Name: in vitro analysis of pdz-dependent cftr macromolecular signaling complexes
Uploaded: 2012-08-13T13:00:00
Description: Watch this Scientific Journal Video about In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes at JoVE.com

Il nostro lavoro è stato supportato anche da finanziamenti American Heart Association (Greater Southeast Affiliate) Inizio-grant-in-aiuto 0765185B, la Fondazione U. Pardee Elsa assegno di ricerca, e Wayne State University intramurale del fondo di avvio e Cardiovascular Research Institute premio Initiative Isis. Questo metodo di assemblaggio in vitro complesso macromolecolare CFTR è stata originariamente introdotta da Dr. AP Naren (University of Tennessee Health Science Center).

1. Espressione e purificazione di proteine ​​ricombinanti in batteri Fusion etichettate <ol><li> Amplifica le regioni definite del C-code (gli ultimi 50-100 aminoacidi contenenti i motivi PDZ a C-terminale) per CFTR, LPA 2, MRP2, MRP4, β 2 AR, e NHERFs (full-length o PDZ1 o PDZ2 domini ) con approccio PCR. </li><li> Clonare i prodotti di PCR in pGEX4T-1 vettore per le proteine ​​di fusione GST-(come GST-NHERFs, GST-MRP4 CT), pMAL-C2 vettore per MBP-fusione proteine ​​(come MBP-β <su...

