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1.
Mol Pharmacol ; 2020 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-32198210

RESUMO

The powerful analgesic effects of opioid drugs have captivated the interest of physicians and scientists for millennia, and the ability of opioid drugs to produce serious undesired effects has been recognized for a similar period of time (Kieffer and Evans, 2009) . Many of these develop progressively with prolonged or repeated drug use and then persist, motivating particular interest in understanding how opioid drugs initiate adaptive or maladaptive modifications in neural function or regulation. Exciting advances have been made over the past several years in elucidating drug-induced changes at molecular, cellular and physiological levels of analysis. The present review will highlight some of these advances, focusing on the cellular level as a bridge across scales, and on imaging approaches which put opioid drug action 'under the microscope'. SIGNIFICANCE STATEMENT: Opioid receptors are major pharmacological targets but their signaling at the cellular level results from a complex interplay between pharmacology, regulation, subcellular localization and membrane trafficking. This minireview discusses recent advances in understanding the cellular biology of opioid receptors, emphasizing particular topics discussed at the 50th anniversary of the INRC meeting. Our goal is to highlight distinct signaling and regulatory properties emerging from the cellular biology of opioid receptors, and discuss potential relevance to therapeutics.

2.
Elife ; 92020 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-32096468

RESUMO

G protein-coupled receptors (GPCRs) signal through allostery, and it is increasingly clear that chemically distinct agonists can produce different receptor-based effects. It has been proposed that agonists selectively promote receptors to recruit one cellular interacting partner over another, introducing allosteric 'bias' into the signaling system. However, the underlying hypothesis - that different agonists drive GPCRs to engage different cytoplasmic proteins in living cells - remains untested due to the complexity of readouts through which receptor-proximal interactions are typically inferred. We describe a cell-based assay to overcome this challenge, based on GPCR-interacting biosensors that are disconnected from endogenous transduction mechanisms. Focusing on opioid receptors, we directly demonstrate differences between biosensor recruitment produced by chemically distinct opioid ligands in living cells. We then show that selective recruitment applies to GRK2, a biologically relevant GPCR regulator, through discrete interactions of GRK2 with receptors or with G protein beta-gamma subunits which are differentially promoted by agonists.

3.
Neuron ; 105(4): 663-677.e8, 2020 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-31837915

RESUMO

A major function of GPCRs is to inhibit presynaptic neurotransmitter release, requiring ligand-activated receptors to couple locally to effectors at terminals. The current understanding of how this is achieved is through receptor immobilization on the terminal surface. Here, we show that opioid peptide receptors, GPCRs that mediate highly sensitive presynaptic inhibition, are instead dynamic in axons. Opioid receptors diffuse rapidly throughout the axon surface and internalize after ligand-induced activation specifically at presynaptic terminals. We delineate a parallel regulated endocytic cycle for GPCRs operating at the presynapse, separately from the synaptic vesicle cycle, which clears activated receptors from the surface of terminals and locally reinserts them to maintain the diffusible surface pool. We propose an alternate strategy for achieving local control of presynaptic effectors that, opposite to using receptor immobilization and enforced proximity, is based on lateral mobility of receptors and leverages the inherent allostery of GPCR-effector coupling.

4.
Nat Protoc ; 14(12): 3471-3505, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31732722

RESUMO

Multiple aspects of neural activity, from neuronal firing to neuromodulator release and signaling, underlie brain function and ultimately shape animal behavior. The recently developed and constantly growing toolbox of genetically encoded sensors for neural activity, including calcium, voltage, neurotransmitter and neuromodulator sensors, allows precise measurement of these signaling events with high spatial and temporal resolution. Here, we describe the engineering, characterization and application of our recently developed dLight1, a suite of genetically encoded dopamine (DA) sensors based on human inert DA receptors. dLight1 offers high molecular specificity, requisite affinity and kinetics and great sensitivity for measuring DA release in vivo. The detailed workflow described in this protocol can be used to systematically characterize and validate dLight1 in increasingly intact biological systems, from cultured cells to acute brain slices to behaving mice. For tool developers, we focus on characterizing five distinct properties of dLight1: dynamic range, affinity, molecular specificity, kinetics and interaction with endogenous signaling; for end users, we provide comprehensive step-by-step instructions for how to leverage fiber photometry and two-photon imaging to measure dLight1 transients in vivo. The instructions provided in this protocol are designed to help laboratory personnel with a broad range of experience (at the graduate or post-graduate level) to develop and utilize novel neuromodulator sensors in vivo, by using dLight1 as a benchmark.


