Agradecemos o generoso apoio de diversas fundações e doadores filantrópicos (ver SakmarLab.org).

1. Site-specific Constituição genética de Unnatural Aminoácidos em GPCRs <ol><li> Manter células HEK293T em DMEM (4,5 g / L de glicose, glutamina 2 mM) suplementado com 10% de soro fetal bovino (FBS) a 37 ° C numa atmosfera de CO2 a 5%. </li><li> Transfectar as células cultivadas a 60-80% de confluência numa placa de 10 cm usando o reagente Lipofectamine Plus. <ol><li> Para 750 mL de DMEM, adicionar 10 ul de reagente Bonés, 3,5 ug de cDNA de GPCR (pMT4. Rho ou pcDNA 3.1. CCR5) contendo o codão de terminação âmbar, numa posição desejada, 3,5 ug de ARNt supressor de cDNA (pSVB.Yam) e 0,35 ug de mutante cDNA ARNt sintetase amino-acilo para o p-azido-L-fenilalanina (pcDNA.RS). Incubar à temperatura ambiente durante 15 min. Também realizar uma transfecção semelhante utilizando o peso de GPCR de cDNA (que não contém um codão de terminação âmbar) para servir como um controlo. Adicionar esta mistura de 750 ul de DMEM com 17 ul de Lipofectamina. Depois de equilibrar 15 min à temperatura ambiente, levar avolume total de 4 ml. </li><li> Aspirar media em 10 cm de placa, aplica mistura de transfecção às células, e regresso a 37 ° C em 5% de CO2. Após 4-6 hr, complementar células com 4 ml de DMEM contendo 20% de FBS e AZF 1 mM. </li></ol></li><li> No dia seguinte, substituir o meio de crescimento por DMEM contendo 10% de FBS e AZF 0,5 mM. </li><li> Células colheita de 48 horas após a transfecção, para analisar a expressão ou prosseguir com os procedimentos fotoreticulação ou rotulagem descritos nas seções seguintes. <ol><li> Confirmar a expressão do mutante de âmbar de GPCR, na presença de SAU nos meios de crescimento. Fazemos isso através da detecção immunoblot ocidental. Lise do sedimento celular em 1% (w / v) de dodecil-β-D-maltósido (DDM) em 20 mM Tris-HCl, pH 7,5, NaCl 100 mM durante 1 hora a 4 ° C. Centrifuga-se o lisado a 16.000 x g e resolver o sobrenadante em condições redutoras, por SDS-PAGE. Transfira para uma membrana de PVDF e realizar análise immunoblot para detectar a expressão do receptor de corpo inteiro usando ªe 1D4 mAb (Cell National Culture Center), que reconhece um epitopo de engenharia na extremidade C-terminal do receptor 31. </li></ol></li></ol> 2. Traçando um local de ligação do ligando Usando alvejado fotoreticulação <ol><li> 48 horas após a transfecção, a mídia aspirado a partir de células e substituir com 4 ml 1x tampão fosfato (PBS). E volta a 37 ° C durante 15 min. </li><li> Ressuspender as células com PBS 1x e transferir para um tubo Falcon. Centrifugar a 1500 rpm durante 2 min para sedimentar as células e aspiração do sobrenadante. </li><li> Ressuspender o sedimento celular em Solução Salina Tamponada de Hank 1x contendo 20 mM de HEPES, pH 7,5, BSA a 0,2% e do ligando tritiado a concentração de saturação de ligação para o comprimento necessário de tempo e temperatura, para assegurar a formação do complexo receptor-ligando. </li><li> Transferência da suspensão de células para uma de 24 poços ou 96 poços por placa de fotoactivação da AZF a 365 nm (por exemplo, utilizar uma lâmpada de UV-A) durante 15 min a 4 ° C. Manutenn a placa em gelo para evitar o aquecimento da amostra e lise celular. </li><li> Transferência das células para um tubo de Eppendorf, centrífuga para sedimentar as células, remove-se o sobrenadante e proceder à análise. Os peletes podem ser armazenadas a -20 ° C para uso futuro. </li><li> Ressuspender as pelotas de células em tampão de