Analysis of CFTR interactome in the macromolecular complexes

Abstract

Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel localized primarily at the apical surface of epithelial cells lining the airway, gut, exocrine glands, etc., where it is responsible for transepithelial salt and water transport. A growing number of proteins have been reported to interact directly or indirectly with CFTR chloride channel, suggesting that CFTR might regulate the activities of other ion channels, receptors, and transporters, in addition to its role as a chloride conductor. Most interactions occur primarily between the opposing terminal tails (N or C) of CFTR and its binding partners, either directly or mediated through various PDZ domain-containing proteins. This chapter describes methods we developed to cross-link CFTR into a macromolecular complex to identify and analyze the assembly and regulation of CFTR-containing complexes in the plasma membrane.

Figure 15.1

CFTR exists as a higher order complex in plasma membrane through cross-linking. (A) Calu-3 cells were cross-linked as described in the text. The cells were lysed in RIPA buffer and blotted for CFTR (R1104). (B) Calu-3 cells cross-linked with DSP were lysed in RIPA buffer, then 50 µg of total proteins were solubilized with sample buffer in the absence (left) or presence (right) of 2.5% β-mercaptoethanol (βME) before immunoblotting for CFTR (R1104). (Reproduced from ref. with permission from American Society for Biochemistry and Molecular Biology.)

Figure 15.2

Stoichiometry of CFTR:NHERF1 in the complex. (A) Polarized Calu-3 cells were cross-linked with 1 mM DSP and then apically biotinylated with sulfo-NHS-LC-biotin. The plasma membrane was isolated, and the apical plasma membrane was captured using streptavidin-agarose beads and then subjected to co-immunoprecipitation using anti-NBD-R IgG. The immunoprecipitated complex was treated with 2.5% βME to dissociate the cross-linked complex and dissolved on 10% SDS-PAGE. Purified CFTR_his10 was used as a standard to quantitate CFTR (upper panel), and GST-PDZ2 of NHERF1 was used as a standard to quantitate the co-immunoprecipitated NHERF1 (bottom panel). (B) Calu-3 cells were cross-linked with 1 mM DSP, lysed in RIPA buffer, and subjected to co-immunoprecipitation using mouse anti-CFTR antibody (MM13-4; bottom panel) or normal mouse IgG (upper panel). The samples were eluted and subjected to 2-D gel electrophoresis (Bio-Rad). IEF (pI: 3–10) was performed in the 1^st dimension, and SDS-PAGE was performed in a 10% polyacrylamide gel in the 2^nd dimension and blotted with affinity-purified rabbit G_β IgG. (Reproduced from ref. with permission from American Society for Biochemistry and Molecular Biology.)

Figure 15.3

LPA₂ forms a macromolecular complex with CFTR mediated through NHERF2. (A) A pictorial representation of the macromolecular complex assay. (B) A macromolecular complex of MBP-C-CFTR, GST-NHERF2, and Flag-tagged LPA₂ that is assembled in vitro. (Reproduced from ref. with permission from Rockefeller University Press.)