RESUMEN
ß-arrestins bind G protein-coupled receptors to terminate G protein signaling and to facilitate other downstream signaling pathways. Using single-molecule fluorescence resonance energy transfer imaging, we show that ß-arrestin is strongly autoinhibited in its basal state. Its engagement with a phosphopeptide mimicking phosphorylated receptor tail efficiently releases the ß-arrestin tail from its N domain to assume distinct conformations. Unexpectedly, we find that ß-arrestin binding to phosphorylated receptor, with a phosphorylation barcode identical to the isolated phosphopeptide, is highly inefficient and that agonist-promoted receptor activation is required for ß-arrestin activation, consistent with the release of a sequestered receptor C tail. These findings, together with focused cellular investigations, reveal that agonism and receptor C-tail release are specific determinants of the rate and efficiency of ß-arrestin activation by phosphorylated receptor. We infer that receptor phosphorylation patterns, in combination with receptor agonism, synergistically establish the strength and specificity with which diverse, downstream ß-arrestin-mediated events are directed.
Asunto(s)
Fosfopéptidos , Receptores Acoplados a Proteínas G , Fosfopéptidos/metabolismo , Fosforilación , Receptores Acoplados a Proteínas G/metabolismo , beta-Arrestina 1/metabolismo , beta-Arrestinas/metabolismoRESUMEN
The ß2-adrenergic receptor (ß2AR), a prototypic G-protein-coupled receptor (GPCR), is a powerful driver of bronchorelaxation, but the effectiveness of ß-agonist drugs in asthma is limited by desensitization and tachyphylaxis. We find that during activation, the ß2AR is modified by S-nitrosylation, which is essential for both classic desensitization by PKA as well as desensitization of NO-based signaling that mediates bronchorelaxation. Strikingly, S-nitrosylation alone can drive ß2AR internalization in the absence of traditional agonist. Mutant ß2AR refractory to S-nitrosylation (Cys265Ser) exhibits reduced desensitization and internalization, thereby amplifying NO-based signaling, and mice with Cys265Ser mutation are resistant to bronchoconstriction, inflammation, and the development of asthma. S-nitrosylation is thus a central mechanism in ß2AR signaling that may be operative widely among GPCRs and targeted for therapeutic gain.
Asunto(s)
Asma , Animales , Asma/inducido químicamente , Asma/genética , Ratones , Transducción de SeñalRESUMEN
G protein-coupled receptors (GPCRs) selectively activate at least one of the four families of heterotrimeric G proteins, but the mechanism of coupling selectivity remains unclear. Structural studies emphasize structural complementarity of GPCRs and nucleotide-free G proteins, but selectivity is likely to be determined by transient intermediate-state complexes that exist before nucleotide release. Here we study coupling to nucleotide-decoupled G protein variants that can adopt conformations similar to receptor-bound G proteins without releasing nucleotide, and are therefore able to bypass intermediate-state complexes. We find that selectivity is degraded when nucleotide release is not required for GPCR-G protein complex formation, to the extent that most GPCRs interact with most nucleotide-decoupled G proteins. These findings demonstrate the absence of absolute structural incompatibility between noncognate receptor-G protein pairs, and are consistent with the hypothesis that transient intermediate states are partly responsible for coupling selectivity.
Asunto(s)
Proteínas de Unión al GTP Heterotriméricas , Receptores Acoplados a Proteínas G , Receptores Acoplados a Proteínas G/metabolismo , Conformación Proteica , Proteínas de Unión al GTP Heterotriméricas/química , Proteínas de Unión al GTP Heterotriméricas/metabolismoRESUMEN
G protein-coupled receptors (GPCRs) are gatekeepers of cellular homeostasis and the targets of a large proportion of drugs. In addition to their signaling activity at the plasma membrane, it has been proposed that their actions may result from translocation and activation of G proteins at endomembranes-namely endosomes. This could have a significant impact on our understanding of how signals from GPCR-targeting drugs are propagated within the cell. However, little is known about the mechanisms that drive G protein movement and activation in subcellular compartments. Using bioluminescence resonance energy transfer (BRET)-based effector membrane translocation assays, we dissected the mechanisms underlying endosomal Gq trafficking and activity following activation of Gq-coupled receptors, including the angiotensin II type 1, bradykinin B2, oxytocin, thromboxane A2 alpha isoform, and muscarinic acetylcholine M3 receptors. Our data reveal that GPCR-promoted activation of Gq at the plasma membrane induces its translocation to endosomes independently of ß-arrestin engagement and receptor endocytosis. In contrast, Gq activity at endosomes was found to rely on both receptor endocytosis-dependent and -independent mechanisms. In addition to shedding light on the molecular processes controlling subcellular Gq signaling, our study provides a set of tools that will be generally applicable to the study of G protein translocation and activation at endosomes and other subcellular organelles, as well as the contribution of signal propagation to drug action.
