RESUMEN
Immobilized protein makes a profound impact on the development of assays for drug discovery, diagnosis and in vivo biological interaction analysis. Traditional methods are enormously challenged by the G-protein coupled receptor ascribed to the loss of receptor functions. We introduced a ß2-adrenergic receptor (ß2-AR) aptamer into the immobilization of the receptor. This was achieved by mixing the receptor conjugated silica gel with cell lysates containing the receptor. We found that the aptamer-directed method makes immobilized ß2-AR good stability in seven days and high specificity of ligand recognition at the subtype receptor level. Feasibility of the immobilized ß2-AR in drug-receptor interaction analysis was evaluated by injection amount-dependent method, nonlinear chromatography, and peak decay analysis. Salbutamol, methoxyphenamine, ephedrine hydrochloride, clorprenaline, tulobuterol, bambuterol, propranolol and ICI 118551 bound to the receptor through one type of binding sites. The association constants presented good agreement within the three methods but exhibited clear differences from the data by radio-ligand binding assay. Regarding these results, we concluded that the aptamer-directed method will probably become an alternative for reversible and site-specific immobilization of GPCRs directly from complex matrices; the immobilized receptor is qualitative for drug-receptor interaction analysis.
Asunto(s)
Técnicas de Química Analítica/métodos , Cromatografía , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/aislamiento & purificación , Agonistas Adrenérgicos beta/metabolismo , Sitios de Unión , Interacciones Farmacológicas , Ligandos , Receptores Adrenérgicos beta 2/metabolismoRESUMEN
ß2-adrenergic receptor (ß2-AR) was expressed efficiently using Bac-to-Bac Baculovirus Expression System in Sf9 cells as a bio-recognition element for multianalyte screening of ß-agonist residues in pork. Sf9 cells were selected as the expression system, and codon optimization of wild-type nucleic acid sequence and time-dependent screening of expression conditions were then carried out for enhancing expression level and biological activity. Under optimum conditions of multiplicity of infection (MOI) = 5 and 48 h post transfection, the protein yield was up to 1.23 mg/ml. After purification by chromatographic techniques, the purified recombinant protein was applied to develop a direct competitive enzyme-linked receptor assay (ELRA) and the efficiency and reliability of the assay was determined. The IC50 values of clenbuterol, salbutamol, and ractopamine were 28.36, 50.70, and 59.57 µg/l, and clenbuterol showed 47.61% and 55.94% cross-reactivities with ractopamine and salbutamol, respectively. The limit of detection (LOD) was 3.2 µg/l and the relevant recoveries in pork samples were in the range of 73.0-91.2%, 69.4-84.6%, and 63.7-80.2%, respectively. The results showed that it had better performance compared with other present nonradioactive receptorbased assays, indicating that the genetically modified ß2-AR would have great application potential in detection of ß-agonist residues.
Asunto(s)
Agonistas Adrenérgicos beta/análisis , Técnicas Biosensibles/métodos , Expresión Génica , Receptores Adrenérgicos beta 2/metabolismo , Carne Roja/análisis , Agonistas Adrenérgicos beta/metabolismo , Animales , Clonación Molecular , Límite de Detección , Unión Proteica , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 2/aislamiento & purificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Reproducibilidad de los Resultados , Células Sf9 , Spodoptera , PorcinosRESUMEN
Large-scale expression of ß2-adrenergic receptor (ß2-AR) in functional form is necessary for establishment of receptor assays for detecting illegally abused ß-adrenergic agonists (ß-agonists). Cell-based heterologous expression systems have manycritical difficulties in synthesizing this membrane protein, such as low protein yields and aberrant folding. To overcome these challenges, the main objective of the present work was to synthesize large amounts of functional ß2-AR in a cell-free system based on Escherichia coli extracts. A codon-optimized porcine ß2-AR gene (codon adaptation index: 0.96) suitable for high expression in E. coli was synthesized and transcribed to the cell-free system, which contributed to increase the expression up to 1.1 mg/ml. After purification using Ni-affinity chromatography, the bioactivity of the purified receptor was measured by novel enzyme-linked receptor assays. It was determined that the relative affinities of the purified ß2-AR for ß-agonists in descending order were as follows: clenbuterol > salbutamol > ractopamine. Moreover, their IC50 values were 45.99, 60.38, and 78.02 µg/liter, respectively. Although activity of the cell-free system was slightly lower than activity of systems based on insect and mammalian cells, this system should allow production of ß2-AR in bulk amounts sufficient for the development of multianalyte screening methods for detecting ß-agonist residues.