lise contendo 1% (w / v) de DDM, 20 mM Tris-HCl, pH 7,5 e os inibidores da protease. Após a incubação de 1 hora a 4 ° C, centrifugar o lisado celular durante 10 min a 16.000 x g, e transferir o sobrenadante para um novo tubo. </li><li> Adicionar resina 2B mAb 1D4-sefarose para o sobrenadante e incubar durante a noite a 4 ° C para o GPCR immunopurify utilizando o terminal C de mAb 1D4 epitopo engenharia. No dia seguinte, lavar as esferas várias vezes com tampão de lise. Amostras eluir com 1% de SDS a 37 ° C durante 1 hora com agitação. </li><li> Transfira uma porção do eluente a um frasco contendo 15 ml de fluido de cintilação e contar num contador de cintilação beta para quantificar a quantidade de ligação específica do ligando titulado para poe do receptor. </li><li> Resolver o restante 1D4 mAb eluente purificado por eletroforese em gel padrão. Usamos um gel de 4-12% SDS-PAGE e depois transferência semi-seca para uma membrana de PVDF para imunoblotting. Nós também incluímos uma pista com escada proteína padrão para orientar aproximação visual de peso molecular. <ol><li> Bloquear a membrana PVDF com leite a 5% em tampão TBS contendo 0,05% de Tween-20. Sondar a membrana para confirmar a expressão do receptor de comprimento completo com o mAb 1D4, seguido por anticorpo secundário anti-IgG de ratinho conjugado com HRP. </li><li> Tratou-se com reagente de quimioluminescência aumentada (ECL) e expor a membrana a película de auto-radiografia para visualizar as bandas. </li></ol></li><li> Cortar a membrana com uma lâmina de barbear para separar cada pista de amostra, seguindo-se o corte em marcadores com pesos moleculares específicos. Transferir os segmentos de membrana para frascos contendo líquido de cintilação. Quantificar a quantidade de trítio em cada segmento de membrana por contagem num contador de beta-cintilação. </li><li> Identificar o positivophotocrosslink com a detecção de trítio na massa molecular aparente igual à soma do que do GPCR e o ligando. </li></ol> 3. Alvejado Epitope Tagging e Superfície Celular ligada ao ensaio imunoenzimático (ELISA) <ol><li> 24 horas pós-transfecção, lavar as células com 1 ml de PBS 1x e trypsinize com 1 ml de tripsina a 0,25% durante 3 min. Suplemento com 9 ml de DMEM contendo 10% de FBS e AZF 0,5 mM. Ressuspender as células e contar a densidade celular. Usamos um hemocitômetro padrão. </li><li> Pré-tratar uma chapa de fundo claro, de ligação de alta, de 96 poços com 100 ml de 0,01 mg / ml de poli-D-lisina por poço. Incubar durante 30 minutos à temperatura ambiente, lavar várias vezes com PBS 1x e ar seco. </li><li> Placa de 200 ul das células transfectadas a uma densidade de 6 x 4-8 outubro 4 x 10 células / poço para a placa de 96 poços e regresso a 37 ° C em 5% de CO2. </li><li> No dia seguinte, preparar células para a rotulagem. Lavar suavemente três vezes com 100 ul / poço de PBS 1x to remover qualquer AZF contendo meio de cultura. Assegurar que as células permanecem aderidas, por exemplo, através da utilização de PBS contendo Ca 2 + e Mg 2 +. </li><li> Prepare 50-200 mM FLAG-triarilfosfína reagente rotulagem em 1x HBSS / PBS a partir de um estoque mantido em 5-20 mM em PBS. Aplicar 60 ml a cada poço (em triplicado para cada mutante âmbar) e regresso a 37 ° C durante 30 minutos a 4 horas. Manter um conjunto de poços sem tratamento rótulo em 1x HBSS / PBS. Também incluem um controle de peso GPCR para comparar com variantes AZF-GPCR. </li><li> Lavar as células 3 vezes com 100 ul / poço de tampão de bloqueio, BB (1x PBS, BSA