RESUMEN
A series of novel tetrazole analogues of resveratrol were synthesized and evaluated for their anti-leukemic activity against an extensive panel of human cancer cell lines and against the MV4-11 AML cell line. These molecules were designed as drug-like derivatives of the resveratrol analogue DMU-212 and its cyano derivatives. Four compounds 8g, 8h, 10a and 10b exhibited LD50 values of 4.60⯵M, 0.02⯵M, 1.46⯵M, and 1.08⯵M, respectively, against MV4-11 leukemia cells. The most potent compounds, 8h and 10b, were also found to be active against an extensive panel of human hematological and solid tumor cell lines; compound 8h was the most potent compound with GI50 values <10â¯nM against more than 90% of the human cancer cell lines in the 60-cell panel. Analogues 8g, 8h, 10a and 10b were also tested for their ability to inhibit the polymerization of tubulin, and compound 8h was found to be the most potent analogue. Molecular modeling studies demonstrated that 8h binds to the colchicine binding site on tubulin. Thus, compound 8h is considered to be a lead druglike molecule from this tetrazole series of compounds.
Asunto(s)
Antineoplásicos/farmacología , Tetrazoles/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Diseño de Fármacos , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Estructura Molecular , Relación Estructura-Actividad , Tetrazoles/síntesis química , Tetrazoles/químicaRESUMEN
Arginine methylation is a common post-translational modification, but its role in regulating protein function is poorly understood. This study demonstrates that, TNF receptor-associated factor 6 (TRAF6), an E3 ubiquitin ligase involved in innate immune signaling, is regulated by reversible arginine methylation in a range of primary and cultured cells. Under basal conditions, TRAF6 is methylated by the methyltransferase PRMT1, and this inhibits its ubiquitin ligase activity, reducing activation of toll-like receptor signaling. In response to toll-like receptor ligands, TRAF6 is demethylated by the Jumonji domain protein JMJD6. Demethylation is required for maximal activation of NF-κB. Loss of JMJD6 leads to reduced response, and loss of PRMT1 leads to basal pathway activation with subsequent desensitization to ligands. In human primary cells, variations in the PRMT1/JMJD6 ratio significantly correlate with TRAF6 methylation, basal activation of NF-κB, and magnitude of response to LPS. Reversible arginine methylation of TRAF6 by the opposing effects of PRMT1 and JMJD6 is, therefore, a novel mechanism for regulation of innate immune pathways.
Asunto(s)
Arginina/metabolismo , Transducción de Señal , Factor 6 Asociado a Receptor de TNF/metabolismo , Receptores Toll-Like/metabolismo , Animales , Western Blotting , Línea Celular , Línea Celular Tumoral , Células Cultivadas , Humanos , Quinasas Asociadas a Receptores de Interleucina-1/genética , Quinasas Asociadas a Receptores de Interleucina-1/metabolismo , Histona Demetilasas con Dominio de Jumonji/genética , Histona Demetilasas con Dominio de Jumonji/metabolismo , Cinética , Ligandos , Masculino , Metilación , Ratones Endogámicos C57BL , FN-kappa B/genética , FN-kappa B/metabolismo , Proteína-Arginina N-Metiltransferasas/genética , Proteína-Arginina N-Metiltransferasas/metabolismo , Interferencia de ARN , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Factor 6 Asociado a Receptor de TNF/genética , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
UNLABELLED: Hepatitis C virus (HCV) infection produces chronic liver injury that is significantly exacerbated by alcohol consumption. While multiple mechanisms contribute to this synergy, a viral-induced loss of antioxidant responses has been shown to play an important role. This study examined the effects of HCV infection and alcohol on the regulation of the transcription factor FOXO3, an important regulator of Mn-superoxide dismutase (SOD2) expression, a tumor suppressor, and a component of the hepatic antioxidant response system. FOXO3 was activated by either HCV or alcohol alone but suppressed by the combination. To understand this paradoxical result, we applied a capillary isoelectric focusing (IEF) method to determine the pattern of FOXO3 posttranslational modifications (PTMs) induced by HCV and alcohol. We observed the presence of multiple different nuclear and cytosolic species of FOXO3 and used antiphosphoserine, acetyl-lysine, methylarginine, and ubiquitin antibodies to identify the PTM patterns present in each species. HCV caused multiple changes including phosphorylation of FOXO3 at S-574, a novel c-Jun N-terminal kinase (JNK) site, which promoted nuclear translocation and transcription. Ethanol suppressed arginine-methylation of FOXO3 promoting nuclear export and degradation of the JNK phosphorylated form. Human liver biopsy samples showed the presence of the HCV-specific form of FOXO3 in HCV-infected livers but not in normal liver or nonalcoholic steatohepatitis. CONCLUSION: The development of this novel IEF method for the simultaneous quantification of differently modified FOXO3 species allowed us to demonstrate how HCV and alcohol combine to modify a complex pattern of FOXO3 PTMs that contribute to pathogenesis. This approach will allow further dissection of the role of protein PTMs in viral liver disease.