Asunto(s)
Agonistas de Receptores Adrenérgicos beta 2/análisis , Sistema Libre de Células/metabolismo , Receptores Adrenérgicos beta 2/biosíntesis , Animales , Escherichia coli/metabolismo , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 2/aislamiento & purificación , Porcinos/metabolismoRESUMEN
Integral membrane proteins are amphipathic molecules crucial for all cellular life. The structural study of these macromolecules starts with protein extraction from the native membranes, followed by purification and crystallisation. Detergents are essential tools for these processes, but detergent-solubilised membrane proteins often denature and aggregate, resulting in loss of both structure and function. In this study, a novel class of agents, designated mannitol-based amphiphiles (MNAs), were prepared and characterised for their ability to solubilise and stabilise membrane proteins. Some of MNAs conferred enhanced stability to four membrane proteins including a G protein-coupled receptor (GPCR), the ß2 adrenergic receptor (ß2 AR), compared to both n-dodecyl-d-maltoside (DDM) and the other MNAs. These agents were also better than DDM for electron microscopy analysis of the ß2 AR. The ease of preparation together with the enhanced membrane protein stabilisation efficacy demonstrates the value of these agents for future membrane protein research.
Asunto(s)
Manitol/química , Proteínas de la Membrana/química , Tensoactivos/química , Antiportadores/química , Antiportadores/aislamiento & purificación , Arabidopsis/química , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/aislamiento & purificación , Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificación , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Complejos de Proteína Captadores de Luz/química , Complejos de Proteína Captadores de Luz/aislamiento & purificación , Proteínas de la Membrana/aislamiento & purificación , Estabilidad Proteica , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/aislamiento & purificación , Rhodobacter capsulatus/química , SolubilidadRESUMEN
ARRDC3 is one of six known human α-arrestins, and has been implicated in the downregulation of the ß2-adrenergic receptor (ß2AR). ARRDC3 consists of a two-lobed arrestin fold and a C-terminal tail containing two PPYX motifs. In the current model for receptor downregulation by ARRDC3, the arrestin fold portion is thought to bind the receptor, while the PPXY motifs recruit ubiquitin ligases of the NEDD4 family. Here we report the crystal structures of the N-terminal lobe of human ARRDC3 in two conformations, at 1.73 and 2.8 Å resolution, respectively. The structures reveal a large electropositive region that is capable of binding phosphate ions of crystallization. Residues within the basic patch were shown to be important for binding to ß2AR, similar to the situation with ß-arrestins. This highlights potential parallels in receptor recognition between α- and ß-arrestins.
Asunto(s)
Arrestinas/química , Arrestinas/metabolismo , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/metabolismo , Arrestinas/aislamiento & purificación , Sitios de Unión , Células HEK293 , Humanos , Modelos Moleculares , Conformación Proteica , Receptores Adrenérgicos beta 2/aislamiento & purificaciónRESUMEN
Oligomerization is one of several mechanisms that can regulate the activity of G protein-coupled receptors (GPCRs), but little is known about the structure of GPCR oligomers. Crystallography and NMR are the only methods able to reveal the details of receptor-receptor interactions at an atomic level, and several GPCR homodimers already have been described from crystal structures. Two clusters of symmetric interfaces have been identified from these structures that concur with biochemical data, one involving helices I, II, and VIII and the other formed mainly by helices V and VI. In this chapter, we describe the protocols used in our laboratory for the crystallization of rhodopsin and the ß2-adrenergic receptor (ß2-AR). For bovine rhodopsin, we developed a new purification strategy including a (NH4)2SO4-induced phase separation that proved essential to obtain crystals of photoactivated rhodopsin containing parallel dimers. Crystallization of native bovine rhodopsin was achieved by the classic vapor-diffusion technique. For ß2-AR, we developed a purification strategy based on previously published protocols employing a lipidic cubic phase to obtain diffracting crystals of a ß2-AR/T4-lysozyme chimera bound to the antagonist carazolol.
