Ringraziamo il generoso sostegno di numerose fondazioni e filantropi (vedi SakmarLab.org).

1. Site-specific Costituzione genetica di Innaturale Aminoacidi in GPCR <ol><li> Mantenere HEK293T cellule in DMEM (4,5 g / L di glucosio, glutammina 2 mM) supplementato con 10% siero bovino fetale (FBS) a 37 ° C in una atmosfera di CO 2 5%. </li><li> Trasfezione delle cellule cresciute al 60-80% di confluenza in una piastra da 10 cm usando Lipofectamine Plus. reagente. <ol><li> A 750 microlitri DMEM, aggiungere 10 microlitri Plus. reagente, 3,5 pg di GPCR cDNA (pMT4. Rho o pcDNA 3.1. CCR5) contenenti l&#39;ambra codone di stop in una posizione desiderata, 3,5 pg di tRNA soppressore cDNA (pSVB.Yam) e 0,35 mg di mutante amino-acil tRNA sintetasi cDNA per p-azido-L-fenilalanina (pcDNA.RS). Incubare a temperatura ambiente per 15 min. Effettuare anche una trasfezione simile usando l&#39;wt GPCR cDNA (non contenente un codone di stop ambra) per servire come controllo. Aggiungi questa miscela a 750 ml di DMEM con 17 microlitri Lipofectamine. Dopo equilibrazione 15 minuti a temperatura ambiente, portare lavolume totale di 4 ml. </li><li> Aspirare il terreno di 10 cm piastra, si applicano miscela di trasfezione per le cellule, e tornare a 37 ° C in 5% di CO 2 nell&#39;atmosfera. Dopo 4-6 ore, integrare le celle con 4 ml di DMEM contenente 20% FBS e 1 AZF mm. </li></ol></li><li> Il giorno seguente, sostituire i mezzi di crescita con DMEM contenente 10% FBS e azf 0,5 mM. </li><li> Celle di raccolta 48 ore dopo la trasfezione, per analizzare l&#39;espressione o procedere procedure photocrosslinking o etichettatura descritti nelle sezioni seguenti. <ol><li> Confermare espressione del mutante ambra GPCR in presenza della SAU nel mezzo di crescita. Facciamo questo rilevamento immunoblot occidentale. Lyse il pellet cellulare in 1% (w / v) dodecil-β-D-maltoside (DDM) in 20 mM Tris-HCl, pH 7,5, NaCl 100 mM per 1 ora a 4 ° C. Centrifugare il lisato a 16.000 xg e risolvere il supernatante in condizioni riducenti mediante SDS-PAGE. Trasferimento a una membrana PVDF e effettuare analisi immunoblot per rilevare l&#39;espressione del recettore full-length usando the 1D4 mAb (Cell Nazionale Culture Center), che riconosce un epitopo ingegnerizzato al C-terminale del recettore. 31 </li></ol></li></ol> 2. Mappatura di rilegatura sito Ligand Uso mirata Photocrosslinking <ol><li> 48 ore dopo la trasfezione, mezzi di aspirare da cellule e sostituirlo con 4 ml di 1x tampone fosfato (PBS). Rientro a 37 ° C per 15 min. </li><li> Risospendere le cellule con PBS 1x e trasferire in una provetta Falcon. Centrifugare a 1500 rpm per 2 minuti per far sedimentare le cellule e aspirare il surnatante. </li><li> Risospendere il pellet di cellule in soluzione salina tamponata di Hank 1x contenente HEPES 20 mM, pH 7,5, 0,2% BSA e il ligando triziato a concentrazioni saturanti vincolante per il periodo di tempo e la temperatura richiesta per garantire recettore-ligando formazione del complesso. </li><li> Trasferire la sospensione cellulare in una piastra a 24 pozzetti o 96 pozzetti per fotoattivazione del AZF a 365 nm (per esempio utilizzare un lampada UV-A) per 15 min a 4 ° C. Manten la piastra sul ghiaccio per evitare il riscaldamento del campione e lisi cellulare. </li><li> Trasferire le cellule in una provetta Eppendorf, centrifugare per