Assuntos
Neurotransmissores/metabolismo , Optogenética/métodos , Receptores Dopaminérgicos/metabolismo , Animais , Dopamina/metabolismo , Engenharia Genética/métodos , Humanos , Proteínas Luminescentes/genética , Neurônios/metabolismo , Fluxo de Trabalho
5.
Traffic ; 20(2): 130-136, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30578610

RESUMO

G protein-coupled receptors (GPCRs) physically connect extracellular information with intracellular signal propagation. Membrane trafficking plays a supportive role by "bookending" signaling events: movement through the secretory pathway delivers GPCRs to the cell surface where receptors can sample the extracellular environment, while endocytosis and endolysosomal membrane trafficking provide a versatile system to titrate cellular signaling potential and maintain homeostatic control. Recent evidence suggests that, in addition to these important effects, GPCR trafficking actively shapes the cellular signaling response by altering the location and timing of specific receptor-mediated signaling reactions. Here, we review key experimental evidence underlying this expanding view, focused on GPCR signaling mediated through activation of heterotrimeric G proteins located in the cytoplasm. We then discuss lingering and emerging questions regarding the interface between GPCR signaling and trafficking.


Assuntos
Endossomos/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Transdução de Sinais , Animais , Humanos , Multimerização Proteica , Transporte Proteico , Receptores Acoplados a Proteínas-G/química
6.
Cell ; 174(3): 505-520, 2018 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-30053424

RESUMO

Although gene discovery in neuropsychiatric disorders, including autism spectrum disorder, intellectual disability, epilepsy, schizophrenia, and Tourette disorder, has accelerated, resulting in a large number of molecular clues, it has proven difficult to generate specific hypotheses without the corresponding datasets at the protein complex and functional pathway level. Here, we describe one path forward-an initiative aimed at mapping the physical and genetic interaction networks of these conditions and then using these maps to connect the genomic data to neurobiology and, ultimately, the clinic. These efforts will include a team of geneticists, structural biologists, neurobiologists, systems biologists, and clinicians, leveraging a wide array of experimental approaches and creating a collaborative infrastructure necessary for long-term investigation. This initiative will ultimately intersect with parallel studies that focus on other diseases, as there is a significant overlap with genes implicated in cancer, infectious disease, and congenital heart defects.


Assuntos
Mapeamento Cromossômico/métodos , Transtornos do Neurodesenvolvimento/genética , Biologia de Sistemas/métodos , Redes Reguladoras de Genes/genética , Predisposição Genética para Doença/genética , Estudo de Associação Genômica Ampla/métodos , Genômica/métodos , Humanos , Neurobiologia/métodos , Neuropsiquiatria
7.
Science ; 360(6396)2018 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-29853555

RESUMO

Neuromodulatory systems exert profound influences on brain function. Understanding how these systems modify the operating mode of target circuits requires spatiotemporally precise measurement of neuromodulator release. We developed dLight1, an intensity-based genetically encoded dopamine indicator, to enable optical recording of dopamine dynamics with high spatiotemporal resolution in behaving mice. We demonstrated the utility of dLight1 by imaging dopamine dynamics simultaneously with pharmacological manipulation, electrophysiological or optogenetic stimulation, and calcium imaging of local neuronal activity. dLight1 enabled chronic tracking of learning-induced changes in millisecond dopamine transients in mouse striatum. Further, we used dLight1 to image spatially distinct, functionally heterogeneous dopamine transients relevant to learning and motor control in mouse cortex. We also validated our sensor design platform for developing norepinephrine, serotonin, melatonin, and opioid neuropeptide indicators.


