The WD5 repeat of RACK1 binds the PDZ1‐domain of NHERF1 in Calu‐3 airway epithelial cells

Regulation of CFTR Cl channel function involves a protein complex of activated protein kinase C (PKC)‐ 1 □ bound to RACK1, a Receptor for Activated C Kinase, and RACK1 bound to human Na + /H + exchanger regulatory factor (NHERF1). Previously, we showed binding of PKC‐ε to the WD6 repeat of RACK1 and binding of NHERF1 via a PDZ1 domain. The goal of this study was to determine the site of interaction of NHERF1 on RACK1 using peptides encoding the 7 WD40 repeat units of RACK1. Protein interactions were analyzed by solution and solid phase binding assays and pulldown assays. WD1‐WD4 peptides and WD6‐WD7 peptides did not inhibit binding of RACK1 to NHERF1 and did not directly bind NHERF1. The WD5 repeat peptide directly binds NHERF1 with an EC50 of 0.93 μg and the PDZ1 domain with an EC50 of 0.71 μg. The WD5 repeat peptide prevents binding of recombinant RACK1 and NHERF1 with an IC50 of 5.9 μg. An 11 amino acid peptide encoding the GYGF motif of the PDZ1 domain blocks binding of the WD5 repeat peptide with the PDZ1 domain with an IC50 of 6.4 μg. The WD5 repeat pulls down endogenous RACK1 from Calu‐3 cell lysate in a dose‐dependent manner. A peptide encoding 12 amino acids of the WD5 repeat binds to the PDZ1 domain with an EC50 of 0.9 μg and acts as an inhibitory peptide to prevent binding of RACK1 and the PDZ1 domain with an IC50 of 8.7μg. These results indicate binding of NHERF1 to RACK1 at the WD5 repeat of RACK1, which is distinct from the PKC‐ε binding site of the WD6 repeat. Supported by a grant from the NIH, RO1‐67190.