Asunto(s)
Receptores Adrenérgicos beta 2/química , Proteínas Recombinantes de Fusión/química , Rodopsina/química , Segmento Externo de la Célula en Bastón/química , Sulfato de Amonio/química , Animales , Bovinos , Centrifugación por Gradiente de Densidad , Cromatografía de Afinidad , Cromatografía en Gel , Cristalización , Cristalografía por Rayos X , Glucósidos/química , Muramidasa/química , Muramidasa/genética , Propanolaminas/química , Multimerización de Proteína , Estructura Secundaria de Proteína , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 2/aislamiento & purificación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Rodopsina/aislamiento & purificación , Células Sf9 , Spodoptera , Proteínas Virales/química , Proteínas Virales/genética , Acetato de Zinc/químicaRESUMEN
G-protein-coupled receptors (GPCRs) are membrane proteins with high significance as therapeutic targets. The GPCR family in humans includes over 800 different proteins, but high-resolution experimental structures of nearly complete structures are limited to only seven different family members. Additional structural data are expected to stimulate drug development efforts and an improved understanding of the basic signal transduction process. Characterization of GPCR three-dimensional structures is slowed by numerous intrinsic challenges, but similarities in the structures of the helical bundles of the currently available structures suggest that alternative methods focused on the more highly variable loop segments connecting the helical regions are of value. This chapter focuses on the design of GPCR segment models that spontaneously fold in solution to allow characterization of individual loop segments from any GPCR sequence, as well as how to establish appropriate conditions for structural studies and integrate the structures that result from such studies into computational models to produce hybrid homology-experimental GPCR structural models.
Asunto(s)
Modelos Moleculares , Receptores Adrenérgicos beta 2/química , Secuencia de Aminoácidos , Animales , Simulación por Computador , Expresión Génica , Humanos , Datos de Secuencia Molecular , Pliegue de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 2/aislamiento & purificación , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Alineación de Secuencia , Homología Estructural de ProteínaRESUMEN
Human beta2-adrenergic receptor is one of the most studied G-protein-coupled receptors. It plays a key role in autonomic nervous system and is a drug target in cardiovascular and pulmonary diseases. Despite the fact that its crystal structure was revealed, a physiological role and molecular mechanisms of its action remain largely unknown. We designed the construct pVR2ADRH, which contained the gene for human beta2-adrenergic receptor with a polyhistidine tag C-terminal extension. The recombinant DNA was used for transformation of the GS115 strain of Pichia pastoris. The heterologous expression level obtained was about 20 mg/l. The receptor was extracted from membrane fraction and was purified by metal-affinity and ion-exchange chromatography. The active receptors were isolated by alprenolol-sepharose CL-4B. The resulting level of purified human beta2-adrenergic receptor was approximately 1 mg per liter of culture. The homogeneity of the protein sample was confirmed by a dynamical light scattering analysis of the receptor's micellar solution.