Assuntos
Técnicas Biossensoriais , Córtex Cerebral/metabolismo , Dopamina/metabolismo , Neuroimagem/métodos , Neurotransmissores/metabolismo , Optogenética , Animais , Cálcio/análise , Cálcio/metabolismo , Córtex Cerebral/química , Corpo Estriado , Dopamina/análise , Engenharia Genética , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Humanos , Aprendizagem , Camundongos , Neurônios/fisiologia , Neurotransmissores/análise , Receptores de Dopamina D1/química , Receptores de Dopamina D1/genética , Serotonina/análise , Serotonina/metabolismo
8.
Nature ; 557(7705): 381-386, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29720660

RESUMO

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.


Assuntos
Receptores Acoplados a Proteínas-G/metabolismo , beta-Arrestinas/metabolismo , Animais , Biocatálise , Células COS , Membrana Celular/metabolismo , Células HEK293 , Humanos , Fosfatidilinositóis/metabolismo , Transporte Proteico , Receptores Acoplados a Proteínas-G/química , beta-Arrestinas/química
9.
Neuron ; 98(5): 963-976.e5, 2018 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-29754753

RESUMO

Opioid receptors (ORs) precisely modulate behavior when activated by native peptide ligands but distort behaviors to produce pathology when activated by non-peptide drugs. A fundamental question is how drugs differ from peptides in their actions on target neurons. Here, we show that drugs differ in the subcellular location at which they activate ORs. We develop a genetically encoded biosensor that directly detects ligand-induced activation of ORs and uncover a real-time map of the spatiotemporal organization of OR activation in living neurons. Peptide agonists produce a characteristic activation pattern initiated in the plasma membrane and propagating to endosomes after receptor internalization. Drugs produce a different activation pattern by additionally driving OR activation in the somatic Golgi apparatus and Golgi elements extending throughout the dendritic arbor. These results establish an approach to probe the cellular basis of neuromodulation and reveal that drugs distort the spatiotemporal landscape of neuronal OR activation.


Assuntos
Analgésicos Opioides/metabolismo , Membrana Celular/metabolismo , Dendritos/metabolismo , Endossomos/metabolismo , Complexo de Golgi/metabolismo , Neurônios/metabolismo , Peptídeos/metabolismo , Receptores Opioides/metabolismo , Animais , Técnicas Biossensoriais , Ala(2)-MePhe(4)-Gly(5)-Encefalina/metabolismo , D-Penicilina (2,5)-Encefalina/metabolismo , Leucina Encefalina-2-Alanina/metabolismo , Células HEK293 , Células HeLa , Humanos , Espaço Intracelular , Microscopia de Fluorescência , Morfina/metabolismo , Naloxona , Antagonistas de Entorpecentes , Ratos , Análise Espaço-Temporal
10.
Cell Rep ; 22(10): 2593-2600, 2018 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-29514089

RESUMO

Growth factor binding to EGFR drives conformational changes that promote homodimerization and transphosphorylation, followed by adaptor recruitment, oligomerization, and signaling through Ras. Whether specific receptor conformations and oligomerization states are necessary for efficient activation of Ras is unclear. We therefore evaluated the sufficiency of a phosphorylated EGFR dimer to activate Ras without growth factor by developing a chemical-genetic strategy to crosslink and "trap" full-length EGFR homodimers on cells. Trapped dimers become phosphorylated and recruit adaptor proteins at stoichiometry equivalent to that of EGF-stimulated receptors. Surprisingly, these phosphorylated dimers do not activate Ras, Erk, or Akt. In the absence of EGF, phosphorylated dimers do not further oligomerize or reorganize on cell membranes. These results suggest that a phosphorylated EGFR dimer loaded with core signaling adapters is not sufficient to activate Ras and that EGFR ligands contribute to conformational changes or receptor dynamics necessary for oligomerization and efficient signal propagation through the SOS-Ras-MAPK pathway.


