RESUMEN
Over half of cutaneous melanoma tumors have BRAFV600E/K mutations. Acquired resistance to BRAF inhibitors (BRAFi) remains a major hurdle in attaining durable therapeutic responses. In this study we demonstrate that ~50-60% of melanoma cell lines with vemurafenib resistance acquired in vitro show activation of RhoA family GTPases. In BRAFi-resistant melanoma cell lines and tumors, activation of RhoA is correlated with decreased expression of melanocyte lineage genes. Using a machine learning approach, we built gene expression-based models to predict drug sensitivity for 265 common anticancer compounds. We then projected these signatures onto the collection of TCGA cutaneous melanoma and found that poorly differentiated tumors were predicted to have increased sensitivity to multiple Rho kinase (ROCK) inhibitors. Two transcriptional effectors downstream of Rho, MRTF and YAP1, are activated in the RhoHigh BRAFi-resistant cell lines, and resistant cells are more sensitive to inhibition of these transcriptional mechanisms. Taken together, these results support the concept of targeting Rho-regulated gene transcription pathways as a promising therapeutic approach to restore sensitivity to BRAFi-resistant tumors or as a combination therapy to prevent the onset of drug resistance.
Asunto(s)
Desdiferenciación Celular/efectos de los fármacos , Melanoma/patología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Quinasas Asociadas a rho/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Línea Celular Tumoral , Activación Enzimática/efectos de los fármacos , Humanos , Melanocitos/efectos de los fármacos , Melanocitos/patología , Factores de Transcripción/metabolismo , Transcripción Genética/efectos de los fármacos , Proteínas Señalizadoras YAPRESUMEN
Gene transcription mechanisms are critical control points for cell function and differentiation as well as disease pathology. It has remained difficult to target gene transcription mechanisms with small molecule drugs due in part to the role of protein-protein interactions in transcription complexes. RhoA/C- GTPase regulation of the serum responsive transcription factor complex involving serum response factor (SRF) and myocardin-related transcription factor (MRTF) plays a key role in cancer and fibrotic mechanisms. In an attempt to disrupt this critical gene transcription mechanism, we undertook a high-throughput ″pathway screen″ using an SRE-Luciferase reporter which was activated by transient transfection of HEK293 cells with Ga13, an up- stream activator of RhoA and RhoC. The Rho/MRTF inhibitor tool compound CCG-1423 was identified in this screen. It and analogs such as CCG-203971 have been used extensively to disrupt myofibroblast activation and tissue fibrosis as well as melanoma cell migration and metastasis. In the present study, we have used immobilized compounds and mass spectroscopy to identify the molecular target of the CCG-203971 series of anti-fibrotic and anti-meta?static agents. It is a poorly studied intranuclear protein that participates in gene transcription regulation by NF-κB and MRTF/SRF mechanisms. This dual mechanism rationalizes the strong efficacy of CCG-203971 and related compounds as anti-fibrotic and anti-metastatic agents. The identification of a molecular target also greatly facilitates future compound development through structure- based drug discovery and target biology evaluation.
RESUMEN
Many biological processes are rhythmic and proper timing is increasingly appreciated as being critical for development and maintenance of physiological functions. To understand how temporal modulation of an input signal influences downstream responses, we employ microfluidic pulsatile stimulation of a G-protein coupled receptor, the muscarinic M3 receptor, in single cells with simultaneous real-time imaging of both intracellular calcium and NFAT nuclear localization. Interestingly, we find that reduced stimulation with pulses of ligand can give more efficient transcription factor activation, if stimuli are timed appropriately. Our experiments and computational analyses show that M3 receptor-induced calcium oscillations form a low pass filter while calcium-induced NFAT translocation forms a high pass filter. The combination acts as a band-pass filter optimized for intermediate frequencies of stimulation. We demonstrate that receptor desensitization and NFAT translocation rates determine critical features of the band-pass filter and that the band-pass may be shifted for different receptors or NFAT dynamics. As an example, we show that the two NFAT isoforms (NFAT4 and NFAT1) have shifted band-pass windows for the same receptor. While we focus specifically on the M3 muscarinic receptor and NFAT translocation, band-pass processing is expected to be a general theme that applies to multiple signaling pathways.