a 0,5%), para remover marcador que não reagiu completamente. </li><li> Aplicar 100 l / poço de paraformaldeído a 4% (preparada a partir de um estoque de 16% em PBS 1x) para as células. Incubar durante 20 minutos à temperatura ambiente. Lavar três vezes com BB. </li><li> Realizar protocolo ELISA superfície celular padrão para detectar receptor marcado e expressão do receptor. Incubar com anticorpo primário durante 1,5 horas em gelo, seguido de um derivadotibody incubação à temperatura ambiente durante 1 hora. <ol><li> Adicionar 100 ul de anticorpo anti-FLAG M2 (por exemplo, diluição de 1:2.000 de anticorpos feitas no BB) para detectar o péptido FLAG epitopo do reagente de marcação FLAG-triarilfosfina. Sonda também não rótulo poços tratados como controle negativo. Adicionar anti-CCR5 2D7 (usamos uma diluição de 1:500) do anticorpo a um conjunto separado de poços para determinar em peso ou AZF-GPCR expressão na superfície celular. </li><li> Lave as células três vezes com BB, e incubar com 100 uL de anticorpo secundário anti-IgG de ratinho conjugado com HRP (1:2000 diluição). </li><li> Lavar cuidadosamente as células cinco vezes com o BB. Adicionar 50 ul de tampão de detecção: Amplex Red, 20 mM H 2 O 2, 1x PBS (proporção 1:10:90) e incubar 15 min a temperatura ambiente. Coletar dados espectrais em um multi-leitor de placas bem fluorescência em λ ex = 530 e λ em = 590. </li></ol></li></ol> 4. Bioorthogonal Labeling fluorescentes de um GPCR <ol><li> Lyse10 7 células colhidas que expressam em peso ou AZF-rodopsina variantes em 1 ml de tampão de solubilização, contendo 1% (w / v), MS, HEPES 50 mM ou Tris-HCl, pH 6,8, NaCl 100 mM, CaCl2 1 mM e inibidores de protease para uma hr a 4 ° C. Centrifuga-se o lisado celular a 15000 x g durante 10 minutos e recolher a fracção sobrenadante. </li><li> Adicionar 100 uL de mAb 1D4-resina de Sepharose 2B para capturar o receptor utilizando o terminal C 1D4 epitopo engenharia. Incubar durante 12 horas a 4 ° C. Lava-se a resina três vezes com 0,1% (w / v) DDM em tampão fosfato de sódio 0,1 M, pH 7,3. </li><li> Adicionar 0,1 mM de fluoresceína-fosfina em um volume total de 0,3-0,5 mL de rotular receptor imobilizado na matriz de imunoafinidade (1D4-mAb Sepharose 2B). Incubar durante 12 horas à temperatura ambiente. Centrífuga e remover o sobrenadante. Lava-se a resina para remover marcador que não reagiu. </li><li> Eluir o receptor marcado com 100 ml de tampão de eluição contendo 0,1% (w / v) de DDM e 0,33 mg / ml de nonapéptido (C9 péptido contra o epitopo de 1D4)em tampão de fosfato 2 mM, pH 6,0, por incubação em gelo durante 1 hr. </li><li> Resolve amostras marcadas por SDS-PAGE sob condições redutoras. Lavar géis brevemente em PBS e, em seguida, visualizar rotulagem de AZF-GPCR por fluorescência em gel. Por exemplo, uso de um scanner de fluorescência confocal a laser com um comprimento de onda de 488 nm para a detecção do receptor modificado com 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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Descrevemos aqui uma metodologia robusta para incorporação site-specific de uma sonda reativa, AZF, em GPCRs e demonstrar três aplicações úteis deste instrumento para estudar a estrutura ea dinâmica de GPCRs. Nosso método para o site especificamente incorporar UAAs contorna um problema fundamental com uma estratégia alternativa baseada na rotulagem quimicamente 32, 33 ou anexar photocrosslinkers 34 para um único mutante cisteína acessível. Embora, químicos que têm como alvo grupos tio...