Asunto(s)
Consumo de Bebidas Alcohólicas/metabolismo , Factores de Transcripción Forkhead/metabolismo , Hepatitis C/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Procesamiento Proteico-Postraduccional , Sustitución de Aminoácidos , Línea Celular Tumoral , Etanol/farmacología , Proteína Forkhead Box O3 , Factores de Transcripción Forkhead/efectos de los fármacos , Humanos , Focalización Isoeléctrica , Metilación/efectos de los fármacos , Fosforilación/efectos de los fármacos , Solventes/farmacologíaRESUMEN
Hepatitis C virus (HCV) infection exacerbates alcoholic liver injury by mechanisms that include enhanced oxidative stress. The forkhead box transcription factor FOXO3 is an important component of the antioxidant stress response that can be altered by HCV. To test whether FOXO3 is protective for alcoholic liver injury, we fed alcohol to FOXO3(-/-) mice. After 3 weeks, one third of these mice developed severe hepatic steatosis, neutrophilic infiltration, and >10-fold alanine aminotransferase (ALT) elevations. In cell culture, either alcohol or HCV infection alone increased FOXO3 transcriptional activity and expression of target genes, but the combination of HCV and alcohol together caused loss of nuclear FOXO3 and decreased its transcriptional activity. This was accompanied by increased phosphorylation of FOXO3. Mice expressing HCV structural proteins on a background of reduced expression of superoxide dismutase 2 (SOD2; Sod2(+/-)) also had increased liver sensitivity to alcohol, with elevated ALT, steatosis, and lobular inflammation. Elevated ALT was associated with an alcohol-induced decrease in SOD2 and redistribution of FOXO3 to the cytosol. These results demonstrate that FOXO3 functions as a protective factor preventing alcoholic liver injury. The combination of HCV and alcohol, but not either condition alone, inactivates FOXO3, causing a decrease in expression of its target genes and an increase in liver injury. Modulation of the FOXO3 pathway is a potential therapeutic approach for HCV-alcohol-induced liver injury.
