The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated chloride channel localized primarily at the apical or luminal surfaces of epithelial cells that line the airway, gut, and exocrine glands; it is well established that CFTR plays a pivotal role in cholera toxin (CTX)-induced secretory diarrhea. Lysophosphatidic acid (LPA), a naturally occurring phospholipid present in blood and foods, has been reported to play a vital role in a variety of conditions involving gastrointestinal wound repair, apoptosis, inflammatory bowel disease, and diarrhea. Here we show, for the first time, that type 2 LPA receptors (LPA2) are expressed at the apical surface of intestinal epithelial cells, where they form a macromolecular complex with Na+/H+ exchanger regulatory factor–2 and CFTR through a PSD95/Dlg/ZO-1–based interaction. LPA inhibited CFTR-dependent iodide efflux through LPA2-mediated Gi pathway, and LPA inhibited CFTR-mediated short-circuit currents in a compartmentalized fashion. CFTR-dependent intestinal fluid secretion induced by CTX in mice was reduced substantially by LPA administration; disruption of this complex using a cell-permeant LPA2-specific peptide reversed LPA2-mediated inhibition. Thus, LPA-rich foods may represent an alternative method of treating certain forms of diarrhea.

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...