Heptahelical receptores acoplados à proteína (GPCRs) compreendem uma superfamília de proteínas de membrana altamente dinâmicos que medeiam sinais extracelulares importantes e diversas. No paradigma clássico, a activação do receptor é acoplada com alterações conformacionais induzidas pelo ligando. 1, 2 avanços recentes em biologia estrutural dos GPCRs têm fornecido uma visão significativa sobre os mecanismos moleculares da sinalização transmembrana. 3-5 No entanto, para compreender com maior precisão o químico mecanismo funcional e dinâmica estrutural de sinalização GPCR, um kit de ferramentas de abordagens é necessária para incorporar sondas moleculares e químicas informativos para interrogar complexos de sinalização ativos. Para este fim, nós adaptamos um método para o site especificamente introduzir não ou minimamente sondas perturbadores em receptores expressos com base no aminoácido não natural mutagênese (SAU), utilizando tecnologia de supressão de âmbar códon anteriormente iniciada por Schultz e coworkers. 6 Nós optimizado a metodologia mutagénese UAA para alcançar uma expressão de alto rendimento e um sistema de mutagénese de proteínas, tais como os GPCRs, que são difíceis de expressar em células de mamífero do que outros sistemas mais heterólogos. Usando um supressor de engenharia ortogonal tRNA e evoluiu aminoacil-tRNA sintetase par para uma SAU específica, local especificamente introduzido UAAs em GPCRs alvo expressas. A incorporação bem sucedida de UAAs, p-acetil-L-fenilalanina (AcF), p-benzoil-L-fenilalanina (BZF), e p-azido-L-fenilalanina (AZF) foi demonstrada no nosso modelo GPCRs - rodopsina e humano receptor de quimiocina CC, CCR5. 7, 8 Em princípio, um UAA podem ser geneticamente codificado em qualquer posição dentro da seqüência de proteínas e esta propriedade é uma ferramenta bioquímica inestimável, pois permite que um único códon de varredura de um GPCR alvo. Nós nos focamos especificamente sobre a versatilidade da UAA, AZF, que tem um azi reativafazer metade. Além de servir como um (IR) da sonda original de infravermelhos, 8, 9 AZF pode também servir como um agente de ligação cruzada por fotoactivável reagir com aminas primárias ou hidrogénios alifáticos vizinhos. Além disso, o grupo azido biologicamente inertes podem participar como pega química seletiva em reações de rotulagem bioorthogonal. Aqui apresenta-se exemplos que ilustram as aplicações úteis de incorporação específica do local de AZF em GPCRs, tais como o fotocruzamento alvo para interceptar um complexo receptor-ligando, e a modificação dos GPCRs por marcação de epitopo bioorthogonal e estratégias de marcação fluorescente. Fotoactiváveis ​​reagentes foram utilizados para estudar sistemas biológicos desde os anos 1960. 10 Neste período, uma abundância de experiências de reticulação ligando-receptor foram relatados para estudar os complexos de GPCR, a maioria dos quais envolve a utilização de ligantes de fotoafinidade 11, 12 No entanto, estes. aplicações são tecnicamente limitados, uma vez que require a síntese de ligandos que possuem um grupo de reticulação 13-15. Além disso, com a porção de agente de ligação cruzada no ligando, isto é difícil de identificar a localização da ligação transversal na GPCR. Site-especificamente a introdução de grupos de fotorreticulação como UAAs em proteínas utilizando o codão âmbar tecnologia de supressão é um avanço valioso 16, 17. Desenvolvemos uma técnica de foto-reticulação para identificar a interface de ligação a um receptor que está envolvida na formação de um complexo receptor-ligando em vivo através da introdução de grupos de células em fotolábeis GPCRs. 18, 19 Aqui, descrevemos o protocolo experimental e método de análise de dados para aplicar esta tecnologia fotorreticulação alvo para identificar o local de ligação de um ligando de molécula pequena, maraviroc tritiada, em CCR5. Este método aproveita a quantificação exacta do identificador radioactivo no ligando, para além de manter a estrutura química nativa do ligando. Técnicas baseadas em fluorescência apoiar a compreensão precisa da base estrutural da activação do receptor por sondando directamente o estado conformacional do receptor. 