Cystic Fibrosis Transmembrane Conductance Regulator Interacts with Multiple Immunoglobulin Domains of Filamin A

Abstract

Mutations of the chloride channel cystic fibrosis transmembrane conductance regulator (CFTR) that impair its apical localization and function cause cystic fibrosis. A previous report has shown that filamin A (FLNa), an actin-cross-linking and -scaffolding protein, interacts directly with the cytoplasmic N terminus of CFTR and that this interaction is necessary for stability and confinement of the channel to apical membranes. Here, we report that the CFTR N terminus has sequence similarity to known FLNa-binding partner-binding sites. FLNa has 24 Ig (IgFLNa) repeats, and a CFTR peptide pulled down repeats 9, 12, 17, 19, 21, and 23, which share sequence similarity yet differ from the other FLNa Ig domains. Using known structures of IgFLNa⅐partner complexes as templates, we generated in silico models of IgFLNa⅐CFTR peptide complexes. Point and deletion mutants of IgFLNa and CFTR informed by the models, including disease-causing mutations L15P and W19C, disrupted the binding interaction. The model predicted that a P5L CFTR mutation should not affect binding, but a synthetic P5L mutant peptide had reduced solubility, suggesting a different disease-causing mechanism. Taken together with the fact that FLNa dimers are elongated (ϳ160 nm) strands, whereas CFTR is compact (6ϳ8 nm), we propose that a single FLNa molecule can scaffold multiple CFTR partners. Unlike previously defined dimeric FLNa⅐partner complexes, the FLNamonomeric CFTR interaction is relatively weak, presumptively facilitating dynamic clustering of CFTR at cell membranes. Finally, we show that deletion of all CFTR interacting domains from FLNa suppresses the surface expression of CFTR on baby hamster kidney cells.

A Novel Structural Unit in the N-terminal Region of Filamins

“…Taken together, these results demonstrate that the CD face of domain 4 is the preferred binding site for ␤-strand forming peptides within the module of FLN domains 3-5 and that this site is accessible for interactions in this module. Functionally, our findings are in consensus with the hypothesis that there are multiple domains in FLNs to which the same ligand binds (18,19,61). In the case of FLNa, this finding is relevant as the interaction between FLNa and GPIb is essential for platelet activation (11,65).…”

“…In this mechanism, the interaction partner forms an additional ␤-strand next to the strand C of the FLN domain and simultaneously interacts with the hydrophobic groove between the strands C and D, called the CD face (11)(12)(13)(14). It is using this mode that ligands like transmembrane receptors of the integrin family (12,15,16), the platelet von Willebrand factor receptor subunit glycoprotein Ib (GPIb) (11), and the cystic fibrosis transmembrane conductance regulator (17,18) interact with FLNa domains 17,19,21, and 23. Also, signaling protein FilGAP uses the same mechanism for interacting with FLNa domain 23 (4) and migfilin with FLNa domain 21 (13,14).…”

The filamins

“…this review focuses on the structure and functions of Flna in cell migration and adhesion. [16][17][18][19][20][21][22][23] and the self-association domain (repeat 24). 4,7 FLN repeats are Ig-like (IgFLN), 8 each a β-barrel structure assembled from seven runs of (A~G) β-strands.…”

Congenital short bowel syndrome as the presenting symptom in male patients with FLNA mutations

“…It is known that FLNA interacts with other transmembrane proteins such as integrin beta and the cystic fibrosis transmembrane conductance regulator. 20 There is also supportive evidence that FLNA plays a role in anchoring transmembrane proteins in the cell membrane. It has been shown, e.g., that for the expression of cystic fibrosis transmembrane conductance regulator on the cell membrane, its interaction with FLNA is important.…”

“…It has been shown, e.g., that for the expression of cystic fibrosis transmembrane conductance regulator on the cell membrane, its interaction with FLNA is important. 20 Therefore, we might suspect that FLNA plays a role in the internalization of CLMP in the plasma membrane as well. It can be speculated that the mutations in FLNA influence the expression of CLMP on the plasma membrane.…”