In vitro-analyse af PDZ-afhængige CFTR Makromolekylære signalering Komplekser
Summary
Cystisk fibrose transmembran konduktans regulator (CFTR), en epitelcelle chlorid-kanal, er blevet rapporteret at interagere med forskellige proteiner og regulere vigtige cellulære processer, deriblandt CFTR PDZ motiv-medierede interaktioner er blevet godt dokumenteret. Denne protokol beskriver de metoder vi har udviklet til at samle en PDZ-afhængig CFTR makromolekylære signalering kompleks<em> In vitro</em>.
Abstract
Cystisk fibrose transmembran konduktans regulator (CFTR), en chlorid-kanal er placeret hovedsageligt på de apikale membraner i epitelceller, spiller en afgørende rolle i transepithelial fluid homøostase 1-3. CFTR har været impliceret i to vigtige sygdomme: cystisk fibrose (CF) 4 og sekretorisk diarré 5. I CF, er syntesen eller funktionelle aktivitet af CFTR Cl-kanal reduceret. Denne lidelse påvirker cirka 1 ud af 2.500 kaukasiere i USA 6. Overdreven CFTR-aktivitet har også været impliceret i tilfælde af toksin-induceret sekretorisk diarré (fx ved koleratoxin og varmestabil E. coli enterotoxin), der stimulerer cAMP eller cGMP-produktion i tarmen 7.
Akkumulerende beviser antyder eksistensen af fysiske og funktionelle interaktioner mellem CFTR og et stigende antal af andre proteiner, herunder transportører, ionkanaler, receptorer, kinaser, phosphataser, signalIng molekyler og cytoskeletale elementer, og disse interaktioner mellem CFTR og dets bindende proteiner har vist sig at være kritisk involveret i reguleringen af CFTR-medieret transepithelial iontransport in vitro og også de vivo 8-19. I denne protokol, fokuserer vi kun på de metoder, som støtte i studiet af samspillet mellem CFTR carboxylterminal hale, som besidder et protein-bindende motiv [benævnt PSD95/Dlg1/ZO-1 (PDZ) motiv], og en gruppe stillads proteiner, som indeholder en specifik bindingspartner modul benævnt PDZ-domæner. Hidtil har flere forskellige PDZ stilladsdele proteiner blevet rapporteret at binde til den carboxylterminale hale af CFTR med forskellige affiniteter, såsom NHERF1 og NHERF2 og PDZK1 og PDZK2 og CAL (CFTR-associeret ligand), Shank2, og gribe 20-27. PDZ motiv i CFTR, der genkendes af PDZ skelettet proteiner er de sidste fire aminosyrer ved C-terminus (dvs. 1477-DTRL-1480 i human CFTR) 20. InteressantCFTR kan binde mere end en PDZ-domæne af både NHERFs og PDZK1, selv med varierende affiniteter 22. Dette multivalens med hensyn til CFTR binding har vist sig at være funktionelle betydning, hvilket antyder, at PDZ stilladsdele proteiner kan lette dannelse af CFTR makromolekylære signalering komplekser til specifikke / selektive og effektive signalering i celler 16-18.
Flere biokemiske assays er blevet udviklet til at studere CFTR-involverer protein-interaktioner, såsom co-immunopræcipitation, pull-down assay parvise bindingsassay, kolorimetrisk parvise bindingsassay, og makromolekylære komplekst assay 16-19,28,29 . Her fokuserer vi på de detaljerede procedurer for at samle et PDZ motiv-afhængig CFTR-holdige makromolekylære kompleks in vitro, som bruges i udstrakt grad af vores laboratorium til at studere protein-protein eller domæne-domæne-interaktioner, der involverer CFTR 16-19,28,29.