Asunto(s)
Expresión Génica , Receptores Adrenérgicos beta 2 , Humanos , Pichia , Receptores Adrenérgicos beta 2/biosíntesis , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 2/aislamiento & purificación , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
Beta2-Adrenoceptors are the members of cell surface receptors which perform their signal transduction to the interior of the cells by coupling to heterotrimeric G proteins. On the foundation of successful clone and expression of beta2-adrenoceptors, a two-step chromatographic method using Ni-chelated Sepharose High Performance affinity media and Quaternary Sepharose Fast Flow anion exchangers was established to prepare recombinant beta2-adrenoceptors expressed in E. coli BL21 (DE3) as histidine-tagged protein. In the affinity chromatographic column, the buffer A was consisted of 20 mmol/L phosphate buffered saline (PBS) containing 500 mmol/L NaCl (pH 7.4), and the buffer B was consisted of buffer A with the addition of 0.5 mol/L imidazole (pH 7.4); in anion chromatographic column, the buffer A was 20 mmol/L PBS (pH 7.4), and the buffer B was consisted of buffer A with 800 mmol/L NaCl (pH 7.4). The analysis results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and high performance size-exclusion chromatography (Shim-pack Diol-300) showed that the purity of obtained beta2-adrenoceptors was about 95%. Furthermore, the bioactivity of beta2-adrenoceptors was studied by receptor ligand combination test, and the results assured the object protein possessed good bioactivity. Finally the conclusion can be reached that the method can effectively separate active recombined beta2-adrenoceptors.
Asunto(s)
Cromatografía/métodos , Receptores Adrenérgicos beta 2/aislamiento & purificación , Proteínas Recombinantes/aislamiento & purificación , Animales , Escherichia coli/genética , Escherichia coli/metabolismo , Vectores Genéticos/genética , Receptores Adrenérgicos beta 2/biosíntesis , Receptores Adrenérgicos beta 2/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , PorcinosRESUMEN
The three beta adrenergic receptor subtypes, beta1-, beta2- and beta3-, were expressed in the methylotrophic yeast Pichia pastoris. These receptors were N-terminally fused to the enhanced green fluorescent protein (EGFP) and the fluorescent properties of EGFP were used: (1) to select the recombinant strains, (2) to monitor the expression of the fluorescent receptors, and (3) to monitor the purification of the receptors by immobilized metal affinity chromatography. We demonstrate here that Pichia pastoris can be an alternative host to express and purify milligram amounts of human beta adrenergic receptors.
Asunto(s)
Proteínas Fluorescentes Verdes/metabolismo , Pichia/metabolismo , Receptores Adrenérgicos beta/aislamiento & purificación , Receptores Adrenérgicos beta/metabolismo , Fluorescencia , Proteínas Fluorescentes Verdes/genética , Humanos , Immunoblotting , Cinética , Receptores Adrenérgicos beta/genética , Receptores Adrenérgicos beta 1/genética , Receptores Adrenérgicos beta 1/aislamiento & purificación , Receptores Adrenérgicos beta 1/metabolismo , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 2/aislamiento & purificación , Receptores Adrenérgicos beta 2/metabolismo , Receptores Adrenérgicos beta 3/genética , Receptores Adrenérgicos beta 3/aislamiento & purificación , Receptores Adrenérgicos beta 3/metabolismo , Nitrato de Plata , SolubilidadRESUMEN
G protein-coupled receptors (GPCRs) respond to a diverse array of ligands, mediating cellular responses to hormones and neurotransmitters, as well as the senses of smell and taste. The structures of the GPCR rhodopsin and several G proteins have been determined by x-ray crystallography, yet the organization of the signaling complex between GPCRs and G proteins is poorly understood. The observations that some GPCRs are obligate heterodimers, and that many GPCRs form both homo- and heterodimers, has led to speculation that GPCR dimers may be required for efficient activation of G proteins. However, technical limitations have precluded a definitive analysis of G protein coupling to monomeric GPCRs in a biochemically defined and membrane-bound system. Here we demonstrate that a prototypical GPCR, the beta2-adrenergic