Spatiotemporal Coupling of cAMP Transporter to CFTR Chloride Channel Function in the Gut Epithelia

Abstract

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated chloride channel localized at apical cell membranes and exists in macromolecular complexes with a variety of signaling and transporter molecules. Here, we report that the multidrug resistance protein 4 (MRP4), a cAMP transporter, functionally and physically associates with CFTR. Adenosine-stimulated CFTR-mediated chloride currents are potentiated by MRP4 inhibition, and this potentiation is directly coupled to attenuated cAMP efflux through the apical cAMP transporter. CFTR single-channel recordings and FRET-based intracellular cAMP dynamics suggest that a compartmentalized coupling of cAMP transporter and CFTR occurs via the PDZ scaffolding protein, PDZK1, forming a macromolecular complex at apical surfaces of gut epithelia. Disrupting this complex abrogates the functional coupling of cAMP transporter activity to CFTR function. Mrp4 knockout mice are more prone to CFTR-mediated secretory diarrhea. Our findings have important implications for disorders such as inflammatory bowel disease and secretory diarrhea.

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

“…After transfection, the dHL-60 cells were used for Ca 2ϩ mobilization, chemotaxis, or transmigration assays. The HEK293 cells and HT-29 human colonic epithelial cells were purchased from ATCC and cultured in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen) supplemented with 10% FBS as described before (31). HEK293 cells were transfected using Lipofectamine 2000 with HA-tagged human CXCR2, murine CXCR2, and FLAG-tagged PLC-␤1, -␤2, -␤3, and -␤4, respectively, for various biochemical assays.…”

“…Pulldown Assay-Freshly isolated human or murine neutrophils, dHL60 cells, or HEK293 cells overexpressing various constructs (3HA-tagged human CXCR2, murine CXCR2, or FLAG-tagged PLC-␤1, -␤2, -␤3, -␤4) were used for the GST pulldown assays as reported (30,31). In brief, the cells were lysed in lysis buffer (PBS, 0.2% Triton) supplemented with a mixture of protease inhibitors (containing 1 mM phenylmethylsulfonyl fluoride, 1 g/ml of aprotinin, 1 g/ml of leupeptin, and 1 g/ml of pepstatin) and phosphatase inhibitor mixture (Sigma), and the clear supernatant (16,000 ϫ g, 15 min) was mixed with various GST-PDZ fusion proteins (GST-NHERF1, GST-NHERF2, or GST-PDZK1) or GST alone at 4 ºC for 3 h. The complex was pulled down by glutathione-agarose beads (BD Biosciences) at 4 ºC for 1 h, washed three times with lysis buffer, and eluted in Laemmli sample buffer containing ␤-mercaptoethanol.…”

“…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. A Co-immunoprecipitation Kit (Pierce) was used to immobilize the anti-CXCR2 IgG to the resin and the co-precipitated protein complex was eluted with Laemmli sample buffer before being subjected to immunoblotting and probed for PLC-␤2 and NHERF1.…”

“…Measurement of Cytosolic Ca 2ϩ Concentration-CXCR2 C-tail peptides (WT and ⌬TTL) were mixed with the Chariot peptide delivery reagent (Active Motif; total volume of 400 l) at 22 ºC for 30 min as reported (30,31). The Chariot-peptide complex was then incubated with freshly isolated neutrophils (or dHL-60 cells) with gentle agitation at 37 ºC for 1 h. The peptide-delivered neutrophils (1 ϫ 10 6 ) were labeled with 1 M Fura-2 (Invitrogen) at 22 ºC for 30 min.…”

Altered cGMP Dynamics at the Plasma Membrane Contribute to Diarrhea in Ulcerative Colitis

“…12 Briefly, 20 mg His-S-iNOS-C-tail protein was mixed with various amounts of GST-NHERF2 (0 to 15 mg) at 4 C for 1 hour, followed by mixing with 20 mL S-beads for another 1 hour. This step, which is called pairwise binding, was performed in 200 mL of lysis buffer (PBS that contained 0.2% Triton X-100 and protease inhibitors).…”