Filamin‐A modulates CFTR surface expression via Filamin‐A Ig repeat 21 and interacts with the scaffold protein RACK1

Abstract

Filamin‐A (FlnA) binds to CFTR through a protein‐protein interaction that is disrupted by the missense mutation S13F in CFTR, causing unstable surface expression of CFTR. We hypothesized that the N‐terminal sequence of CFTR binds to immunoglobulin repeat 21 of FlnA (Ig21), based on previously studied binding preferences of Ig21. We confirmed binding of a CFTR N‐terminal peptide to Ig21 and solved the structure of the peptide bound to Ig21 in a novel staggered 2:1 complex, in which the single CFTR N‐terminal peptide completes an extended beta sheet spanning two parallel Ig21 domains. The structure reveals how the disease‐causing CF mutation S13F disrupts the FlnA:CFTR interaction. To test functional effects of Ig21, we delivered Ig21 into Calu‐3 human airway epithelial cells and examined CFTR apical membrane expression by biotinylation. Ig21 caused a dose‐dependent decrease in biotinylated CFTR. CoIP experiments demonstrated an interaction between CFTR and FlnA. We also detected RACK1 in IPs of FlnA and vice versa and colocalization of RACK1 and FlnA by IF. To study RACK1 binding to FlnA, FlnA or CFTR were IPed from Calu‐3 cells, washed under high stringent conditions, and incubated with recombinant RACK1. RACK1 was shown to bind to FlnA in a dose dependent manner, but not to CFTR. Our results suggest that the important FlnA:CFTR interaction is indeed mediated by FlnA‐Ig21 and that FlnA:RACK1 interaction may be important for RACK1‐mediated recruitment of PKCε to CFTR. Supported by the Ralph Wilson Medical Research Foundation (SM), NIH R01‐58598 and ‐67190 (CML) and an RDP grant from the Cystic Fibrosis Foundation (CML).