agglomerare le cellule, rimuovere il supernatante e procedere all&#39;analisi. I pellet possono essere conservati a -20 ° C per un uso futuro. </li><li> Risospendere il pellet di cellule in tampone di lisi contenente 1% (w / v) DDM, 20 mM Tris-HCl, pH 7.5 e inibitori della proteasi. Dopo 1 ora di incubazione a 4 ° C, centrifugare il lisato cellulare per 10 minuti a 16.000 xg, e trasferire il surnatante in una nuova provetta. </li><li> Aggiungere 1D4 mAb-Sepharose resina 2B al supernatante e incubare una notte a 4 ° C per immunopurify GRCP usando l&#39;ingegnerizzato C-terminale 1D4 mAb epitopo. Il giorno dopo lavare le perline più volte con tampone di lisi. Campioni Eluire con 1% SDS a 37 ° C per 1 ora con agitazione. </li><li> Trasferire una porzione dell&#39;eluente in una fiala contenente 15 ml di liquido di scintillazione e contare su un contatore a scintillazione beta per quantificare la quantità di legame specifico del ligando triziato al secoloe recettore. </li><li> Risolvere il restante 1D4 mAb eluente purificato mediante elettroforesi su gel standard. Utilizziamo un 4-12% SDS-PAGE gel e poi trasferimento semi-dry ad una membrana di PVDF per immunoblotting. Abbiamo anche una corsia con scaletta proteine ​​standard per guidare l&#39;approssimazione visiva di peso molecolare. <ol><li> Bloccare la membrana PVDF con 5% di latte in tampone TBS contenente 0,05% Tween-20. Sondare la membrana per confermare l&#39;espressione del recettore full-length con 1D4 mAb seguito da anticorpo secondario anti-topo IgG HRP-coniugato. </li><li> Trattare con chemiluminescenza (ECL) Reattivo ed esporre membrana per autoradiografia pellicola per visualizzare bande. </li></ol></li><li> Tagliare la membrana con una lama di rasoio di separare ogni corsia campione e quindi tagliato a specifici marcatori di peso molecolare. Trasferimento segmenti di membrana di flaconi contenenti liquido di scintillazione. Quantificare la quantità di trizio in ogni segmento membrana contando su un contatore beta-scintillazione. </li><li> Identificare il positivophotocrosslink con il rilevamento di trizio alla massa molecolare apparente pari alla somma di quella del GPCR e il ligando. </li></ol> 3. Mirata epitopi Tagging e Cell Surface immunoenzimatico Assay (ELISA) <ol><li> 24 ore dopo la trasfezione, lavare le cellule con 1 ml di PBS 1X e trypsinize con 1 ml 0,25% tripsina per 3 min. Integrare con 9 ml DMEM contenente 10% FBS e AZF 0,5 mm. Risospendere le cellule e contare la densità cellulare. Usiamo un emocitometro standard. </li><li> Pre-trattare una, chiara-bottom piastra a 96 pozzetti alto legame con 100 ml di 0,01 mg / ml poli-D-lisina per pozzetto. Incubare per 30 minuti alla temperatura ambiente, lavate ripetutamente con PBS 1x e aria secca. </li><li> Piastra 200 ml di cellule trasfettate con una densità di 6 x ottobre 04-08 x 10 4 cellule / pozzetto di piastra a 96 pozzetti e ritorno a 37 ° C in 5% CO 2 nell&#39;atmosfera. </li><li> Il giorno dopo preparare le cellule per l&#39;etichettatura. Lavare delicatamente tre volte con 100 microlitri / pozzetto 1x PBS to rimuovere AZF contenente terreno di coltura. Assicurarsi che le cellule rimangono attaccati, per esempio, utilizzando PBS contenente Ca 2 + e Mg 2 +. </li><li> Preparare 50-200 micron FLAG-triarylphosphine etichettatura reagente in 1x HBSS / PBS da uno stock mantenuto