Protocol
Discussion
I denne protokol vi demonstreret en fremgangsmåde til in vitro-samling og påvisning af en CFTR indeholdende makromolekylært signalering kompleks under anvendelse af oprensede proteiner (eller proteinfragmenter) og / eller cellelysater, som beskrevet tidligere 16-19,29,30. For at opnå det bedste resultat de følgende kritiske punkter under fremstillingsprocessen kræver særlig opmærksomhed:
- Det er vigtigt, at pH i elueringspufferen være justeret til 8,0 efter tilsætning af reducer…
Declarações
The authors have nothing to disclose.
Acknowledgements
Vores arbejde er blevet støttet af tilskud fra American Heart Association (Greater Sydøst Affiliate) Beginning-tilskud-in-støtte 0765185B, at Elsa U. Pardee Foundation forskningsbevilling, og Wayne State University murene opstart fond og Cardiovascular Research Institute Isis Initiative pris. Denne fremgangsmåde til in vitro CFTR makromolekylært kompleks samling blev oprindeligt udviklet af Dr. AP Naren (University of Tennessee Health Science Center).
Materials
| Name of the reagent | Company | Catalog number | Comments |
| pGEX4T-1 vector | GE Healthcare | 28-9545-49 | formerly Amersham Biosciences |
| pMAL-C2 vector | New England BioLabs | ||
| pET30 vector | EMD Chemicals | 69077-3 | formerly Novagen |
| Glutathione agarose beads | BD Biosciences | 554780 | |
| Amylose resin | New England BioLabs | E8021S | |
| Talon beads | Clontech | 635501 | |
| reduced glutathione | BD Biosciences | 554782 | |
| imidazole | Fisher | BP305-50 | |
| maltose | Fisher | BP684-500 | |
| S-protein agarose | EMD Chemicals | 69704-3 | formerly Novagen |
| Anti-Flag HRP | Sigma | A8592 | |
| Anti-CFTR IgG | Custom-made | R1104 | mAb recognizing CFTR epitope at a.a. 722-734 |
| Anti-MRP2 IgG | Chemicon International | MAB4148 | Now a part of Millipore |
Table 2. Specific reagents and equipment.
Referências
- Anderson, M. P. Demonstration that CFTR is a chloride channel by alteration of its anion selectivity. Science. 253, 202-205 (1991).
- Bear, C. E. Purification and functional reconstitution of the cystic fibrosis transmembrane conductance regulator (CFTR. Cell. 68, 809-818 (1992).
- Quinton, P. M. Chloride impermeability in cystic fibrosis. Nature. 301, 421-422 (1983).
- Cheng, S. H. Defective intracellular transport and processing of CFTR is the molecular basis of most cystic fibrosis. Cell. 63, 827-834 (1990).
- Gabriel, S. E., Brigman, K. N., Koller, B. H., Boucher, R. C., Stutts, M. J. Cystic fibrosis heterozygote resistance to cholera toxin in the cystic fibrosis mouse model. Science. 266, 107-109 (1994).
- Li, C., Naren, A. P. CFTR chloride channel in the apical compartments: spatiotemporal coupling to its interacting partners. Integr. Biol (Camb). 2, 161-177 (2010).
- Chao, A. C. Activation of intestinal CFTR Cl- channel by heat-stable enterotoxin and guanylin via cAMP-dependent protein kinase. Embo. J. 13, 1065-1072 (1994).
- Gabriel, S. E., Clarke, L. L., Boucher, R. C., Stutts, M. J. CFTR and outward rectifying chloride channels are distinct proteins with a regulatory relationship. Nature. 363, 263-268 (1993).
- McNicholas, C. M. 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. Proc. Natl. Acad. Sci. USA. 93, 8083-8088 (1996).
- Schreiber, R., Nitschke, R., Greger, R., Kunzelmann, K. The cystic fibrosis transmembrane conductance regulator activates aquaporin 3 in airway epithelial cells. J. Biol. Chem. 274, 11811-11816 (1999).
- Shumaker, H., Amlal, H., Frizzell, R., Ulrich, C. D., Soleimani, M. CFTR drives Na+-nHCO-3 cotransport in pancreatic duct cells: a basis for defective HCO-3 secretion in CF. Am. J. Physiol. 276, 16-25 (1999).
