Damos las gracias al generoso apoyo de varias fundaciones y donantes filantrópicos (ver SakmarLab.org).

1. Sitio específico de incorporación genética de Unnatural Aminoácidos en GPCRs <ol><li> Mantener las células HEK293T en medio DMEM (4,5 g / l de glucosa, glutamina 2 mM) suplementado con suero bovino fetal al 10% (FBS) a 37 ° C en una atmósfera de CO2 al 5%. </li><li> Transfectar las células cultivadas a 60-80% de confluencia en una placa de 10 cm usando reactivo Lipofectamine Plus. <ol><li> Para 750 l de DMEM, añadir 10 l de reactivo Plus, 3,5 g de ADNc de GPCR (pMT4. Rho o pcDNA 3.1. CCR5) que contiene el codón de parada ámbar en una posición deseada, 3,5 g de ADNc supresor de ARNt (pSVB.Yam) y 0,35 g de ADNc sintetasa mutante-amino acil ARNt para p-azido-L-fenilalanina (pcDNA.RS). Incubar a temperatura ambiente durante 15 min. También realizar una transfección similar utilizando el ADNc de GPCR en peso (que no contiene un codón de parada ámbar) para servir como un control. Añadir esta mezcla a 750 l de DMEM con 17 l Lipofectamine. Después de equilibrar 15 min a temperatura ambiente, llevar elvolumen total de 4 ml. </li><li> Aspirar los medios sobre 10 cm de placa, se aplican mezcla de transfección a las células, y volver a 37 ° C en el 5% atmósfera de CO 2. Después de 4-6 horas, complementar las células con 4 ml de DMEM que contiene 20% de SFB y 1 mM AZF. </li></ol></li><li> Al día siguiente, vuelva a colocar el medio de cultivo por DMEM que contiene 10% de SFB y 0,5 mM AZF. </li><li> Células de cosecha 48 horas después de la transfección, para analizar la expresión o proceder a procedimientos fotorreticulación o de etiquetado descritos en las siguientes secciones. <ol><li> Confirmar la expresión de la mutante ámbar de GPCR en presencia de la SAU en el medio de crecimiento. Hacemos esto mediante la detección de inmunotransferencia Western. Lyse el sedimento celular en 1% (w / v) de dodecil-β-D-maltósido (DDM) en 20 mM de Tris-HCl, pH 7,5, NaCl 100 mM durante 1 hora a 4 ° C. Centrifugar el lisado a 16.000 xg y resolver el sobrenadante en condiciones reductoras por SDS-PAGE. Transferencia a una membrana de PVDF y llevar a cabo el análisis de inmunoblot para detectar la expresión del receptor de longitud completa utilizando the 1D4 mAb (Nacional de Cultivo Celular Center), que reconoce un epítopo de ingeniería en el extremo C-terminal del receptor. 31 </li></ol></li></ol> 2. Asignación de un sitio de unión al ligando Usando Targeted fotorreticulación <ol><li> 48 horas después de la transfección, los medios de comunicación el aspirado de células y reemplazar con 4 ml 1x Tampón fosfato salino (PBS). Volver a 37 ° C durante 15 min. </li><li> Resuspender las células con PBS 1x y transferir a un tubo Falcon. Centrifugar a 1500 rpm durante 2 min para sedimentar las células y aspirar el sobrenadante. </li><li> Resuspender el sedimento de células en solución salina tamponada de Hank 1x que contenía HEPES, pH 7,5, 0,2% de BSA y el ligando tritiado a concentraciones saturantes de unión para la longitud requerida de tiempo y temperatura para asegurar la formación del complejo receptor-ligando 20. </li><li> Transferir la suspensión celular a una placa de 24 pocillos o de 96 pocillos para la fotoactivación del AZF a 365 nm (por ejemplo, usar una lámpara UV-A) durante 15 min a 4 ° C. Mantenienn de la placa en hielo para evitar el calentamiento de la muestra y la lisis celular. </li><li> Transfiera las células a un tubo Eppendorf, centrifugar para sedimentar las células, separar el sobrenadante y proceder a su análisis. Los gránulos se pueden almacenar a -20 ° C para su uso futuro. </li><li> Resuspender los sedimentos celulares en tampón de lisis que contiene 1% (w / v) de DDM, mM de Tris-HCl 20, pH 7,5 y los inhibidores de la proteasa. Después de la incubación de 1 hora a 4 ° C, centrifugar el lisado de células durante 10 min a 16.000 xg, y transferir el sobrenadante a un nuevo tubo. </li><li> Añadir resina 2B mAb 1D4-Sepharose al sobrenadante y se incuba durante la noche a 4 ° C a immunopurify el GPCR usando el C-terminal 1D4 mAb epítopo de ingeniería. Al día siguiente, lavar las perlas varias veces con tampón de lisis. Muestras se eluye con 1% SDS a 37 º C durante 1 hora con agitación. </li><li> Transferir una porción del eluyente a un vial que contiene 15 ml de fluido de centelleo y contar en un contador beta de centelleo para cuantificar la cantidad de unión específica del ligando tritiado a thcorreo del receptor. </li><li> Resolver el restante 1D4 mAb eluyente purificado por electroforesis en gel estándar. Nosotros usamos un gel SDS-PAGE al 4-12% y luego la transferencia semiseco a una membrana de PVDF para inmunotransferencia. También se incluyen un carril con la escala estándar de proteínas para guiar aproximación visual de peso molecular. <ol><li> Bloquear la membrana de PVDF con leche 5% en tampón TBS que contiene 0,05% de Tween-20. Sonda de la membrana para confirmar la expresión del receptor de longitud completa con mAb 1D4 seguido por el anticuerpo secundario anti-IgG de ratón conjugado con HRP. </li><li> Tratar con un aumento de quimioluminiscencia reactivo (ECL) y exponer la membrana a autorradiografía película para visualizar las bandas. </li></ol></li><li> Cortar la membrana con una hoja de afeitar para separar cada carril muestra, seguido por el corte en marcadores específicos de peso molecular. Transferir los segmentos de membrana a viales que contenían fluido de centelleo. Cuantificar la cantidad de tritio en cada segmento de la membrana mediante el recuento en un contador de centelleo beta. </li><li> Identificar lo positivophotocrosslink con la detección de tritio en la masa molecular aparente igual a la suma de la del GPCR y el ligando. </li></ol> 3. Targeted Epitope Etiquetado y ensayo inmunoabsorbente ligado a enzimas Superficie Celular (ELISA) <ol><li> 24 horas después de la transfección, lavar las células con 1 ml de PBS 1X y trypsinize con 1 ml de tripsina al 0,25% durante 3 min. Complementar con 9 ml de DMEM que contiene 10% de SFB y 0,5 mM AZF. Resuspender las células y contar la densidad celular. Utilizamos un hemocitómetro estándar. </li><li> Pre-tratamiento de una placa de 96 pocillos de alto vinculante, de fondo claro con 100 ml de 0,01 mg / ml de poli-D-lisina por pocillo. Incubar durante 30 minutos a temperatura ambiente, se lava varias veces con 1x PBS y secar al aire. </li><li> Placa de 200 l de las células transfectadas a una densidad de 6 x 4 al 08 10 x 10 4 células / pocillo a la placa de 96 pocillos y se vuelven a 37 ° C en 5% de CO 2 atmósfera. </li><li> Al día siguiente preparar células para el etiquetado. Lave suavemente tres veces con 100 l / pocillo de PBS 1x to eliminar cualquier que contiene medio de crecimiento del AZF. Asegúrese de células permanecen adheridas, por ejemplo, mediante el uso de PBS que contenía Ca 2 + y Mg 2 +. </li><li> Preparar 50-200 M reactivo marcador FLAG-triarilfosfina en 1x HBSS / PBS a partir de una acción mantenida a 5-20 mM en PBS. Aplicar 60 ml a cada pocillo (por triplicado para cada mutante ámbar) y volver a 37 ° C durante 30 min a 4 h. Mantener un conjunto de pozos sin tratamiento en la etiqueta 1x HBSS / PBS. También incluya un control de GPCR en peso para comparar con variantes AZF-GPCR. </li><li> Lavar las células 3 veces con 100 l / pocillo de tampón de bloqueo, BB (1x PBS, 0,5% de BSA), para eliminar por completo la etiqueta sin reaccionar. </li><li> Aplicar 100 l / pocillo de 4% de paraformaldehído (preparado a partir de una población 16% en 1x PBS) a las células. Incubar durante 20 min a temperatura ambiente. Lavar tres veces con BB. </li><li> Realizar el protocolo de ELISA de la superficie celular estándar para detectar receptor marcado y la expresión del receptor. Incubar con el anticuerpo primario durante 1,5 h en hielo seguido de una secundariatibody incubación a temperatura ambiente durante 1 h. <ol><li> Añadir 100 l de anticuerpo anti-FLAG M2 (por ejemplo, 1:2.000 dilución de anticuerpo realizados en BB) para detectar el epítopo Flag péptido del reactivo etiquetado con FLAG triarilfosfina. También sondear los pocillos tratados que no tiene etiqueta como un control negativo. Añadir anti-CCR5 2D7 (usamos una dilución 1:500) de anticuerpos a un conjunto separado de pozos para determinar en peso o azf-GPCR expresión en la superficie celular. </li><li> Lavar las células tres veces con BB, y se incuba con 100 l de anticuerpo secundario anti-IgG de ratón conjugado con HRP (1:2000 dilución). </li><li> Lave cuidadosamente las células cinco veces con BB. Añadir 50 l de tampón de detección: Amplex Red, 20 mM de H 2 O 2, 1x PBS (1:10:90 relación) e incubar 15 min a temperatura ambiente. Recopilar datos espectrales en un lector de placas de pocillos múltiples fluorescencia a λ ex = 530 y λ em = 590. </li></ol></li></ol> 4. Bioorthogonal Marcaje fluorescente de un GPCR <ol><li> Lyse10 7 células cosechadas expresan en peso o azf-rodopsina variantes en 1 ml de tampón de solubilización que contiene 1% (w / v) de DM, HEPES 50 mM o Tris-HCl, pH 6,8, NaCl 100 mM, CaCl2 1 mM e inhibidores de proteasa para 1 horas a 4 ° C. Centrifugar el lisado de células a 15.000 xg durante 10 min y recoger la fracción sobrenadante. </li><li> Añadir 100 l de resina Sepharose 2B 1D4-mAb para capturar el receptor usando el C-terminal epítopo 1D4 de ingeniería. Incubar durante 12 horas a 4 ° C. La resina se lava tres veces con 0,1% (w / v) de DDM en 0,1 M de tampón de fosfato de sodio, pH 7,3. </li><li> Añadir 0,1 mM de fluoresceína-fosfina en un volumen total de 0,3 a 0,5 ml para etiquetar receptor inmovilizado en la matriz de inmunoafinidad (1D4-mAb Sepharose 2B). Incubar durante 12 horas a temperatura ambiente. Centrifugar y eliminar el sobrenadante. La resina se lava para quitar la etiqueta que no ha reaccionado. </li><li> Eluir el receptor marcado con tampón de elución que contiene 100 ml de 0,1% (w / v) de DDM y 0,33 mg / ml de nonapéptido (C9 péptido contra el epítopo 1D4)en tampón de fosfato 2 mM, pH 6,0 mediante incubación en hielo durante 1 h. </li><li> Resolver muestras marcadas por SDS-PAGE en condiciones reductoras. Lavar geles brevemente en PBS y luego visualizar etiquetado de azf-GPCR por fluorescencia en gel. Por ejemplo, utilizar un escáner de fluorescencia confocal con un láser de longitud de onda de 488 nm para detectar el receptor modificado con fluoresceína. </li></ol>