Asunto(s)
Transferencia de Energía por Resonancia de Bioluminiscencia/métodos , Endocitosis/fisiología , Endosomas/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/fisiología , Receptores Acoplados a Proteínas G/fisiología , Células HEK293 , Humanos , Factores de Intercambio de Guanina Nucleótido Rho/fisiología , Transducción de Señal/fisiología , beta-Arrestinas/fisiologíaRESUMEN
Apyrase from potato (Solanum tuberosum) is a divalent metal ion-dependent enzyme that catalyzes the hydrolysis of nucleoside di- and tri-phosphates with broad substrate specificity. The enzyme is widely used to manipulate nucleotide levels such as in the G protein-coupled receptor (GPCR) field where it is used to deplete guanine nucleotides to stabilize nucleotide-free ternary agonist-GPCR-G protein complexes. Potato apyrase is available commercially as the native enzyme purified from potatoes or as a recombinant protein, but these are prohibitively expensive for some research applications. Here, we report a relatively simple method for the bacterial production of soluble, active potato apyrase. Apyrase has several disulfide bonds, so we co-expressed the enzyme bearing a C-terminal (His)6 tag with the E. coli disulfide isomerase DsbC at low temperature (18 °C) in the oxidizing cytoplasm of E. coli Origami B (DE3). This allowed low level production of soluble apyrase. A two-step purification procedure involving Ni-affinity followed by Cibacron Blue-affinity chromatography yielded highly purified apyrase at a level of â¼0.5 mg per L of bacterial culture. The purified enzyme was functional for ATP hydrolysis in an ATPase assay and for GTP/GDP hydrolysis in a GPCR-G protein coupling assay. This methodology enables the time- and cost-efficient production of recombinant apyrase for various research applications.
Asunto(s)
Apirasa , Solanum tuberosum , Apirasa/genética , Apirasa/química , Escherichia coli/metabolismo , Proteínas de Unión al GTP/química , Proteínas de Unión al GTP/metabolismo , Proteínas Recombinantes/química , Solanum tuberosum/genética , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
Agonist binding promotes activation of G protein-coupled receptors (GPCRs) and association of active receptors with G protein heterotrimers. The resulting active-state ternary complex is the basis for conventional stimulus-response coupling. Although GPCRs can also associate with G proteins before agonist binding, the impact of such preassociated complexes on agonist-induced signaling is poorly understood. Here we show that preassociation of 5-HT7 serotonin receptors with Gs heterotrimers is necessary for agonist-induced signaling. 5-HT7 receptors in their inactive state associate with Gs, as these complexes are stabilized by inverse agonists and receptor mutations that favor the inactive state. Inactive-state 5-HT7-Gs complexes dissociate in response to agonists, allowing the formation of conventional agonist-5-HT7-Gs ternary complexes and subsequent Gs activation. Inactive-state 5-HT7-Gs complexes are required for the full dynamic range of agonist-induced signaling, as 5-HT7 receptors spontaneously activate Gs variants that cannot form inactive-state complexes. Therefore, agonist-induced signaling in this system involves two distinct receptor-G protein complexes, a conventional ternary complex that activates G proteins and an inverse-coupled binary complex that maintains the inactive state when agonist is not present.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Complejos Multiproteicos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal , Relación Dosis-Respuesta a Droga , Proteínas de Unión al GTP/química , Cinética , Ligandos , Modelos Biológicos , Complejos Multiproteicos/química , Unión Proteica , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/química , Receptores de Serotonina/química , Receptores de Serotonina/metabolismo , Antagonistas de la Serotonina , Agonistas de Receptores de Serotonina , Transducción de Señal/efectos de los fármacosRESUMEN
