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To build a just, equitable, and diverse academy, scientists and institutions must address systemic barriers that sex and gender minorities face. This Commentary summarizes (1) critical context informing the contemporary oppression of transgender people, (2) how this shapes extant research on sex and gender, and (3) actions to build an inclusive and rigorous academy for all.
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Minorías Sexuales y de Género , Personas Transgénero , Masculino , Femenino , Humanos , Identidad de GéneroRESUMEN
Synthetic and chimeric receptors capable of recognizing and responding to user-defined antigens have enabled "smart" therapeutics based on engineered cells. These cell engineering tools depend on antigen sensors which are most often derived from antibodies. Advances in the de novo design of proteins have enabled the design of protein binders with the potential to target epitopes with unique properties and faster production timelines compared to antibodies. Building upon our previous work combining a de novo-designed minibinder of the Spike protein of SARS-CoV-2 with the synthetic receptor synNotch (SARSNotch), we investigated whether minibinders can be readily adapted to a diversity of cell engineering tools. We show that the Spike minibinder LCB1 easily generalizes to a next-generation proteolytic receptor SNIPR that performs similarly to our previously reported SARSNotch. LCB1-SNIPR successfully enables the detection of live SARS-CoV-2, an improvement over SARSNotch which can only detect cell-expressed Spike. To test the generalizability of minibinders to diverse applications, we tested LCB1 as an antigen sensor for a chimeric antigen receptor (CAR). LCB1-CAR enabled CD8+ T cells to cytotoxically target Spike-expressing cells. Our findings suggest that minibinders represent a novel class of antigen sensors that have the potential to dramatically expand the sensing repertoire of cell engineering tools.
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Vesicle fusion at the plasma membrane is critical for releasing hormones and neurotransmitters and for delivering the cognate G protein-coupled receptors (GPCRs) to the cell surface. The SNARE fusion machinery that releases neurotransmitters has been well characterized. In contrast, the fusion machinery that delivers GPCRs is still unknown. Here, using high-speed multichannel imaging to simultaneously visualize receptors and v-SNAREs in real time in individual fusion events, we identify VAMP2 as a selective v-SNARE for GPCR delivery. VAMP2 was preferentially enriched in vesicles that mediate the surface delivery of µ opioid receptor (MOR), but not other cargos, and was required selectively for MOR recycling. Interestingly, VAMP2 did not show preferential localization on MOR-containing endosomes, suggesting that v-SNAREs are copackaged with specific cargo into separate vesicles from the same endosomes. Together, our results identify VAMP2 as a cargo-selective v-SNARE and suggest that surface delivery of specific GPCRs is mediated by distinct fusion events driven by distinct SNARE complexes.
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Fusión de Membrana , Receptores Acoplados a Proteínas G , Proteínas SNARE , Proteína 2 de Membrana Asociada a Vesículas , Membrana Celular/metabolismo , Neurotransmisores/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Proteínas SNARE/metabolismo , Proteína 2 de Membrana Asociada a Vesículas/genética , Proteína 2 de Membrana Asociada a Vesículas/metabolismoRESUMEN
Postdoctoral training enables research independence and professional readiness. National reports have emphasized professional development as a critical component of this training period. In response, many institutions are establishing transferable skills training workshops for postdocs; however, the lack of structured programs and an absence of methods to assess outcomes beyond participant satisfaction surveys are critical gaps in postdoctoral training. To address these shortcomings, we took the approach of structured programming and developed a method for controlled assessment of outcomes. Our program You3 (You, Your Team, Your Project), co-designed by postdoctoral fellows, focused on discussing specific management and leadership skills agnostic of ultimate career path(s) in a structured manner. We then measured outcomes in a controlled manner, by systematically comparing perceived knowledge and growth as indicators of awareness and confidence in participants against that of non-participants as the control group. You3 participants self-rated greater growth in targeted competencies compared to non-participants independent of the number of years of training. This growth was shown by multiple criteria including self-reporting and associative analysis. Correspondingly, You3 participants reported greater knowledge in 75% of the modules when compared to controls. These data indicate that structured learning, where postdocs commit to a curriculum via a cohort-structure, leads to positive outcomes and provides a framework for programs to assess outcomes in a rigorous manner.