Assuntos
Receptores ErbB/metabolismo , Multimerização Proteica , Proteínas ras/metabolismo , Vesículas Revestidas por Clatrina/efeitos dos fármacos , Vesículas Revestidas por Clatrina/metabolismo , Reagentes para Ligações Cruzadas/química , Reagentes para Ligações Cruzadas/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/química , Células HEK293 , Humanos , Ligantes , Fosforilação/efeitos dos fármacos , Fosfotirosina/metabolismo , Conformação Proteica , Transdução de Sinais/efeitos dos fármacos
11.
Trends Pharmacol Sci ; 39(2): 200-208, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29478570

RESUMO

G protein-coupled receptors (GPCRs) comprise a large and diverse class of signal-transducing receptors that undergo dynamic and isoform-specific membrane trafficking. GPCRs thus have an inherent potential to initiate or regulate signaling reactions from multiple membrane locations. This review discusses emerging insights into the subcellular organization of GPCR function in mammalian cells, focusing on signaling transduced by heterotrimeric G proteins and ß-arrestins. We summarize recent evidence indicating that GPCR-mediated activation of G proteins occurs not only from the plasma membrane (PM) but also from endosomes and Golgi membranes and that ß-arrestin-dependent signaling can be transduced from the PM by ß-arrestin trafficking to clathrin-coated pits (CCPs) after dissociation from a ligand-activated GPCR.


Assuntos
Membranas Intracelulares/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Transdução de Sinais , Animais , Humanos , Transporte Proteico , Receptores Acoplados a Proteínas-G/química , beta-Arrestinas/metabolismo
12.
Nat Genet ; 50(2): 180-185, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29311635

RESUMO

Most monogenic cases of obesity in humans have been linked to mutations in genes encoding members of the leptin-melanocortin pathway. Specifically, mutations in MC4R, the melanocortin-4 receptor gene, account for 3-5% of all severe obesity cases in humans1-3. Recently, ADCY3 (adenylyl cyclase 3) gene mutations have been implicated in obesity4,5. ADCY3 localizes to the primary cilia of neurons 6 , organelles that function as hubs for select signaling pathways. Mutations that disrupt the functions of primary cilia cause ciliopathies, rare recessive pleiotropic diseases in which obesity is a cardinal manifestation 7 . We demonstrate that MC4R colocalizes with ADCY3 at the primary cilia of a subset of hypothalamic neurons, that obesity-associated MC4R mutations impair ciliary localization and that inhibition of adenylyl cyclase signaling at the primary cilia of these neurons increases body weight. These data suggest that impaired signaling from the primary cilia of MC4R neurons is a common pathway underlying genetic causes of obesity in humans.


Assuntos
Adenilil Ciclases/genética , Cílios/metabolismo , Neurônios/metabolismo , Obesidade/genética , Receptor Tipo 4 de Melanocortina/genética , Adenilil Ciclases/metabolismo , Animais , Células Cultivadas , Cílios/genética , Feminino , Predisposição Genética para Doença , Células HEK293 , Humanos , Espaço Intracelular/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Mutação , Células NIH 3T3 , Neurônios/citologia , Obesidade/metabolismo , Receptor Tipo 4 de Melanocortina/metabolismo , Transdução de Sinais/genética
13.
Elife ; 62017 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-29189201

RESUMO

Transcriptional assays, such as yeast two-hybrid and TANGO, that convert transient protein-protein interactions (PPIs) into stable expression of transgenes are powerful tools for PPI discovery, screens, and analysis of cell populations. However, such assays often have high background and lose information about PPI dynamics. We have developed SPARK (Specific Protein Association tool giving transcriptional Readout with rapid Kinetics), in which proteolytic release of a membrane-tethered transcription factor (TF) requires both a PPI to deliver a protease proximal to its cleavage peptide and blue light to uncage the cleavage site. SPARK was used to detect 12 different PPIs in mammalian cells, with 5 min temporal resolution and signal ratios up to 37. By shifting the light window, we could reconstruct PPI time-courses. Combined with FACS, SPARK enabled 51 fold enrichment of PPI-positive over PPI-negative cells. Due to its high specificity and sensitivity, SPARK has the potential to advance PPI analysis and discovery.


