113.824 not only delayed normal parturition in mice but also that it inhibited both PGF2alpha- and lipopolysaccharide-induced preterm labor. PDC113.824 inhibited PGF2alpha-mediated, G(alpha)(12)-dependent activation of the Rho/ROCK signaling pathways, actin remodeling, and contraction of human myometrial cells likely by acting as a non-competitive, allosteric modulator of PGF2alpha binding. In contrast to its negative allosteric modulating effects on Rho/ROCK signaling, PDC113.824 acted as a positive allosteric modulator on PGF2alpha-mediated protein kinase C and ERK1/2 signaling. This bias in receptor-dependent signaling was explained by an increase in FP receptor coupling to G(alpha)(q), at the expense of coupling to G(alpha)(12). Our findings regarding the allosteric and biased nature of PDC113.824 offer new mechanistic insights into FP receptor signaling relevant to parturition and suggest novel therapeutic opportunities for the development of new tocolytic drugs.

PALB2 links BRCA1 and BRCA2 in homologous recombinational repair of DNA double strand breaks (DSBs). Mono-allelic mutations in PALB2 increase the risk of breast, pancreatic, and other cancers, and biallelic mutations cause Fanconi anemia (FA). Like Brca1 and Brca2, systemic knock-out of Palb2 in mice results in embryonic lethality. In this study, we generated a hypomorphic Palb2 allele expressing a mutant PALB2 protein unable to bind BRCA1. Consistent with an FA-like phenotype, cells from the mutant mice showed hypersensitivity and chromosomal breakage when treated with mitomycin C, a DNA interstrand crosslinker. Moreover, mutant males showed reduced fertility due to impaired meiosis and increased apoptosis in germ cells. Interestingly, mutant meiocytes showed a significant defect in sex chromosome synapsis, which likely contributed to the germ cell loss and fertility defect. Our results underscore the in vivo importance of the PALB2-BRCA1 complex formation in DSB repair and male meiosis.