<ol>
	<li>Anderson, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Demonstration+that+CFTR+is+a+chloride+channel+by+alteration+of+its+anion+selectivity.">Demonstration that CFTR is a chloride channel by alteration of its anion selectivity.</a> Science. 253, 202-205 (1991).</li><li>Bear, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+functional+reconstitution+of+the+cystic+fibrosis+transmembrane+conductance+regulator+(CFTR.">Purification and functional reconstitution of the cystic fibrosis transmembrane conductance regulator (CFTR.</a> Cell. 68, 809-818 (1992).</li><li>Quinton, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chloride+impermeability+in+cystic+fibrosis.">Chloride impermeability in cystic fibrosis.</a> Nature. 301, 421-422 (1983).</li><li>Cheng, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defective+intracellular+transport+and+processing+of+CFTR+is+the+molecular+basis+of+most+cystic+fibrosis.">Defective intracellular transport and processing of CFTR is the molecular basis of most cystic fibrosis.</a> Cell. 63, 827-834 (1990).</li><li>Gabriel, S. E., Brigman, K. N., Koller, B. H., Boucher, R. C., Stutts, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cystic+fibrosis+heterozygote+resistance+to+cholera+toxin+in+the+cystic+fibrosis+mouse+model.">Cystic fibrosis heterozygote resistance to cholera toxin in the cystic fibrosis mouse model.</a> Science. 266, 107-109 (1994).</li><li>Li, C., Naren, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CFTR+chloride+channel+in+the+apical+compartments:+spatiotemporal+coupling+to+its+interacting+partners.">CFTR chloride channel in the apical compartments: spatiotemporal coupling to its interacting partners.</a> Integr. Biol (Camb). 2, 161-177 (2010).</li><li>Chao, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+intestinal+CFTR+Cl-+channel+by+heat-stable+enterotoxin+and+guanylin+via+cAMP-dependent+protein+kinase.">Activation of intestinal CFTR Cl- channel by heat-stable enterotoxin and guanylin via cAMP-dependent protein kinase.</a> Embo. J. 13, 1065-1072 (1994).</li><li>Gabriel, S. E., Clarke, L. L., Boucher, R. C., Stutts, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CFTR+and+outward+rectifying+chloride+channels+are+distinct+proteins+with+a+regulatory+relationship.">CFTR and outward rectifying chloride channels are distinct proteins with a regulatory relationship.</a> Nature. 363, 263-268 (1993).</li><li>McNicholas, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitivity+of+a+renal+K++channel+(ROMK2)+to+the+inhibitory+sulfonylurea+compound+glibenclamide+is+enhanced+by+coexpression+with+the+ATP-binding+cassette+transporter+cystic+fibrosis+transmembrane+regulator.">Sensitivity of a renal K+ channel (ROMK2) to the inhibitory sulfonylurea compound glibenclamide is enhanced by coexpression with the ATP-binding cassette transporter cystic fibrosis transmembrane regulator.</a> Proc. Natl. Acad. Sci. USA. 93, 8083-8088 (1996).</li><li>Schreiber, R., Nitschke, R., Greger, R., Kunzelmann, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cystic+fibrosis+transmembrane+conductance+regulator+activates+aquaporin+3+in+airway+epithelial+cells.">The cystic fibrosis transmembrane conductance regulator activates aquaporin 3 in airway epithelial cells.</a> J. Biol. Chem. 274, 11811-11816 (1999).</li><li>Shumaker, H., Amlal, H., Frizzell, R., Ulrich, C. D., Soleimani, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CFTR+drives+Na+-nHCO-3+cotransport+in+pancreatic+duct+cells:+a+basis+for+defective+HCO-3+secretion+in+CF.">CFTR drives Na+-nHCO-3 cotransport in pancreatic duct cells: a basis for defective HCO-3 secretion in CF.</a> Am. J. Physiol. 276, 16-25 (1999).</li><li>Ahn, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulatory+interaction+between+the+cystic+fibrosis+transmembrane+conductance+regulator+and+HCO3-+salvage+mechanisms+in+model+systems+and+the+mouse+pancreatic+duct.">Regulatory interaction between the cystic fibrosis transmembrane conductance regulator and HCO3- salvage mechanisms in model systems and the mouse pancreatic duct.</a> J. Biol. Chem. 276, 17236-17243 (2001).</li><li>Sugita, M., Yue, Y., Foskett, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CFTR+Cl-+channel+and+CFTR-associated+ATP+channel:+distinct+pores+regulated+by+common+gates.">CFTR Cl- channel and CFTR-associated ATP channel: distinct pores regulated by common gates.</a> Embo. J. 17, 898-908 (1998).</li><li>Naren, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+CFTR+chloride+channels+by+syntaxin+and+Munc18+isoforms.">Regulation of CFTR chloride channels by syntaxin and Munc18 isoforms.</a> Nature. 390, 302-305 (1997).</li><li>Naren, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Syntaxin+1A+is+expressed+in+airway+epithelial+cells,+where+it+modulates+CFTR+Cl(-)+currents.">Syntaxin 1A is expressed in airway epithelial cells, where it modulates CFTR Cl(-) currents.</a> J. Clin. Invest. 105, 377-386 (2000).</li><li>Naren, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+macromolecular+complex+of+beta+2+adrenergic+receptor,+CFTR,+and+ezrin/radixin/moesin-binding+phosphoprotein+50+is+regulated+by+PKA.">A macromolecular complex of beta 2 adrenergic receptor, CFTR, and ezrin/radixin/moesin-binding phosphoprotein 50 is regulated by PKA.</a> Proc. Natl. Acad. Sci. USA. 100, 342-346 (1073).</li><li>Li, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lysophosphatidic+acid+inhibits+cholera+toxin-induced+secretory+diarrhea+through+CFTR-dependent+protein+interactions.">Lysophosphatidic acid inhibits cholera toxin-induced secretory diarrhea through CFTR-dependent protein interactions.</a> J. Exp. Med. 202, 975-986 (2005).</li><li>Li, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatiotemporal+coupling+of+cAMP+transporter+to+CFTR+chloride+channel+function+in+the+gut+epithelia.">Spatiotemporal coupling of cAMP transporter to CFTR chloride channel function in the gut epithelia.</a> Cell. 131, 940-951 (2007).</li><li>Li, C., Schuetz, J. D., Naren, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tobacco+carcinogen+NNK+transporter+MRP2+regulates+CFTR+function+in+lung+epithelia:+implications+for+lung+cancer.">Tobacco carcinogen NNK transporter MRP2 regulates CFTR function in lung epithelia: implications for lung cancer.</a> Cancer Lett. 292, 246-253 (2010).</li><li>Hall, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+C-terminal+motif+found+in+the+beta2-adrenergic+receptor,+P2Y1+receptor+and+cystic+fibrosis+transmembrane+conductance+regulator+determines+binding+to+the+Na+/H++exchanger+regulatory+factor+family+of+PDZ+proteins.">A C-terminal motif found in the beta2-adrenergic receptor, P2Y1 receptor and cystic fibrosis transmembrane conductance regulator determines binding to the Na+/H+ exchanger regulatory factor family of PDZ proteins.</a> Proc. Natl. Acad. Sci. U.S.A. 95, 8496-8501 (1998).</li><li>Short, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+apical+PDZ+protein+anchors+the+cystic+fibrosis+transmembrane+conductance+regulator+to+the+cytoskeleton.">An apical PDZ protein anchors the cystic fibrosis transmembrane conductance regulator to the cytoskeleton.</a> J. Biol. Chem. 273, 19797-19801 (1998).</li><li>Wang, S., Yue, H., Derin, R. B., Guggino, W. B., Li, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accessory+protein+facilitated+CFTR-CFTR+interaction,+a+molecular+mechanism+to+potentiate+the+chloride+channel+activity.">Accessory protein facilitated CFTR-CFTR interaction, a molecular mechanism to potentiate the chloride channel activity.</a> Cell. 103, 169-179 (2000).</li><li>Sun, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=E3KARP+mediates+the+association+of+ezrin+and+protein+kinase+A+with+the+cystic+fibrosis+transmembrane+conductance+regulator+in+airway+cells.">E3KARP mediates the association of ezrin and protein kinase A with the cystic fibrosis transmembrane conductance regulator in airway cells.</a> J. Biol. Chem. 275, 29539-29546 (2000).</li><li>Cheng, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Golgi-associated+PDZ+domain+protein+modulates+cystic+fibrosis+transmembrane+regulator+plasma+membrane+expression.">A Golgi-associated PDZ domain protein modulates cystic fibrosis transmembrane regulator plasma membrane expression.</a> J. Biol. Chem. 277, 3520-3529 (1074).</li><li>Scott, R. O., Thelin, W. R., Milgram, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+PDZ+protein+regulates+the+activity+of+guanylyl+cyclase+C,+the+heat-stable+enterotoxin+receptor.">A novel PDZ protein regulates the activity of guanylyl cyclase C, the heat-stable enterotoxin receptor.</a> The Journal of biological chemistry. 277, 22934-22941 (1074).</li><li>Lee, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+regulation+of+cystic+fibrosis+transmembrane+conductance+regulator+by+competitive+interactions+of+molecular+adaptors.">Dynamic regulation of cystic fibrosis transmembrane conductance regulator by competitive interactions of molecular adaptors.</a> The Journal of biological chemistry. 282, 10414-10422 (2007).</li><li>Gee, H. Y., Noh, S. H., Tang, B. L., Kim, K. H., Lee, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rescue+of+DeltaF508-CFTR+trafficking+via+a+GRASP-dependent+unconventional+secretion+pathway.">Rescue of DeltaF508-CFTR trafficking via a GRASP-dependent unconventional secretion pathway.</a> Cell. 146, 746-760 (2011).</li><li>Naren, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+the+study+of+intermolecular+and+intramolecular+interactions+regulating+CFTR+function.">Methods for the study of intermolecular and intramolecular interactions regulating CFTR function.</a> Met. Molecul. Med. 70, 175-186 (2002).</li><li>Li, C., Roy, K., Dandridge, K., Naren, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+assembly+of+cystic+fibrosis+transmembrane+conductance+regulator+in+plasma+membrane.">Molecular assembly of cystic fibrosis transmembrane conductance regulator in plasma membrane.</a> The Journal of biological chemistry. 279, 24673-24684 (2004).</li><li>Li, C., Naren, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+CFTR+Interactome+in+the+Macromolecular+Complexes.">Analysis of CFTR Interactome in the Macromolecular Complexes.</a> Met. Molecul. Med. 741, 255-270 (2011).</li><li>Wu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chemokine+receptor+CXCR2+macromolecular+complex+regulates+neutrophil+functions+in+inflammatory+diseases.">A chemokine receptor CXCR2 macromolecular complex regulates neutrophil functions in inflammatory diseases.</a> J. Biol. Chem. , (2011).</li></ol>