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S., MacKenzie, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+antibodies+to+rhodopsin:+characterization,+cross-reactivity,+and+application+as+structural+probes.">Monoclonal antibodies to rhodopsin: characterization, cross-reactivity, and application as structural probes.</a> Biochemistry. 22, 653-660 (1983).</li><li>Gether, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agonists+induce+conformational+changes+in+transmembrane+domains+III+and+VI+of+the+beta2+adrenoceptor.">Agonists induce conformational changes in transmembrane domains III and VI of the beta2 adrenoceptor.</a> The EMBO journal. 16, 6737-6747 (1997).</li><li>Hubbell, W. L., Altenbach, C., Hubbell, C. M., Khorana, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rhodopsin+structure,+dynamics,+and+activation:+A+perspective+from+crystallography,+site-directed+spin+labeling,+sulfhydryl+reactivity,+and+disulfide+cross-linking.">Rhodopsin structure, dynamics, and activation: A perspective from crystallography, site-directed spin labeling, sulfhydryl reactivity, and disulfide cross-linking.</a> Advances in Protein Chemistry; Membrane Proteins. 63, 243-290 (2003).</li><li>Cai, K., Itoh, Y., Khorana, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+of+contact+sites+in+complex+formation+between+transducin+and+light-activated+rhodopsin+by+covalent+crosslinking:+use+of+a+photoactivatable+reagent.">Mapping of contact sites in complex formation between transducin and light-activated rhodopsin by covalent crosslinking: use of a photoactivatable reagent.</a> Proceedings of the National Academy of Sciences of the United States of America. 98, 4877-4882 (2001).</li><li>Dorman, G., Prestwich, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Benzophenone+photophores+in+biochemistry.">Benzophenone photophores in biochemistry.</a> Biochemistry. 33, 5661-5673 (1994).</li><li>Zhang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+strategy+for+the+site-specific+modification+of+proteins+in+vivo.">A new strategy for the site-specific modification of proteins in vivo.</a> Biochemistry. 42, 6735-6746 (2003).</li><li>Dirksen, A., Hackeng, T. M., Dawson, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleophilic+catalysis+of+oxime+ligation.">Nucleophilic catalysis of oxime ligation.</a> Angew. Chem. Int. Ed. 45, 7581-7584 (2006).</li><li>Yanagisawa, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multistep+engineering+of+pyrrolysyl-tRNA+synthetase+to+genetically+encode+N(epsilon)-(o-azidobenzyloxycarbonyl)+lysine+for+site-specific+protein+modification.">Multistep engineering of pyrrolysyl-tRNA synthetase to genetically encode N(epsilon)-(o-azidobenzyloxycarbonyl) lysine for site-specific protein modification.</a> Chemistry &amp; biology. 15, 1187-1197 (2008).</li></ol>