receptor (beta2AR), can be incorporated into a reconstituted high-density lipoprotein (rHDL) phospholipid bilayer particle together with the stimulatory heterotrimeric G protein, Gs. Single-molecule fluorescence imaging and FRET analysis demonstrate that a single beta2AR is incorporated per rHDL particle. The monomeric beta2AR efficiently activates Gs and displays GTP-sensitive allosteric ligand-binding properties. These data suggest that a monomeric receptor in a lipid bilayer is the minimal functional unit necessary for signaling, and that the cooperativity of agonist binding is due to G protein association with a receptor monomer and not receptor oligomerization.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Lipoproteínas HDL/metabolismo , Receptores Adrenérgicos beta 2/aislamiento & purificación , Receptores Adrenérgicos beta 2/metabolismo , Animales , Bovinos , Transferencia Resonante de Energía de Fluorescencia , Humanos , Lipoproteínas HDL/química , Lipoproteínas HDL/ultraestructura , Microscopía Electrónica de Transmisión , Modelos Moleculares , Unión Proteica , Estructura Cuaternaria de Proteína , Receptores Adrenérgicos beta 2/químicaRESUMEN
Human beta2-adrenergic receptor is a G-protein-coupled receptor with seven transmembrane helices, and is important in pharmaceutical targeting on pulmonary and cardiovascular diseases. N-terminal histidine-tagged gene constructs with optimized codon usage were designed so as to obtain Pichia pastoris transformants with a high expression level. The constructs were inserted into the pPIC9 vector, and then electroporated into the SMD1168 strains. The highest expression level obtained was about 4 mg/liter-culture broth. The dissociation constant of the receptor in the membrane fraction was 1.2 nM toward CGP-12177 antagonist. The receptor was solubilized with sucrose monolaurate and purified with a series of chromatography steps including anion-exchange, Ni-Sepharose, alprenolol-Agarose, and hydroxyapatite columns. The receptor was heterogeneously glycosylated, showing broad SDS-PAGE bands around 70-90 kDa. After endoglycosidase treatment, the receptor appeared as a single band around 45 kDa, and was further purified with hydroxyapatite and gel-filtration columns. The receptor was eluted as a sharp peak at the gel-filtration elution volume corresponding to a molecular mass of 117 kDa. The saccharide-trimmed receptor thus purified is homogeneous as analyzed with SDS-PAGE, shows the dissociation constant of 4.7 nM toward CGP-12177 antagonist, and is suitable for crystallization experiments.
Asunto(s)
Pichia/metabolismo , Receptores Adrenérgicos beta 2/aislamiento & purificación , Cromatografía en Gel , Electroporación , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidasa/metabolismo , Metanol/farmacología , Pichia/efectos de los fármacos , Propanolaminas/metabolismo , Receptores Adrenérgicos beta 2/biosíntesis , Receptores Adrenérgicos beta 2/metabolismoRESUMEN
Integral membrane G protein-coupled receptors (GPCRs) compose the single most prolific class of drug targets, yet significant functional and structural questions remain unanswered for this superfamily. A primary reason for this gap in understanding arises from the difficulty of forming soluble, monodisperse receptor membrane preparations that maintain the transmembrane signaling activity of the receptor and provide robust biophysical and biochemical assay systems. Here we report a technique for self-assembling functional beta2-adrenergic receptor (beta2AR) into a nanoscale phospholipid bilayer system (Nanodisc) that is highly soluble in aqueous solution. The approximately 10-nm nanobilayer particles contain beta2AR in a native-like phospholipid bilayer domain of approximately 100 phospholipid molecules circumferentially bound by a membrane scaffold protein (MSP). The resulting construct allows for access to the physiologically intracellular and extracellular faces of the receptor and thus allows unrestricted access of antagonists, agonists, and G proteins. These Nanodisc-solubilized GPCRs can be directly purified by normal chromatographic procedures. We define the resultant Nanodisc-embedded monomeric beta2AR by antagonist and agonist binding isotherms and demonstrate faithful G protein coupling.