Asunto(s)
Depresores del Sistema Nervioso Central/efectos adversos , Etanol/efectos adversos , Factores de Transcripción Forkhead , Hepacivirus/metabolismo , Hepatitis C , Hepatopatías Alcohólicas , Animales , Depresores del Sistema Nervioso Central/farmacología , Etanol/farmacología , Proteína Forkhead Box O3 , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Hepacivirus/genética , Hepatitis C/genética , Hepatitis C/metabolismo , Hepatitis C/patología , Humanos , Hepatopatías Alcohólicas/genética , Hepatopatías Alcohólicas/metabolismo , Hepatopatías Alcohólicas/patología , Ratones , Ratones Noqueados , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Factores de Tiempo , Transcripción Genética/efectos de los fármacos , Transcripción Genética/genéticaRESUMEN
G-protein heterotrimers, composed of a guanine nucleotide-binding G alpha subunit and an obligate G betagamma dimer, regulate signal transduction pathways by cycling between GDP- and GTP-bound states. Signal deactivation is achieved by G alpha-mediated GTP hydrolysis (GTPase activity) which is enhanced by the GTPase-accelerating protein (GAP) activity of "regulator of G-protein signaling" (RGS) proteins. In a cellular context, RGS proteins have also been shown to speed up the onset of signaling, and to accelerate deactivation without changing amplitude or sensitivity of the signal. This latter paradoxical activity has been variably attributed to GAP/enzymatic or non-GAP/scaffolding functions of these proteins. Here, we validated and exploited a G alpha switch-region point mutation, known to engender increased GTPase activity, to mimic in cis the GAP function of RGS proteins. While the transition-state, GDP x AlF(4)(-)-bound conformation of the G202A mutant was found to be nearly identical to wild-type, G alpha(i1)(G202A) x GDP assumed a divergent conformation more closely resembling the GDP x AlF(4)(-)-bound state. When placed within Saccharomyces cerevisiae G alpha subunit Gpa1, the fast-hydrolysis mutation restored appropriate dose-response behaviors to pheromone signaling in the absence of RGS-mediated GAP activity. A bioluminescence resonance energy transfer (BRET) readout of heterotrimer activation with high temporal resolution revealed that fast intrinsic GTPase activity could recapitulate in cis the kinetic sharpening (increased onset and deactivation rates) and blunting of sensitivity also engendered by RGS protein action in trans. Thus G alpha-directed GAP activity, the first biochemical function ascribed to RGS proteins, is sufficient to explain the activation kinetics and agonist sensitivity observed from G-protein-coupled receptor (GPCR) signaling in a cellular context.
Asunto(s)
GTP Fosfohidrolasas/química , Proteínas de Unión al GTP/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Alanina/química , Relación Dosis-Respuesta a Droga , Glicina/química , Humanos , Hidrólisis , Cinética , Luminiscencia , Modelos Moleculares , Mutación , Feromonas/metabolismo , Saccharomyces cerevisiae/metabolismo , Transducción de SeñalRESUMEN
Papillary thyroid carcinoma (PTC) demonstrates significantly reduced patient survival with metastatic progression. Tumor progression can be influenced by metabolism, including antioxidant glutathione (GSH). Glutathione peroxidase 4 (GPX4) is a selenoenzyme that uses GSH as a co-factor to regulate lipid peroxidation of cell membranes during increased oxidative stress. GPX4 suppression in tumor cells can induce ferroptosis. This study aims to examine ferroptosis as a potentially critical pathway in effective targeting of thyroid cancer (TC) cells. We treated human TC cells (K1, MDA-T68, MDA-T32, TPC1) with (1S,3R)-RSL3 (RSL3), a small-molecule inhibitor of GPX4 and examined the effects on ferroptosis, tumor cell survival and migration, spheroid formation, oxidative stress, DNA damage repair response, and mTOR signaling pathway in vitro. GPX4 inhibition activated ferroptosis, inducing TC cell death, rapid rise in reactive oxygen species and effectively arrested cell migration in vitro. Suppression of mTOR signaling pathway triggered autophagy. GPX4 genetic knockdown mirrored RSL3 effect on mTOR pathway suppression. RSL3 subdued DNA damage repair response by suppressing phosphorylation of nucleophosmin 1 (NPM1). Thus, observed potent induction of ferroptosis, GPX4-dependent novel suppression of mTOR pathway and DNA damage repair response in preclinical in vitro model of TC supports GPX4 targeting for therapeutic benefit in advanced therapy-resistant thyroid cancers.