In questo protocollo abbiamo dimostrato un metodo in vitro per l&#39;assemblaggio e il rilevamento di una CFTR contenente complesso macromolecolare segnalazione utilizzando proteine ​​purificate (o frammenti di proteine) e / o lisati di cellule come riportato precedentemente 16-19,29,30. Per ottenere i migliori risultati i seguenti punti critici durante il processo di preparazione richiedono una particolare attenzione: <ul><li> È importante che il pH del tampone di eluizione essere regolata ...

<table border="1"><tbody><tr><td> Nome del reattivo </td><td> Azienda </td><td> Numero di catalogo </td><td> Comments </td></tr><tr><td> pGEX4T-1 vettore </td><td> GE Healthcare </td><td> 28-9545-49 </td><td> già Amersham Biosciences </td></tr><tr><td> pMAL-C2 vettoriale </td><td> New England BioLabs </td><td></td><td></td></tr><tr><td> pET30 vettore </td><td> EMD Chemicals </td><td> 69077-3 </td><td> ex Novagen </td></tr><tr><td> Glutatione agarosio perline </td><td> BD Biosciences </td><td> 554780 </td><td></td></tr><tr><td> Amilosio resina </td><td> New England BioLabs </td><td> E8021S </td><td></td></tr><tr><td> Talon perline </td><td> Clontech </td><td> 635501 </td><td></td></tr><tr><td> glutatione ridotto </td><td> BD Biosciences </td><td> 554782 </td><td></td&gt; </tr><tr><td> imidazolo </td><td> Pescatore </td><td> BP305-50 </td><td></td></tr><tr><td> maltosio </td><td> Pescatore </td><td> BP684-500 </td><td></td></tr><tr><td> S-proteina agarosio </td><td> EMD Chemicals </td><td> 69704-3 </td><td> ex Novagen </td></tr><tr><td> Anti-Flag HRP </td><td> Sigma </td><td> A8592 </td><td></td></tr><tr><td> Anti-IgG CFTR </td><td> Su misura </td><td> R1104 </td><td> mAb riconoscendo CFTR epitopo al aa 722-734 </td></tr><tr><td> Anti-IgG MRP2 </td><td> Chemicon International </td><td> MAB4148 </td><td> Ora una parte di Millipore </td></tr></tbody></table> Tabella 2. Reagenti e attrezzature specifiche.

