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

Vårt arbeid har blitt støttet med tilskudd fra American Heart Association (Greater Sørøst Affiliate) Beginning-stipend-i-hjelp 0765185B, den Elsa U. Pardee Foundation forskningsstipend, og Wayne State University egenutført oppstart fond og Cardiovascular Research Institute Isis Initiative prisen. Denne metoden for in vitro CFTR macromolecular kompleks forsamlingen ble opprinnelig utviklet av Dr. AP Naren (University of Tennessee Health Science Center).

1. Uttrykk og rensing av rekombinant tagget Fusion Proteiner i Bakterier <ol><li> Forsterke definerte regioner i C-haler (de siste 50-100 aminosyrene som inneholder PDZ motivene ved C-terminus) for CFTR, 2 år 2. LPA, MRP2, MRP4, β, og NHERFs (full-lengde eller PDZ1 eller PDZ2 domener ) ved PCR tilnærming. </li><li> Klone PCR produktene til pGEX4T-en vektor for GST-fusion proteiner (for eksempel GST-NHERFs, GST-MRP4 CT), pMAL-C2 vektor for MBP-fusion proteiner (som MBP-β 2 AR CT, MB...

<ol>
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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. 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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. 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I denne protokollen viste vi en metode for in vitro montering og påvisning av en CFTR inneholder macromolecular signalisere kompleks med rensede proteiner (eller protein fragmenter) og / eller celle lysates som rapportert tidligere 16-19,29,30. For å oppnå best mulig resultat følgende kritiske punkter under forberedelsen prosessen krever spesiell oppmerksomhet: <ul><li> Det er viktig at pH i eluering buffer bli justert til 8,0 etter at redusert glutation når rense GST-fusion protein som besk...

<table border="1"><tbody><tr><td> Navn på reagensen </td><td> Firma </td><td> Katalognummer </td><td> Kommentarer </td></tr><tr><td> pGEX4T-1 vektor </td><td> GE Healthcare </td><td> 28-9545-49 </td><td> tidligere Amersham Biosciences </td></tr><tr><td> pMAL-C2 vektor </td><td> New England BioLabs </td><td></td><td></td></tr><tr><td> pET30 vektor </td><td> EMD Kjemikalier </td><td> 69077-3 </td><td> tidligere Novagen </td></tr><tr><td> Glutation agarose perler </td><td> BD Biosciences </td><td> 554780 </td><td></td></tr><tr><td> Amylose harpiks </td><td> New England BioLabs </td><td> E8021S </td><td></td></tr><tr><td> Talon perler </td><td> Clontech </td><td> 635501 </td><td></td></tr><tr><td> redusert glutation </td><td> BD Biosciences </td><td> 554782 </td><td></td&gt; </tr><tr><td> imidazole </td><td> Fisher </td><td> BP305-50 </td><td></td></tr><tr><td> maltose </td><td> Fisher </td><td> BP684-500 </td><td></td></tr><tr><td> S-protein agarose </td><td> EMD Kjemikalier </td><td> 69704-3 </td><td> tidligere Novagen </td></tr><tr><td> Anti-Flag HRP </td><td> Sigma </td><td> A8592 </td><td></td></tr><tr><td> Anti-CFTR IgG </td><td> Custom-made </td><td> R1104 </td><td> MAB erkjenner CFTR epitop på aa 722-734 </td></tr><tr><td> Anti-MRP2 IgG </td><td> Chemicon International </td><td> MAB4148 </td><td> Nå en del av Millipore </td></tr></tbody></table> Tabell 2. Spesifikke reagenser og utstyr.

in vitro analysis of pdz-dependent cftr macromolecular signaling complexes

Cystisk fibrose transmembrane konduktans regulator (CFTR), en klorid kanal ligger hovedsakelig i den apikale membraner i epitelceller, spiller en avgjørende rolle i transepithelial væske homeostase 1-3. CFTR har vært innblandet i to alvorlige sykdommer: cystisk fibrose (CF) 4 og sekretoriske diaré fem. I CF, er syntesen eller funksjonell aktivitet av CFTR Cl-kanalen redusert. Denne lidelsen påvirker ca 1 i 2500 kaukasiere i USA seks. Overdreven CFTR aktivitet har også vært innblandet i tilfeller av toksin-indusert sekretorisk diaré (for eksempel ved koleratoksin og varme stabil E. coli enterotoksin) som stimulerer cAMP eller cGMP produksjon i tarmen 7. Innsamling av bevismateriale tyder eksistensen av fysiske og funksjonelle samspillet mellom CFTR og et økende antall andre proteiner, inkludert transport, ionekanaler, reseptorer, kinaser, fosfataser, signaling molekyler, og cytoskeletal elementer, og disse interaksjoner mellom CFTR og dets proteiner har vist seg å være kritisk involvert i regulering av CFTR-mediert transepithelial ion transport in vitro og in vivo også 8-19. I denne protokollen, fokuserer vi kun på de metoder som hjelpemiddel i studiet av samspillet mellom CFTR carboxyl terminal hale, som besitter en protein-bindende motiv [referert til som PSD95/Dlg1/ZO-1 (PDZ) motiv], og en gruppe av stillas proteiner, som inneholder en spesifikk binding modul kalt PDZ domener. Så langt har flere forskjellige PDZ stillaset proteiner er rapportert å binde til carboxyl terminal hale av CFTR med ulike slektskap, som NHERF1 og NHERF2 og PDZK1 og PDZK2, Cal (CFTR-assosiert ligand), Shank2, og ta tak 20-27. Den PDZ motiv innenfor CFTR som er anerkjent av PDZ stillaset proteiner er de siste fire aminosyrene på C endestasjonen (dvs. 1477-DTRL-1480 i human CFTR) 20. Interessant,CFTR kan binde mer enn ett PDZ domene av både NHERFs og PDZK1, dog med varierende slektskap 22. Dette multivalency med hensyn til CFTR bindende har vist seg å være av funksjonell betydning, noe som tyder på at PDZ stillaset proteiner kan fremme dannelsen av CFTR macromolecular signalisere komplekser for spesifikke / selektiv og effektiv signalering i cellene 16-18. Flere biokjemiske analyser har blitt utviklet for å studere CFTR-involverer protein interaksjoner, for eksempel co-immunoprecipitation, pull-down analysen, par-klok bindende analysen, kolorimetrisk par-klok bindende analysen, og macromolecular kompleks montering analysen 16-19,28,29 . Her fokuserer vi på de detaljerte prosedyrene og sammenstille et PDZ motiv-avhengig CFTR-holdig macromolecular kompleks in vitro, som brukes mye av vårt laboratorium for å studere protein-protein eller domene-domene interaksjoner som involverer 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

Cystisk fibrose transmembrane konduktans regulator (CFTR), en epitelial klorid kanal, har blitt rapportert å samhandle med ulike proteiner og regulere viktige cellulære prosesser; blant dem CFTR PDZ motiv-medierte interaksjoner er godt dokumentert. Denne protokollen beskriver metoder vi utviklet for å montere en PDZ-avhengig CFTR macromolecular signaliserte kompleks<em&gt; In vitro</em&gt;.

In Vitro Analyse av PDZ-avhengige CFTR makromolekylær signalveier Komplekser

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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