Asunto(s)
Membrana Dobles de Lípidos/metabolismo , Nanoestructuras/química , Nanotecnología , Receptores Adrenérgicos beta 2/aislamiento & purificación , Receptores Adrenérgicos beta 2/metabolismo , Línea Celular , Cromatografía en Gel , Detergentes/farmacología , Electroforesis en Gel de Poliacrilamida , Humanos , Membrana Dobles de Lípidos/química , Modelos Biológicos , Tamaño de la Partícula , Fosfatidilcolinas/química , Unión Proteica , Receptores Adrenérgicos beta 2/genética , Proteínas Recombinantes/metabolismo , Solubilidad , Agua/químicaRESUMEN
High-level expression of G-protein-coupled receptors (GPCRs) in functional form is required for structure-function studies. The main goal of the present work was to improve expression levels of beta2-adrenergic receptor (beta2-AR) so that biophysical studies involving EPR, NMR, and crystallography can be pursued. Toward this objective, the total synthesis of a codon-optimized hamster beta2-AR gene suitable for high-level expression in mammalian systems has been accomplished. Transient expression of the gene in COS-1 cells resulted in 18 +/- 3 pmol beta2-AR/mg of membrane protein, as measured by saturation binding assay using the beta2-AR antagonist [3H] dihydroalprenolol. Previously, we reported the development of an HEK293S tetracycline-inducible system for high-level expression of rhodopsin. Here, we describe construction of beta2-AR stable cell lines using the HEK293S-TetR-inducible system, which, after induction, express wild-type beta2-AR at levels of 220 +/- 40 pmol/mg of membrane protein corresponding to 50 +/- 8 microg/15-cm plate. This level of expression is the highest reported so far for any wild-type GPCR, other than rhodopsin. The yield of functional receptor using the single-step affinity purification is 12 +/- 3 microg/15-cm plate. This level of expression now makes it feasible to pursue structure-function studies using EPR. Furthermore, scale-up of beta2-AR expression using suspension cultures in a bioreactor should now allow production of enough beta2-AR for the application of biophysical techniques such as NMR spectroscopy and crystallography.
Asunto(s)
Ingeniería de Proteínas/métodos , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 2/metabolismo , Tetraciclina/farmacología , Antagonistas de Receptores Adrenérgicos beta 2 , Antagonistas Adrenérgicos beta/metabolismo , Animales , Secuencia de Bases , Línea Celular/efectos de los fármacos , Chlorocebus aethiops , Codón , Cricetinae , Dihidroalprenolol/metabolismo , Regulación de la Expresión Génica , Humanos , Ligandos , Mamíferos , Datos de Secuencia Molecular , Receptores Adrenérgicos beta 2/aislamiento & purificación , SolubilidadRESUMEN
In Escherichia coli and other cell-based expression systems, there are critical difficulties in synthesizing membrane proteins, such as the low protein expression levels and the formation of insoluble aggregates. However, structure determinations by X-ray crystallography require the purification of milligram quantities of membrane proteins. In this study, we tried to solve these problems by using cell-free protein expression with an E. coli S30 extract, with G protein coupled receptors (GPCRs) as the target integral membrane proteins. In this system, the thioredoxin-fusion vector induced high protein expression levels as compared with the non-fusion and hexa-histidine-tagged proteins. Two detergents, Brij35 and digitonin, effectively solubilized the produced GPCRs, with little or no effect on the protein yields. The synthesized proteins were detected by Coomassie brilliant blue staining within 1h of reaction initiation, and were easily reconstituted within phospholipid vesicles. Surprisingly, the unpurified, reconstituted thioredoxin-fused receptor proteins had functional activity, in that a specific affinity binding value of an antagonist was obtained for the receptor. This cell-free translation system (about 1mg/ml of reaction volume for 6-8 h) has biophysical and biochemical advantages for the synthesis of integral membrane proteins.