20, 21 No entanto, as técnicas que possuem a flexibilidade de introduzir marcadores fluorescentes em GPCRs local especificamente são limitados. Estamos interessados ​​em empregar estratégias de modificação química bioorthogonal para facilitar a detecção de moléculas individuais (SMD) de GPCR complexos de sinalização. 22 O grupo azido podem participar químicas bioorthogonal como Staudinger-Bertozzi ligadura, 23, 24 azida-promovido-deformação sem cobre cicloadição alcino (SpAAC) 25 e azida-alcino cicloadição catalisada por cobre (CuAAC). 26 Nós nos focamos na reação ligadura Staudinger-Bertozzi, que envolve a reação específica entre uma azida e uma fosfina. Nós demonstramos a utilização de dois derivados diferentes de fosfina, conjugados com um péptido epitopo (péptido FLAG) ou uma etiqueta fluorescente(F luoresceína) para conseguir a modificação específica do local de GPCRs. Nós já otimizada as condições para a rotulagem específica do site da rodopsina AZF variantes usando a estrutura de cristal de raio-X e simulações dinâmicas para escolher os locais alvo que são expostos solvente. 27, 28 Também ilustrou a viabilidade de alcançar rotulagem sem fundo por Staudinger- bertozzi ligadura. 29 Demonstramos aqui o procedimento geral empregue para alcançar marcação fluorescente de um receptor de detergente solubilizado que é imobilizado sobre uma matriz de imunoafinidade e posteriormente visualizados por fluorescência em gel. Além disso, nós demonstramos uma extensão útil sobre esta estratégia de rotulagem para identificar posições passíveis de rotulagem em um receptor de estrutura desconhecida, CCR5. Esta é levada a cabo usando uma estratégia de marcação péptido epitopo específico, que se baseia na modificação bioorthogonal de AZF-GPCR variantes em um formato semi-alto rendimento com base em células. 30 Estemétodo explora as propriedades de detecção multi-passo de um ELISA da superfície celular para monitorar eventos de rotulagem. As metodologias que discutimos aqui são gerais e, em princípio, pode ser aplicado a qualquer GPCR incorporado com AZF usando o âmbar códon tecnologia de supressão. Nos protocolos apresentados aqui pormenorizadamente as etapas envolvidas na expressão das células de mamíferos de receptores incorporados com UAAs como AZF utilizando o método de mutagénese de aminoácido não natural e seus pedidos subsequentes para facilitar os estudos estruturais e dinâmicas de 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 os estudos estruturais e dinâmicas de G do receptor acoplado à proteína (GPCR) de sinalização complexos, novas abordagens são necessários para introduzir sondas informativas ou etiquetas em receptores expressos que não perturbam a função do receptor. Usamos âmbar tecnologia de supressão de codões para codificar geneticamente o aminoácido não natural, p-azido-L-fenilalanina (AZF) em várias posições orientadas nos GPCRs heterologamente expressos em células de mamíferos. A versatilidade do grupo azido é aqui ilustrado em diferentes aplicações para estudar GPCRs no seu ambiente celular nativo, ou sob condições de detergentes solubilizadas. Em primeiro lugar, nós demonstramos uma tecnologia de foto-reticulação com base nas células-alvo para identificar os resíduos no bolso de ligação ao ligando de GPCR em que um marcado com trítio pequena molécula ligante é reticulado com um ácido azido-amino geneticamente codificado. Nós, então, demonstrar a modificação específica do site de GPCRs pelo bioorthogonal Staudinger-Bertozzi ligadura reaçãoção que tem como alvo o grupo azido utilização de derivados de fosfina. Discute-se uma estratégia geral para o alvo de marcação de péptidos com epitopo das proteínas de membrana expressos em cultura e a sua detecção usando uma abordagem baseada em ELISA de célula inteira. Finalmente, mostra-se que AZF-GPCRs pode ser marcado selectivamente com sondas fluorescentes. As metodologias são discutidos em geral, na medida em que podem, em princípio, ser aplicada a qualquer posição do aminoácido em qualquer expressa GPCR para interrogar os complexos de sinalização activas.