Assuntos
Mapeamento de Interação de Proteínas/métodos , Proteínas/metabolismo , Células HEK293 , Humanos , Fatores de Tempo , Transcrição Genética
14.
Sci Signal ; 10(484)2017 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-28634209

RESUMO

The ß2-adrenergic receptor (ß2AR) has provided a paradigm to elucidate how G protein-coupled receptors (GPCRs) control intracellular signaling, including the discovery that ß-arrestins, which bind to ligand-activated GPCRs, are central for GPCR function. We used genome editing, conditional gene deletion, and small interfering RNAs (siRNAs) to determine the roles of ß-arrestin 1 (ß-arr1) and ß-arr2 in ß2AR internalization, trafficking, and signaling to ERK. We found that only ß-arr2 was essential for ß2AR internalization. Unexpectedly, ß-arr1 and ß-arr2 and receptor internalization were dispensable for ERK activation. Instead, ß2AR signaled through Gαs and Gßγ subunits through a pathway that involved the tyrosine kinase SRC, the adaptor protein SHC, the guanine nucleotide exchange factor SOS, the small GTPase RAS, and the kinases RAF and MEK, which led to ERK activation. These findings provide a molecular framework for ß2AR signaling through ß-arrestin-independent pathways in key physiological functions and under pathological conditions.


Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Transdução de Sinais , beta-Arrestina 1/metabolismo , beta-Arrestina 2/metabolismo , Animais , Endocitose , GTP Fosfo-Hidrolases/metabolismo , Células HEK293 , Humanos , Ligantes , Camundongos , Camundongos Knockout , Fosforilação , Ligação Proteica , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição
15.
Nat Chem Biol ; 13(7): 799-806, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28553949

RESUMO

G-protein-coupled receptors (GPCRs) are increasingly recognized to operate from intracellular membranes as well as the plasma membrane. The ß2-adrenergic GPCR can activate Gs-linked cyclic AMP (Gs-cAMP) signaling from endosomes. We show here that the homologous human ß1-adrenergic receptor initiates an internal Gs-cAMP signal from the Golgi apparatus. By developing a chemical method to acutely squelch G-protein coupling at defined membrane locations, we demonstrate that Golgi activation contributes significantly to the overall cellular cAMP response. Golgi signaling utilizes a preexisting receptor pool rather than receptors delivered from the cell surface, requiring separate access of extracellular ligands. Epinephrine, a hydrophilic endogenous ligand, accesses the Golgi-localized receptor pool by facilitated transport requiring the organic cation transporter 3 (OCT3), whereas drugs can access the Golgi pool by passive diffusion according to hydrophobicity. We demonstrate marked differences, among both agonist and antagonist drugs, in Golgi-localized receptor access and show that ß-blocker drugs currently used in the clinic differ markedly in ability to antagonize the Golgi signal. We propose 'location bias' as a new principle for achieving functional selectivity of GPCR-directed drug action.


Assuntos
Antagonistas Adrenérgicos beta/farmacologia , Dobutamina/farmacologia , Epinefrina/farmacologia , Receptores Adrenérgicos beta 1/metabolismo , Antagonistas Adrenérgicos beta/química , Dobutamina/química , Epinefrina/química , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/metabolismo , Células HeLa , Humanos , Ligantes , Relação Estrutura-Atividade
16.
Cell ; 169(2): 350-360.e12, 2017 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-28388416

RESUMO

Cells operate through protein interaction networks organized in space and time. Here, we describe an approach to resolve both dimensions simultaneously by using proximity labeling mediated by engineered ascorbic acid peroxidase (APEX). APEX has been used to capture entire organelle proteomes with high temporal resolution, but its breadth of labeling is generally thought to preclude the higher spatial resolution necessary to interrogate specific protein networks. We provide a solution to this problem by combining quantitative proteomics with a system of spatial references. As proof of principle, we apply this approach to interrogate proteins engaged by G-protein-coupled receptors as they dynamically signal and traffic in response to ligand-induced activation. The method resolves known binding partners, as well as previously unidentified network components. Validating its utility as a discovery pipeline, we establish that two of these proteins promote ubiquitin-linked receptor downregulation after prolonged activation.


