Gαs is dispensable for ß-arrestin coupling but dictates GRK selectivity and is predominant for gene expression regulation by ß2-adrenergic receptor.

ß-arrestins play a key role in G protein-coupled receptor (GPCR) internalization, trafficking, and signaling. Whether ß-arrestins act independently of G protein-mediated signaling has not been fully elucidated. Studies using genome-editing approaches revealed that whereas G proteins are essential for mitogen-activated protein kinase activation by GPCRs., ß-arrestins play a more prominent role in signal compartmentalization. However, in the absence of G proteins, GPCRs may not activate ß-arrestins, thereby limiting the ability to distinguish G protein from ß-arrestin-mediated signaling events. We used ß2-adrenergic receptor (ß2AR) and its ß2AR-C tail mutant expressed in human embryonic kidney 293 cells wildtype or CRISPR-Cas9 gene edited for Gαs, ß-arrestin1/2, or GPCR kinases 2/3/5/6 in combination with arrestin conformational sensors to elucidate the interplay between Gαs and ß-arrestins in controlling gene expression. We found that Gαs is not required for ß2AR and ß-arrestin conformational changes, ß-arrestin recruitment, and receptor internalization, but that Gαs dictates the GPCR kinase isoforms involved in ß-arrestin recruitment. By RNA-Seq analysis, we found that protein kinase A and mitogen-activated protein kinase gene signatures were activated by stimulation of ß2AR in wildtype and ß-arrestin1/2-KO cells but absent in Gαs-KO cells. These results were validated by re-expressing Gαs in the corresponding KO cells and silencing ß-arrestins in wildtype cells. These findings were extended to cellular systems expressing endogenous levels of ß2AR. Overall, our results support that Gs is essential for ß2AR-promoted protein kinase A and mitogen-activated protein kinase gene expression signatures, whereas ß-arrestins initiate signaling events modulating Gαs-driven nuclear transcriptional activity.

Effect of the Metabotropic Glutamate Receptor Type 5 Negative Allosteric Modulator Dipraglurant on Motor and Non-Motor Symptoms of Parkinson's Disease.

Parkinson's disease (PD) patients suffer not only from the primary motor symptoms of the disease but also from a range of non-motor symptoms (NMS) that cause disability and low quality of life. Excessive glutamate activity in the basal ganglia resulting from degeneration of the nigrostriatal dopamine pathway has been implicated in the motor symptoms, NMS and dyskinesias in PD patients. In this study, we investigated the effects of a selective mGlu5 negative allosteric modulator (NAM), dipraglurant, in a rodent motor symptoms model of PD, but also in models of anxiety, depression and obsessive-compulsive disorder, all of which are among the most prevalent NMS symptoms. Dipraglurant is rapidly absorbed after oral administration, readily crosses the blood-brain barrier, and exhibits a high correlation between plasma concentration and efficacy in behavioral models. In vivo, dipraglurant dose-dependently reduced haloperidol-induced catalepsy, increased punished licks in the Vogel conflict-drinking model, decreased immobility time in the forced swim test, decreased the number of buried marbles in the marble-burying test, but had no effect on rotarod performance or locomotor activity. These findings suggest that dipraglurant may have benefits to address some of the highly problematic comorbid non-motor symptoms of PD, in addition to its antidyskinetic effect demonstrated in PD-LID patients.

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.

The RanBP2/RanGAP1-SUMO complex gates ß-arrestin2 nuclear entry to regulate the Mdm2-p53 signaling axis.

Mdm2 antagonizes the tumor suppressor p53. Targeting the Mdm2-p53 interaction represents an attractive approach for the treatment of cancers with functional p53. Investigating mechanisms underlying Mdm2-p53 regulation is therefore important. The scaffold protein ß-arrestin2 (ß-arr2) regulates tumor suppressor p53 by counteracting Mdm2. ß-arr2 nucleocytoplasmic shuttling displaces Mdm2 from the nucleus to the cytoplasm resulting in enhanced p53 signaling. ß-arr2 is constitutively exported from the nucleus, via a nuclear export signal, but mechanisms regulating its nuclear entry are not completely elucidated. ß-arr2 can be SUMOylated, but no information is available on how SUMO may regulate ß-arr2 nucleocytoplasmic shuttling. While we found ß-arr2 SUMOylation to be dispensable for nuclear import, we identified a non-covalent interaction between SUMO and ß-arr2, via a SUMO interaction motif (SIM), that is required for ß-arr2 cytonuclear trafficking. This SIM promotes association of ß-arr2 with the multimolecular RanBP2/RanGAP1-SUMO nucleocytoplasmic transport hub that resides on the cytoplasmic filaments of the nuclear pore complex. Depletion of RanBP2/RanGAP1-SUMO levels result in defective ß-arr2 nuclear entry. Mutation of the SIM inhibits ß-arr2 nuclear import, its ability to delocalize Mdm2 from the nucleus to the cytoplasm and enhanced p53 signaling in lung and breast tumor cell lines. Thus, a ß-arr2 SIM nuclear entry checkpoint, coupled with active ß-arr2 nuclear export, regulates its cytonuclear trafficking function to control the Mdm2-p53 signaling axis.

Synthetic Lethal Screens Reveal Cotargeting FAK and MEK as a Multimodal Precision Therapy for GNAQ-Driven Uveal Melanoma.

