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1.
Cell ; 185(24): 4560-4573.e19, 2022 11 23.
Artículo en Inglés | MEDLINE | ID: mdl-36368322

RESUMEN

Binding of arrestin to phosphorylated G protein-coupled receptors (GPCRs) is crucial for modulating signaling. Once internalized, some GPCRs remain complexed with ß-arrestins, while others interact only transiently; this difference affects GPCR signaling and recycling. Cell-based and in vitro biophysical assays reveal the role of membrane phosphoinositides (PIPs) in ß-arrestin recruitment and GPCR-ß-arrestin complex dynamics. We find that GPCRs broadly stratify into two groups, one that requires PIP binding for ß-arrestin recruitment and one that does not. Plasma membrane PIPs potentiate an active conformation of ß-arrestin and stabilize GPCR-ß-arrestin complexes by promoting a fully engaged state of the complex. As allosteric modulators of GPCR-ß-arrestin complex dynamics, membrane PIPs allow for additional conformational diversity beyond that imposed by GPCR phosphorylation alone. For GPCRs that require membrane PIP binding for ß-arrestin recruitment, this provides a mechanism for ß-arrestin release upon translocation of the GPCR to endosomes, allowing for its rapid recycling.


Asunto(s)
Arrestinas , Fosfatidilinositoles , beta-Arrestinas/metabolismo , Fosfatidilinositoles/metabolismo , Arrestinas/metabolismo , beta-Arrestina 1/metabolismo , Receptores Acoplados a Proteínas G/metabolismo
2.
J Biol Chem ; 299(11): 105293, 2023 11.
Artículo en Inglés | MEDLINE | ID: mdl-37774973

RESUMEN

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.


Asunto(s)
Proteínas de Unión al GTP , Regulación de la Expresión Génica , Receptores Adrenérgicos beta 2 , beta-Arrestinas , Humanos , beta-Arrestina 1/genética , beta-Arrestina 1/metabolismo , Arrestina beta 2/genética , Arrestina beta 2/metabolismo , beta-Arrestinas/genética , beta-Arrestinas/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Regulación de la Expresión Génica/genética , Proteínas de Unión al GTP/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosforilación , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 2/metabolismo , Células HEK293 , Subunidades alfa de la Proteína de Unión al GTP/genética , Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Estructura Terciaria de Proteína , Isoformas de Proteínas , Activación Enzimática/genética
3.
Nature ; 557(7705): 381-386, 2018 05.
Artículo en Inglés | MEDLINE | ID: mdl-29720660

RESUMEN

ß-arrestins are critical regulator and transducer proteins for G-protein-coupled receptors (GPCRs). ß-arrestin is widely believed to be activated by forming a stable and stoichiometric GPCR-ß-arrestin scaffold complex, which requires and is driven by the phosphorylated tail of the GPCR. Here we demonstrate a distinct and additional mechanism of ß-arrestin activation that does not require stable GPCR-ß-arrestin scaffolding or the GPCR tail. Instead, it occurs through transient engagement of the GPCR core, which destabilizes a conserved inter-domain charge network in ß-arrestin. This promotes capture of ß-arrestin at the plasma membrane and its accumulation in clathrin-coated endocytic structures (CCSs) after dissociation from the GPCR, requiring a series of interactions with membrane phosphoinositides and CCS-lattice proteins. ß-arrestin clustering in CCSs in the absence of the upstream activating GPCR is associated with a ß-arrestin-dependent component of the cellular ERK (extracellular signal-regulated kinase) response. These results delineate a discrete mechanism of cellular ß-arrestin function that is activated catalytically by GPCRs.


Asunto(s)
Receptores Acoplados a Proteínas G/metabolismo , beta-Arrestinas/metabolismo , Animales , Biocatálisis , Células COS , Membrana Celular/metabolismo , Chlorocebus aethiops , Células HEK293 , Humanos , Fosfatidilinositoles/metabolismo , Transporte de Proteínas , Receptores Acoplados a Proteínas G/química , beta-Arrestinas/química
4.
Elife ; 112022 10 17.
Artículo en Inglés | MEDLINE | ID: mdl-36250629