G proteins are activated when they associate with G protein-coupled receptors (GPCRs), often in response to agonist-mediated receptor activation. It is generally thought that agonist-induced receptor-G protein association necessarily promotes G protein activation and, conversely, that activated GPCRs do not interact with G proteins that they do not activate. Here we show that GPCRs can form agonist-dependent complexes with G proteins that they do not activate. Using cell-based bioluminescence resonance energy transfer (BRET) and luminescence assays we find that vasopressin V2 receptors (V2R) associate with both Gs and G12 heterotrimers when stimulated with the agonist arginine vasopressin (AVP). However, unlike V2R-Gs complexes, V2R-G12 complexes are not destabilized by guanine nucleotides and do not promote G12 activation. Activating V2R does not lead to signaling responses downstream of G12 activation, but instead inhibits basal G12-mediated signaling, presumably by sequestering G12 heterotrimers. Overexpressing G12 inhibits G protein receptor kinase (GRK) and arrestin recruitment to V2R and receptor internalization. Formyl peptide (FPR1 and FPR2) and Smoothened (Smo) receptors also form complexes with G12 that are insensitive to nucleotides, suggesting that unproductive GPCR-G12 complexes are not unique to V2R. These results indicate that agonist-dependent receptor-G protein association does not always lead to G protein activation and may in fact inhibit G protein activation.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP G12-G13/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismo , Transferencia de Energía por Resonancia de Bioluminiscencia/métodos , AMP Cíclico/metabolismo , Subunidades alfa de la Proteína de Unión al GTP G12-G13/fisiología , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gs/fisiología , Proteínas de Unión al GTP/metabolismo , Células HEK293 , Humanos , Ligandos , Unión Proteica/fisiología , Receptores de Vasopresinas/metabolismo , Transducción de Señal/fisiología , Vasopresinas/metabolismo , beta-Arrestinas/metabolismoRESUMEN
Phosphodiesterase-5 inhibitors (PDE5i) are under investigation for repurposing for colon cancer prevention. A drawback to conventional PDE5i are their side-effects and drug-drug interactions. We designed an analog of the prototypical PDE5i sildenafil by replacing the methyl group on the piperazine ring with malonic acid to reduce lipophilicity, and measured its entry into the circulation and effects on colon epithelium. This modification did not affect pharmacology as malonyl-sildenafil had a similar IC50 to sildenafil but exhibited an almost 20-fold reduced EC50 for increasing cellular cGMP. Using an LC-MS/MS approach, malonyl-sildenafil was negligible in mouse plasma after oral administration but was detected at high levels in the feces. No bioactive metabolites of malonyl-sildenafil were detected in the circulation by measuring interactions with isosorbide mononitrate. The treatment of mice with malonyl-sildenafil in the drinking water resulted in a suppression of proliferation in the colon epithelium that is consistent with results previously published for mice treated with PDE5i. A carboxylic-acid-containing analog of sildenafil prohibits the systemic delivery of the compound but maintains sufficient penetration into the colon epithelium to suppress proliferation. This highlights a novel approach to generating a first-in-class drug for colon cancer chemoprevention.
Asunto(s)
Neoplasias del Colon , Inhibidores de Fosfodiesterasa 5 , Ratones , Animales , Inhibidores de Fosfodiesterasa 5/farmacología , Citrato de Sildenafil/farmacología , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 5 , Cromatografía Liquida , Espectrometría de Masas en Tándem , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/prevención & control , Proliferación Celular , GMP Cíclico/metabolismoRESUMEN
The G protein signaling cascade is a key player in cell signaling. Cascade activation leads to a redistribution of its members in various cellular compartments. These changes are likely related to the "second wave" of signaling from endosomes. Here, we set out to determine whether Gs signaling cascade members expressed at very low levels exhibit altered mobility and localize in clathrin-coated structures (CCSs) or caveolae upon activation by ß2 -adrenergic receptors (ß2 AR). Activated ß2 AR showed decreased mobility and sustained accumulation in CCSs but not in caveolae. Arrestin 3 translocated to the plasma membrane after ß2 AR activation and showed very low mobility and pronounced accumulation in CCSs. In contrast, Gαs and Gγ2 exhibited a modest reduction in mobility but no detectable accumulation in or exclusion from CCSs or caveolae. The effector adenylyl cyclase 5 (AC5) showed a slight mobility increase upon ß2 AR stimulation, no redistribution to CCSs, and weak activation-independent accumulation in caveolae. Our findings show an overall decrease in the mobility of most activated Gs signaling cascade members and confirm that ß2 AR and arrestin 3 accumulate in CCSs, while Gαs , Gγ2 and AC5 can transiently enter CCSs and caveolae but do not accumulate in and are not excluded from these domains.
