सिस्टिक फाइब्रोसिस transmembrane प्रवाहकत्त्व नियामक (CFTR), एक उपकला क्लोराइड चैनल, विभिन्न प्रोटीन के साथ बातचीत और महत्वपूर्ण सेलुलर प्रक्रियाओं को विनियमित करने के लिए सूचित किया गया है, उन के बीच में CFTR PDZ आकृति की मध्यस्थता बातचीत अच्छी तरह से प्रलेखित किया गया है. इस प्रोटोकॉल हम CFTR PDZ पर निर्भर जटिल संकेत macromolecular इकट्ठा विकसित तरीकों का वर्णन<em> इन विट्रो में</em>.
Cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel located primarily at the apical membranes of epithelial cells, plays a crucial role in transepithelial fluid homeostasis1-3. CFTR has been implicated in two major diseases: cystic fibrosis (CF)4 and secretory diarrhea5. In CF, the synthesis or functional activity of the CFTR Cl- channel is reduced. This disorder affects approximately 1 in 2,500 Caucasians in the United States6. Excessive CFTR activity has also been implicated in cases of toxin-induced secretory diarrhea (e.g., by cholera toxin and heat stable E. coli enterotoxin) that stimulates cAMP or cGMP production in the gut7.
Accumulating evidence suggest the existence of physical and functional interactions between CFTR and a growing number of other proteins, including transporters, ion channels, receptors, kinases, phosphatases, signaling molecules, and cytoskeletal elements, and these interactions between CFTR and its binding proteins have been shown to be critically involved in regulating CFTR-mediated transepithelial ion transport in vitro and also in vivo8-19. In this protocol, we focus only on the methods that aid in the study of the interactions between CFTR carboxyl terminal tail, which possesses a protein-binding motif [referred to as PSD95/Dlg1/ZO-1 (PDZ) motif], and a group of scaffold proteins, which contain a specific binding module referred to as PDZ domains. So far, several different PDZ scaffold proteins have been reported to bind to the carboxyl terminal tail of CFTR with various affinities, such as NHERF1, NHERF2, PDZK1, PDZK2, CAL (CFTR-associated ligand), Shank2, and GRASP20-27. The PDZ motif within CFTR that is recognized by PDZ scaffold proteins is the last four amino acids at the C terminus (i.e., 1477-DTRL-1480 in human CFTR)20. Interestingly, CFTR can bind more than one PDZ domain of both NHERFs and PDZK1, albeit with varying affinities22. This multivalency with respect to CFTR binding has been shown to be of functional significance, suggesting that PDZ scaffold proteins may facilitate formation of CFTR macromolecular signaling complexes for specific/selective and efficient signaling in cells16-18.
Multiple biochemical assays have been developed to study CFTR-involving protein interactions, such as co-immunoprecipitation, pull-down assay, pair-wise binding assay, colorimetric pair-wise binding assay, and macromolecular complex assembly assay16-19,28,29. Here we focus on the detailed procedures of assembling a PDZ motif-dependent CFTR-containing macromolecular complex in vitro, which is used extensively by our laboratory to study protein-protein or domain-domain interactions involving CFTR16-19,28,29.
इस प्रोटोकॉल में हम में इन विट्रो कोडांतरण और एक macromolecular संकेत जटिल शुद्ध प्रोटीन (या प्रोटीन टुकड़े) और / या सेल lysates का उपयोग युक्त CFTR का पता लगाने के लिए एक विधि का प्रदर्शन के रूप में 16-19,29,30 पहले की रि?…
The authors have nothing to disclose.
हमारा काम अमेरिकन हार्ट एसोसिएशन (ग्रेटर दक्षिण पूर्व संबद्ध) शुरुआत अनुदान सहायता 0765185B, Elsa Pardee अमेरिकी फाउंडेशन के अनुसंधान अनुदान, और वेन राज्य विश्वविद्यालय अंदर स्टार्टअप निधि और हृदय अनुसंधान संस्थान Isis पहल पुरस्कार से अनुदान द्वारा समर्थित किया गया है. इन विट्रो CFTR macromolecular जटिल विधानसभा में यह विधि मूल रूप से डा. एपी नरेन (टेनेसी स्वास्थ्य विज्ञान केंद्र के विश्वविद्यालय) द्वारा बीड़ा उठाया है.
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.