a 5-20 mM in PBS. Applicare 60 ml in ciascun pozzetto (in triplicato per ogni mutante ambra) e ritorno a 37 ° C per 30 min a 4 ore. Mantenere una serie di pozzi senza trattamento etichetta in 1x HBSS / PBS. Anche un controllo peso GPCR confrontare con varianti AZF-GPCR. </li><li> Lavare le cellule 3 volte con 100 microlitri / pozzetto di tampone di bloccaggio, BB (1x PBS, 0.5% BSA), per rimuovere completamente l&#39;etichetta che non ha reagito. </li><li> Applicare 100 microlitri / pozzetto del 4% paraformaldeide (preparato da uno stock di 16% in PBS 1x) alle cellule. Incubare per 20 minuti a temperatura ambiente. Lavare tre volte con BB. </li><li> Eseguire protocollo ELISA superficie cellulare standard per rilevare recettore etichettati e l&#39;espressione del recettore. Incubare con anticorpo primario per 1,5 ore su ghiaccio seguita da una secondariatibody incubazione a temperatura ambiente per 1 ora. <ol><li> Aggiungere 100 ml di anticorpi anti-FLAG M2 (es. 1:2.000 diluizione di anticorpo fatto in BB) per rilevare FLAG peptide epitopo di etichettatura reagente FLAG-triarylphosphine. Sondare anche non-label pozzetti trattati come controllo negativo. Aggiungi anti-CCR5 2D7 (usiamo una diluizione 1:500) anticorpo ad una serie separata di pozzi per determinare in peso o superficie cellulare espressione AZF-GPCR. </li><li> Lavare le cellule tre volte con BB, e incubare con 100 ml di HRP-coniugato anticorpo secondario anti-topo IgG (1:2.000 diluizione). </li><li> Lavare accuratamente le cellule cinque volte con BB. Aggiungere rilevamento buffer 50 microlitri: Amplex Rosso, 20 mM H 2 O 2, 1x PBS (1:10:90 ratio) e incubare per 15 min a temperatura ambiente. Raccogliere dati spettrali su un lettore di fluorescenza piatto multi-bene a λ ex = 530 e λ em = 590. </li></ol></li></ol> 4. Bioorthogonal Etichettatura fluorescente di un GPCR <ol><li> Lyse10 7 cellule raccolte esprimono wt o AZF-Rhodopsin varianti in 1 ml di tampone di solubilizzazione contenente 1% (w / v) DM, HEPES 50 mM o Tris-HCl, pH 6,8, 100 mM NaCl, 1 mM CaCl 2 e inibitori della proteasi per 1 ore a 4 ° C. Centrifugare il lisato cellulare a 15.000 xg per 10 minuti e raccogliere il surnatante. </li><li> Aggiungere 100 microlitri 1D4-mAb resina sepharose 2B per catturare recettore usando l&#39;ingegneria C terminus 1D4 epitopo. Incubare per 12 ore a 4 ° C. Lavare la resina tre volte con 0,1% (w / v) DDM in 0,1 M tampone fosfato di sodio, pH 7,3. </li><li> Aggiungere 0,1 mM fluoresceina-fosfina in un volume totale di 0,3 - 0,5 etichettare recettore immobilizzato sulla matrice immunoaffinità (1D4-mAb sefarosio 2B). Incubare per 12 ore a temperatura ambiente. Centrifuga e si preleva il surnatante. Lavare la resina per rimuovere etichetta non reagito. </li><li> Eluire il recettore marcato con tampone di eluizione 100 ml contenente 0,1% (w / v) DDM e 0,33 mg / ml nonapeptide (C9 peptide contro l&#39;epitopo 1D4)in 2 mM tampone fosfato, pH 6,0 mediante incubazione in ghiaccio per 1 ora. </li><li> Risolvere campioni etichettati mediante SDS-PAGE in condizioni riducenti. Lavare gel brevemente in PBS e quindi visualizzare l&#39;etichettatura di AZF-GPCR da in-gel fluorescenza. Ad esempio, utilizzare uno scanner a fluorescenza confocale con un laser di lunghezza d&#39;onda 488 nm per rilevare recettore modificato con fluoresceina. </li></ol>