Asunto(s)
Ferroptosis , Neoplasias de la Tiroides , Humanos , Fosfolípido Hidroperóxido Glutatión Peroxidasa , Muerte Celular , Glutatión Peroxidasa/metabolismo , Glutatión/metabolismo , Neoplasias de la Tiroides/tratamiento farmacológico , Serina-Treonina Quinasas TORRESUMEN
Gene fusions are known to drive many human cancers. Therefore, the functional characterization of newly discovered fusions is critical to understanding the oncobiology of these tumors and to enable therapeutic development. NPM1-TYK2 is a novel fusion identified in CD30 + lymphoproliferative disorders, and here we present the functional evaluation of this fusion gene as an oncogene. The chimeric protein consists of the amino-terminus of nucleophosmin 1 (NPM1) and the carboxyl-terminus of tyrosine kinase 2 (TYK2), including the kinase domain. Using in vitro lymphoid cell transformation assays and in vivo tumorigenic xenograft models we present direct evidence that the fusion gene is an oncogene. NPM1 fusion partner provides the critical homodimerization needed for the fusion kinase constitutive activation and downstream signaling that are responsible for cell transformation. As a result, our studies identify NPM1-TYK2 as a novel fusion oncogene and suggest that inhibition of fusion homodimerization could be a precision therapeutic approach in cutaneous T-cell lymphoma patients expressing this chimera.
RESUMEN
Anaplastic large cell lymphoma (ALCL) is an aggressive type of non-Hodgkin lymphoma. More than three-fourths of anaplastic lymphoma kinase (ALK)-positive ALCL cases express the nucleophosmin 1 (NPM1)-ALK fusion gene as a result of t(2;5) chromosomal translocation. The homodimerization of NPM1-ALK fusion protein mediates constitutive activation of the chimeric tyrosine kinase activity and downstream signaling pathways responsible for lymphoma cell proliferation and survival. Gilteritinib is a tyrosine kinase inhibitor recently approved by the FDA for the treatment of FMS-like tyrosine kinase mutation-positive acute myeloid leukemia. In this study, we demonstrate for the first time gilteritinib-mediated growth inhibitory effects on NPM1-ALK-driven ALCL cells. We utilized a total of five ALCL model cell lines, including both human and murine. Gilteritinib treatment inhibits NPM1-ALK fusion kinase phosphorylation and downstream signaling, resulting in induced apoptosis. Gilteritinib-mediated apoptosis was associated with caspase 3/9, PARP cleavage, the increased expression of proapoptotic protein BAD, and decreased expression of antiapoptotic proteins, survivin and MCL-1. We also found downregulation of fusion kinase activity resulted in decreased c-Myc protein levels. Furthermore, cell-cycle analysis indicated gilteritinib induced G0-G1-phase cell-cycle arrest and reduced CD30 expression. In summary, our preclinical studies explored the novel therapeutic potential of gilteritinib in the treatment of ALCL cells expressing NPM1-ALK and potentially in other ALK or ALK fusion-driven hematologic or solid malignancies. IMPLICATIONS: Our preclinical results explore the use of gilteritinib for the treatment of NPM1-ALK-driven ALCL cells and pave a path for developing future clinical trials. VISUAL OVERVIEW: http://mcr.aacrjournals.org/content/molcanres/19/5/913/F1.large.jpg.
Asunto(s)
Quinasa de Linfoma Anaplásico/metabolismo , Compuestos de Anilina/uso terapéutico , Linfoma Anaplásico de Células Grandes/tratamiento farmacológico , Pirazinas/uso terapéutico , Compuestos de Anilina/farmacología , Humanos , Linfoma Anaplásico de Células Grandes/genética , Pirazinas/farmacologíaRESUMEN
Many of the molecules that mediate G-protein signaling are thought to constitutively associate with each other in variably stable signaling complexes. Much of the evidence for signaling complexes has come from Förster resonance energy transfer and bioluminescence resonance energy transfer (BRET) studies. However, detection of constitutive protein association with these methods is hampered by nonspecific energy transfer that occurs when donor and acceptor molecules are in close proximity by chance. We show that chemically-induced recruitment of local third-party BRET donors or acceptors reliably separates nonspecific and specific BRET. We use this method to reexamine the constitutive association of class A G-protein-coupled receptors (GPCRs) with other GPCRs and with heterotrimeric G-proteins. We find that beta2 adrenoreceptors constitutively associate with each other and with several other class A GPCRs. In contrast, GPCRs and G-proteins are unlikely to exist in stable constitutive preassembled complexes.