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Se describe aquí una metodología sólida para la incorporación específica de sitio de una sonda reactiva, AZF, en GPCRs y demostrar tres aplicaciones útiles de esta herramienta para el estudio de la estructura y dinámica de los GPCRs. Nuestro método para sitios específicos incorporar UAAs elude un problema fundamental con una estrategia alternativa basada en el etiquetado químicamente 32, 33 o adjuntar photocrosslinkers 34 a un solo mutante cisteína accesible. Aunque, las químicas que se...

Receptores acoplados a proteínas G (GPCRs heptahelical) comprenden una superfamilia de proteínas de la membrana altamente dinámicos que median señales extracelulares importantes y diversos. En el paradigma clásico, la activación del receptor se acopla con los cambios conformacionales inducidos por ligando. 1, 2 avances recientes en biología estructural de los GPCR han proporcionado información significativa sobre los mecanismos moleculares de la señalización transmembrana. 3-5 Sin embargo, para entender con mayor precisión la química mecanismo funcional y dinámica estructural de señalización de GPCR, se requiere un conjunto de herramientas de enfoques para incorporar sondas moleculares y químicas informativas para interrogar a los complejos de señalización activo. Para este fin, se adaptó un método para introducir específicamente en el sitio no o mínimamente sondas perturbadores en receptores expresados ​​sobre la base de aminoácido no natural (SAU) mutagénesis usando codón ámbar tecnología de supresión previamente por primera vez por Schultz y C. oworkers 6 Hemos optimizado la metodología de mutagénesis SAU para lograr una expresión de alto rendimiento y sistema de mutagénesis para las proteínas tales como los GPCR, que son difíciles de expresar en sistemas más heterólogas distintas de las células de mamíferos. El uso de un supresor ARNt ortogonal diseñado y desarrollado aminoacil-ARNt-sintetasa par de UAA específica, hemos introducido específicamente en el sitio UAAs en GPCRs objetivo expresado. La incorporación con éxito de UAAs, p-acetil-L-fenilalanina (ACF), p-benzoil-L-fenilalanina (BZF), y p-azido-L-fenilalanina (AZF) se ha demostrado en nuestros GPCR modelo - rodopsina humana y receptor de quimiocinas CC, CCR5. 7, 8 En principio, un UAA puede ser codificada genéticamente en cualquier posición dentro de la secuencia de la proteína, y esta propiedad es una herramienta bioquímica invaluable, ya que permite el escaneo de un solo codón de un GPCR diana. Nos centramos aquí específicamente en la versatilidad de la UAA, AZF, que tiene un azi reactivahacer fracción. Además de servir como un (IR) de la sonda única de infrarrojos, 8, 9 azf también puede servir como un agente de reticulación fotoactivable por reacción con aminas primarias vecinas o hidrógenos alifáticos. Además, el grupo azido biológicamente inerte puede participar como un asa química selectiva en las reacciones de etiquetado bioorthogonal. Aquí se presentan ejemplos que ilustran las aplicaciones útiles de la incorporación específica de sitio de AZF en GPCRs, como fotorreticulación dirigida a atrapar a un complejo receptor-ligando, y la modificación de los GPCRs de etiquetado epítopo bioorthogonal y estrategias de marcaje fluorescente. Reactivos fotoactivables se han utilizado para estudiar los sistemas biológicos desde la década de 1960. 10 En este período, se ha informado de una gran cantidad de experimentos de reticulación de receptor-ligando para estudiar complejos de GPCR, la mayoría de las cuales implicó el uso de ligandos de fotoafinidad. 11, 12 Sin embargo, estos las aplicaciones son técnicamente limitados, ya que requiere síntesis de ligandos que portan un grupo de reticulación. 13-15 Además, con el resto reticulante en el ligando, es un reto para identificar la ubicación de la reticulación en el GPCR. La introducción específica de sitio grupos fotorreticulación como UAAs en proteínas utilizando el codón ámbar tecnología de supresión es un avance valioso. 16, 17 Hemos desarrollado una técnica fotorreticulación para identificar la interfaz de unión sobre un receptor que está implicado en la formación de un complejo receptor-ligando en células vivas mediante la introducción de grupos fotolábiles en los GPCR. 18, 19 Aquí se describe el protocolo experimental y método de análisis de datos para la aplicación de esta tecnología fotorreticulación dirigido a identificar el sitio de unión de un ligando de molécula pequeña, maraviroc tritiada, en el CCR5. Este método aprovecha la cuantificación precisa de la manija radiactivo en el ligando, además de retener la estructura química nativa del ligando. Las técnicas basadas en la fluorescencia apoyan la comprensión precisa de la base estructural de la activación del receptor mediante el sondeo directamente el estado conformacional del receptor. 20, 21 Sin embargo, las técnicas que poseen la flexibilidad para introducir etiquetas fluorescentes en los GPCR específica de sitio son limitados. Estamos interesados ​​en el empleo de estrategias de modificación química bioorthogonal para facilitar la detección de moléculas individuales (SMD) de complejos de señalización de GPCR. 22 El grupo azido puede participar en las químicas bioorthogonal como la ligadura de Staudinger-Bertozzi, 23, 24 azida-deformación-promovido sin cobre cicloadición alquino (SpAAC) 25 y azida alquino cicloadición catalizada por cobre (CuAAC). 26 Nos centramos aquí en la reacción de ligación Staudinger-Bertozzi que implica la reacción específica entre una azida y una fosfina. Se demuestra el uso de dos derivados de fosfina diferentes, conjugados a un epítopo de péptido (péptido FLAG) o un marcador fluorescente(Fluoresceína) para lograr la modificación específica de sitio de los GPCR. Previamente Hemos optimizado las condiciones para el etiquetado específico del sitio de la rodopsina AZF variantes utilizando la estructura cristalina de rayos X y simulaciones dinámicas para elegir los sitios de destino que estén expuestos al disolvente. 27, 28 también ilustra la factibilidad de lograr un etiquetado fondo libre por Staudinger- ligadura Bertozzi. 29 Se demuestra aquí el procedimiento general empleado para conseguir el marcaje fluorescente de un receptor solubilizada con detergente que está inmovilizado sobre una matriz de inmunoafinidad y posteriormente visualizada por fluorescencia en gel. Además, demostramos una útil extensión de esta estrategia de etiquetado para identificar posiciones susceptibles de etiquetado en un receptor de estructura desconocida, CCR5. Esto se lleva a cabo utilizando una estrategia de marcado epítopo de péptido dirigido que se basa en la modificación de bioorthogonal azf-GPCR variantes en un formato semi-alto rendimiento basado en células. 30 Estamétodo explota las propiedades de detección de varios pasos de un ELISA de la superficie celular para monitorear eventos de etiquetado. Las metodologías que se discuten aquí son generales y, en principio, se pueden aplicar a cualquier GPCR incorporado con AZF utilizando el codón ámbar tecnología de supresión. En los protocolos presentados aquí detallamos los pasos involucrados en la expresión de células de mamíferos de los receptores incorporados con UAAs como AZF utilizando el método de mutagénesis de aminoácidos no naturales y sus aplicaciones posteriores para facilitar los estudios estructurales y dinámicas de los GPCRs.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Plasmid pSVB. Yam</td>