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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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Descriviamo qui una solida metodologia per site-specific inserimento di una sonda reattiva, AZF, in GPCR e dimostriamo tre applicazioni utili di questo strumento per studiare la struttura e la dinamica dei GPCR. Il nostro metodo al sito-specifico incorporano UAAs elude un problema fondamentale con una strategia alternativa basata sulla etichettatura chimicamente 32, 33 o allegando photocrosslinkers 34 per una singola cisteina mutante accessibile. Sebbene, chimiche che colpiscono gruppi tiolo della ...

Recettori accoppiati alla proteina G (GPCR Heptahelical) comprendono una superfamiglia di proteine ​​di membrana altamente dinamici che mediano i segnali extracellulari importanti e diverse. Nel paradigma classico, l&#39;attivazione del recettore è accoppiato con i cambiamenti conformazionali ligando-indotta. 1, 2 recenti progressi nel campo della biologia strutturale di GPCR hanno fornito informazioni significative sui meccanismi molecolari della segnalazione transmembrana. 3-5 Tuttavia, per comprendere con maggiore precisione chimica meccanismo funzionale e dinamica strutturale della segnalazione GPCR, un toolkit di approcci sono necessari per incorporare sonde molecolari e chimiche informativi per interrogare complessi di segnalazione attiva. A tal fine, abbiamo adattato un metodo per sito-specifico di introdurre non o poco sonde perturbanti nella recettori espressi sulla base di aminoacidi innaturale (SAU) mutagenesi con ambra codone tecnologia di soppressione precedentemente introdotta da Schultz ecoworkers. 6 Abbiamo ottimizzato la metodologia mutagenesi SAU per ottenere una espressione ad alto rendimento e sistema di mutagenesi per proteine ​​come GPCR, che sono difficili da esprimere in sistemi eterologhi più diversi dalle cellule di mammifero. L&#39;utilizzo di un silenziatore costruito ortogonale tRNA e si è evoluta-tRNA sintetasi pair per una specifica SAU, noi del sito specificamente introdotto UAAs in GPCR bersaglio espresse. L&#39;incorporazione di successo UAAs, p-acetil-L-fenilalanina (ACF), p-benzoil-L-fenilalanina (BZF), e p-azido-L-fenilalanina (AZF) è stata dimostrata nei nostri GPCR modello - rodopsina e umana recettore CC chemochine, CCR5. 7, 8 In linea di principio, una SAU può essere geneticamente codificato in qualsiasi posizione all&#39;interno della sequenza della proteina e questa proprietà è uno strumento biochimico prezioso in quanto consente la scansione singolo codone di un GPCR bersaglio. Ci concentriamo qui in particolare sulla versatilità della SAU, AZF, che ha una azi reattivafare parte. Oltre a servire come infrarossi (IR) sonda unico, 8, 9 AZF può anche servire come un photoactivatable reticolante per reazione con ammine primarie vicini o idrogeni alifatici. Inoltre, il gruppo azido biologicamente inerte può partecipare come un manico chimica selettiva nelle reazioni di etichettatura bioorthogonal. Qui vi presentiamo esempi che illustrano le applicazioni utili del sito-specifica incorporazione di AZF in GPCR, come photocrosslinking mirato ad intrappolare un complesso recettore-ligando, e la modifica dei GPCR da bioorthogonal codifica epitopo e le strategie in materia di etichettatura fluorescenti. Photoactivatable reagenti sono stati utilizzati per studiare i sistemi biologici dal 1960. 10 In questo periodo, l&#39;abbondanza di esperimenti reticolazione recettore-ligando sono stati segnalati per studiare complessi GPCR, la maggior parte dei quali ha coinvolto l&#39;uso di leganti fotoaffinità. 