- Ahn, W. Regulatory interaction between the cystic fibrosis transmembrane conductance regulator and HCO3- salvage mechanisms in model systems and the mouse pancreatic duct. J. Biol. Chem. 276, 17236-17243 (2001).
- Sugita, M., Yue, Y., Foskett, J. K. CFTR Cl- channel and CFTR-associated ATP channel: distinct pores regulated by common gates. Embo. J. 17, 898-908 (1998).
- Naren, A. P. Regulation of CFTR chloride channels by syntaxin and Munc18 isoforms. Nature. 390, 302-305 (1997).
- Naren, A. P. Syntaxin 1A is expressed in airway epithelial cells, where it modulates CFTR Cl(-) currents. J. Clin. Invest. 105, 377-386 (2000).
- Naren, A. P. A macromolecular complex of beta 2 adrenergic receptor, CFTR, and ezrin/radixin/moesin-binding phosphoprotein 50 is regulated by PKA. Proc. Natl. Acad. Sci. USA. 100, 342-346 (1073).
- Li, C. Lysophosphatidic acid inhibits cholera toxin-induced secretory diarrhea through CFTR-dependent protein interactions. J. Exp. Med. 202, 975-986 (2005).
- Li, C. Spatiotemporal coupling of cAMP transporter to CFTR chloride channel function in the gut epithelia. Cell. 131, 940-951 (2007).
- Li, C., Schuetz, J. D., Naren, A. P. Tobacco carcinogen NNK transporter MRP2 regulates CFTR function in lung epithelia: implications for lung cancer. Cancer Lett. 292, 246-253 (2010).
- Hall, R. A. 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. Proc. Natl. Acad. Sci. U.S.A. 95, 8496-8501 (1998).
- Short, D. B. An apical PDZ protein anchors the cystic fibrosis transmembrane conductance regulator to the cytoskeleton. J. Biol. Chem. 273, 19797-19801 (1998).
- Wang, S., Yue, H., Derin, R. B., Guggino, W. B., Li, M. Accessory protein facilitated CFTR-CFTR interaction, a molecular mechanism to potentiate the chloride channel activity. Cell. 103, 169-179 (2000).
- Sun, F. E3KARP mediates the association of ezrin and protein kinase A with the cystic fibrosis transmembrane conductance regulator in airway cells. J. Biol. Chem. 275, 29539-29546 (2000).
- Cheng, J. A Golgi-associated PDZ domain protein modulates cystic fibrosis transmembrane regulator plasma membrane expression. J. Biol. Chem. 277, 3520-3529 (1074).
- Scott, R. O., Thelin, W. R., Milgram, S. L. A novel PDZ protein regulates the activity of guanylyl cyclase C, the heat-stable enterotoxin receptor. The Journal of biological chemistry. 277, 22934-22941 (1074).
- Lee, J. H. Dynamic regulation of cystic fibrosis transmembrane conductance regulator by competitive interactions of molecular adaptors. The Journal of biological chemistry. 282, 10414-10422 (2007).
- Gee, H. Y., Noh, S. H., Tang, B. L., Kim, K. H., Lee, M. G. Rescue of DeltaF508-CFTR trafficking via a GRASP-dependent unconventional secretion pathway. Cell. 146, 746-760 (2011).
- Naren, A. P. Methods for the study of intermolecular and intramolecular interactions regulating CFTR function. Met. Molecul. Med. 70, 175-186 (2002).
- Li, C., Roy, K., Dandridge, K., Naren, A. P. Molecular assembly of cystic fibrosis transmembrane conductance regulator in plasma membrane. The Journal of biological chemistry. 279, 24673-24684 (2004).
- Li, C., Naren, A. P. Analysis of CFTR Interactome in the Macromolecular Complexes. Met. Molecul. Med. 741, 255-270 (2011).
- Wu, Y. A chemokine receptor CXCR2 macromolecular complex regulates neutrophil functions in inflammatory diseases. J. Biol. Chem. , (2011).