Asunto(s)
Factores de Transcripción NFATC/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal , Transporte Activo de Núcleo Celular , Área Bajo la Curva , Calcio/metabolismo , Simulación por Computador , Células HEK293 , Humanos , Ligandos , Microfluídica , Modelos TeóricosRESUMEN
We develop a mathematical model of nanoparticles depositing onto and penetrating into a biofilm grown in a parallel-plate flow cell. We carry out deposition experiments in a flow cell to support the modeling. The modeling and the experiments are motivated by the potential use of polymer nanoparticles as part of a treatment strategy for killing biofilms infecting the deep passages in the lungs. In the experiments and model, a fluid carrying polymer nanoparticles is injected into a parallel-plate flow cell in which a biofilm has grown over the bottom plate. The model consists of a system of transport equations describing the deposition and diffusion of nanoparticles. Standard asymptotic techniques that exploit the aspect ratio of the flow cell are applied to reduce the model to two coupled partial differential equations. We perform numerical simulations using the reduced model. We compare the experimental observations with the simulation results to estimate the nanoparticle sticking coefficient and the diffusion coefficient of the nanoparticles in the biofilm. The distributions of nanoparticles through the thickness of the biofilm are consistent with diffusive transport, and uniform distributions through the thickness are achieved in about four hours. Nanoparticle deposition does not appear to be strongly influenced by the flow rate in the cell for the low flow rates considered.
Asunto(s)
Biopelículas , Sistemas de Liberación de Medicamentos , Modelos Teóricos , Nanopartículas/administración & dosificación , Pseudomonas aeruginosa/fisiología , Quitosano/análogos & derivados , Quitosano/química , Difusión , Pulmón/microbiología , Moco , Nanopartículas/química , Organofosfatos/administración & dosificación , Organofosfatos/química , Polietilenglicoles/química , Polímeros/administración & dosificación , Polímeros/químicaRESUMEN
Regulators of G-protein signaling (RGS) proteins form a multifunctional signaling family. A key role of RGS proteins is binding to the G-protein Galpha-subunit and acting as GTPase-activating proteins (GAPs), thereby rapidly terminating G protein-coupled receptor (GPCR) signaling. Using the published RGS4-Gialpha1 X-ray structure we have designed and synthesized a series of cyclic peptides, modeled on the Gialpha Switch I region, that inhibit RGS4 GAP activity. These compounds should prove useful for elucidating RGS-mediated activity and serve as a starting point for the development of a novel class of therapeutic agent.
Asunto(s)
Péptidos Cíclicos/química , Péptidos Cíclicos/farmacología , Proteínas RGS/antagonistas & inhibidores , Animales , Células CHO , Cricetinae , Cricetulus , Proteínas Activadoras de GTPasa/metabolismo , Concentración 50 Inhibidora , Péptidos Cíclicos/síntesis química , Proteínas RGS/metabolismo , Relación Estructura-ActividadRESUMEN
Many drugs of abuse signal through receptors that couple to G proteins (GPCRs), so the factors that control GPCR signaling are likely to be important to the understanding of drug abuse. Contributions by the recently identified protein family, regulators of G protein signaling (RGS) to the control of GPCR function are just beginning to be understood. RGS proteins can accelerate the deactivation of G proteins by 1000-fold and in cell systems they profoundly inhibit signaling by many receptors, including mu-opioid receptors. Coupled with the known dynamic regulation of RGS protein expression and function, they are of obvious interest in understanding tolerance and dependence mechanisms. Furthermore, drugs that could inhibit their activity could be useful in preventing the development of or in treating drug dependence.