Asunto(s)
Transferencia de Energía/fisiología , Transferencia Resonante de Energía de Fluorescencia/métodos , Proteínas de Unión al GTP/fisiología , Mediciones Luminiscentes/métodos , Proteínas de la Membrana/fisiología , Receptores Acoplados a Proteínas G/fisiología , Transducción de Señal/fisiología , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/fisiología , Proteínas de Unión al GTP/metabolismo , Unión Proteica , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
Epstein-Barr virus (EBV) is directly implicated in several B-cell lymphoid malignancies. EBV-associated lymphomas are characterized by prominent activation of the NF-κB pathway and targeting this pathway establishes a rationale for a therapeutic approach. The ubiquitin/proteasome signaling plays an essential role in the regulation of the NF-κB pathway. Ixazomib is an FDA-approved, orally bioavailable proteasome inhibitor. Here we report the first preclinical evaluation of ixazomib-mediated growth-inhibitory effects on EBV-infected B-lymphoblastoid cell lines Raji and Daudi. Ixazomib induced apoptosis in these cell lines in a dose-dependent manner. Cell-cycle analysis demonstrated ixazomib treatment induced cell-cycle arrest at the G2-M phase with a concomitant decrease in G0-G1 and S phases. The results further revealed an increase in p53, p21, and p27 levels and a decrease in survivin and c-Myc protein levels. Mechanistically, ixazomib treatment resulted in the accumulation of polyubiquitinated proteins, including phosphorylated IκBα with a significant reduction of p65 subunit nuclear translocation. Altogether, our preclinical data support the rationale for in vivo testing of ixazomib in EBV-associated B-cell neoplasms. IMPLICATIONS: This preclinical study supports the use of oral proteasome inhibitor ixazomib for targeting NF-κB signaling in the treatment of EBV-associated B-cell neoplasms.Visual Overview: http://mcr.aacrjournals.org/content/molcanres/17/4/839/F1.large.jpg.
Asunto(s)
Compuestos de Boro/farmacología , Infecciones por Virus de Epstein-Barr/patología , Glicina/análogos & derivados , Linfoma de Células B/tratamiento farmacológico , Linfoma de Células B/virología , Antineoplásicos/farmacología , Línea Celular Tumoral , Infecciones por Virus de Epstein-Barr/metabolismo , Infecciones por Virus de Epstein-Barr/virología , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Glicina/farmacología , Humanos , Linfoma de Células B/metabolismo , Linfoma de Células B/patología , Puntos de Control de la Fase M del Ciclo Celular/efectos de los fármacos , Terapia Molecular Dirigida , Inhibidor NF-kappaB alfa/metabolismo , FN-kappa B/metabolismo , Fosforilación , Inhibidores de Proteasas/farmacología , Complejo de la Endopetidasa Proteasomal/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Nucleophosmin 1 (NPM1) is a nucleolar phosphoprotein that performs diverse biological functions including molecular chaperoning, ribosome biogenesis, DNA repair, and genome stability. Acute myeloid leukemia (AML) is a heterogeneous disease, more than half of the AML cases exhibit normal karyotype (NK). Approximately 50-60 percent of patients with NK-AML carry NPM1 mutations which are characterized by cytoplasmic dislocation of the NPM1 protein. In AML, mutant NPM1 (NPM1c+) acts in a dominant negative fashion and also blocks the differentiation of myeloid cells through gain-of-function for the AML phenotype. Currently, there is limited knowledge on the gain-of-function mechanism of mutant NPM1. Here, we review the known mechanisms of mutant NPM1 in the pathogenesis of AML. We describe genetic abnormalities, the clinical significance of exon-12 mutations in the NPM1 gene, and chromosomal translocations including the recently discovered NPM1-TYK2, and NPM1-HAUS1. Also, we outline the possible therapeutic interventions for the treatment of AML by targeting NPM1. Overall, the review will summarize present knowledge on mutant NPM1 origin, pathogenesis, and therapy in AML.