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Curriculum , Educación Profesional , Movilidad Laboral , Humanos , Conocimiento , Liderazgo , Investigadores , AutoinformeRESUMEN
The prevailing model for the variety in drug responses is that different drugs stabilize distinct active states of their G protein-coupled receptor (GPCR) targets, allowing coupling to different effectors. However, whether the same ligand generates different GPCR active states based on the immediate environment of receptors is not known. Here we address this question using spatially resolved imaging of conformational biosensors that read out distinct active conformations of the δ-opioid receptor (DOR), a physiologically relevant GPCR localized to Golgi and the surface in neuronal cells. We have shown that Golgi and surface pools of DOR both inhibit cAMP, but engage distinct conformational biosensors in response to the same ligand in rat neuroendocrine cells. Further, DOR recruits arrestins on the surface but not on the Golgi. Our results suggest that the local environment determines the active states of receptors for any given drug, allowing GPCRs to couple to different effectors at different subcellular locations.
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Benzamidas/farmacología , Membrana Celular/efectos de los fármacos , Aparato de Golgi/efectos de los fármacos , Neuronas/efectos de los fármacos , Piperazinas/farmacología , Receptores Opioides delta/agonistas , Animales , Técnicas Biosensibles , Membrana Celular/metabolismo , AMP Cíclico/metabolismo , Aparato de Golgi/metabolismo , Ligandos , Microscopía Fluorescente , Neuronas/metabolismo , Células PC12 , Conformación Proteica , Ratas , Receptores Opioides delta/química , Receptores Opioides delta/genética , Receptores Opioides delta/metabolismo , Relación Estructura-Actividad , beta-Arrestinas/metabolismoRESUMEN
The COVID-19 pandemic has demonstrated the need for exploring different diagnostic and therapeutic modalities to tackle future viral threats. In this vein, we propose the idea of sentinel cells, cellular biosensors capable of detecting viral antigens and responding to them with customizable responses. Using SARS-CoV-2 as a test case, we developed a live cell sensor (SARSNotch) using a de novo-designed protein binder against the SARS-CoV-2 Spike protein. SARSNotch is capable of driving custom genetically-encoded payloads in immortalized cell lines or in primary T lymphocytes in response to purified SARS-CoV-2 Spike or in the presence of Spike-expressing cells. Furthermore, SARSNotch is functional in a cellular system used in directed evolution platforms for development of better binders or therapeutics. In keeping with the rapid dissemination of scientific knowledge that has characterized the incredible scientific response to the ongoing pandemic, we extend an open invitation for others to make use of and improve SARSNotch sentinel cells in the hopes of unlocking the potential of the next generation of smart antiviral therapeutics.
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G protein-coupled receptor (GPCR) kinases (GRKs) play a key role in terminating signals initiated by agonist-bound GPCRs. However, chronic stimulation of GPCRs, such as that which occurs during heart failure, leads to the overexpression of GRKs and maladaptive downregulation of GPCRs on the cell surface. We previously reported the discovery of potent and selective families of GRK inhibitors based on either the paroxetine or GSK180736A scaffold. A new inhibitor, CCG258747, which is based on paroxetine, demonstrates increased potency against the GRK2 subfamily and favorable pharmacokinetic parameters in mice. CCG258747 and the closely related compound CCG258208 also showed high selectivity for the GRK2 subfamily in a kinome panel of 104 kinases. We developed a cell-based assay to screen the ability of CCG258747 and 10 other inhibitors with different GRK subfamily selectivities and with either the paroxetine or GSK180736A scaffold to block internalization of the µ-opioid receptor (MOR). CCG258747 showed the best efficacy in blocking MOR internalization among the compounds tested. Furthermore, we show that compounds based on paroxetine had much better cell permeability than those based on GSK180736A, which explains why GSK180736A-based inhibitors, although being potent in vitro, do not always show efficacy in cell-based assays. This study validates the paroxetine scaffold as the most effective for GRK inhibition in living cells, confirming that GRK2 predominantly drives internalization of MOR in the cell lines we tested and underscores the utility of high-resolution cell-based assays for assessment of compound efficacy. SIGNIFICANCE STATEMENT: G protein-coupled receptor kinases (GRKs) are attractive targets for developing therapeutics for heart failure. We have synthesized a new GRK2 subfamily-selective inhibitor, CCG258747, which has nanomolar potency against GRK2 and excellent selectivity over other kinases. A live-cell receptor internalization assay was used to test the ability of GRK2 inhibitors to impart efficacy on a GRK-dependent process in cells. Our data indicate that CCG258747 blocked the internalization of the µ-opioid receptor most efficaciously because it has the ability to cross cell membranes.