Asunto(s)
Receptores Acoplados a Proteínas G/biosíntesis , Receptores Acoplados a Proteínas G/genética , Animales , Sistema Libre de Células , ADN/genética , Detergentes , Digitonina , Escherichia coli/genética , Escherichia coli/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/biosíntesis , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/genética , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/aislamiento & purificación , Subunidades alfa de la Proteína de Unión al GTP Gs/biosíntesis , Subunidades alfa de la Proteína de Unión al GTP Gs/genética , Subunidades alfa de la Proteína de Unión al GTP Gs/aislamiento & purificación , Vectores Genéticos , Humanos , Técnicas In Vitro , Cinética , Fusión de Membrana , Polidocanol , Polietilenglicoles , Biosíntesis de Proteínas , Ratas , Receptor Muscarínico M2/biosíntesis , Receptor Muscarínico M2/genética , Receptor Muscarínico M2/aislamiento & purificación , Receptores Adrenérgicos beta 2/biosíntesis , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 2/aislamiento & purificación , Receptores Acoplados a Proteínas G/aislamiento & purificación , Receptores de Neurotensina/biosíntesis , Receptores de Neurotensina/genética , Receptores de Neurotensina/aislamiento & purificación , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Solubilidad , Tiorredoxinas/biosíntesis , Tiorredoxinas/genética , Tiorredoxinas/aislamiento & purificaciónRESUMEN
Cell fractionation, SDS-PAGE, quantitative Western blot, confocal immunolocalization and immunogold labelling were performed to find an interpretation of the physiological response of the unicellular eukaryote Paramecium to beta-adrenergic ligands. The 69 kDa polypeptide separated by SDS-PAGE in S2 and P2 Paramecium subcellular fractions cross-reacted with antibody directed against human beta2-adrenergic receptor. This was detected by Western blotting followed by chemiluminescent detection. Quantitative image analysis showed that beta-selective adrenergic agonist (-)-isoproterenol--previously shown to enhance phagocytic activity--evoked redistribution of the adrenergic receptor analogue from membraneous (P2) to cytosolic (S2) fraction. The relative increase in immunoreactive band intensity in S2 reached 80% and was paralleled by a 59% decrease in P2 fraction. Confocal immunofluorescence revealed beta2-adrenergic receptor sites on the cell surface and at the ridge of the cytopharynx--where nascent phagosomes are formed. This localization was confirmed by immunoelectron microscopy. These results indicate that the 69 kDa Paramecium polypeptide immunorelated to vertebrate beta2-adrenergic receptor appeared in this evolutionary ancient cell as a nutrient receptor.
Asunto(s)
Paramecium/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Animales , Sitios de Unión , Western Blotting , Fraccionamiento Celular , Membrana Celular/química , Membrana Celular/inmunología , Electroforesis en Gel de Poliacrilamida , Humanos , Isoproterenol/farmacología , Microscopía Confocal , Microscopía Inmunoelectrónica , Peso Molecular , Paramecium/inmunología , Paramecium/ultraestructura , Receptores Adrenérgicos beta 2/inmunología , Receptores Adrenérgicos beta 2/aislamiento & purificación , Vesículas Transportadoras/química , Vesículas Transportadoras/inmunología , Vertebrados/inmunología , Vertebrados/metabolismoAsunto(s)
Receptores Adrenérgicos beta 2/química , Rodopsina/metabolismo , Espectrometría de Fluorescencia/métodos , Secuencia de Aminoácidos , Cromatografía Liquida/métodos , Colorantes Fluorescentes/química , Datos de Secuencia Molecular , Conformación Proteica , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 2/aislamiento & purificación , Receptores Adrenérgicos beta 2/metabolismoRESUMEN