Asunto(s)
Caveolas/metabolismo , Membrana Celular/metabolismo , Transducción de Señal , Proteínas de Unión al GTP/metabolismo , Receptores Adrenérgicos beta 2/metabolismoRESUMEN
The Golgi apparatus (GA) is a cellular organelle that plays a critical role in the processing of proteins for secretion. Activation of G protein-coupled receptors at the plasma membrane (PM) induces the translocation of G protein ßγ dimers to the GA. However, the functional significance of this translocation is largely unknown. Here, we study PM-GA translocation of all 12 Gγ subunits in response to chemokine receptor CXCR4 activation and demonstrate that Gγ9 is a unique Golgi-translocating Gγ subunit. CRISPR-Cas9-mediated knockout of Gγ9 abolishes activation of extracellular signal-regulated kinase 1 and 2 (ERK1/2), two members of the mitogen-activated protein kinase family, by CXCR4. We show that chemically induced recruitment to the GA of Gßγ dimers containing different Gγ subunits activates ERK1/2, whereas recruitment to the PM is ineffective. We also demonstrate that pharmacological inhibition of phosphoinositide 3-kinase γ (PI3Kγ) and depletion of its subunits p110γ and p101 abrogate ERK1/2 activation by CXCR4 and Gßγ recruitment to the GA. Knockout of either Gγ9 or PI3Kγ significantly suppresses prostate cancer PC3 cell migration, invasion, and metastasis. Collectively, our data demonstrate a novel function for Gßγ translocation to the GA, via activating PI3Kγ heterodimers p110γ-p101, to spatiotemporally regulate mitogen-activated protein kinase activation by G protein-coupled receptors and ultimately control tumor progression.
Asunto(s)
Fosfatidilinositol 3-Quinasa Clase Ib/genética , Subunidades beta de la Proteína de Unión al GTP/genética , Subunidades gamma de la Proteína de Unión al GTP/genética , Aparato de Golgi/genética , Receptores CXCR4/genética , Membrana Celular/genética , Dimerización , Células HEK293 , Humanos , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Fosfatidilinositol 3-Quinasas/genética , Transporte de Proteínas/genética , Receptores Acoplados a Proteínas G/genética , Transducción de Señal/genéticaRESUMEN
The adhesion G-protein-coupled receptor (GPCR) latrophilin 3 (ADGRL3) has been associated with increased risk of attention deficit hyperactivity disorder (ADHD) and substance use in human genetic studies. Knockdown in multiple species leads to hyperlocomotion and altered dopamine signaling. Thus, ADGRL3 is a potential target for treatment of neuropsychiatric disorders that involve dopamine dysfunction, but its basic signaling properties are poorly understood. Identification of adhesion GPCR signaling partners has been limited by a lack of tools to acutely activate these receptors in living cells. Here, we design a novel acute activation strategy to characterize ADGRL3 signaling by engineering a receptor construct in which we could trigger acute activation enzymatically. Using this assay, we found that ADGRL3 signals through G12/G13 and Gq, with G12/13 the most robustly activated. Gα12/13 is a new player in ADGRL3 biology, opening up unexplored roles for ADGRL3 in the brain. Our methodological advancements should be broadly useful in adhesion GPCR research.