RESUMEN

ß-Arrestins are master regulators of cellular signaling that operate by desensitizing ligand-activated G-protein-coupled receptors (GPCRs) at the plasma membrane and promoting their subsequent endocytosis. The endocytic activity of ß-arrestins is ligand dependent, triggered by GPCR binding, and increasingly recognized to have a multitude of downstream signaling and trafficking consequences that are specifically programmed by the bound GPCR. However, only one biochemical 'mode' for GPCR-mediated triggering of the endocytic activity is presently known - displacement of the ß-arrestin C-terminus (CT) to expose clathrin-coated pit-binding determinants that are masked in the inactive state. Here, we revise this view by uncovering a second mode of GPCR-triggered endocytic activity that is independent of the ß-arrestin CT and, instead, requires the cytosolic base of the ß-arrestin C-lobe (CLB). We further show each of the discrete endocytic modes is triggered in a receptor-specific manner, with GPCRs that bind ß-arrestin transiently ('class A') primarily triggering the CLB-dependent mode and GPCRs that bind more stably ('class B') triggering both the CT and CLB-dependent modes in combination. Moreover, we show that different modes have opposing effects on the net signaling output of receptors - with the CLB-dependent mode promoting rapid signal desensitization and the CT-dependent mode enabling prolonged signaling. Together, these results fundamentally revise understanding of how ß-arrestins operate as efficient endocytic adaptors while facilitating diversity and flexibility in the control of cell signaling.


Asunto(s)
Arrestinas , Transducción de Señal , beta-Arrestinas/metabolismo , Arrestinas/metabolismo , Ligandos , Transducción de Señal/fisiología , Receptores Acoplados a Proteínas G/metabolismo , Arrestina/metabolismo
5.
Science ; 370(6523): 1473-1479, 2020 12 18.
Artículo en Inglés | MEDLINE | ID: mdl-33154106

RESUMEN

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus enters host cells via an interaction between its Spike protein and the host cell receptor angiotensin-converting enzyme 2 (ACE2). By screening a yeast surface-displayed library of synthetic nanobody sequences, we developed nanobodies that disrupt the interaction between Spike and ACE2. Cryo-electron microscopy (cryo-EM) revealed that one nanobody, Nb6, binds Spike in a fully inactive conformation with its receptor binding domains locked into their inaccessible down state, incapable of binding ACE2. Affinity maturation and structure-guided design of multivalency yielded a trivalent nanobody, mNb6-tri, with femtomolar affinity for Spike and picomolar neutralization of SARS-CoV-2 infection. mNb6-tri retains function after aerosolization, lyophilization, and heat treatment, which enables aerosol-mediated delivery of this potent neutralizer directly to the airway epithelia.


Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Anticuerpos de Dominio Único/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Enzima Convertidora de Angiotensina 2/química , Enzima Convertidora de Angiotensina 2/inmunología , Animales , Anticuerpos Neutralizantes/química , Anticuerpos Antivirales/química , Afinidad de Anticuerpos , Chlorocebus aethiops , Microscopía por Crioelectrón , Humanos , Pruebas de Neutralización , Unión Proteica , Estabilidad Proteica , Anticuerpos de Dominio Único/química , Glicoproteína de la Espiga del Coronavirus/química , Células Vero
6.
bioRxiv ; 2020 Aug 17.
Artículo en Inglés | MEDLINE | ID: mdl-32817938

RESUMEN

Without an effective prophylactic solution, infections from SARS-CoV-2 continue to rise worldwide with devastating health and economic costs. SARS-CoV-2 gains entry into host cells via an interaction between its Spike protein and the host cell receptor angiotensin converting enzyme 2 (ACE2). Disruption of this interaction confers potent neutralization of viral entry, providing an avenue for vaccine design and for therapeutic antibodies. Here, we develop single-domain antibodies (nanobodies) that potently disrupt the interaction between the SARS-CoV-2 Spike and ACE2. By screening a yeast surface-displayed library of synthetic nanobody sequences, we identified a panel of nanobodies that bind to multiple epitopes on Spike and block ACE2 interaction via two distinct mechanisms. Cryogenic electron microscopy (cryo-EM) revealed that one exceptionally stable nanobody, Nb6, binds Spike in a fully inactive conformation with its receptor binding domains (RBDs) locked into their inaccessible down-state, incapable of binding ACE2. Affinity maturation and structure-guided design of multivalency yielded a trivalent nanobody, mNb6-tri, with femtomolar affinity for SARS-CoV-2 Spike and picomolar neutralization of SARS-CoV-2 infection. mNb6-tri retains stability and function after aerosolization, lyophilization, and heat treatment. These properties may enable aerosol-mediated delivery of this potent neutralizer directly to the airway epithelia, promising to yield a widely deployable, patient-friendly prophylactic and/or early infection therapeutic agent to stem the worst pandemic in a century.

