The current dopamine (DA) hypothesis of schizophrenia postulates striatal hyperdopaminergia and cortical hypodopaminergia. Although partial agonists at DA D2 receptors (D2Rs), like aripiprazole, were developed to simultaneously target both phenomena, they do not effectively improve cortical dysfunction. In this study, we investigate the potential for newly developed β-arrestin2 (βarr2)-biased D2R partial agonists to simultaneously target hyperand hypodopaminergia. Using neuron-specific βarr2-KO mice, we show that the antipsychotic-like effects of a βarr2-biased D2R ligand are driven through both striatal antagonism and cortical agonism of D2R-βarr2 signaling. Furthermore, βarr2-biased D2R agonism enhances firing of cortical fast-spiking interneurons. This enhanced cortical agonism of the biased ligand can be attributed to a lack of G-protein signaling and elevated expression of βarr2 and G proteincoupled receptor (GPCR) kinase 2 in the cortex versus the striatum. Therefore, we propose that βarr2-biased D2R ligands that exert region-selective actions could provide a path to develop more effective antipsychotic therapies.arrestin | antipsychotics | biased signaling | dopamine D2R | fast-spiking interneurons G protein-coupled receptors (GPCRs) represent the largest family of receptors in the human genome and are one of the most common targets of pharmaceutical drugs (1, 2). Upon ligand binding, GPCRs activate downstream G protein-dependent signaling pathways followed by phosphorylation of the receptor by G protein-coupled receptor kinases (GRKs) (3). Phosphorylation enhances association of the GPCR with β-arrestins (βarrs), and this combined process mediates desensitization of G-protein signaling (4) and internalization of GPCRs (5-7). Two isoforms of βarrs, βarr1 and βarr2, are widely coexpressed in most tissues in mammals and are 80% identical, but they can have either overlapping or distinct functions (8, 9). It is now firmly established that GPCRs activate downstream signaling pathways through not only canonical G-protein pathways but also, the ability of βarrs to scaffold distinct intracellular signaling complexes (10-12). Elucidation of these distinct G-protein and βarr signaling pathways has provided support for the concept of functional selectivity or biased signaling, wherein each signaling pathway has the ability to mediate distinct physiological responses (13). There are now several physiologically relevant examples of selective engagement of signaling pathways or selective GPCR ligands that target these different signaling pathways (13-15). Therefore, leveraging the concept of GPCR functional selectivity holds promise for the development of more selective therapeutic approaches. Dopamine (DA) is a catecholamine neurotransmitter that has been implicated in movement, reward, and cognition (16-19) as well as CNS disorders, such as schizophrenia, attention deficit hyperactivity disorder, Parkinson's disease, and obsessive-compulsive disorder (20-23). DA mediates its effects via GPCRs belonging to two major ...

The neuromodulator dopamine signals through the dopamine D2 receptor (D 2 R) to modulate central nervous system functions through diverse signal transduction pathways. D 2 R is a prominent target for drug treatments in disorders where dopamine function is aberrant, such as schizophrenia. D 2 R signals through distinct G-protein and β-arrestin pathways, and drugs that are functionally selective for these pathways could have improved therapeutic potential. How D 2 R signals through the two pathways is still not well defined, and efforts to elucidate these pathways have been hampered by the lack of adequate tools for assessing the contribution of each pathway independently. To address this, Evolutionary Trace was used to produce D 2 R mutants with strongly biased signal transduction for either the G-protein or β-arrestin interactions. These mutants were used to resolve the role of G proteins and β-arrestins in D 2 R signaling assays. The results show that D 2 R interactions with the two downstream effectors are dissociable and that G-protein signaling accounts for D 2 R canonical MAP kinase signaling cascade activation, whereas β-arrestin only activates elements of this cascade under certain conditions. Nevertheless, when expressed in mice in GABAergic medium spiny neurons of the striatum, the β-arrestin-biased D 2 R caused a significant potentiation of amphetamine-induced locomotion, whereas the G proteinbiased D 2 R had minimal effects. The mutant receptors generated here provide a molecular tool set that should enable a better definition of the individual roles of G-protein and β-arrestin signaling pathways in D 2 R pharmacology, neurobiology, and associated pathologies.