Asunto(s)
Predisposición Genética a la Enfermedad , Leucemia Mieloide Aguda/genética , Mutación , Proteínas Nucleares/genética , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Biomarcadores de Tumor , Mutación con Ganancia de Función , Estudios de Asociación Genética , Humanos , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/metabolismo , Terapia Molecular Dirigida , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Nucleofosmina , Pronóstico , Procesamiento Proteico-Postraduccional , Transducción de Señal/efectos de los fármacos , Translocación GenéticaAsunto(s)
Antineoplásicos/uso terapéutico , Proteínas de Ciclo Celular/antagonistas & inhibidores , Chaperoninas/antagonistas & inhibidores , Expresión Génica , Linfoma Anaplásico de Células Grandes/tratamiento farmacológico , Linfoma Anaplásico de Células Grandes/genética , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Tirosina Quinasas/genética , Antineoplásicos/administración & dosificación , Antineoplásicos/efectos adversos , Humanos , Linfoma Anaplásico de Células Grandes/metabolismo , Nucleofosmina , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/efectos adversos , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/metabolismo , Resultado del TratamientoRESUMEN
G protein-coupled receptors (GPCRs) self-associate as dimers or higher-order oligomers in living cells. The stability of associated GPCRs has not been extensively studied, but it is generally thought that these receptors move between the plasma membrane and intracellular compartments as intact dimers or oligomers. Here we show that ß(2)-adrenergic receptors (ß(2)ARs) that self-associate at the plasma membrane can dissociate during agonist-induced internalization. We use bioluminescence-resonance energy transfer (BRET) to monitor movement of ß(2)ARs between subcellular compartments. BRET between ß(2)ARs and plasma membrane markers decreases in response to agonist activation, while at the same time BRET between ß(2)ARs and endosome markers increases. Energy transfer between ß(2)ARs is decreased in a similar manner if either the donor- or acceptor-labeled receptor is mutated to impair agonist binding and internalization. These changes take place over the course of 30 minutes, persist after agonist is removed, and are sensitive to several inhibitors of arrestin- and clathrin-mediated endocytosis. The magnitude of the decrease in BRET between donor- and acceptor-labeled ß(2)ARs suggests that at least half of the receptors that contribute to the BRET signal are physically segregated by internalization. These results are consistent with the possibility that ß(2)ARs associate transiently with each other in the plasma membrane, or that ß(2)AR dimers or oligomers are actively disrupted during internalization.
Asunto(s)
Receptores Adrenérgicos beta 2/metabolismo , Animales , Células Cultivadas , Endocitosis/genética , Endocitosis/fisiología , Transferencia de Energía/genética , Transferencia de Energía/fisiología , Humanos , Modelos Biológicos , Unión Proteica/genética , Unión Proteica/fisiología , Multimerización de Proteína/genética , Multimerización de Proteína/fisiología , Transporte de Proteínas/genética , Transporte de Proteínas/fisiología , Receptores Adrenérgicos beta 2/genética , TransfecciónRESUMEN
Signals mediated by heterotrimeric G proteins often develop over the course of tens of milliseconds, and could require either conformational rearrangement or complete physical dissociation of Galphabetagamma heterotrimers. Although it is known that some active heterotrimers are dissociated (into Galpha and Gbetagamma) at steady-state, it is not clear that dissociation occurs quickly enough to participate in rapid signaling. Here we show that fusion proteins containing the c-terminus of GPCR kinase 3 (GRK3ct) and either the fluorescent protein cerulean or Renilla luciferase bind to venus-labeled Gbetagamma dimers (Gbetagamma-V), resulting in Förster or bioluminescence resonance energy transfer (FRET or BRET). GRK3ct fusion proteins are freely-diffusible, and do not form preassembled complexes with G proteins. GRK3ct fusion proteins bind to free Gbetagamma-V dimers but not to rearranged heterotrimers, and thus can report G protein dissociation with high temporal resolution. We find that heterotrimer dissociation can occur in living cells in less than 100 ms. Under the conditions of these experiments diffusion and collision of masGRK3ct fusion proteins and Gbetagamma-V were not rate-limiting. These results indicate that G protein heterotrimers can dissociate quickly enough to participate in rapid signaling.