			<td></td>
			<td></td>
			<td>(Ye et al., 2008)</td>
		</tr>
		<tr>
			<td>Plasmid pcDNA.RS for azF</td>
			<td></td>
			<td></td>
			<td>(Ye et al., 2009)</td>
		</tr>
		<tr>
			<td>Plasmids pMT4.Rho and pcDNA 3.1.CCR5</td>
			<td></td>
			<td></td>
			<td>Optionally contain the amber stop codon (TAG) at a desired position</td>
		</tr>
		<tr>
			<td>HEK 293T cells</td>
			<td></td>
			<td></td>
			<td>Adherent cells</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM)</td>
			<td>Gibco</td>
			<td>10566</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Gibco</td>
			<td>14200</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gemini Bio-products</td>
			<td>100-106</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine Plus</td>
			<td>Invitrogen</td>
			<td>Lipofectamine: 18324-012 
			Plus: 11514-015</td>
			<td></td>
		</tr>
		<tr>
			<td>p-azido-L-phenylalanine</td>
			<td>Chem-Impex International</td>
			<td>6162</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 1. Site-specific genetic incorporation of unnatural amino acids into GPCRs materials</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>[header]</td>
		</tr>
		<tr>
			<td>Maxima ML-3500S UV-A lamp</td>
			<td>Spectronics Corporation</td>
			<td></td>
			<td>azF is activated by 365-nm light</td>
		</tr>
		<tr>
			<td>Hank&#39;s Buffered Salt Solution (HBSS)</td>
			<td>Gibco</td>
			<td>14065</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Irvine Scientific</td>
			<td>9319</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Roche</td>
			<td>3117405001</td>
			<td></td>
		</tr>
		<tr>
			<td>Tritium-labeled ligand</td>
			<td></td>
			<td></td>
			<td>From collaborator (Grunbeck et al., 2012)</td>
		</tr>
		<tr>
			<td>1% (w/v) n-dodecyl-&#946;-D-maltoside</td>
			<td>Anatrace</td>
			<td>D310LA</td>
			<td></td>
		</tr>
		<tr>
			<td>1D4-sepharose resin</td>
			<td></td>
			<td></td>
			<td>1D4 mAb immobilized on CNBr-activated sepharose 2B resin</td>
		</tr>
		<tr>
			<td>1% (w/v) sodium dodecyl sulfate (SDS)</td>
			<td>Fisher Scientific</td>
			<td>BP166</td>
			<td></td>
		</tr>
		<tr>
			<td>NuPAGE Novex 4 - 12% SDS gels</td>
			<td>Invitrogen</td>
			<td>NP0322BOX</td>
			<td>SDS-PAGE performed on NuPAGE apparatus</td>
		</tr>
		<tr>
			<td>Trans-Blot SD apparatus</td>
			<td>Biorad</td>
			<td></td>
			<td>Apparatus for semi-dry transfer</td>
		</tr>
		<tr>
			<td>Immobilon polyvinylidene difluoride (PVDF) membrane</td>
			<td>Millipore</td>
			<td>IPVH00010</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-fat powered milk</td>
			<td>Fisher Scientific</td>
			<td>NC9934262</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Aldrich</td>
			<td>274348</td>
			<td></td>
		</tr>
		<tr>
			<td>Ecoscint A</td>
			<td>National Diagnostics</td>
			<td>LS-273</td>
			<td>Scintillation fluid</td>
		</tr>
		<tr>
			<td>Scintillation vials</td>
			<td>Fisher Scientific</td>
			<td>333726</td>
			<td></td>
		</tr>
		<tr>
			<td>LKB Wallac 1209 Rackbeta Liquid Scintillation Counter</td>
			<td>Perkin Elmer</td>
			<td></td>
			<td>Beta-Scintillation counter</td>
		</tr>
		<tr>
			<td>Anti-rhodopsin 1D4 mAb</td>
			<td>National Cell Culture Center</td>
			<td>custom</td>
			<td></td>
		</tr>
		<tr>
			<td>Horseradish peroxidase (HRP)-conjugated anti-mouse IgG</td>
			<td>KPL, Inc.</td>
			<td>474-1806</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperSignal West Pico Chemiluminescent Substrate</td>
			<td>Thermo Scientific</td>
			<td>34080</td>
			<td></td>
		</tr>
		<tr>
			<td>Hyblot CL AR film</td>
			<td>Denville</td>
			<td>E3018</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 2. Targeted photocrosslinking materials</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>[header]</td>
		</tr>
		<tr>
			<td>0.25% Trypsin</td>
			<td>Invitrogen</td>
			<td>15050065</td>
			<td></td>
		</tr>
		<tr>
			<td>FLAG-triarylphosphine</td>
			<td>Sigma</td>
			<td>GPHOS1</td>
			<td></td>
		</tr>
		<tr>
			<td>M2 FLAG mAb</td>
			<td>Sigma</td>
			<td>F1804</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-CCR5 2D7 mAb</td>
			<td>BD Biosciences</td>
			<td>555990</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-lysine</td>
			<td>Sigma</td>
			<td>P6407</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate</td>
			<td>Costar</td>
			<td>3601</td>
			<td>Clear bottom, high binding EIA/RIA</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (Calcium, Magnesium)</td>
			<td>Gibco</td>
			<td>14040</td>
			<td></td>
		</tr>
		<tr>
			<td>16% Paraformaldehyde</td>
			<td>EMS</td>
			<td>28908</td>
			<td></td>
		</tr>
		<tr>
			<td>HRP-conjugated</td>
			<td>KPL, Inc.</td>
			<td>474-1516</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-rabbit IgG</td>
			<td>KPL, Inc.</td>
			<td>474-1516</td>
			<td></td>
		</tr>
		<tr>
			<td>Amplex Red</td>
			<td>Invitrogen</td>
			<td>A12222</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen peroxide</td>
			<td>DE Healthcare Products</td>
			<td>97-93399</td>
			<td></td>
		</tr>
		<tr>
			<td>CytoFluor II fluorescence multi-well plate reader</td>
			<td>Perseptive Biosystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 3. Targeted peptide-epitope tagging and cell surface ELISA materials</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>[header]</td>
		</tr>
		<tr>
			<td>Fluorescein-phosphine</td>
			<td></td>
			<td></td>
			<td>(Huber et al., submitted 2012)</td>
		</tr>
		<tr>
			<td>Nonapeptide (C9 peptide)</td>
			<td>AnaSpec</td>
			<td>62190</td>
			<td>Peptide mimicking the 1D4-epitope NH2-TETSQVAPA-COOH</td>
		</tr>
		<tr>
			<td>1% (w/v) n-dodecyl-&#946;-D-maltoside</td>
			<td>Anatrace</td>
			<td>D310LA</td>
			<td></td>
		</tr>
		<tr>
			<td>1D4-sepharose resin</td>
			<td></td>
			<td></td>
			<td>1D4 mAb immobilized on CNBr-activated sepharose 2B resin</td>
		</tr>
		<tr>
			<td>NuPAGE Novex 4 - 12% SDS gels</td>
			<td>Invitrogen</td>
			<td>NP0322BOX</td>
			<td>SDS-PAGE performed on NuPAGE apparatus</td>
		</tr>
		<tr>
			<td>Confocal Typhoon 9400 fluorescence scanner</td>
			<td>GE Healthcare</td>
			<td>discontinued</td>
			<td>Scanner with 488-nm wavelength laser</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 4. Fluorescent labeling materials</td>
		</tr>
	</tbody>
</table>