Asunto(s)
Factor de Transcripción Activador 6/metabolismo , Subunidades alfa de la Proteína de Unión al GTP G12-G13/metabolismo , Péptidos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Péptidos/metabolismo , Factor de Transcripción Activador 6/agonistas , Factor de Transcripción Activador 6/química , Factor de Transcripción Activador 6/genética , Animales , Arrestina/química , Arrestina/genética , Arrestina/metabolismo , Sistemas CRISPR-Cas , Ingeniería Celular , Subunidades alfa de la Proteína de Unión al GTP G12-G13/química , Subunidades alfa de la Proteína de Unión al GTP G12-G13/genética , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/química , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/genética , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Expresión Génica , Células HEK293 , Humanos , Cinética , Ratones , Proteína Quinasa 1 Activada por Mitógenos/química , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/química , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Péptidos/química , Péptidos/farmacología , Unión Proteica , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Receptores de Péptidos/química , Receptores de Péptidos/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción de SeñalRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
G protein-coupled receptors (GPCRs) activate four families of heterotrimeric G proteins, and individual receptors must select a subset of G proteins to produce appropriate cellular responses. Although the precise mechanisms of coupling selectivity are uncertain, the Gα subunit C terminus is widely believed to be the primary determinant recognized by cognate receptors. Here, we directly assess coupling between 14 representative GPCRs and 16 Gα subunits, including one wild-type Gα subunit from each of the four families and 12 chimeras with exchanged C termini. We use a sensitive bioluminescence resonance energy transfer (BRET) assay that provides control over both ligand and nucleotide binding, and allows direct comparison across G protein families. We find that the Gs- and Gq-coupled receptors we studied are relatively promiscuous and always couple to some extent to Gi1 heterotrimers. In contrast, Gi-coupled receptors are more selective. Our results with Gα subunit chimeras show that the Gα C terminus is important for coupling selectivity, but no more so than the Gα subunit core. The relative importance of the Gα subunit core and C terminus is highly variable and, for some receptors, the Gα core is more important for selective coupling than the C terminus. Our results suggest general rules for GPCR-G protein coupling and demonstrate that the critical G protein determinants of selectivity vary widely, even for different receptors that couple to the same G protein.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Línea Celular , Células HEK293 , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Humanos , Ligandos , Subunidades de Proteína/metabolismo , Transducción de Señal/fisiologíaRESUMEN
Craniofacial morphogenesis is regulated in part by signaling from the Endothelin receptor type A (EDNRA). Pathogenic variants in EDNRA signaling pathway components EDNRA, GNAI3, PCLB4, and EDN1 cause Mandibulofacial Dysostosis with Alopecia (MFDA), Auriculocondylar syndrome (ARCND) 1, 2, and 3, respectively. However, cardiovascular development is normal in MFDA and ARCND individuals, unlike Ednra knockout mice. One explanation may be that partial EDNRA signaling remains in MFDA and ARCND, as mice with reduced, but not absent, EDNRA signaling also lack a cardiovascular phenotype. Here we report an individual with craniofacial and cardiovascular malformations mimicking the Ednra -/- mouse phenotype, including a distinctive micrognathia with microstomia and a hypoplastic aortic arch. Exome sequencing found a novel homozygous missense variant in EDNRA (c.1142A>C; p.Q381P). Bioluminescence resonance energy transfer assays revealed that this amino acid substitution in helix 8 of EDNRA prevents recruitment of G proteins to the receptor, abrogating subsequent receptor activation by its ligand, Endothelin-1. This homozygous variant is thus the first reported loss-of-function EDNRA allele, resulting in a syndrome we have named Oro-Oto-Cardiac Syndrome. Further, our results illustrate that EDNRA signaling is required for both normal human craniofacial and cardiovascular development, and that limited EDNRA signaling is likely retained in ARCND and MFDA individuals. This work illustrates a straightforward approach to identifying the functional consequence of novel genetic variants in signaling molecules associated with malformation syndromes.