in vitro analysis of pdz-dependent cftr macromolecular signaling complexes

Transmembrana della fibrosi cistica regolatore della conduttanza (CFTR), un canale del cloro si trova soprattutto a livello delle membrane apicali delle cellule epiteliali, gioca un ruolo cruciale nella omeostasi del liquido transepiteliale 1-3. CFTR è stata implicata in due importanti malattie: fibrosi cistica (CF) 4 e 5 secretoria diarrea. In CF, la sintesi o attività funzionale della CFTR Cl-canale è ridotta. Questo disturbo colpisce circa 1 su 2500 caucasici negli Stati Uniti 6. Attività eccessiva CFTR è stata anche implicata in casi di tossina indotta diarrea secretoria (ad esempio, da tossina del colera e stabile al calore enterotossina E. coli) che stimola la produzione di cAMP o cGMP nell&#39;intestino 7. Accumulare le prove suggeriscono l&#39;esistenza di interazioni fisiche e funzionali tra CFTR e un numero crescente di altre proteine, tra trasportatori, canali ionici, recettori, chinasi, fosfatasi, segnalemolecole zione ed elementi del citoscheletro, e queste interazioni tra CFTR e le sue proteine ​​leganti hanno dimostrato di essere criticamente coinvolte nella regolazione CFTR trasporto mediato transepiteliale ionico in vitro e in vivo 8-19. In questo protocollo, ci concentriamo solo sui metodi che gli aiuti nello studio delle interazioni tra CFTR carbossile terminale della coda, che possiede una proteina di legame motivo [denominato PSD95/Dlg1/ZO-1 (PDZ) motivo], e un gruppo di proteine ​​scaffold, che contengono un modulo di legame specifico denominato domini PDZ. Finora, molte diverse proteine ​​PDZ ponteggi sono stati riportati da associare alla coda terminale carbossile di CFTR con affinità diverse, ad esempio, i NHERF1 NHERF2 e PDZK1 e PDZK2 e CAL (CFTR-associata ligando), Shank2, e GRASP 20-27. Il motivo PDZ all&#39;interno CFTR che è riconosciuto da PDZ proteine ​​scaffold è ultimi quattro amminoacidi al C terminale (cioè, 1477-DTRL-1480 in umano CFTR) 20. Interessante,CFTR può legare più di un dominio PDZ sia NHERFs e PDZK1, pur in diversi affinità 22. Questa polivalenza rispetto CFTR legame ha dimostrato di essere di importanza funzionale, suggerendo che le proteine ​​PDZ impalcatura può facilitare la formazione di complessi macromolecolari CFTR segnalazione per la segnalazione specifici / selettivo ed efficace in cellule 16-18. Più saggi biochimici sono stati sviluppati per studiare CFTR, che coinvolge interazioni proteiche, come la co-immunoprecipitazione, pull-down assay, pair-wise saggio di legame, colorimetrico pair-wise saggio di legame, e il dosaggio di assemblaggio macromolecolare complessa 16-19,28,29 . Qui ci concentriamo sulle procedure dettagliate di assemblaggio di un motivo PDZ CFTR-dipendente contenenti macromolecole complesse in vitro, che è ampiamente utilizzato dal nostro laboratorio per studiare proteina-proteina o di dominio nel dominio delle interazioni che coinvolgono CFTR 16-19,28,29.

Watch this Scientific Journal Video about In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes at JoVE.com

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

Transmembrana della fibrosi cistica regolatore conduttanza (CFTR), un canale cloruro epiteliale, è stata riportata per interagire con varie proteine ​​e regolano importanti processi cellulari, tra i quali i CFTR PDZ motivo mediate da interazioni sono stati ben documentati. Questo protocollo descrive i metodi che abbiamo sviluppato per assemblare un PDZ-dipendente macromolecolare CFTR segnalazione complessa<em&gt; In vitro</em&gt;.

L&#39;analisi in vitro dei complessi CFTR PDZ-dipendenti macromolecolari di segnalazione

Cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel located primarily at the apical membranes of epithelial cells, plays a ...

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In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

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