PURPOSE: Uveal melanoma is the most common eye cancer in adults. Approximately 50% of patients with uveal melanoma develop metastatic uveal melanoma (mUM) in the liver, even after successful treatment of the primary lesions. mUM is refractory to current chemo- and immune-therapies, and most mUM patients die within a year. Uveal melanoma is characterized by gain-of-function mutations in GNAQ/GNA11, encoding Gαq proteins. We have recently shown that the Gαq-oncogenic signaling circuitry involves a noncanonical pathway distinct from the classical activation of PLCß and MEK-ERK. GNAQ promotes the activation of YAP1, a key oncogenic driver, through focal adhesion kinase (FAK), thereby identifying FAK as a druggable signaling hub downstream from GNAQ. However, targeted therapies often activate compensatory resistance mechanisms leading to cancer relapse and treatment failure. EXPERIMENTAL DESIGN: We performed a kinome-wide CRISPR-Cas9 sgRNA screen to identify synthetic lethal gene interactions that can be exploited therapeutically. Candidate adaptive resistance mechanisms were investigated by cotargeting strategies in uveal melanoma and mUM in vitro and in vivo experimental systems. RESULTS: sgRNAs targeting the PKC and MEK-ERK signaling pathways were significantly depleted after FAK inhibition, with ERK activation representing a predominant resistance mechanism. Pharmacologic inhibition of MEK and FAK showed remarkable synergistic growth-inhibitory effects in uveal melanoma cells and exerted cytotoxic effects, leading to tumor collapse in uveal melanoma xenograft and liver mUM models in vivo. CONCLUSIONS: Coupling the unique genetic landscape of uveal melanoma with the power of unbiased genetic screens, our studies reveal that FAK and MEK-ERK cotargeting may provide a new network-based precision therapeutic strategy for mUM treatment.See related commentary by Harbour, p. 2967.

A new inhibitor of the ß-arrestin/AP2 endocytic complex reveals interplay between GPCR internalization and signalling.

In addition to G protein-coupled receptor (GPCR) desensitization and endocytosis, ß-arrestin recruitment to ligand-stimulated GPCRs promotes non-canonical signalling cascades. Distinguishing the respective contributions of ß-arrestin recruitment to the receptor and ß-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 ß-arrestin and the ß2-adaptin subunit of the clathrin adaptor protein AP2 without interfering with the formation of receptor/ß-arrestin complexes. This selective ß-arrestin/ß2-adaptin inhibitor (Barbadin) blocks agonist-promoted endocytosis of the prototypical ß2-adrenergic (ß2AR), V2-vasopressin (V2R) and angiotensin-II type-1 (AT1R) receptors, but does not affect ß-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 ß2AR, supporting the concept of ß-arrestin/AP2-dependent signalling for both G protein-dependent and -independent pathways.

Receptor sequestration in response to ß-arrestin-2 phosphorylation by ERK1/2 governs steady-state levels of GPCR cell-surface expression.

MAPKs are activated in response to G protein-coupled receptor (GPCR) stimulation and play essential roles in regulating cellular processes downstream of these receptors. However, very little is known about the reciprocal effect of MAPK activation on GPCRs. To investigate possible crosstalk between the MAPK and GPCRs, we assessed the effect of ERK1/2 on the activity of several GPCR family members. We found that ERK1/2 activation leads to a reduction in the steady-state cell-surface expression of many GPCRs because of their intracellular sequestration. This subcellular redistribution resulted in a global dampening of cell responsiveness, as illustrated by reduced ligand-mediated G-protein activation and second-messenger generation as well as blunted GPCR kinases and ß-arrestin recruitment. This ERK1/2-mediated regulatory process was observed for GPCRs that can interact with ß-arrestins, such as type-2 vasopressin, type-1 angiotensin, and CXC type-4 chemokine receptors, but not for the prostaglandin F receptor that cannot interact with ß-arrestin, implicating this scaffolding protein in the receptor's subcellular redistribution. Complementation experiments in mouse embryonic fibroblasts lacking ß-arrestins combined with in vitro kinase assays revealed that ß-arrestin-2 phosphorylation on Ser14 and Thr276 is essential for the ERK1/2-promoted GPCR sequestration. This previously unidentified regulatory mechanism was observed after constitutive activation as well as after receptor tyrosine kinase- or GPCR-mediated activation of ERK1/2, suggesting that it is a central node in the tonic regulation of cell responsiveness to GPCR stimulation, acting both as an effector and a negative regulator.

A biosensor to monitor dynamic regulation and function of tumour suppressor PTEN in living cells.

Tumour suppressor PTEN is a phosphatase that negatively regulates the PI3K/AKT pathway. The ability to directly monitor PTEN conformation and function in a rapid, sensitive manner is a key step towards developing anti-cancer drugs aimed at enhancing or restoring PTEN-dependent pathways. Here we developed an intramolecular bioluminescence resonance energy transfer (BRET)-based biosensor, capable of detecting signal-dependent PTEN conformational changes in live cells. The biosensor retains intrinsic properties of PTEN, enabling structure-function and kinetic analyses. BRET shifts, indicating conformational change, were detected following mutations that disrupt intramolecular PTEN interactions, promoting plasma membrane targeting and also following physiological PTEN activation. Using the biosensor as a reporter, we uncovered PTEN activation by several G protein-coupled receptors, previously unknown as PTEN regulators. Trastuzumab, used to treat ERBB2-overexpressing breast cancers also elicited activation-associated PTEN conformational rearrangement. We propose the biosensor can be used to identify pathways regulating PTEN or molecules that enhance its anti-tumour activity.