Asunto(s)
Anomalías Craneofaciales/genética , Enfermedades del Oído/genética , Oído/anomalías , Predisposición Genética a la Enfermedad , Disostosis Mandibulofacial/genética , Receptor de Endotelina A/genética , Animales , Anomalías Craneofaciales/fisiopatología , Oído/fisiopatología , Enfermedades del Oído/fisiopatología , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/genética , Regulación del Desarrollo de la Expresión Génica/genética , Humanos , Mutación con Pérdida de Función/genética , Disostosis Mandibulofacial/fisiopatología , Ratones , Ratones Noqueados , Morfogénesis/genética , Cresta Neural/crecimiento & desarrollo , Cresta Neural/patología , Fenotipo , Transducción de Señal/genéticaRESUMEN
G protein-coupled receptors (GPCRs) are key signaling proteins that regulate nearly every aspect of cell function. Studies of GPCRs have benefited greatly from the development of molecular tools to monitor receptor activation and downstream signaling. Here, we show that mini G proteins are robust probes that can be used in a variety of assay formats to report GPCR activity in living cells. Mini G (mG) proteins are engineered GTPase domains of Gα subunits that were developed for structural studies of active-state GPCRs. Confocal imaging revealed that mG proteins fused to fluorescent proteins were located diffusely in the cytoplasm and translocated to sites of receptor activation at the cell surface and at intracellular organelles. Bioluminescence resonance energy transfer (BRET) assays with mG proteins fused to either a fluorescent protein or luciferase reported agonist, superagonist, and inverse agonist activities. Variants of mG proteins (mGs, mGsi, mGsq, and mG12) corresponding to the four families of Gα subunits displayed appropriate coupling to their cognate GPCRs, allowing quantitative profiling of subtype-specific coupling to individual receptors. BRET between luciferase-mG fusion proteins and fluorescent markers indicated the presence of active GPCRs at the plasma membrane, Golgi apparatus, and endosomes. Complementation assays with fragments of NanoLuc luciferase fused to GPCRs and mG proteins reported constitutive receptor activity and agonist-induced activation with up to 20-fold increases in luminescence. We conclude that mG proteins are versatile tools for studying GPCR activation and coupling specificity in cells and should be useful for discovering and characterizing G protein subtype-biased ligands.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Sondas Moleculares/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Sitios de Unión , Compartimento Celular , Transferencia de Energía , Células HEK293 , Humanos , Luciferasas/metabolismo , Microscopía Confocal , Mutación , Unión Proteica , Receptores Acoplados a Proteínas G/genéticaRESUMEN
Heterotrimeric G proteins are localized to the plasma membrane where they transduce extracellular signals to intracellular effectors. G proteins also act at intracellular locations, and can translocate between cellular compartments. For example, Gαs can leave the plasma membrane and move to the cell interior after activation. However, the mechanism of Gαs translocation and its intracellular destination are not known. Here we use bioluminescence resonance energy transfer (BRET) to show that after activation, Gαs rapidly associates with the endoplasmic reticulum, mitochondria, and endosomes, consistent with indiscriminate sampling of intracellular membranes from the cytosol rather than transport via a specific vesicular pathway. The primary source of Gαs for endosomal compartments is constitutive endocytosis rather than activity-dependent internalization. Recycling of Gαs to the plasma membrane is complete 25 min after stimulation is discontinued. We also show that an acylation-deacylation cycle is important for the steady-state localization of Gαs at the plasma membrane, but our results do not support a role for deacylation in activity-dependent Gαs internalization.
Asunto(s)
Cromograninas/metabolismo , Endocitosis/fisiología , Retículo Endoplásmico/enzimología , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Membranas Intracelulares/enzimología , Acilación , Transferencia de Energía por Resonancia de Bioluminiscencia/métodos , Cromograninas/genética , Retículo Endoplásmico/genética , Activación Enzimática/fisiología , Subunidades alfa de la Proteína de Unión al GTP Gs/genética , Células HEK293 , Humanos , Transporte de Proteínas/fisiologíaRESUMEN
Arrestins mediate desensitization and internalization of G protein-coupled receptors and also direct receptor signaling toward heterotrimeric G protein-independent signaling pathways. We previously identified a four-residue segment (residues 212-215) of the dopamine D2 receptor that is necessary for arrestin binding in an in vitro heterologous expression system but that also impairs receptor expression. We now describe the characterization of additional mutations at that arrestin binding site in the third intracellular loop. Mutating two (residues 214 and 215) or three (residues 213-215) of the four residues to alanine partially decreased agonist-induced recruitment of arrestin3 without altering activation of a G protein. Arrestin-dependent receptor internalization, which requires arrestin binding to ß2-adaptin (the ß2 subunit of the clathrin-associated adaptor protein AP2) and clathrin, was disproportionately affected by the three-residue mutation, with no agonist-induced internalization observed even in the presence of overexpressed arrestin or G protein-coupled receptor kinase 2. The disjunction between arrestin recruitment and internalization could not be explained by alterations in the time course of the receptor-arrestin interaction, the recruitment of G protein-coupled receptor kinase 2, or the receptor-induced interaction between arrestin and ß2-adaptin, suggesting that the mutation impairs a property of the internalization complex that has not yet been identified.