Asunto(s)
Fármacos del Sistema Nervioso Central/farmacología , Proteínas de Unión al GTP/metabolismo , Proteínas RGS/fisiología , Animales , Proteínas Activadoras de GTPasa/metabolismo , Expresión Génica/fisiología , Humanos , Estructura Terciaria de Proteína , Receptores de Superficie Celular/antagonistas & inhibidores , Receptores de Superficie Celular/metabolismo , Transducción de Señal/fisiología , Trastornos Relacionados con Sustancias/metabolismoRESUMEN
G protein coupled receptors activate signal transducing guanine nucleotide-binding proteins (G proteins), which consist of an alpha subunit and a betagamma dimer. Whole cell studies have reported that receptors signal through specific betagamma subtypes. Membrane reconstitution studies with the adenosine A(1) and alpha(2A) adrenergic receptors have reached a similar conclusion. We aimed to test the generality of this finding by comparing the gamma subtype specificity for four G(i)-coupled receptors: alpha(2A) adrenergic; A1 adenosine (A(1)-R); 5-hydroxytryptamine(1A) (5-HT(1A)-R); mu opioid. Membranes were reconstituted with Galpha(i)(1) and five gamma subtypes (dimerized to beta1). Using a sensitive alpha-betagamma binding assay, we show that all recombinant betagamma (except beta1gamma1) had comparable affinity for alpha(i)(1). Using high affinity agonist binding as a measure of receptor-G protein coupling, betagamma-containing gamma11 was the most potent for A(1)-R and 5-HT(1A)-R (p < 0.05, one way ANOVA) while gamma7 was most potent for the other two receptors. gamma11 was 3-8-fold more potent for the A(1)-R than were the other gamma subtypes. Also, gamma11 was 2-8-fold more potent for A(1)-R than at the other receptors, suggesting a unique coupling specificity of the A(1)-R for gamma11. In contrast, the discrimination by receptors for the other betagamma subtypes (beta1 and gamma1, gamma2, gamma7, and gamma10) was limited (2-3-fold). Thus the exquisite betagamma specificity of individual receptors reported in whole cell studies may depend on in vivo mechanisms beyond direct receptor recognition of betagamma subtypes.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Receptores Purinérgicos P1/metabolismo , Receptores de Serotonina/metabolismo , Animales , Células CHO , Línea Celular , Cricetinae , Proteínas de Unión al GTP Heterotriméricas/agonistas , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Receptores Adrenérgicos alfa 2/metabolismo , Receptores Opioides mu/metabolismo , Receptores de Serotonina 5-HT1 , Porcinos , Células Tumorales CultivadasRESUMEN
ANG II type 1 (AT(1)) receptors respond to sustained exposure to ANG II by undergoing downregulation of absolute receptor numbers. It has been assumed previously that downregulation involves endocytosis. The present study hypothesized that AT(1) receptor downregulation occurs independently of receptor endocytosis or G protein coupling. Mutant AT(1) receptors with carboxy-terminal deletions internalized <5% of radioligand compared with 65% for wild-type AT(1) receptors. The truncated AT(1) receptors retained the ability to undergo downregulation. These data suggest the existence of an alternative pathway to AT(1) receptor degradation that does not require endocytosis, per se. Point mutations in either the second transmembrane region or second intracellular loop impaired G protein (G(q)) coupling. These receptors exhibited a biphasic pattern of downregulation. The earliest phase of downregulation (0-2 h) was independent of coupling to G(q), but no additional downregulation was observed after 2 h of ANG II exposure in the receptors with impaired coupling to G(q). These data suggest that coupling to G(q) is required for the later phase (2-24 h) of AT(1) receptor downregulation.
Asunto(s)
Regulación hacia Abajo/fisiología , Proteínas de Unión al GTP/fisiología , Receptores de Angiotensina/fisiología , Sustitución de Aminoácidos , Angiotensina II/metabolismo , Animales , Sitios de Unión , Células COS , Calcio/metabolismo , Chlorocebus aethiops , Clonación Molecular , Endocitosis , Radioisótopos de Yodo , Cinética , Mutagénesis Sitio-Dirigida , Fosfatidilinositoles/metabolismo , Ensayo de Unión Radioligante , Ratas , Receptor de Angiotensina Tipo 1 , Receptor de Angiotensina Tipo 2 , Receptores de Angiotensina/química , Receptores de Angiotensina/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Eliminación de Secuencia , TransfecciónRESUMEN
G protein-coupled receptors (GPCRs) play a major role in signal transduction and are targets of many therapeutic drugs. The regulator of G protein signaling (RGS) proteins form a recently identified protein family, and they strongly modulate the activity of G proteins. Their best known function is to inhibit G protein signaling by accelerating GTP hydrolysis [GTPase activating protein (GAP)] thus turning off G protein signals. RGS proteins also possess non-GAP functions, through both their RGS domains and various non-RGS domains and motifs (e.g., GGL, DEP, DH/PH, PDZ domains and a cysteine string motif). They are a highly diverse protein family, have unique tissue distributions, are strongly regulated by signal transduction events, and will likely play diverse functional roles in living cells. Thus they represent intriguing, novel pharmacological/therapeutic targets. Drugs targeting RGS proteins can be divided into five groups: 1) potentiators of endogenous agonist function, 2) potentiators/desensitization blockers of exogenous GPCR agonists, 3) specificity enhancers of exogenous agonists, 4) antagonists of effector signaling by an RGS protein, and 5) RGS agonists. In addition, a novel subsite distinction within the RGS domain has been proposed with significant functional implications and defined herein as "A-site" and "B-site". Therefore, RGS proteins should provide exciting new opportunities for drug development.
