The Relative Binding Affinities of PDZ Partners for CFTR: A Biochemical Basis for Efficient Endocytic Recycling

Abstract

The cystic fibrosis transmembrane conductance regulator (CFTR)1 is an epithelial chloride channel mutated in patients with cystic fibrosis. Its expression and functional interactions in the apical membrane are regulated by several PDZ (PSD-95, discs large, zonula occludens-1) proteins, which mediate protein-protein interactions, typically by binding C-terminal recognition motifs. In particular, the CFTR-associated ligand (CAL) limits cell-surface levels of the most common diseaseassociated mutant ΔF508-CFTR. CAL also mediates degradation of wild-type CFTR, targeting it to lysosomes following endocytosis. Nevertheless, wild-type CFTR survives numerous cycles of uptake and recycling. In doing so, how does it repeatedly avoid CAL-mediated degradation? One mechanism may involve competition between CAL and other PDZ proteins including Na + /H + Exchanger-3 Regulatory Factors 1 and 2 (NHERF1 and NHERF2), which functionally stabilize cell-surface CFTR. Thus, to understand the biochemical basis of WT-CFTR persistence, we need to know the relative affinities of these partners. However, no quantitative binding data are available for CAL or the individual NHERF2 PDZ domains, and published estimates for the NHERF1 PDZ domains conflict. Here we demonstrate that the affinity of the CAL PDZ domain for the CFTR C-terminus is much weaker than those of NHERF1 and NHERF2 domains, enabling wild-type CFTR to avoid premature entrapment in the lysosomal pathway. At the same time, CAL's affinity is evidently sufficient to capture and degrade more rapidly cycling mutants, such as ΔF508-CFTR. The relatively weak affinity of the CAL:CFTR interaction may provide a pharmacological window for stabilizing rescued ΔF508-CFTR in patients with cystic fibrosis.CFTR is a cAMP-activated, ATP-gated chloride channel. It plays a central role in maintaining fluid and ion homeostasis in epithelial tissues and is mutated in patients with cystic fibrosis (CF) (1). Although CFTR is subject to rapid endocytosis (2), this appears to be coupled with a highly efficient constitutive recycling mechanism (e.g refs. 3,4). As a result, mature CFTR exhibits a long functional half-life (5,6), requiring individual molecules to cycle through the endocytic pathway dozens or even hundreds of times. † This work was supported in part by grants from the Cystic Fibrosis Foundation (MADDEN06P0 and STANTO97R0) and the NIH (grants P20-RR018787 from the Institutional Development Award (IDeA) Program of the NCRR and R01-DK075309 from NIDDK). P.B. was supported by the Deutsche Forschungsgemeinschaft (DFG grant VO 885/3-1). 1 The abbreviations used are: CFTR, cystic fibrosis transmembrane conductance regulator; PDZ, PSD-95, discs large, zonula occludens-1; CAL, CFTR-Associated Ligand; NHERF1, Na + /H + Exchanger-3 Regulatory Factor-1; NHERF2, Na + /H + Exchanger-3 Regulatory Factor-2; CF, cystic fibrosis; DTT, dithiothreitol; TCEP, Tris(2-carboxyethyl)phosphine hydrochloride; SPR, surface-plasmon resonance; ITC, isothermal titration calorimetry; FP, fluorescence polarizatio...

Engineering Peptide Inhibitors To Overcome PDZ Binding Promiscuity

“…However, other studies have shown that upstream residues can contribute to PDZ binding affinity [14]. Our hypothesis was that different C-terminal anchor sequences might have distinct upstream preferences, washing out signals in a global motif analysis.…”

“…As promising extensions or sequence modifications are identified, FP [14] assays are used to determine binding constants for all five PDZ domains of the CFTR trafficking system. Each modification is then evaluated for its contribution to the affinity for CAL and to the loss of affinity for the individual NHERF domains.…”

“…To investigate the context dependence of the amino-acid preferences, we performed substitutional analyses (SubAna) on a series of peptides from the 80 best HumLib-peptides, including the C-terminus of the somatostatin receptor type 5 (SSR5 10 ) which has the highest affinity for CALP among a series of known binding sequences [14] (Figures 1, 2B and S1-S2). The SubAnas confirm the difference in the amino-acid preferences at P 0 between the five PDZ domains, and show a clear tolerance of CALP for I at P 0 .…”

MAST205 Competes with Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)-associated Ligand for Binding to CFTR to Regulate CFTR-mediated Fluid Transport

“…Recent studies suggested that TC10, a small Rho-GTPase interacting with CAL and inhibiting CFTR-CAL binding, increased CFTR expression at the plasma membrane (36). When NHERF1 was coexpressed with CAL, the inhibitory effect of CAL on CFTR surface expression was overcome (18). Purified GST-His 6 -S-CFTR protein (1 g) was incubated with purified His 6 -S-CAL protein (1 g), to which increasing amounts of His 6 -S-MAST205 protein were added.…”

“…In contrast, silencing of CAL has been shown to restore function to CFTR and rescue the mutant F508del-CFTR (14,16,17). The CAL-induced reduction in CFTR expression and distribution in cells can be restored by the overexpression of NHERF-1, acting through a competition mechanism for binding to the PDZ motif of CFTR (18). These findings suggest that CAL negatively regulates CFTR intracellular processing and trafficking.…”