11, 12 Tuttavia, questi applicazioni sono tecnicamente limitati, in quanto Require sintesi di leganti recanti un gruppo reticolante. 13-15 Inoltre, con la frazione di reticolante nel ligando, è impegnativo per identificare la posizione della reticolazione sul GPCR. Sito-specifico introducendo photocrosslinking gruppi come UAAs in proteine ​​utilizzando la tecnologia di soppressione ambra codone è un avanzamento importante. 16, 17 Abbiamo sviluppato una tecnica photocrosslinking per identificare l&#39;interfaccia di legame su un recettore che è coinvolto nella formazione di un complesso recettore-ligando in cellule vive introducendo gruppi fotolabili in GPCR. 18, 19 Qui si descrive il protocollo sperimentale e metodo di analisi dei dati per applicare questa tecnologia photocrosslinking mirato per identificare il sito di legame di un ligando piccola molecola, maraviroc triziata, su CCR5. Questo metodo sfrutta la quantificazione precisa del manico sul ligando radioattivo, oltre ad avere la struttura chimica nativa del ligando. Tecniche basate fluorescenza supportano la precisa comprensione della base strutturale del recettore sondando direttamente lo stato conformazionale del recettore. 20, 21 Tuttavia, le tecniche che hanno la flessibilità necessaria per introdurre etichette fluorescenti in GPCR sito-specifica sono limitati. Siamo interessati a impiegare strategie di modificazione chimica bioorthogonal per facilitare l&#39;individuazione singola molecola (SMD) di complessi di segnalazione GPCR. 22 Il gruppo azido può partecipare a chimiche bioorthogonal come Staudinger-Bertozzi legatura, 23, 24 strain-promosso azide-senza rame cicloaddizione alkyne (SpAAC) 25 e azide-alchino cicloaddizione rame-catalizzato (CuAAC) 26. Ci concentriamo qui sulla reazione legatura Staudinger-Bertozzi che coinvolge la reazione specifica tra un azide e fosfina. Abbiamo dimostrato l&#39;utilizzo di due differenti derivati ​​fosfina, coniugati ad un epitopo peptide (peptide FLAG) o un&#39;etichetta fluorescente(Fluoresceina) per raggiungere sito-specifica modifica del GPCR. Abbiamo già ottimizzato le condizioni per l&#39;etichettatura sito-specifica della rodopsina varianti AZF utilizzando la struttura cristallina ai raggi X e simulazioni dinamiche di scegliere siti di destinazione che sono solventi esposte. 27, 28 Abbiamo anche illustrato la possibilità di conseguire l&#39;etichettatura libero-background da Staudinger- Bertozzi legatura. 29 dimostriamo qui la procedura generale impiegato per raggiungere marcatura fluorescente di un recettore detergente-solubilizzato che viene immobilizzato su una matrice immunoaffinità e seguito evidenziata in-gel fluorescenza. Inoltre, abbiamo dimostrato un utile estensione di questa strategia di etichettatura per identificare le posizioni suscettibili di etichettatura su un recettore di struttura sconosciuta, CCR5. Questo avviene tramite una mirata strategia di codifica-peptide epitopo che si basa sulla modificazione bioorthogonal di AZF-GPCR varianti in un formato semi-high throughput base cellulare. 30 Questometodo sfrutta le proprietà di rilevamento multi-step di un ELISA superficie cellulare per monitorare gli eventi di etichettatura. Le metodologie di cui parliamo qui sono generali e in linea di principio può essere applicato a qualsiasi GPCR incorporata con AZF usando l&#39;ambra codone tecnologia di soppressione. Nei protocolli qui presentati dettaglio i passi necessari per l&#39;espressione cellulare di mammifero di recettori incorporato con UAAs come AZF utilizzando il metodo di mutagenesi amminoacido non naturale e successive applicazioni di facilitare studi strutturali e dinamiche di GPCR.