genetically-encoded molecular probes to study g protein-coupled receptors

Para facilitar los estudios estructurales y dinámicas de proteína G-receptor acoplado (GPCR) de señalización complejos, se necesitan nuevos enfoques para introducir sondas o etiquetas informativas en receptores expresados ​​que no perturban la función del receptor. Se utilizó tecnología de supresión de ámbar codón para genéticamente a codificar el aminoácido no natural, p-azido-L-fenilalanina (azf) en varias posiciones específicas en los GPCRs heteróloga expresadas en células de mamífero. La versatilidad del grupo azido se ilustra aquí en diferentes aplicaciones para estudiar los GPCR en su ambiente celular nativo o bajo condiciones de detergente solubilizadas. En primer lugar, demostramos una tecnología fotorreticulación específica basada en células para identificar los residuos en el bolsillo de unión a ligando de GPCR en donde un ligando de molécula pequeña marcada con tritio se reticula a un aminoácido codificado genéticamente azido-. Se demuestra a continuación, la modificación específica de sitio de los GPCR por la bioorthogonal ligadura de reacción de Staudinger-Bertozzición que se dirige el grupo azido el uso de derivados de fosfina. Se discute una estrategia general para el objetivo marcado péptido epítopo de las proteínas de membrana expresadas en la cultura y su detección mediante un enfoque basado en ELISA de células enteras. Por último, se muestra que AZF-GPCR pueden ser etiquetados de forma selectiva con sondas fluorescentes. Las metodologías discutidos son generales, en que pueden, en principio, aplicarse a cualquier posición del aminoácido en cualquiera de GPCR expresado para interrogar a los complejos de señalización activo.

Se empleó la metodología de mutagénesis de aminoácidos no naturales a sitios específicos introducir sondas moleculares en un GPCR utilizando el codón ámbar tecnología de supresión. El esquema de la figura 1 resume los pasos más destacados de la metodología y de las diversas aplicaciones de la incorporación de la SAU versátil, p-azido-L-fenilalanina (AZF), en GPCRs. Expresión de AZF-GPCR en células de mamíferos permite dirigido fotorreticulación a ligandos, y dirigido etiquetado...