7.
Genetics ; 213(1): 59-77, 2019 09.
Artículo en Inglés | MEDLINE | ID: mdl-31331946

RESUMEN

cGMP plays a role in sensory signaling and plasticity by regulating ion channels, phosphodiesterases, and kinases. Studies that primarily used genetic and biochemical tools suggest that cGMP is spatiotemporally regulated in multiple sensory modalities. FRET- and GFP-based cGMP sensors were developed to visualize cGMP in primary cell culture and Caenorhabditis elegans to corroborate these findings. While a FRET-based sensor has been used in an intact animal to visualize cGMP, the requirement of a multiple emission system limits its ability to be used on its own as well as with other fluorophores. Here, we demonstrate that a C. elegans codon-optimized version of the cpEGFP-based cGMP sensor FlincG3 can be used to visualize rapidly changing cGMP levels in living, behaving C. elegans We coexpressed FlincG3 with the blue-light-activated guanylyl cyclases BeCyclOp and bPGC in body wall muscles, and found that the rate of change in FlincG3 fluorescence correlated with the rate of cGMP production by each cyclase. Furthermore, we show that FlincG3 responds to cultivation temperature, NaCl concentration changes, and sodium dodecyl sulfate in the sensory neurons AFD, ASEL/R, and PHB, respectively. Intriguingly, FlincG3 fluorescence in ASEL and ASER decreased in response to a NaCl concentration upstep and downstep, respectively, which is opposite in sign to the coexpressed calcium sensor jRGECO1a and previously published calcium recordings. These results illustrate that FlincG3 can be used to report rapidly changing cGMP levels in an intact animal, and that the reporter can potentially reveal unexpected spatiotemporal landscapes of cGMP in response to stimuli.


Asunto(s)
GMP Cíclico/metabolismo , Transferencia Resonante de Energía de Fluorescencia/métodos , Proteínas Fluorescentes Verdes/metabolismo , Optogenética/métodos , Animales , Caenorhabditis elegans , Células Cultivadas , Proteínas Fluorescentes Verdes/genética , Guanilato Ciclasa/genética , Guanilato Ciclasa/metabolismo , Opsinas/genética , Opsinas/metabolismo , Imagen Óptica/métodos , Células Receptoras Sensoriales/citología , Células Receptoras Sensoriales/metabolismo
8.
Nat Commun ; 7: 11046, 2016 Mar 18.
Artículo en Inglés | MEDLINE | ID: mdl-26988139

RESUMEN

In addition to the popular method of fluorescent protein fusion, live cell protein imaging has now seen more and more application of epitope tags. The small size of these tags may reduce functional perturbation and enable signal amplification. To address their background issue, we adapt self-complementing split fluorescent proteins as epitope tags for live cell protein labelling. The two tags, GFP11 and sfCherry11 are derived from the eleventh ß-strand of super-folder GFP and sfCherry, respectively. The small size of FP11-tags enables a cost-effective and scalable way to insert them into endogenous genomic loci via CRISPR-mediated homology-directed repair. Tandem arrangement FP11-tags allows proportional enhancement of fluorescence signal in tracking intraflagellar transport particles, or reduction of photobleaching for live microtubule imaging. Finally, we show the utility of tandem GFP11-tag in scaffolding protein oligomerization. These experiments illustrate the versatility of FP11-tag as a labelling tool as well as a multimerization-control tool for both imaging and non-imaging applications.


Asunto(s)
Proteínas Fluorescentes Verdes/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Supervivencia Celular , Fluorescencia , Humanos , Multimerización de Proteína , Coloración y Etiquetado
9.
Genetics ; 194(1): 175-87, 2013 May.
Artículo en Inglés | MEDLINE | ID: mdl-23475988

RESUMEN

The organization of neurons and the maintenance of that arrangement are critical to brain function. Failure of these processes in humans can lead to severe birth defects, mental retardation, and epilepsy. Several kinesins have been shown to play important roles in cell migration in vertebrate systems, but few upstream and downstream pathway members have been identified. Here, we utilize the genetic model organism Caenorhabditis elegans to elucidate the pathway by which the C. elegans Kinesin-1 Heavy Chain (KHC)/KIF5 ortholog UNC-116 functions to maintain neuronal cell body position in the PHB sensory neurons. We find that UNC-116/KHC acts in part with the cell and axon migration molecules UNC-6/Netrin and UNC-40/DCC in this process, but in parallel to SAX-3/Robo. We have also identified several potential adaptor, cargo, and regulatory proteins that may provide insight into the mechanism of UNC-116/KHC's function in this process. These include the cargo receptor UNC-33/CRMP2, the cargo adaptor protein UNC-76/FEZ and its regulator UNC-51/ULK, the cargo molecule UNC-69/SCOCO, and the actin regulators UNC-44/Ankyrin and UNC-34/Enabled. These genes also act in cell migration and axon outgrowth; however, many proteins that function in these processes do not affect PHB position. Our findings suggest an active posterior cell migration mediated by UNC-116/KHC occurs throughout development to maintain proper PHB cell body position and define a new pathway that mediates maintenance of neuronal cell body position.


Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/citología , Caenorhabditis elegans/metabolismo , Moléculas de Adhesión Celular/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cinesinas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Células Receptoras Sensoriales/citología , Células Receptoras Sensoriales/metabolismo , Actinas/metabolismo , Animales , Factores de Crecimiento de Fibroblastos/metabolismo , Ganglios de Invertebrados/citología , Ganglios de Invertebrados/metabolismo , Proteínas Motoras Moleculares/metabolismo , Mutación/genética , Netrinas , Prohibitinas , Receptores Inmunológicos/metabolismo , Vía de Señalización Wnt , Proteínas Roundabout
10.
Neural Dev ; 6: 28, 2011 Jun 10.
Artículo en Inglés | MEDLINE | ID: mdl-21663630

RESUMEN

BACKGROUND: An essential stage of neural development involves the assembly of neural circuits via formation of inter-neuronal connections. Early steps in neural circuit formation, including cell migration, axon guidance, and the localization of synaptic components, are well described. However, upon reaching their target region, most neurites still contact many potential partners. In order to assemble functional circuits, it is critical that within this group of cells, neurons identify and form connections only with their appropriate partners, a process we call synaptic partner recognition (SPR). To understand how SPR is mediated, we previously developed a genetically encoded fluorescent trans-synaptic marker called NLG-1 GRASP, which labels synaptic contacts between individual neurons of interest in dense cellular environments in the genetic model organism Caenorhabditis elegans. RESULTS: Here, we describe the first use of NLG-1 GRASP technology, to identify SPR genes that function in this critical process. The NLG-1 GRASP system allows us to assess synaptogenesis between PHB sensory neurons and AVA interneurons instantly in live animals, making genetic analysis feasible. Additionally, we employ a behavioral assay to specifically test PHB sensory circuit function. Utilizing this approach, we reveal a new role for the secreted UNC-6/Netrin ligand and its transmembrane receptor UNC-40/Deleted in colorectal cancer (DCC) in SPR. Synapses between PHB and AVA are severely reduced in unc-6 and unc-40 animals despite normal axon guidance and subcellular localization of synaptic components. Additionally, behavioral defects indicate a complete disruption of PHB circuit function in unc-40 mutants. Our data indicate that UNC-40 and UNC-6 function in PHB and AVA, respectively, to specify SPR. Strikingly, overexpression of UNC-6 in postsynaptic neurons is sufficient to promote increased PHB-AVA synaptogenesis and to potentiate the behavioral response beyond wild-type levels. Furthermore, an artificially membrane-tethered UNC-6 expressed in the postsynaptic neurons promotes SPR, consistent with a short-range signal between adjacent synaptic partners. CONCLUSIONS: These results indicate that the conserved UNC-6/Netrin-UNC-40/DCC ligand-receptor pair has a previously unknown function, acting in a juxtacrine manner to specify recognition of individual postsynaptic neurons. Furthermore, they illustrate the potential of this new approach, combining NLG-1 GRASP and behavioral analysis, in gene discovery and characterization.


Asunto(s)
Vías Nerviosas , Reconocimiento en Psicología , Transducción de Señal/fisiología , Sinapsis/fisiología , Animales , Animales Modificados Genéticamente , Conducta Animal/fisiología , Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiología , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Moléculas de Adhesión Celular Neuronal/genética , Moléculas de Adhesión Celular Neuronal/metabolismo , Comunicación Celular , Movimiento Celular , Regulación de la Expresión Génica/genética , Proteínas Fluorescentes Verdes/genética , Interneuronas/fisiología , Mutación/genética , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Netrinas , Vías Nerviosas/anatomía & histología , Vías Nerviosas/crecimiento & desarrollo , Neuritas/fisiología , Terminales Presinápticos/fisiología , Células Receptoras Sensoriales/citología , Células Receptoras Sensoriales/fisiología , Transducción de Señal/genética , Sinapsis/genética
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