Molecular Assembly of Cystic Fibrosis Transmembrane Conductance Regulator in Plasma Membrane

Abstract

Based on electrophysiological measurements, it has been argued that the active form of cystic fibrosis transmembrane conductance regulator (CFTR) Cl ؊ channel is a multimer. It has also been demonstrated that this multimerization is likely due to PDZ domain-interacting partners. Here we demonstrate that although CFTR in vitro can self-associate into multimers, which depends on PDZ-based interactions, this may not be the case in cell membrane. Using chemical cross-linking, we demonstrated that CFTR exists as a higher order complex in cell membrane. However, this higher order complex is predominantly CFTR dimers, and the PDZ-interacting partners (Na ؉ /H ؉ exchanger regulatory factor-1 (NHERF1) and NHERF2) constitute ϳ2% of this complex. Interestingly solubilizing membrane expressing CFTR in detergents such as Triton X-100, Nonidet P-40, deoxycholate, and SDS tended to destabilize the CFTR dimers and dissociate them into monomeric form. The dimerization of CFTR was tightly regulated by cAMP-dependent protein kinase-dependent phosphorylation and did not depend on the active form of the channel. In addition, the dimerization was not influenced by either the PDZ motif or its interacting partners (NHERF1 and NHERF2). We also demonstrated that other signalingrelated proteins such as G␤ and syntaxin 1A can be in this higher order complex of CFTR as well. Our studies provide a deeper understanding of how the CFTR assembly takes place in native cell membrane.Many studies suggest that PDZ 1 domain-containing proteins can cluster different proteins (such as ion channels, transporters, receptors, cytoskeletal proteins, and cytosolic signaling proteins) into functional complexes at synapses, cellular junctions, and polarized membrane microdomains (1). The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated Cl Ϫ channel localized primarily on the apical membrane of epithelial cells in the airway, gastrointestinal tract, reproductive organs, sweat duct, etc. where it is responsible for the transepithelial ion and fluid transport (2). An elegant functional study using electrophysiological measurements to identify microdomains under the apical membrane that transmit the signaling of CFTR via the adenosine receptor has been reported by Huang et al. (3) who have demonstrated that signaling elements compartmentalized at both extracellular and intracellular surfaces of apical cell membrane activate apical CFTR-dependent Cl Ϫ conductance in airway epithelial cells. We recently have shown that ␤ 2 adrenergic receptor interacts with CFTR via Na ϩ /H ϩ exchanger regulatory factor-1 (NHERF1, also called EBP50; Ref. 4) through their PDZ (PSD-95/Dlg/ZO-1) motifs, forming a macromolecular complex present at the apical surface of airway epithelial cells (5). Deleting the PDZ motif from CFTR uncouples the channel from ␤ 2 adrenergic receptor both physically and functionally, and this uncoupling is specific to ␤ 2 adrenergic receptor and does not affect CFTR coupling to other receptors (e.g. adenosine receptor pathwa...

Monomeric CFTR in Plasma Membranes in Live Cells Revealed by Single Molecule Fluorescence Imaging

“…These data are in accord with high resolution crystal structures of bacterial ATP-binding cassettetype transporters showing unit cells containing two transmembrane-nucleotide binding domains (11). Biochemical approaches including velocity-gradient centrifugation, coimmunoprecipitation, gel filtration, and cross-linking have generated conflicting data suggesting monomeric CFTR (9,12), dimeric CFTR (13), and mixed monomeric/dimeric CFTR (14,15). Data supporting dimeric CFTR have also come from patch clamp of CFTR in the presence of the PDZ domain proteins CAP70 and EBP50 (16 -18), from freezefracture electron microscopy (19), and from atomic force microscopy (20).…”

A Chemokine Receptor CXCR2 Macromolecular Complex Regulates Neutrophil Functions in Inflammatory Diseases

“…Co-immunoprecipitation-Fresh cells (dHL-60 or murine bone marrow neutrophils) were cross-linked with 1 mM dithiobis(succinimidyl propionate) as described before (36). Thereafter, the cells were solubilized in PBS, 0.2% Triton, and cleared lysates (16,000 ϫ g, 15 min) were processed for co-immunoprecipitation and immunoblotting as described before (30,31) using the respective antibodies against PLC-␤2, CXCR2, or NHERF1.…”

“…4A (upper panel). Using this in vitro macromolecular complex assembly assay (29,30,36,42), we detected a complex consisting of CXCR2, PDZ scaffold proteins (NHERF1 or NHERF2), and PLC-␤2 C-tail (Fig. 4A).…”

“…To address this issue, freshly isolated murine bone marrow neutrophils were cross-linked as reported previously (36), and co-immunoprecipitation was performed using anti-murine CXCR2 antibody. We detected the endogenous PLC-␤2 and NHERF1 that were co-precipitated with CXCR2 from freshly isolated neutrophils (Fig.…”

“…1 and 2) does not provide information whether the interactions between CXCR2 or PLC-␤2 and NHERF1 are direct, as cell lysates contain large numbers of other proteins as well. To test if CXCR2 or PLC-␤2 binds NHERF1 directly or by other intermediary proteins, and to test the PDZ motif dependence, we performed a pairwise binding assay that detects a direct interaction between purified proteins in vitro (29,30,36,42). Purified His-S fusion proteins containing the C-tail fragments (last 45 amino acids) of CXCR2 WT, C-tail ⌬TTL (with PDZ motif TTL deleted), or C-tail AAA or ATA (with PDZ motif TTL mutated to AAA or ATA) were mixed with GST-NHERF1, and the mixtures were pulled down by S-protein-agarose (the S-protein can specifically bind to the S-tag within the fusion proteins).…”