Watch this Scientific Journal Video about Genetically-encoded Molecular Probes to Study G Protein-coupled Receptors at JoVE.com

Genetically-encoded Molecular Probes to Study G Protein-coupled Receptors

Nos genéticamente-codificar el aminoácido no natural, p-azido-L-fenilalanina en varias posiciones específicas en los GPCR y mostrar la versatilidad del grupo azido en diferentes aplicaciones. Estos incluyen una tecnología fotorreticulación dirigido a identificar los residuos en el bolsillo de unión a ligando de un GPCR, y la modificación bioorthogonal específica de sitio de los GPCR con una etiqueta de epítopo de péptido o de la sonda fluorescente.

Codificados genéticamente-sondas moleculares para Estudiar G receptores acoplados a proteínas

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce ...

genetically-encoded-molecular-probes-to-study-g-protein-coupled

Research

JoVE Journal

Biology

15.1K vistas.  The Rockefeller University.  Nos genéticamente-codificar el aminoácido no natural, p-azido-L-fenilalanina en varias posiciones específicas en los GPCR y mostrar la versatilidad del grupo azido en diferentes aplicaciones. Estos incluyen una tecnología fotorreticulación dirigido a identificar los residuos en el bolsillo de unión a ligando de un GPCR, y la modificación bioorthogonal específica de sitio de los GPCR con una etiqueta de epítopo de péptido o de la sonda fluorescente. 

Video: Genetically-encoded Molecular Probes to Study G Protein-coupled Receptors

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Prueba de sensibilidad visual a la velocidad y dirección del movimiento en lagartos

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Investigación

Biología

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite morphogenesis at the functional and molecular levels. To aid in these analyses, we have developed a strategy for the isolation of a subclass of PNS neurons called dendritic arborization (da) neurons that have been widely used for studying dendrite morphogenesis1,2. These neurons are very difficult to isolate as a pure population, due in part to their extremely low occurrence and their difficult-to-reach location below the tough chitinous larval cuticle. Our newly developed method overcomes these challenges, and is based on a fast and specific cell enrichment using antibody-coated magnetic beads. For our magnetic bead sorting studies, we have used age-matched third instar larvae expressing a mouse CD8 tagged GFP fusion protein (UAS-mCD8-GFP)3 under the control of either the class IV dendritic arborization (da) neuron-specific pickpocket (ppk)-GAL4 driver4 or the control of the pan-da neuron-specific GAL421-7 driver5. Although this protocol has been optimized for isolating PNS cells which are attached to the inner wall of the larval cuticle, by varying a few parameters, the same protocol could be used to isolate many different cell types attached to the cuticle at larval or pupal stages of development (e.g. epithelia, muscle, oenocytes etc.), or other cell types from larval organs depending upon the GAL4-specific driver expression pattern. The RNA isolated by this method is of high quality and can be readily used for downstream genomic analyses such as microarray gene expression profiling studies. This approach offers a powerful new tool to perform studies on isolated Drosophila dendritic arborization (da) neurons thereby providing novel insights into the molecular mechanisms underlying dendrite morphogenesis.

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

In this video-article we present a method for the isolation and purification of Drosophila peripheral neurons using a fast magnetic bead assisted cell sorting strategy. RNA obtained from the isolated cells can be readily used for downstream applications including microarray analyses.

Aislamiento y purificación de Drosophila Clasificación de las neuronas periféricas por bolas magnéticas

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite ...

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial applications as well as increases basic fundamental biological knowledge. F&ouml;rster (Fluorescence) Resonance Energy Transfer (FRET) between a donor molecule in an electronically excited state and a nearby acceptor molecule has been frequently utilized for studies of protein-protein interactions in living cells. The proteins of interest are tagged with two different types of fluorescent probes and expressed in biological cells. The fluorescent probes are then excited, typically using laser light, and the spectral properties of the fluorescence emission emanating from the fluorescent probes is collected and analyzed. Information regarding the degree of the protein interactions is embedded in the spectral emission data. Typically, the cell must be scanned a number of times in order to accumulate enough spectral information to accurately quantify the extent of the protein interactions for each region of interest within the cell. However, the molecular composition of these regions may change during the course of the acquisition process, limiting the spatial determination of the quantitative values of the apparent FRET efficiencies to an average over entire cells. By means of a spectrally resolved two-photon microscope, we are able to obtain a full set of spectrally resolved images after only one complete excitation scan of the sample of interest. From this pixel-level spectral data, a map of FRET efficiencies throughout the cell is calculated. By applying a simple theory of FRET in oligomeric complexes to the experimentally obtained distribution of FRET efficiencies throughout the cell, a single spectrally resolved scan reveals stoichiometric and structural information about the oligomer complex under study. Here we describe the procedure of preparing biological cells (the yeast Saccharomyces cerevisiae) expressing membrane receptors (sterile 2 &alpha;-factor receptors) tagged with two different types of fluorescent probes. Furthermore, we illustrate critical factors involved in collecting fluorescence data using the spectrally resolved two-photon microscopy imaging system. The use of this protocol may be extended to study any type of protein which can be expressed in a living cell with a fluorescent marker attached to it.

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

By employing a spectrally resolved two-photon microscopy imaging system, pixel-level maps of F&ouml;rster Resonance Energy Transfer (FRET) efficiencies are obtained for cells expressing membrane receptors hypothesized to form homo-oligomeric complexes. From the FRET efficiency maps, we are able to estimate stoichiometric information about the oligomer complex under study.

En la cuantificación in vivo de interacciones de proteínas G del receptor acoplado con espectralmente resuelve de dos fotones de Microscopía

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial ...