Asunto(s)
Diseño de Fármacos , Proteínas RGS/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Secuencias de Aminoácidos/fisiología , Animales , Sitios de Unión/efectos de los fármacos , Proteínas de Unión al GTP/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Estructura Terciaria de Proteína/efectos de los fármacos , Estructura Terciaria de Proteína/fisiología , Proteínas RGS/agonistas , Proteínas RGS/antagonistas & inhibidores , Especificidad por Sustrato/efectos de los fármacosRESUMEN
Receptor based signaling mechanisms are the primary source of cellular regulation. The superfamily of G protein-coupled receptors is the largest and most ubiquitous of the receptor mediated processes. We describe here the analysis in real-time of the assembly and disassembly of soluble G protein-coupled receptor-G protein complexes. A fluorometric method was utilized to determine the dissociation of a fluorescent ligand from the receptor solubilized in detergent. The ligand dissociation rate differs between a receptor coupled to a G protein and the receptor alone. By observing the sensitivity of the dissociation of a fluorescent ligand to the presence of guanine nucleotide, we have shown a time- and concentration-dependent reconstitution of the N-formyl peptide receptor with endogenous G proteins. Furthermore, after the clearing of endogenous G proteins, purified Galpha subunits premixed with bovine brain Gbetagamma subunits were also able to reconstitute with the solubilized receptors. The solubilized N-formyl peptide receptor and Galpha(i3) protein interacted with an affinity of approximately 10(-6) m with other alpha subunits exhibiting lower affinities (Galpha(i3) > Galpha(i2) > Galpha(i1) Galpha(o)). The N-formyl peptide receptor-G protein interactions were inhibited by peptides corresponding to the Galpha(i) C-terminal regions, by Galpha(i) mAbs, and by a truncated form of arrestin-3. This system should prove useful for the analysis of the specificity of receptor-G protein interactions, as well as for the elucidation and characterization of receptor molecular assemblies and signal transduction complexes.
Asunto(s)
Proteínas de Unión al GTP Heterotriméricas/metabolismo , Receptores de Superficie Celular/metabolismo , Animales , Arrestina/metabolismo , Encéfalo/metabolismo , Bovinos , Colorantes Fluorescentes , Humanos , Unión Proteica , Solubilidad , Espectrometría de Fluorescencia , Células U937RESUMEN
Three BODIPY GTPgammaS analogs (FL, 515, and TR), BODIPY FL GppNHp and BODIPY FL GTP molecules were synthesized as possible fluorescent probes to study guanine nucleotide binding spectroscopically. Binding to G(alphao) increases baseline analog fluorescence by 6-, 8.5-, 2.8-, 3.5-, and 3.0-fold, respectively. Binding of GTPgammaS and GppNHp analogs to G(alphao) is of high affinity (K(D) 11, 17, 55, and 110 nM, respectively) and reaches a stable plateau while fluorescence of BODIPY FL GTP shows a transient increase which returns to baseline. Furthermore, BODIPY FL GTPgammaS shows varying affinities for alpha(o), alpha(s), alpha(i1), and alpha(i2) (6, 58, 150, and 300 nM). The affinities of BODIPY FL GppNHp for all four G(alpha) subunits are 10-fold lower than for BODIPY FL GTPgammaS. Half-times for the fluorescence increase are consistent with known GDP release rates for those proteins. Enhancement of fluorescence upon binding the G(alpha) subunit is most likely due to a rotation around the gamma-thiol (GTPgammaS) or the 3' ribose-hydroxyl (GppNHp) bond to relieve the quenching of BODIPY fluorescence by the guanine base. Binding to G(alpha) exposes the BODIPY moiety to the external environment, as seen by an increase in sodium iodide quenching. The visible excitation and emission spectra and high fluorescence levels of these probes permit robust real-time detection of nucleotide binding.
