Cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial chloride channel, has been reported to interact with various proteins and regulate important cellular processes; among them the CFTR PDZ motif-mediated interactions have been well documented. This protocol describes methods we developed to assemble a PDZ-dependent CFTR macromolecular signaling complex in vitro.
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.
1. Expression and Purification of Recombinant Tagged Fusion Proteins in Bacteria
2. Cell Culture and Cell Lysate Preparation
3. In Vitro Assembly of a CFTR-containing Macromolecular Complex (CFTR-PDZK1-MRP4)
4. Representative Results
An example of CFTR-containing macromolecular signaling complex that was assembled in vitro is shown in Figure 1. A macromolecular complex was formed between MRP4 C-terminal 50 amino acids (MRP4-CT50), PDZK1, and full-length CFTR (Figure 1, bottom). The complex formation increased dose-dependently with increasing amounts of the intermediary protein, PDZK1 (Figure 1, bottom)18.
Figure 1. A pictorial representation of the macromolecular complex assay (top). A macromolecular complex was detected in vitro with three proteins (GST-MRP4-CT50, His-S-PDZK1, and Flag-CFTRwt) in a dose-dependent manner (bottom)18.
CFTR-NHERF1-β2AR (ref.14) | CFTR-NHERF2-LPA2 (ref. 15) | CFTR-PDZ proteins-MRP2 (ref. 17) | CFTR-PDZK1-MRP4 (ref. 16) | |
Affinity beads | Amylose resin | S-protein agarose | Amylose resin | Glutathione agarose |
Purified protein-1 | MBP-β2AR CT | His-S-CFTR CT | MBP-CFTR CT | GST-MRP4 CT |
Purified protein-2 | GST-NHERF1 | GST-NHERF2 | GST-PDZ proteins | His-S-PDZK1 |
Purified protein-3 (or cell lysates) | CFTR-wt or CFTR-his10 (BHK or HEK cell lysates) | Flag-LPA2-wt or Flag-LPA2-ΔSTL (BHK cell lysates) | MRP2 (MDCK cell lysates) | Purified Flag-CFTR-wt or CFTR-his10 (or cell lysates) |
Antibody | Anti-CFTR IgG | Anti-Flag HRP | Anti-MRP2 IgG | Anti-Flag HRP |
Table 1. Summary of various CFTR-containing macromolecular complexes assembled in vitro.
In this protocol we demonstrated a method for in vitro assembling and detection of a CFTR containing macromolecular signaling complex using purified proteins (or protein fragments) and/or cell lysates as reported previously16-19,29,30. To achieve best results the following critical points during the preparation process require special attention:
The technique demonstrated here provides a convenient and reproducible assay to biochemically define a CFTR-containing tertiary protein complex. There are multiple ways to assemble the in vitro CFTR macromolecular complexes as outlined in Table-1.
However, detection of the CFTR macromolecular complex in vitro using purified proteins (or protein fragments) or lysates from cells that overexpress CFTR or the interacting proteins does not indicate whether the macromolecular signaling complex exists in cells that endogenously express these interacting proteins. To address this issue, co-immunoprecipitation can be performed to assess the endogenous CFTR complexes in cells such as airway epithelial cells16 and gut epithelial cells17,18. Moreover, mass spectrometry analysis and 2-dimensional gel electrophoresis29 can be performed to identify unknown binding partners for CFTR. However, it is beyond the scope of this protocol to demonstrate these techniques.
In addition, the subcellular localization of interacting proteins in vivo may also affect molecular interactions. For instance, MRP4 has been shown to be expressed in both apical and basolateral membranes, whereas CFTR is localized primarily to the apical membrane, though they have been demonstrated to interact physically and functionally with each other via the PDZ scaffold protein PDZK118. Furthermore, overexpression of the recombinant proteins, as used in the current protocol, may affect their subcellular localization and thus enable interactions that would otherwise not occur. These possibility should also be taken into consideration when interpreting the data and extrapolating the findings.
Although the current protocol focuses on the procedure to assemble PDZ-dependent CFTR-containing macromolecular complexes, this protocol also has potential applications in assembling non-CFTR involved multi-protein complex (either PDZ-dependent or independent). Most recently, using this macromolecular assembly assay, we have demonstrated that a chemokine receptor CXCR2 physically forms a protein complex with its downstream effector PLCβ2 mediated via NHERF1 in neutrophils, and this CXCR2 macromolecular complex has functional relevance as disrupting the complex (via using an exogenous CXCR2 peptide) significantly attenuated neutrophil functions31.
The authors have nothing to disclose.
Our work has been supported by grants from American Heart Association (Greater Southeast Affiliate) Beginning-grant-in-aid 0765185B, the Elsa U. Pardee Foundation research grant, and Wayne State University intramural startup fund and Cardiovascular Research Institute Isis Initiative award. This method of in vitro CFTR macromolecular complex assembly was originally pioneered by Dr. A.P. Naren (University of Tennessee Health Science Center).
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.