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, differentiation, and apoptosis1. During apoptosis, calcium accumulation in mitochondria promotes the release of pro-apoptotic factors from the mitochondria into the cytosol2. It is therefore of interest to directly measure mitochondrial calcium in living cells in situ during apoptosis. High-resolution fluorescent imaging of cells loaded with dual-excitation ratiometric and non-ratiometric synthetic calcium indicator dyes has been proven to be a reliable and versatile tool to study various aspects of intracellular calcium signaling. Measuring cytosolic calcium fluxes using these techniques is relatively straightforward. However, measuring intramitochondrial calcium levels in intact cells using synthetic calcium indicators such as rhod-2 and rhod-FF is more challenging. Synthetic indicators targeted to mitochondria have blunted responses to repetitive increases in mitochondrial calcium, and disrupt mitochondrial morphology3. Additionally, synthetic indicators tend to leak out of mitochondria over several hours which makes them unsuitable for long-term experiments. Thus, genetically encoded calcium indicators based upon green fluorescent protein (GFP)4 or aequorin5 targeted to mitochondria have greatly facilitated measurement of mitochondrial calcium dynamics. Here, we describe a simple method for real-time measurement of mitochondrial calcium fluxes in response to different stimuli. The method is based on fluorescence microscopy of 'ratiometric-pericam' which is selectively targeted to mitochondria. Ratiometric pericam is a calcium indicator based on a fusion of circularly permuted yellow fluorescent protein and calmodulin4. Binding of calcium to ratiometric pericam causes a shift of its excitation peak from 415 nm to 494 nm, while the emission spectrum, which peaks around 515 nm, remains unchanged. Ratiometric pericam binds a single calcium ion with a dissociation constant in vitro of ~1.7 &mu;M4. These properties of ratiometric pericam allow the quantification of rapid and long-term changes in mitochondrial calcium concentration. Furthermore, we describe adaptation of this methodology to a standard wide-field calcium imaging microscope with commonly available filter sets. Using two distinct agonists, the purinergic agonist ATP and apoptosis-inducing drug staurosporine, we demonstrate that this method is appropriate for monitoring changes in mitochondrial calcium concentration with a temporal resolution of seconds to hours. Furthermore, we also demonstrate that ratiometric pericam is also useful for measuring mitochondrial fission/fragmentation during apoptosis. Thus, ratiometric pericam is particularly well suited for continuous long-term measurement of mitochondrial calcium dynamics during apoptosis.

This protocol describes a method for real-time measurement of mitochondrial calcium fluxes by fluorescent imaging. The method takes advantage of a circularly permutated YFP-based dual-excitation ratiometric calcium sensor (ratiometric pericam-mt) selectively expressed in mitochondria.

Seguimiento de los cambios dinámicos en niveles de calcio mitocondrial de la apoptosis mediante un sensor de calcio genéticamente codificados

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, ...

Imaging G-protein Coupled Receptor (GPCR)-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly 1. This guided cell migration is referred as chemotaxis, which is essential for various cells to carry out their functions such as trafficking of immune cells and patterning of neuronal cells 2, 3. A large family of G-protein coupled receptors (GPCRs) detects variable small peptides, known as chemokines, to direct cell migration in vivo 4. The final goal of chemotaxis research is to understand how a GPCR machinery senses chemokine gradients and controls signaling events leading to chemotaxis. To this end, we use imaging techniques to monitor, in real time, spatiotemporal concentrations of chemoattractants, cell movement in a gradient of chemoattractant, GPCR mediated activation of heterotrimeric G-protein, and intracellular signaling events involved in chemotaxis of eukaryotic cells 5-8. The simple eukaryotic organism, Dictyostelium discoideum, displays chemotaxic behaviors that are similar to those of leukocytes, and D. discoideum is a key model system for studying eukaryotic chemotaxis. As free-living amoebae, D. discoideum cells divide in rich medium. Upon starvation, cells enter a developmental program in which they aggregate through cAMP-mediated chemotaxis to form multicullular structures. Many components involved in chemotaxis to cAMP have been identified in D. discoideum. The binding of cAMP to a GPCR (cAR1) induces dissociation of heterotrimeric G-proteins into G&gamma; and G&beta;&gamma; subunits 7, 9, 10. G&beta;&gamma; subunits activate Ras, which in turn activates PI3K, converting PIP2 into PIP3 on the cell membrane 11-13. PIP3 serve as binding sites for proteins with pleckstrin Homology (PH) domains, thus recruiting these proteins to the membrane 14, 15. Activation of cAR1 receptors also controls the membrane associations of PTEN, which dephosphorylates PIP3 to PIP2 16, 17. The molecular mechanisms are evolutionarily conserved in chemokine GPCR-mediated chemotaxis of human cells such as neutrophils 18. We present following methods for studying chemotaxis of D. discoideum cells. 1. Preparation of chemotactic component cells. 2. Imaging chemotaxis of cells in a cAMP gradient. 3. Monitoring a GPCR induced activation of heterotrimeric G-protein in single live cells. 4. Imaging chemoattractant-triggered dynamic PIP3 responses in single live cells in real time. Our developed imaging methods can be applied to study chemotaxis of human leukocytes.

Imaging G-protein Coupled Receptor GPCR-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

Here, we describe detailed live cell imaging methods for investigating chemotaxis. We present fluorescence microscopic methods to monitor spatiotemporal dynamics of signaling events in migrating cells. Measurement of signaling events permits us to further understand how a GPCR-signaling network achieves gradient sensing of chemoattractants and controls directional migration of eukaryotic cells.

Receptor de imagen acoplados a proteína G (GPCR) mediada por eventos de señalización que la quimiotaxis de control de Dictyostelium discoideum</em

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly 1.  This guided cell migration is referred as ...

Quantifying Agonist Activity at G Protein-coupled Receptors

When an agonist activates a population of G protein-coupled receptors (GPCRs), it elicits a signaling pathway that culminates in the response of the cell or tissue. This process can be analyzed at the level of a single receptor, a population of receptors, or a downstream response. Here we describe how to analyze the downstream response to obtain an estimate of the agonist affinity constant for the active state of single receptors.
Receptors behave as quantal switches that alternate between active and inactive states (Figure 1). The active state interacts with specific G proteins or other signaling partners. In the absence of ligands, the inactive state predominates. The binding of agonist increases the probability that the receptor will switch into the active state because its affinity constant for the active state (Kb) is much greater than that for the inactive state (Ka). The summation of the random outputs of all of the receptors in the population yields a constant level of receptor activation in time. The reciprocal of the concentration of agonist eliciting half-maximal receptor activation is equivalent to the observed affinity constant (Kobs), and the fraction of agonist-receptor complexes in the active state is defined as efficacy (&#949;) (Figure 2).
Methods for analyzing the downstream responses of GPCRs have been developed that enable the estimation of the Kobs and relative efficacy of an agonist 1,2. In this report, we show how to modify this analysis to estimate the agonist Kb value relative to that of another agonist. For assays that exhibit constitutive activity, we show how to estimate Kb in absolute units of M-1.
Our method of analyzing agonist concentration-response curves 3,4 consists of global nonlinear regression using the operational model 5. We describe a procedure using the software application, Prism (GraphPad Software, Inc., San Diego, CA). The analysis yields an estimate of the product of Kobs and a parameter proportional to efficacy (&tau;). The estimate of &tau;Kobs of one agonist, divided by that of another, is a relative measure of Kb (RAi) 6. For any receptor exhibiting constitutive activity, it is possible to estimate a parameter proportional to the efficacy of the free receptor complex (&tau;sys). In this case, the Kb value of an agonist is equivalent to &tau;Kobs/&tau;sys 3.
Our method is useful for determining the selectivity of an agonist for receptor subtypes and for quantifying agonist-receptor signaling through different G proteins.