Asunto(s)
Compuestos de Boro/síntesis química , Colorantes Fluorescentes/química , Proteínas de Unión al GTP/química , Guanosina Trifosfato/análogos & derivados , Guanosina Trifosfato/química , Escherichia coli , Proteínas de Unión al GTP/metabolismo , Guanosina Difosfato/química , Unión Proteica , Proteínas RGS/metabolismoRESUMEN
G-protein-coupled receptors activate signal-transducing G-proteins, which consist of an alpha subunit and a betagamma dimer. Membrane extraction with 5-7 M urea has been used to uncouple receptors from endogenous G-proteins to permit reconstitution with purified G-proteins. We show that alpha(i) subunits are inactivated with 5 M urea whereas the betagamma dimer requires at least 7 M urea for its inactivation. There is no significant loss of receptors. Surprisingly, Western-blot analysis indicates that the urea-denatured alpha(i) subunit remains mostly membrane-bound and that beta is only partially removed. After 7 M urea treatment, both alpha(i1) and betagamma subunits are required to restore high-affinity agonist binding and receptor-catalysed guanosine 5'-[gamma-thio]triphosphate binding. We demonstrate the generality of this approach for four G(i)-coupled receptors (alpha(2A)-adrenergic, adenosine A1, 5-hydroxytryptamine(1A) and mu-opioid) expressed in insect cells and two mammalian cell lines. Thus a selectivity of urea for G-protein alpha versus betagamma subunits is established in both concentration and mechanism.
Asunto(s)
Proteínas de Unión al GTP Heterotriméricas/antagonistas & inhibidores , Urea/farmacología , Animales , Western Blotting , Células CHO , Catálisis , Cricetinae , Dimerización , Relación Dosis-Respuesta a Droga , Encefalina Ala(2)-MeFe(4)-Gli(5)/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Subunidades de Proteína , Relación Estructura-Actividad , Células Tumorales Cultivadas , Urea/administración & dosificaciónRESUMEN
Constitutive activation of G protein-coupled receptors (GPCRs) is now well recognized and many classical GPCR antagonists have been found to be inverse agonists. For the alpha(2A)-adrenergic receptor (AR) we determine the relative inverse efficacies of a series of antagonists and utilize the extended ternary complex model to estimate the fraction of constitutively active mutant (CAM) receptors in the active state. Stable Chinese hamster ovary cell lines expressing the porcine alpha(2A)-AR in its wild-type (WT) and constitutively activated (CAM-T373K) form were isolated. Activation of both G(i) and G(s) was enhanced for CAM receptors. cAMP production was suppressed in cells with the CAM alpha(2A)-AR and this suppression was reversed by alpha(2)-adrenergic antagonists with an order of inverse efficacy of rauwolscine > yohimbine > RX821002 > MK912, whereas phentolamine and idazoxan were essentially neutral antagonists. This striking difference in inverse efficacy between idazoxan and RX821002 may account for in vivo pharmacological differences between these two alpha(2)-adrenergic antagonists. Agonist binding affinity to the non-G protein-coupled CAM receptor was 3- to 9-fold higher than to WT, whereas binding of the most efficacious inverse agonists, yohimbine and rauwolscine, was 1.7- and 2.1-fold weaker. Analysis of this difference by the extended ternary complex model indicates that approximately 50% of the CAM alpha(2A)-AR is in the active (R*) state although there is no detectable constitutive activity of the WT receptor in the absence of agonist.