Palmitoylation is unique among lipid modifications in that it is reversible. In recent years, dynamic palmitoylation of G protein alpha subunits and of their cognate receptors has attracted considerable attention. However, very little is known concerning the acylation/deacylation cycle of the proteins in relation to their activity status. In particular, the relative contribution of the activation and desensitization of the signaling unit to the regulation of the receptors and G proteins palmitoylation state is unknown. To address this issue, we took advantage of the fact that a fusion protein composed of the stimulatory alpha subunit of trimeric G protein (Galpha(s)) covalently attached to the beta(2)-adrenergic receptor (beta(2)AR) as a carboxyl-terminal extension (beta(2)AR-Galpha(s)) can be stimulated by agonists but does not undergo rapid inactivation, desensitization, or internalization. When expressed in Sf9 cells, both the receptor and the Galpha(s) moieties of the fusion protein were found to be palmitoylated via thioester linkage. Stimulation with the beta-adrenergic agonist isoproterenol led to a rapid depalmitoylation of both the beta(2)AR and Galpha(s) and inhibited repalmitoylation. The extent of depalmitoylation induced by a series of agonists was correlated (0.99) with their intrinsic efficacy to stimulate the adenylyl cyclase activity. However, forskolin-stimulated cAMP production did not affect the palmitoylation state of beta(2)AR-Galpha(s), indicating that the agonist-promoted depalmitoylation is linked to conformational changes and not to second messenger generation. Given that, upon activation, the fusion protein mimics the activated receptor-G protein complex but cannot undergo desensitization, the data demonstrate that early steps in the activation process lead to the depalmitoylation of both receptor and G protein and that repalmitoylation requires later events that cannot be accommodated by the activated fusion protein.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Isoproterenol/farmacología , Ácido Palmítico/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Animales , Línea Celular , Clonación Molecular , Bromuro de Cianógeno , Subunidades alfa de la Proteína de Unión al GTP Gs/química , Subunidades alfa de la Proteína de Unión al GTP Gs/aislamiento & purificación , Humanos , Hidroxilamina/farmacología , Cinética , Sustancias Macromoleculares , Fragmentos de Péptidos/química , Fragmentos de Péptidos/aislamiento & purificación , Procesamiento Proteico-Postraduccional , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/aislamiento & purificación , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Spodoptera , TransfecciónRESUMEN
Salmeterol is a long-acting beta2-adrenergic receptor (beta 2AR) agonist used clinically to treat asthma. In addition to binding at the active agonist site, it has been proposed that salmeterol also binds with very high affinity at a second site, termed the "exosite", and that this exosite contributes to the long duration of action of salmeterol. To determine the position of the phenyl ring of the aralkyloxyalkyl side chain of salmeterol in the beta 2AR binding site, we designed and synthesized the agonist photoaffinity label [(125)I]iodoazidosalmeterol ([125I]IAS). In direct adenylyl cyclase activation, in effects on adenylyl cyclase after pretreatment of intact cells, and in guinea pig tracheal relaxation assays, IAS and the parent drug salmeterol behave essentially the same. Significantly, the photoreactive azide of IAS is positioned on the phenyl ring at the end of the molecule which is thought to be involved in exosite binding. Carrier-free radioiodinated [125I]IAS was used to photolabel epitope-tagged human beta 2AR in membranes prepared from stably transfected HEK 293 cells. Labeling with [(125)I]IAS was blocked by 10 microM (-)-alprenolol and inhibited by addition of GTP gamma S, and [125I]IAS migrated at the same position on an SDS-PAGE gel as the beta 2AR labeled by the antagonist photoaffinity label [125I]iodoazidobenzylpindolol ([125I]IABP). The labeled receptor was purified on a nickel affinity column and cleaved with factor Xa protease at a specific sequence in the large loop between transmembrane segments 5 and 6, yielding two peptides. While the control antagonist photoaffinity label [125I]IABP labeled both the large N-terminal fragment [containing transmembranes (TMs) 1-5] and the smaller C-terminal fragment (containing TMs 6 and 7), essentially all of the [125I]IAS labeling was on the smaller C-terminal peptide containing TMs 6 and 7. This direct biochemical evidence demonstrates that when salmeterol binds to the receptor, its hydrophobic aryloxyalkyl tail is positioned near TM 6 and/or TM 7. A model of IAS binding to the beta 2AR is proposed.