A method for estimating the affinity constant of an agonist for the active state (Kb) of a G protein-coupled receptor is described. The analysis provides absolute or relative measures of Kb depending on whether constitutive receptor activation is measurable. Our method applies to various responses downstream from receptor activation.

La cuantificación de la actividad agonista en el G receptores acoplados a proteínas

When an agonist activates a population of G protein-coupled receptors (GPCRs), it elicits a signaling pathway that culminates in the response of the cell ...

Characterization of G Protein-coupled Receptors by a Fluorescence-based Calcium Mobilization Assay

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors (GPCRs). The onset of a reverse pharmacology assay is the cloning and subsequent transfection of a GPCR of interest in a cellular expression system. The heterologous expressed receptor is then challenged with a compound library of candidate ligands to identify the receptor-activating ligand(s). Receptor activation can be assessed by measuring changes in concentration of second messenger reporter molecules, like calcium or cAMP. The fluorescence-based calcium mobilization assay described here is a frequently used medium-throughput reverse pharmacology assay. The orphan GPCR is transiently expressed in human embryonic kidney 293T (HEK293T) cells and a promiscuous G&#945;16 construct is co-transfected. Following ligand binding, activation of the G&#945;16 subunit induces the release of calcium from the endoplasmic reticulum. Prior to ligand screening, the receptor-expressing cells are loaded with a fluorescent calcium indicator, Fluo-4 acetoxymethyl. The fluorescent signal of Fluo-4 is negligible in cells under resting conditions, but can be amplified more than a 100-fold upon the interaction with calcium ions that are released after receptor activation. The described technique does not require the time-consuming establishment of stably transfected cell lines in which the transfected genetic material is integrated into the host cell genome. Instead, a transient transfection, generating temporary expression of the target gene, is sufficient to perform the screening assay. The setup allows medium-throughput screening of hundreds of compounds. Co-transfection of the promiscuous G&#945;16, which couples to most GPCRs, allows the intracellular signaling pathway to be redirected towards the release of calcium, regardless of the native signaling pathway in endogenous settings. The HEK293T cells are easy to handle and have proven their efficacy throughout the years in receptor deorphanization assays. However, optimization of the assay for specific receptors may remain necessary.

The here described fluorescence-based calcium mobilization assay is a medium-throughput reverse pharmacology screening system for the identification of functionally activating ligand(s) of orphan G protein-coupled receptors (GPCRs).

Caracterización de G receptores acoplados a proteína por un ensayo de movilización de calcio basado en fluorescencia

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors ...

Visualizing Clathrin-mediated Endocytosis of G Protein-coupled Receptors at Single-event Resolution via TIRF Microscopy

Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological assays, measuring global changes in endocytosis, have identified over 30 known components participating in CME, and biochemical studies have generated an interaction map of many of these components. It is becoming increasingly clear, however, that CME is a highly dynamic process whose regulation is complex and delicate. In this manuscript, we describe the use of Total Internal Reflection Fluorescence (TIRF) microscopy to directly visualize the dynamics of components of the clathrin-mediated endocytic machinery, in real time in living cells, at the level of individual events that mediate this process. This approach is essential to elucidate the subtle changes that can alter endocytosis without globally blocking it, as is seen with physiological regulation. We will focus on using this technique to analyze an area of emerging interest, the role of cargo composition in modulating the dynamics of distinct clathrin-coated pits (CCPs). This protocol is compatible with a variety of widely available fluorescence probes, and may be applied to visualizing the dynamics of many cargo molecules that are internalized from the cell surface.

Clathrin-mediated endocytosis, a rapid and highly dynamic process internalizes many proteins, including signaling receptors. The protocol described here directly visualizes the kinetics of individual endocytic events. This is essential for understanding how core members of the endocytic machinery coordinate with each other, and how protein cargo influence this process.

Visualización de endocitosis mediada por clatrina de G receptores acoplados a proteínas en la Resolución Individual de eventos a través de TIRF de Microscopía

Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological ...

Hydroponics: A Versatile System to Study Nutrient Allocation and Plant Responses to Nutrient Availability and Exposure to Toxic Elements

Hydroponic systems have been utilized as one of the standard methods for plant biology research and are also used in commercial production for several crops, including lettuce and tomato. Within the plant research community, numerous hydroponic systems have been designed to study plant responses to biotic and abiotic stresses. Here we present a hydroponic protocol that can be easily implemented in laboratories interested in pursuing studies on plant mineral nutrition.
This protocol describes the hydroponic system set up in detail and the preparation of plant material for successful experiments. Most of the materials described in this protocol can be found outside scientific supply companies, making the set up for hydroponic experiments less expensive and convenient.
The use of a hydroponic growth system is most advantageous in situations where the nutrient media need to be well controlled and when intact roots need to be harvested for downstream applications. We also demonstrate how nutrient concentrations can be modified to induce plant responses to both essential nutrients and toxic non-essential elements.

Here, we present an easy-to-follow protocol to establish a successful hydroponic system for plant nutrition studies. This protocol has been extensively tested in Arabidopsis and can easily be adapted to other plant species to study specific nutritional requirements or the effect of non-essential elements on plant growth and development.

La hidroponía: un sistema versátil para el Estudio de nutrientes Las respuestas de las plantas a la asignación y disponibilidad de nutrientes y la exposición a elementos tóxicos

Hydroponic systems have been utilized as one of the standard methods for plant biology research and are also used in commercial production for several ...

Imaging G Protein-coupled Receptor-mediated Chemotaxis and its Signaling Events in Neutrophil-like HL60 Cells

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many physiological processes, such as embryogenesis, neuron patterning, metastasis of cancer cells, recruitment of neutrophils to sites of inflammation, and the development of the model organism Dictyostelium discoideum. Eukaryotic cells sense chemo-attractants using G protein-coupled receptors. Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.

Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.

Imaging G acoplados a proteínas mediada por el receptor de quimiotaxis y sus eventos de señalización en células HL60 neutrófilos como

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many ...