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Indazoles/química , Paroxetina/química , Pirimidinas/química , Receptores Opioides mu/antagonistas & inhibidores , Receptores Opioides mu/metabolismo , Animales , Western Blotting , Permeabilidad de la Membrana Celular , Cristalografía por Rayos X , Femenino , Células HEK293 , Humanos , Indazoles/farmacología , Ratones , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/metabolismo , Estructura Molecular , Pirimidinas/farmacologíaRESUMEN
Membrane trafficking and receptor signaling are two fundamental cellular processes that interact constantly. Although how trafficking regulates signaling is well studied, how signaling pathways regulate trafficking is less well understood. Here, we use the mu opioid receptor (MOR), the primary target for opioid analgesics, to define a signaling pathway that dynamically regulates postendocytic receptor recycling. By directly visualizing individual MOR recycling events, we show that agonist increases MOR recycling. Inhibition of G ßγ, phospholipase C, or protein kinase C mimicked agonist removal, whereas activation of G ßγ increased recycling even after agonist removal. Phosphorylation of serine 363 on the C-terminal tail of MOR was required and sufficient for agonist-mediated regulation of MOR recycling. Our results identify a feedback loop that regulates MOR recycling via G ßγ , protein kinase C, and receptor phosphorylation. This could serve as a general model for how signaling regulates postendocytic trafficking of G protein-coupled receptors. SIGNIFICANCE STATEMENT: G protein-coupled receptor (GPCR) localization in the endosome is being increasingly recognized as an important and distinct component of GPCR signaling and physiology. This study identifies a G protein-dependent and protein kinase C-dependent signaling pathway that dynamically regulates the endosomal localization of the mu opioid receptor, the primary target of opioid analgesics and abused drugs. This pathway could provide a mechanism to manipulate spatial encoding of opioid signaling and physiology.
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Endocitosis/fisiología , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Proteína Quinasa C/metabolismo , Receptores Opioides mu/metabolismo , Analgésicos Opioides/farmacología , Endocitosis/efectos de los fármacos , Encefalina Ala(2)-MeFe(4)-Gli(5)/farmacología , Células HEK293 , Humanos , Fosforilación/efectos de los fármacos , Fosforilación/fisiologíaRESUMEN
Several GPCRs, including receptors previously thought to signal primarily from the cell surface, have been recently shown to signal from many intracellular compartments. This raises the idea that signaling by any given receptor is spatially encoded in the cell, with distinct sites of signal origin dictating distinct downstream consequences. We will discuss recent developments that address this novel facet of GPCR physiology, focusing on the spatial segregation of signaling from the cell surface, endosomes, and the Golgi by receptors relevant to the nervous system.