Asunto(s)
Antagonistas de Receptores Adrenérgicos alfa 2 , Antagonistas Adrenérgicos alfa/farmacología , Idazoxan/análogos & derivados , Idazoxan/farmacología , Alcaloides/farmacología , Animales , Células CHO , Cricetinae , AMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Receptores de Imidazolina , Modelos Biológicos , Mutación , Conformación Proteica , Ensayo de Unión Radioligante , Receptores Adrenérgicos alfa 2/química , Receptores Adrenérgicos alfa 2/genética , Receptores Adrenérgicos alfa 2/metabolismo , Receptores de Droga/antagonistas & inhibidoresRESUMEN
We generated mutants of the transporter associated with antigen-processing subunits TAP1 and TAP2 that were altered at the conserved lysine residue in the Walker A motifs of the nucleotide binding domains (NBD). In other ATP binding cassette transporters, mutations of the lysine have been shown to reduce or abrogate the ATP hydrolysis activity and in some cases impair nucleotide binding. Mutants TAP1(K544M) and TAP2(K509M) were expressed in insect cells, and the effects of the mutations on nucleotide binding, peptide binding, and peptide translocation were assessed. The mutant TAP1 subunit is significantly impaired for nucleotide binding relative to wild type TAP1. The identical mutation in TAP2 does not significantly impair nucleotide binding relative to wild type TAP2. Using fluorescence quenching assays to measure the binding of fluorescent peptides, we show that both mutants, in combination with their wild type partners, can bind peptides. Since the mutant TAP1 is significantly impaired for nucleotide binding, these results indicate that nucleotide binding to TAP1 is not a requirement for peptide binding to TAP complexes. Peptide translocation is undetectable for TAP1.TAP2(K509M) complexes, but low levels of translocation are detectable with TAP1(K544M).TAP2 complexes. These results suggest an impairment in nucleotide hydrolysis by TAP complexes containing either mutant TAP subunit and indicate that the presence of one intact TAP NBD is insufficient for efficient catalysis of peptide translocation. Taken together, these results also suggest the possibility of distinct functions for TAP1 and TAP2 NBD during a single translocation cycle.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/química , Mutación , Miembro 3 de la Subfamilia B de Transportadores de Casetes de Unión a ATP , Transportadoras de Casetes de Unión a ATP/genética , Adenosina Difosfato/metabolismo , Secuencias de Aminoácidos , Animales , Apirasa/metabolismo , Línea Celular , ADN Complementario/metabolismo , Relación Dosis-Respuesta a Droga , Hidrólisis , Insectos , Cinética , Lisina/química , Microsomas/metabolismo , Mutagénesis Sitio-Dirigida , Péptidos/metabolismo , Pruebas de Precipitina , Unión Proteica , Transporte de Proteínas , Espectrometría de Fluorescencia , Temperatura , Factores de TiempoRESUMEN
A novel regulator of G-protein signaling (RGS) has been isolated from a highly purified population of mouse long-term hematopoietic stem cells, and designated RGS18. It has 234 amino acids consisting of a central RGS box and short divergent NH(2) and COOH termini. The calculated molecular weight of RGS18 is 27,610 and the isoelectric point is 8.63. Mouse RGS18 is expressed from a single gene and shows tissue specific distribution. It is most highly expressed in bone marrow followed by fetal liver, spleen, and then lung. In bone marrow, RGS18 level is highest in long-term and short-term hematopoietic stem cells, and is decreased as they differentiate into more committed multiple progenitors. The human RGS18 ortholog has a tissue-specific expression pattern similar to that of mouse RGS18. Purified RGS18 interacts with the alpha subunit of both G(i) and G(q) subfamilies. The results of in vitro GTPase single-turnover assays using Galpha(i) indicated that RGS18 accelerates the intrinsic GTPase activity of Galpha(i). Transient overexpression of RGS18 attenuated inositol phosphates production via angiotensin receptor and transcriptional activation through cAMP-responsive element via M1 muscarinic receptor. This suggests RGS18 can act on G(q)-mediated signaling pathways in vivo.
Asunto(s)
Proteínas Portadoras/fisiología , Proteínas de Unión al GTP/fisiología , Células Madre Hematopoyéticas/fisiología , Péptidos y Proteínas de Señalización Intracelular , Transducción de Señal , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas Portadoras/química , Proteínas Portadoras/genética , Línea Celular , Clonación Molecular , GTP Fosfohidrolasas/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Variación Genética , Humanos , Ratones , Datos de Secuencia Molecular , Peso Molecular , Especificidad de Órganos , Proteínas RGS , Alineación de Secuencia , Homología de Secuencia de Aminoácido , TransfecciónRESUMEN
A radioligand is a radioactively labeled drug that can associate with a receptor, transporter, enzyme, or any protein of interest. Measuring the rate and extent of binding provides information on the number of binding sites, and their affinity and accessibility for various drugs. Radioligand binding experiments are easy to perform, and provide useful data in many fields. For example, radioligand binding studies are used to study receptor regulation, investigate receptor localization in different organs or regions using autoradiography, categorize receptor subtypes, and probe mechanisms of receptor signaling. This unit reviews the theory of receptor binding and explains how to analyze experimental data. Since binding data are usually best analyzed using nonlinear regression, this unit also explains the principles of curve fitting with nonlinear regression.