Assuntos
Ascorbato Peroxidases/química , Mapas de Interação de Proteínas , Coloração e Rotulagem/métodos , Animais , Humanos , Lisossomos/metabolismo , Transporte Proteico , Receptores Acoplados a Proteínas-G/metabolismo , Receptores Opioides/metabolismo , Ubiquitina/metabolismo
17.
Mol Pharmacol ; 91(1): 65-73, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27821547

RESUMO

G protein-coupled receptors (GPCRs), the largest family of signaling receptors, are critically regulated by endosomal trafficking, suggesting that endosomes might provide new strategies for manipulating GPCR signaling. Here we test this hypothesis by focusing on class III phosphatidylinositol 3-kinase (Vps34), which is an essential regulator of endosomal trafficking. We verify that Vps34 is required for recycling of the ß2-adrenoceptor (ß2AR), a prototypical GPCR, and then investigate the effects of Vps34 inhibition on the canonical cAMP response elicited by ß2AR activation. Vps34 inhibition impairs the ability of cells to recover this response after prolonged activation, which is in accord with the established role of recycling in GPCR resensitization. In addition, Vps34 inhibition also attenuates the short-term cAMP response, and its effect begins several minutes after initial agonist application. These results establish Vps34 as an essential determinant of both short-term and long-term canonical GPCR signaling, and support the potential utility of the endosomal system as a druggable target for signaling.


Assuntos
Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Endossomos/enzimologia , Receptores Adrenérgicos beta 2/metabolismo , Transdução de Sinais , AMP Cíclico/metabolismo , Células HEK293 , Humanos , Modelos Biológicos , Fosfatos de Fosfatidilinositol/metabolismo
18.
Mol Pharmacol ; 91(2): 145-156, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27879340

RESUMO

The ability of chemically distinct ligands to produce different effects on the same G protein-coupled receptor (GPCR) has interesting therapeutic implications, but, if excessively propagated downstream, would introduce biologic noise compromising cognate ligand detection. We asked whether cells have the ability to limit the degree to which chemical diversity imposed at the ligand-GPCR interface is propagated to the downstream signal. We carried out an unbiased analysis of the integrated cellular response elicited by two chemically and pharmacodynamically diverse ß-adrenoceptor agonists, isoproterenol and salmeterol. We show that both ligands generate an identical integrated response, and that this stereotyped output requires endocytosis. We further demonstrate that the endosomal ß2-adrenergic receptor signal confers uniformity on the downstream response because it is highly sensitive and saturable. Based on these findings, we propose that GPCR signaling from endosomes functions as a biologic noise filter to enhance reliability of cognate ligand detection.


Assuntos
Endocitose , Receptores Acoplados a Proteínas-G/metabolismo , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Células HEK293 , Humanos , Isoproterenol/farmacologia , Ligantes , Espectrometria de Massas , Modelos Biológicos , Análise de Sequência com Séries de Oligonucleotídeos , Fosfoproteínas/metabolismo , Fosforilação/efeitos dos fármacos , Proteoma/metabolismo , Proteômica , Receptores Adrenérgicos beta 2/metabolismo , Xinafoato de Salmeterol/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transcrição Genética/efeitos dos fármacos
19.
Curr Biol ; 26(23): 3129-3142, 2016 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-27839977