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Sistema Nervioso/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal , Animales , Membrana Celular/metabolismo , Endosomas/metabolismo , Aparato de Golgi/metabolismo , Humanos , Transporte de ProteínasRESUMEN
BACKGROUND AND PURPOSE: The δ-opioid receptor is an emerging target for the management of chronic pain and depression. Biased signalling, the preferential activation of one signalling pathway over another downstream of δ-receptors, may generate better therapeutic profiles. BMS 986187 is a positive allosteric modulator of δ-receptors. Here, we ask if BMS 986187 can directly activate the receptor from an allosteric site, without an orthosteric ligand, and if a signalling bias is generated. EXPERIMENTAL APPROACH: We used several clonal cell lines expressing δ-receptors, to assess effects of BMS 986187 on events downstream of δ-receptors by measuring G-protein activation, ß-arrestin 2 recruitment, receptor phosphorylation, loss of surface receptor expression, ERK1/ERK2 phosphorylation, and receptor desensitization. KEY RESULTS: BMS 986187 is a G protein biased allosteric agonist, relative to ß-arrestin 2 recruitment. Despite showing direct and potent G protein activation, BMS 986187 has a low potency to recruit ß-arrestin 2. This appears to reflect the inability of BMS 986187 to elicit any significant receptor phosphorylation, consistent with low receptor internalization and a slower onset of desensitization, compared with the full agonist SNC80. CONCLUSIONS AND IMPLICATIONS: This is the first evidence of biased agonism mediated through direct binding to an allosteric site on an opioid receptor, without a ligand at the orthosteric site. Our data suggest that agonists targeting δ-receptors, or indeed any GPCR, through allosteric sites may be a novel way to promote signalling bias and thereby potentially produce a more specific pharmacology than can be observed by activation via the orthosteric site.
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Proteínas de Unión al GTP/agonistas , Receptores Opioides delta/metabolismo , Xantonas/farmacología , Sitio Alostérico , Animales , Células CHO , Cricetulus , Proteínas de Unión al GTP/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Células HEK293 , Humanos , Masculino , RatonesRESUMEN
The trafficking of G protein coupled-receptors (GPCRs) is one of the most exciting areas in cell biology because of recent advances demonstrating that GPCR signaling is spatially encoded. GPCRs, acting in a diverse array of physiological systems, can have differential signaling consequences depending on their subcellular localization. At the plasma membrane, GPCR organization could fine-tune the initial stages of receptor signaling by determining the magnitude of signaling and the type of effectors to which receptors can couple. This organization is mediated by the lipid composition of the plasma membrane, receptor-receptor interactions, and receptor interactions with intracellular scaffolding proteins. GPCR organization is subsequently changed by ligand binding and the regulated endocytosis of these receptors. Activated GPCRs can modulate the dynamics of their own endocytosis through changing clathrin-coated pit dynamics, and through the scaffolding adaptor protein ß-arrestin. This endocytic regulation has signaling consequences, predominantly through modulation of the MAPK cascade. This review explores what is known about receptor sorting at the plasma membrane, protein partners that control receptor endocytosis, and the ways in which receptor sorting at the plasma membrane regulates downstream trafficking and signaling.
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Membrana Celular/metabolismo , Endocitosis , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal , Animales , HumanosRESUMEN
Functional selectivity at the µ opioid receptor (µR), a prototypical G-protein-coupled receptor that is a physiologically relevant target for endogenous opioid neurotransmitters and analgesics, has been a major focus for drug discovery in the recent past. Functional selectivity is a cumulative effect of the magnitudes of individual signaling pathways, e.g., the Gαi-mediated and the arrestin-mediated pathways for µR. The present work tested the hypothesis that lifetimes of agonist-induced receptor-arrestin clusters at the cell surface control the magnitude of arrestin signaling, and therefore functional selectivity, at µR. We show that endomorphin-2 (EM2), an arrestin-biased ligand for µR, lengthens surface lifetimes of receptor-arrestin clusters significantly compared with morphine. The lengthening of lifetimes required two specific leucines on the C-terminal tail of µR. Mutation of these leucines to alanines decreased the magnitude of arrestin-mediated signaling by EM2 without affecting G-protein signaling, suggesting that lengthened endocytic lifetimes were required for arrestin-biased signaling by EM2. Lengthening surface lifetimes by pharmacologically slowing endocytosis was sufficient to increase arrestin-mediated signaling by both EM2 and the clinically relevant agonist morphine. Our findings show that distinct ligands can leverage specific sequence elements on µR to regulate receptor endocytic lifetimes and the magnitude of arrestin-mediated signaling, and implicate these sequences as important determinants of functional selectivity in the opioid system.