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Ensayo de Unión Radioligante/métodos , Sitios de Unión , Unión Competitiva , Intervalos de Confianza , Semivida , Cinética , Radioisótopos , Receptores de Superficie Celular/metabolismo , Análisis de RegresiónRESUMEN
Regulator of G-protein signaling (RGS) proteins accelerate GTP hydrolysis by Galpha subunits speeding deactivation. Galpha deactivation kinetics mediated by RGS are too fast to be directly studied using conventional radiochemical methods. We describe a stopped-flow spectroscopic approach to visualize these rapid kinetics by measuring the intrinsic tryptophan fluorescence decrease of Galpha accompanying GTP hydrolysis and Galpha deactivation on the millisecond time scale. Basal k(cat) values for Galpha(o), Galpha(i1), and Galpha(i2) at 20 degrees C were similar (0.025-0.033 s(-1)). Glutathione S-transferase fusion proteins containing RGS4 and an RGS7 box domain (amino acids 305-453) enhanced the rate of Galpha deactivation in a manner linear with RGS concentration. RGS4-stimulated rates could be measured up to 5 s(-1) at 3 microm, giving a catalytic efficiency of 1.7-2.8 x 10(6) m(-1) s(-1) for all three Galpha subunits. In contrast, RGS7 showed catalytic efficiencies of 0.44, 0.10, and 0.02 x 10(6) m(-1) s(-1) toward Galpha(o), Galpha(i2), and Galpha(i1), respectively. Thus RGS7 is a weaker GTPase activating protein than RGS4 toward all Galpha subunits tested, but it is specific for Galpha(o) over Galpha(i1) or Galpha(i2). Furthermore, the specificity of RGS7 for Galpha(o) does not depend on N- or C-terminal extensions or a Gbeta(5) subunit but resides in the RGS domain itself.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Proteínas RGS/fisiología , Fluorescencia , GTP Fosfohidrolasas/metabolismo , Guanosina Trifosfato/metabolismo , Hidrólisis , Cinética , Subunidades de ProteínaRESUMEN
Two constructs encoding the human micro-opioid receptor (hMOR) fused at its C terminus to either one of two Galpha subunits, Galpha(o1) (hMOR-Galpha(o1)) and Galpha(i2) (hMOR-Galpha(i2)), were expressed in Escherichia coli at levels suitable for pharmacological studies (0.4-0.5 pmol/mg). Receptors fused to Galpha(o1) or to Galpha(i2) maintained high-affinity binding of the antagonist diprenorphine. Affinities of the micro-selective agonists morphine, [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]enkephalin (DAMGO), and endomorphins as well as their potencies and intrinsic activities in stimulating guanosine 5'-O-(3-[(35)S]thiotriphosphate) ([(35)S]GTPgammaS) binding were assessed in the presence of added purified Gbetagamma subunits. Both fusion proteins displayed high-affinity agonist binding and agonist-stimulated [(35)S]GTPgammaS binding. In the presence of Gbetagamma dimers, the affinities of DAMGO and endomorphin-1 and -2 were higher at hMOR-Galpha(i2) than at hMOR-Galpha(o1), whereas morphine displayed similar affinities at the two chimeras. Potencies of the four agonists in stimulating [(35)S]GTPgammaS binding at hMOR-Galpha(o1) were similar, whereas at hMOR-Galpha(i2), endomorphin-1 and morphine were more potent than DAMGO and endomorphin-2. The intrinsic activities of the four agonists at the two fusion constructs were similar. The results confirm hMOR coupling to Galpha(o1) and Galpha(i2) and support the hypothesis of the existence of multiple receptor conformational states, depending on the nature of the G protein to which it is coupled.