<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

Per facilitare gli studi strutturali e dinamiche di G protein-coupled recettore (GPCR) complessi di segnalazione, sono necessari nuovi approcci per introdurre sonde informativi o etichette in recettori espressi che non perturbano la funzione del recettore. Abbiamo usato ambra tecnologia di soppressione codone geneticamente codificare l&#39;amminoacido non naturale, p-azido-L-fenilalanina (AZF) in diverse posizioni mirate nei GPCR eterologamente espressi in cellule di mammifero. La versatilità del gruppo azido è illustrato qui in applicazioni diverse per studiare GPCR nel loro ambiente nativo cellulare o detergenti in condizioni solubilizzate. In primo luogo, abbiamo dimostrato una tecnologia photocrosslinking mirata basata celle per identificare i residui nella tasca di legame del ligando di GPCR dove una piccola molecola ligando marcato con trizio viene reticolato di un acido ammino azido geneticamente codificati. Abbiamo poi Dimostriamo sito-specifica modifica del GPCR dal bioorthogonal Staudinger-Bertozzi legatura reazionizione che gli obiettivi del gruppo azido mediante derivati ​​fosfina. Discutiamo di una strategia generale per la mirata peptide-epitopo tagging delle proteine ​​di membrana espresse in-cultura e la sua individuazione utilizzando un metodo ELISA basato whole-cell. Infine, dimostriamo che AZF-GPCR possono essere selettivamente contrassegnati con sonde fluorescenti. Le metodologie sono discusse generale, in quanto possono in principio essere applicate a qualsiasi posizione amminoacido in qualsiasi espresso GPCR interrogare complessi di segnalazione attive.

Abbiamo utilizzato la metodologia mutagenesi amminoacido non naturale al sito-specifico introdurre sonde molecolari in un GPCR usando l&#39;ambra codone tecnologia di soppressione. Lo schema in figura 1 illustra le fasi salienti della metodologia e le varie applicazioni di incorporare la versatile UAA, p-azido-L-fenilalanina (AZF), in GPCR. Espressione di AZF-GPCR in cellule di mammifero permette mirato photocrosslinking ai ligandi, e mirata peptide-epitopo codifica o modifiche fluorescenti di ...

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

Abbiamo geneticamente codificare l&#39;amminoacido non naturale, p-azido-L-fenilalanina in varie posizioni mirate a GPCR e mostra la versatilità del gruppo azido in diverse applicazioni. Questi includono una tecnologia photocrosslinking mirato a identificare i residui nella tasca-ligando di un GPCR, e site-specific modifica bioorthogonal di GPCR con un tag peptide-epitopo o sonda fluorescente.

Molecular Probes geneticamente codificati a studiare G recettori accoppiati alle proteine

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

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Research

JoVE Journal

Biology

15.1K Views.  The Rockefeller University.  Abbiamo geneticamente codificare l'amminoacido non naturale, p-azido-L-fenilalanina in varie posizioni mirate a GPCR e mostra la versatilità del gruppo azido in diverse applicazioni. Questi includono una tecnologia photocrosslinking mirato a identificare i residui nella tasca-ligando di un GPCR, e site-specific modifica bioorthogonal di GPCR con un tag peptide-epitopo o 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.

Test di sensibilità visiva alla velocità e direzione del movimento in lucertole

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

Ricerca

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 purificazione di Drosophila Neuroni periferici da Magnetic Ordinamento Bead

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.

Quantificazione in vivo di G interazioni proteina recettore accoppiato con spettralmente risolte microscopia a due fotoni

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.

Monitoraggio Cambiamenti dei livelli di calcio mitocondriale durante l&#39;apoptosi mediante un sensore di calcio geneticamente codificato

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 Imaging proteina G accoppiata (GPCR)-mediata eventi di segnalazione che chemiotassi di controllo di 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.

Quantificare attività agonista a G recettori accoppiati alla proteina

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).

Caratterizzazione di G recettori accoppiati alla proteina da un calcio di Mobilitazione Assay Basato sulla fluorescenza

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.

Visualizzazione Clathrin endocitosi mediata da G recettori accoppiati a proteine ​​a risoluzione singolo evento tramite 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.

Hydroponics: un sistema versatile per studiare nutrienti allocazione e vegetali risposte a disponibilità di nutrienti e l&#39;esposizione a elementi tossici

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 protein-coupled recettore-mediata chemiotassi ed i suoi eventi di segnalazione nelle cellule HL60 neutrofili-like

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