RESUMO

Retromer mediates sequence-directed cargo exit from endosomes to support both endosome-to-Golgi (retrograde transport) and endosome-to-plasma membrane (recycling) itineraries. It is not known whether these trafficking functions require cargos to exit endosomes separately via distinct transport intermediates or whether the same retromer-coated carriers can support both itineraries. We addressed this question by comparing human Wntless (Wls) and ß2 adrenergic receptor (ß2AR), which require retromer physiologically for retrograde transport and recycling, respectively. We show here by direct visualization in living cells that both cargos transit primarily the same endosomes and exit via shared transport vesicles generated from a retromer-coated endosome domain. While both Wls and ß2AR clearly localize to the same retromer-coated endosome domains, Wls is consistently enriched more strongly. This enrichment difference is determined by distinct motifs present in the cytoplasmic tail of each cargo, with Wls using tandem Φ-X-[L/M] motifs and ß2AR using a PDZ motif. Exchanging these determinants reverses the enrichment phenotype of each cargo but does not change cargo itinerary, verifying the multifunctional nature of retromer and implying that additional sorting must occur downstream. Quantitative differences in the degree of cargo enrichment instead underlie a form of kinetic sorting that impacts the rate of cargo delivery via both itineraries and determines the ability of ß2AR to activate its cognate G protein transducer locally from endosomes. We propose that mammalian retromer forms a multifunctional membrane coat that supports shared cargo exit for divergent trafficking itineraries and regulates signaling from endosomes.


Assuntos
Endossomos/fisiologia , Proteínas de Ligação ao GTP/metabolismo , Transporte Proteico/fisiologia , Receptores Acoplados a Proteínas-G/fisiologia , Proteínas de Ligação ao GTP/genética , Células HEK293 , Células HeLa , Humanos
20.
Proc Natl Acad Sci U S A ; 113(39): E5721-30, 2016 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-27621449

RESUMO

We previously showed that guanine nucleotide-binding (G) protein α subunit (Gα)-interacting vesicle-associated protein (GIV), a guanine-nucleotide exchange factor (GEF), transactivates Gα activity-inhibiting polypeptide 1 (Gαi) proteins in response to growth factors, such as EGF, using a short C-terminal motif. Subsequent work demonstrated that GIV also binds Gαs and that inactive Gαs promotes maturation of endosomes and shuts down mitogenic MAPK-ERK1/2 signals from endosomes. However, the mechanism and consequences of dual coupling of GIV to two G proteins, Gαi and Gαs, remained unknown. Here we report that GIV is a bifunctional modulator of G proteins; it serves as a guanine nucleotide dissociation inhibitor (GDI) for Gαs using the same motif that allows it to serve as a GEF for Gαi. Upon EGF stimulation, GIV modulates Gαi and Gαs sequentially: first, a key phosphomodification favors the assembly of GIV-Gαi complexes and activates GIV's GEF function; then a second phosphomodification terminates GIV's GEF function, triggers the assembly of GIV-Gαs complexes, and activates GIV's GDI function. By comparing WT and GIV mutants, we demonstrate that GIV inhibits Gαs activity in cells responding to EGF. Consequently, the cAMP→PKA→cAMP response element-binding protein signaling axis is inhibited, the transit time of EGF receptor through early endosomes are accelerated, mitogenic MAPK-ERK1/2 signals are rapidly terminated, and proliferation is suppressed. These insights define a paradigm in G-protein signaling in which a pleiotropically acting modulator uses the same motif both to activate and to inhibit G proteins. Our findings also illuminate how such modulation of two opposing Gα proteins integrates downstream signals and cellular responses.


Assuntos
Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Proliferação de Células/efeitos dos fármacos , Quimiotaxia/efeitos dos fármacos , AMP Cíclico/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Quinase 5 Dependente de Ciclina/metabolismo , Regulação para Baixo/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Fator de Crescimento Epidérmico/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Transferência Ressonante de Energia de Fluorescência , Subunidades beta da Proteína de Ligação ao GTP , Subunidades gama da Proteína de Ligação ao GTP , Guanosina Trifosfato/metabolismo , Células HeLa , Humanos , Proteínas dos Microfilamentos/química , Proteínas Mutantes/metabolismo , Fosforilação/efeitos dos fármacos , Ligação Proteica , Proteína Quinase C-theta/metabolismo , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Proteínas de Transporte Vesicular/química
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