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Subunidades alfa de la Proteína de Unión al GTP Gi-Go , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Receptores Opioides mu/metabolismo , Animales , Encéfalo/metabolismo , Bovinos , Diprenorfina/farmacocinética , Encefalina Ala(2)-MeFe(4)-Gli(5)/farmacología , Escherichia coli , Subunidad alfa de la Proteína de Unión al GTP Gi2 , Proteínas de Unión al GTP/química , Proteínas de Unión al GTP/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Proteínas de Unión al GTP Heterotriméricas/genética , Humanos , Cinética , Morfina/farmacología , Oligopéptidos/farmacología , Proteínas Proto-Oncogénicas/genética , Ensayo de Unión Radioligante , Receptores Opioides mu/agonistas , Receptores Opioides mu/genética , Proteínas Recombinantes de Fusión/agonistas , Proteínas Recombinantes de Fusión/metabolismo , TritioRESUMEN
In this study, we investigated the hypothesis of agonist-directed trafficking of receptor signaling for the alpha(2A)-adrenergic receptor (alpha(2A)-AR). alpha(2A)-ARs couple to both G(s) and G(i) to stimulate or inhibit adenylyl cyclase activity. Chinese hamster ovary-K1 cell lines expressing the porcine alpha(2A)-AR at high (alpha(2A)-H) and low (alpha(2A)-L) levels were used to estimate the relative efficacies (R.e.s) of a series of agonists for the G(s) and G(i) pathways. G(s)-mediated responses were measured after pertussis toxin treatment to inactivate G(i) in alpha(2A)-H, whereas G(i) responses were measured in alpha(2A)-L, where G(s) responses were absent. The full agonist UK-14,304 showed a large receptor reserve for G(i) responses in alpha(2A)-H but little receptor reserve for G(s) responses in alpha(2A)-H or for G(i) responses in alpha(2A)-L. With the exception of l-isoproterenol (ISO), all agonists showed similar R.e.s at the alpha(2A)-AR for G(s) and G(i) responses, with rank orders of R.e.s as follows: l-epinephrine = l-norepinephrine = UK-14,304 > p-aminoclonidine > or = BHT-920 > or = BHT-933 > clonidine = p-iodoclonidine > or = xylazine > or = guanabenz. Interestingly, ISO had the highest efficacy at the alpha(2A)-AR for activating G(s) versus G(i) (9-fold higher); however, it had low potency for both. By several criteria, the ISO response was mediated by the alpha(2A)-AR, supporting the hypothesis of agonist-directed trafficking of receptor signaling or agonist-specific G protein selectivity. In contrast, the apparent G(i) pathway selectivity of oxymetazoline appears to be mediated by an endogenous serotonergic receptor. It is intriguing that a classic beta-AR agonist that activates G(s) through beta(2)-ARs also appears to produce a G(s)-selective conformation of the G(i)-coupled alpha(2A)-AR.
Asunto(s)
Agonistas de Receptores Adrenérgicos alfa 2 , Agonistas alfa-Adrenérgicos/farmacología , Agonistas Adrenérgicos beta/farmacología , Subunidades alfa de la Proteína de Unión al GTP Gs/fisiología , Isoproterenol/farmacología , Transducción de Señal/fisiología , Antagonistas Adrenérgicos alfa/farmacología , Animales , Sitios de Unión , Tartrato de Brimonidina , Células CHO , Cricetinae , Interacciones Farmacológicas , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/agonistas , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/fisiología , Subunidades alfa de la Proteína de Unión al GTP Gs/agonistas , Cinética , Oximetazolina/antagonistas & inhibidores , Oximetazolina/farmacología , Pindolol/análogos & derivados , Pindolol/farmacología , Unión Proteica , Quinoxalinas/farmacología , Receptores Adrenérgicos alfa 2/metabolismo , Receptores Adrenérgicos alfa 2/fisiología , Antagonistas de la Serotonina/farmacología , Transducción de Señal/efectos de los fármacos , Porcinos , Yohimbina/farmacologíaRESUMEN
Era is an essential Escherichia coli guanine nucleotide binding protein that appears to play a number of cellular roles. Although the kinetics of Era guanine nucleotide binding and hydrolysis have been described, guanine nucleotide exchange rates have never been reported. Here we describe a kinetic analysis of guanine nucleotide binding, exchange, and hydrolysis by Era using the fluorescent mant (N-methyl-3'-O-anthraniloyl) guanine nucleotide analogs. The equilibrium binding constants (K(D)) for mGDP and mGTP (0.61 +/- 0. 12 microgM and 3.6 +/- 0.80 microM, respectively) are similar to those of the unmodified nucleotides. The single turnover rates for mGTP hydrolysis by Era were 3.1 +/- 0.2 mmol of mGTP hydrolyzed/min/mol in the presence of 5 mM MgCl(2) and 5.6 +/- 0.3 mmol of mGTP hydrolyzed/min/mol in the presence of 0.2 mM MgCl(2). Moreover, Era associates with and exchanges guanine nucleotide rapidly (on the order of seconds) in both the presence and absence of Mg(2+). We suggest that models of Era function should reflect the rapid exchange of nucleotides in addition to the GTPase activity inherent to Era.