Biased agonism, the ability of different ligands for the same receptor to selectively activate some signaling pathways while blocking others, is now an established paradigm for G protein-coupled receptor signaling. One group of receptors in which endogenous bias is critical is the chemokine system, consisting of over 50 ligands and 20 receptors that bind one another with significant promiscuity. We have previously demonstrated that ligands for the same receptor can cause biased signaling responses. The goal of this study was to identify mechanisms that could underlie biased signaling between different receptor splice variants. The C-X-C motif chemokine receptor 3 (CXCR3) has two splice variants, CXCR3A and CXCR3B, which differ by 51 amino acids at its N-terminus. Consistent with an earlier study, we found that C-X-C motif chemokine ligands 4, 9, 10, and 11 all activated G at CXCR3A, while at CXCR3B these ligands demonstrated no measurable G or G activity.-arrestin (arr) was recruited at a reduced level to CXCR3B relative to CXCR3A, which was also associated with differences in arr2 conformation.arr2 recruitment to CXCR3A was attenuated by both G protein receptor kinase (GRK) 2/3 and GRK5/6 knockdown, while only GRK2/3 knockdown blunted recruitment to CXCR3B. Extracellular regulated kinase 1/2 phosphorylation downstream from CXCR3A and CXCR3B was increased and decreased, respectively, by arr1/2 knockout. The splice variants also differentially activated transcriptional reporters. These findings demonstrate that differential splicing of CXCR3 results in biased responses associated with distinct patterns ofarr conformation and recruitment. Differential splicing may serve as a common mechanism for generating biased signaling and provides insights into how chemokine receptor signaling can be modulated post-transcriptionally.

The COVID-19 pandemic has created a high demand on personal protective equipment, including disposable N95 masks. Given the need for mask reuse, we tested the feasibility of vaporized hydrogen peroxide (VHP), ultraviolet light (UV), and ethanol decontamination strategies on N95 mask integrity and the ability to remove the infectious potential of SARS-CoV-2. FIT test data showed functional degradation by both ethanol and UV decontamination to different degrees. VHP treated masks showed no significant change in function after two treatments. We also report a single SARS-CoV-2 virucidal experiment using Vero E6 cell infection. We hope our data will guide further research for evidenced-based decisions for disposable N95 mask reuse and help protect caregivers from SARS-CoV-2 and other pathogens.

Introduction: The COVID-19 pandemic has created a high demand on personal protective equipment, including disposable N95 masks. Given the need for mask reuse, we tested the feasibility of vaporized hydrogen peroxide (VHP), ultraviolet light (UV), and ethanol decontamination strategies on N95 mask integrity and the ability to remove the infectious potential of SARS-CoV-2. Methods: Disposable N95 masks, including medical grade (1860, 1870+) and industrial grade (8511) masks, were treated by vaporized hydrogen peroxide (VHP), ultraviolet light (UV), and ethanol decontamination. Mask degradation was tested using a quantitative respirator fit testing. Pooled clinical samples of SARS-CoV-2 were applied to mask samples, treated and and then either sent immediately for real-time reverse transcriptase–polymerase chain reaction (RT-PCR) or incubated with Vero E6 cells to assess for viracidal effect. Results: Both ethanol and UV decontamination showed functional degradation to different degrees while VHP treatment showed no significant change after 2 treatments.We also report a single SARS-CoV-2 virucidal experiment using Vero E6 cell infection in which only ethanol treatment eliminated detectable SARS-CoV-2 RNA. Conclusions: We hope our data will guide further research for evidenced-based decisions for disposable N95 mask reuse and help protect caregivers from SARS-CoV-2 and other pathogens.

Heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) are common drug targets and canonically couple to specific Gα protein subtypes and β-arrestin adaptor proteins. G protein- and β-arrestin-mediated signaling have been considered separable. We show GPCRs promote a direct interaction between Gαi protein subtype family members and β-arrestins, regardless of their canonical Gαi protein subtype coupling. Gαi:β-arrestin complexes bound extracellular signal-regulated kinase (ERK) and their disruption impaired both ERK activation and cell migration, consistent with β-arrestins requiring a functional interaction with Gαi for certain signaling events. These results introduce a GPCR signaling mechanism distinct from canonical G protein activation in which GPCRs cause the formation of Gαi:β-arrestin signaling complexes.

The dopamine D2 receptor (D2R) is a G protein-coupled receptor (GPCR) that is critical for many central nervous system functions. The D2R carries out these functions by signaling through two transducers: G proteins and b-arrestins (barrs). Selectively engaging either the G protein or barr pathway may be a way to improve drugs targeting GPCRs. The current model of GPCR signal transduction posits a chain of events where G protein activation ultimately leads to barr recruitment. GPCR kinases (GRKs), which are regulated by G proteins and whose kinase action facilitates barr recruitment, bridge these pathways. Therefore barr recruitment appears to be intimately tied to G protein activation via GRKs. Here, we sought to understand how GRK2 action at the D2R would be disrupted when G protein activation is eliminated and the effect of this on barr recruitment. We used two recently developed biased D2R mutants that can preferentially interact either with G proteins or barrs as well as a barr-biased D2R ligand, UNC9994. With these functionally selective tools, we investigated the mechanism whereby the barr-preferring D2R achieves barr pathway activation in the complete absence of G protein activation. We describe how direct, G protein-independent recruitment of GRK2 drives interactions at the barr-preferring D2R and also contributes to barr recruitment at the WT D2R. Additionally, we found an additive interaction between the barr-preferring D2R mutant and UNC9994. These results reveal that the D2R can directly recruit GRK2 without G protein activation and that this mechanism may have relevance to achieving barr-biased signaling.

BackgroundMembrane proteins regulate a diversity of physiological processes and are the most successful class of targets in drug discovery. However, the number of targets adequately explored in chemical space and the limited resources available for screening are significant problems shared by drug-discovery centers and small laboratories. Therefore, a low-cost and universally applicable screen for membrane protein trafficking was developed.ResultsThis high-throughput screen (HTS), termed IRFAP-HTS, utilizes the recently described MarsCy1-fluorogen activating protein and the near-infrared and membrane impermeant fluorogen SCi1. The cell surface expression of MarsCy1 epitope-tagged receptors can be visualized by simple addition of SCi1. User-friendly, rapid, and quantitative detection occurs on a standard infrared western-blotting scanner. The reliability and robustness of IRFAP-HTS was validated by confirming human vasopressin-2 receptor and dopamine receptor-2 trafficking in response to agonist or antagonist. The IRFAP-HTS screen was deployed against the leucine-rich G protein-coupled receptor-5 (Lgr5). Lgr5 is expressed in stem cells, modulates Wnt/ß-catenin signaling, and is therefore a promising drug target. However, small molecule modulators have yet to be reported. The constitutive internalization of Lgr5 appears to be one primary mode through which its function is regulated. Therefore, IRFAP-HTS was utilized to screen 11,258 FDA-approved and drug-like small molecules for those that antagonize Lgr5 internalization. Glucocorticoids were found to potently increase Lgr5 expression at the plasma membrane.ConclusionThe IRFAP-HTS platform provides a versatile solution for screening more targets with fewer resources. Using only a standard western-blotting scanner, we were able to screen 5,000 compounds per hour in a robust and quantitative assay. Multi-purposing standardly available laboratory equipment eliminates the need for idiosyncratic and more expensive high-content imaging systems. The modular and user-friendly IRFAP-HTS is a significant departure from current screening platforms. Small laboratories will have unprecedented access to a robust and reliable screening platform and will no longer be limited by the esoteric nature of assay development, data acquisition, and post-screening analysis. The discovery of glucocorticoids as modulators for Lgr5 trafficking confirms that IRFAP-HTS can accelerate drug-discovery and drug-repurposing for even the most obscure targets.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-015-0216-3) contains supplementary material, which is available to authorized users.