Utilizamos a metodologia de mutagênese aminoácido não natural para o site especificamente introduzir sondas moleculares em um GPCR usando o âmbar códon tecnologia de supressão. O esquema da Figura 1 esboça os passos salientes da metodologia e os vários pedidos de incorporar o versátil UAA, p-azido-L-fenilalanina (AZF), para GPCRs. Expressão da AZF-GPCRs em células de mamíferos permite fotoreticulação alvejado a ligantes, e marcação peptídeo epítopo ou modificações fluorescen...

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

Nós geneticamente codificam o aminoácido não natural, p-azido-L-fenilalanina em várias posições orientadas nos GPCRs e mostram a versatilidade do grupo azido em diferentes aplicações. Estes incluem uma tecnologia de fotorreticulação alvo para identificar resíduos na bolsa de ligação ao ligando de um GPCR, e específica do local de modificação bioorthogonal de GPCRs com uma etiqueta de péptido epitopo ou sonda fluorescente.

Geneticamente codificados Molecular Probes para estudar G receptores acoplados à proteína

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 visualizações.  The Rockefeller University.  Nós geneticamente codificam o aminoácido não natural, p-azido-L-fenilalanina em várias posições orientadas nos GPCRs e mostram a versatilidade do grupo azido em diferentes aplicações. Estes incluem uma tecnologia de fotorreticulação alvo para identificar resíduos na bolsa de ligação ao ligando de um GPCR, e específica do local de modificação bioorthogonal de GPCRs com uma etiqueta de péptido epitopo ou sonda fluorescente. 

Vídeo: 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.

Teste de sensibilidade visual para a velocidade e direção do movimento em Lagartos

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

Biologia

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.

Isolamento e purificação de Drosophila Neurônios periféricos por classificação Bead Magnética

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.

Em Quantificação in vivo de proteínas G Interações Receptor Acoplado usando espectral Resolvido Microscopia de dois fótons

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.

Mudanças Dinâmicas de monitoramento dos níveis de cálcio mitocondrial durante a apoptose através de um sensor de cálcio geneticamente codificado

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 imagem proteína G-Coupled (GPCR) mediada por eventos de sinalização que Quimiotaxia de Controle 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.

Quantificar atividade agonista no G receptores acoplados à proteína

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).

Caracterização dos receptores acoplados à proteína por uma mobilização de cálcio Ensaio baseado em fluorescência

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.

Visualizando Endocitose clatrina mediada por receptores acoplados à proteína a resolução de evento único através de microscopia TIRF

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.

Hidroponia: um sistema versátil para estudar nutrientes de alocação e de plantas As respostas a disponibilidade de nutrientes e exposição 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.

Imagiologia G protein-coupled receptor mediada por quimiotaxia e seus eventos de sinalização em células de HL60 de Neutrófilos-like

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