Polarization of CFTR, a cAMP-activated Cl^– channel to the apical plasma membrane in epithelial cells is critical for vectorial Cl^– transport. Previously, we reported that the C-terminus of CFTR constitutes a PDZ interacting domain (DTRL using the single letter code) that is required for CFTR polarization to the apical plasma membrane and interaction with the PDZ domain-containing protein EBP50/NHERF (Moyer et al. 1999). In addition, the PDZ proteins CAP70 and EBP50 regulate CFTR channel activity (Wang et al. 2000; Raghuram et al. 2001). PDZ domains, which are named for three proteins in which this domain was first described (PSD-95, Dlg and ZO-1), play an essential role is assembling macromolecular signalling complexes, in determining cell polarity and in regulating intracellular trafficking. Recently, we identified, by yeast two hybrid (YTH), a novel PDZ protein, CAL (CFTR Associated Ligand) that binds to the PDZ interacting domain of CFTR. Co-immunoprecipitation confirmed YTH studies that CAL and CFTR interact via the PDZ interacting domain of CFTR and demonstrated that CAL is a homodimer. CAL has one PDZ domain, two coiled-coil domains and is expressed primarily in the trans-Golgi network (TGN). CAL has a broad tissue distribution in mammalian cells and a CAL homologue has been identified in C. elegans. The coiled-coil domains localize CAL to the cytoplasmic face of the TGN. Overexpression of CAL significantly reduced plasma membrane expression of CFTR and induced intracellular aggregation of CFTR and CAL in COS and HEK cells. Overexpression of CAL also reduced CFTR Cl^– currents in Xenopus oocytes. EBP50/NHERF, a PDZ protein that also interacts with the PDZ interacting domain in CFTR and the actin-based cytoskeleton (Moyer et al. 2000), is co-expressed with CFTR in the apical plasma membrane of epithelial cells. Overexpression of EBP50 had no effect on the plasma membrane expression of CFTR. However, EBP50/NHERF reversed the CAL-induced intracellular accumulation of CFTR. In pulse-chase studies overexpression of CAL reduced the rate of appearance of newly synthesized CFTR in the plasma membrane and also reduced the half-life of CFTR in the plasma membrane. Our data support a model whereby CAL is involved in the trafficking of CFTR from the TGN to the plasma membrane. We speculate that at the plasma membrane CFTR releases CAL and binds to EBP50/NHERF, which, via its interaction with the actin-based cytoskeleton, retains CFTR in the apical plasma membrane. CAL recycles back to the TGN. The broad tissue distribution suggests that CAL may regulate the trafficking of other proteins containing PDZ interacting domains between the TGN and plasma membrane. Thus CAL, a novel PDZ protein, regulates the intracellular trafficking of CFTR.

This work was supported by the Cystic Fibrosis Foundation and the National Institutes of Health.

Trafficking and Targeting of Transporters and ChannelsTrafficking and Targeting of Transporters and ChannelsTrafficking and Targeting of Transporters and ChannelsTrafficking and Targeting of Transporters and Channels>This work was supported by the Cystic Fibrosis Foundation and the National Institutes of Health.

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