Asunto(s)
Mutación , Receptores de Dopamina D2/metabolismo , Subunidades beta de Complejo de Proteína Adaptadora/genética , Subunidades beta de Complejo de Proteína Adaptadora/metabolismo , Quinasa 2 del Receptor Acoplado a Proteína-G/genética , Quinasa 2 del Receptor Acoplado a Proteína-G/metabolismo , Células HEK293 , Humanos , Estructura Secundaria de Proteína , Transporte de Proteínas/fisiología , Receptores de Dopamina D2/genéticaRESUMEN
Allosteric modulators are an attractive approach to achieve receptor subtype-selective targeting of G protein-coupled receptors. Benzyl quinolone carboxylic acid (BQCA) is an unprecedented example of a highly selective positive allosteric modulator of the M1 muscarinic acetylcholine receptor (mAChR). However, despite favorable pharmacological characteristics of BQCA in vitro and in vivo, there is limited evidence of the impact of allosteric modulation on receptor regulatory mechanisms such as ß-arrestin recruitment or receptor internalization and endocytic trafficking. In the present study we investigated the impact of BQCA on M1 mAChR regulation. We show that BQCA potentiates agonist-induced ß-arrestin recruitment to M1 mAChRs. Using a bioluminescence resonance energy transfer approach to monitor intracellular trafficking of M1 mAChRs, we show that once internalized, M1 mAChRs traffic to early endosomes, recycling endosomes and late endosomes. We also show that BQCA potentiates agonist-induced subcellular trafficking. M1 mAChR internalization is both ß-arrestin and G protein-dependent, with the third intracellular loop playing an important role in the dynamics of ß-arrestin recruitment. As the global effect of receptor activation ultimately depends on the levels of receptor expression at the cell surface, these results illustrate the need to extend the characterization of novel allosteric modulators of G protein-coupled receptors to encapsulate the consequences of chronic exposure to this family of ligands.
Asunto(s)
Endocitosis/fisiología , Endosomas/metabolismo , Receptor Muscarínico M1/química , Receptor Muscarínico M1/metabolismo , Regulación Alostérica/fisiología , Animales , Arrestinas/metabolismo , Células CHO , Liberación de Peligros Químicos , Cricetulus , Quinasa 2 del Receptor Acoplado a Proteína-G/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Células HEK293 , Humanos , Ligandos , Transporte de Proteínas/fisiología , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/fisiología , beta-ArrestinasRESUMEN
Diffusion can enhance Förster resonance energy transfer (FRET) when donors or acceptors diffuse distances that are similar to the distances separating them during the donor's excited state lifetime. Lanthanide donors remain in the excited state for milliseconds, which makes them useful for time-resolved FRET applications but also allows time for diffusion to enhance energy transfer. Here we show that diffusion dramatically enhances FRET between membrane proteins labeled with lanthanide donors. This phenomenon complicates interpretation of experiments that use long-lived donors to infer association or proximity of mobile membrane proteins, but also offers a method of monitoring diffusion in membrane domains in real time in living cells.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia/normas , Elementos de la Serie de los Lantanoides/química , Receptores Adrenérgicos beta 2/química , Proteínas Recombinantes de Fusión/química , Proteínas SNARE/química , Difusión , Expresión Génica , Células HEK293 , Humanos , Elementos de la Serie de los Lantanoides/metabolismo , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 2/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas SNARE/genética , Proteínas SNARE/metabolismo , Coloración y EtiquetadoRESUMEN
Peripheral and integral membrane proteins can be located in several different subcellular compartments, and it is often necessary to determine the location of such proteins or to track their movement in living cells. Image-based colocalization of labeled membrane proteins and compartment markers is frequently used for this purpose, but this method is limited in terms of throughput and resolution. Here we show that bioluminescence resonance energy transfer (BRET) between membrane proteins of interest and compartment-targeted BRET partners can report subcellular location and movement of membrane proteins in live cells. The sensitivity of the method is sufficient to localize a few hundred protein copies per cell. The spatial resolution can be sufficient to determine membrane topology, and the temporal resolution is sufficient to track changes that occur in less than 1 second. BRET requires little user intervention, and is thus amenable to large-scale experimental designs with standard instruments.