title: PDZ domain interaction controls the endocytic recycling of the cystic fibrosis transmembrane conductance regulator.
abstract: The C terminus of CFTR contains a PDZ interacting domain that is required for the polarized expression of cystic fibrosis transmembrane conductance regulator (CFTR) in the apical plasma membrane of polarized epithelial cells. To elucidate the mechanism whereby the PDZ interacting domain mediates the polarized expression of CFTR, Madin-Darby canine kidney cells were stably transfected with wild type (wt-CFTR) or C-terminally truncated human CFTR (CFTR-DeltaTRL). We tested the hypothesis that the PDZ interacting domain regulates sorting of CFTR from the Golgi to the apical plasma membrane. Pulse-chase studies in combination with domain-selective cell surface biotinylation revealed that newly synthesized wt-CFTR and CFTR-DeltaTRL were targeted equally to the apical and basolateral membranes in a nonpolarized fashion. Thus, the PDZ interacting domain is not an apical sorting motif. Deletion of the PDZ interacting domain reduced the half-life of CFTR in the apical membrane from approximately 24 to approximately 13 h but had no effect on the half-life of CFTR in the basolateral membrane. Thus, the PDZ interacting domain is an apical membrane retention motif. Next, we examined the hypothesis that the PDZ interacting domain affects the apical membrane half-life of CFTR by altering its endocytosis and/or endocytic recycling. Endocytosis of wt-CFTR and CFTR-DeltaTRL did not differ. However, endocytic recycling of CFTR-DeltaTRL was decreased when compared with wt-CFTR. Thus, deletion of the PDZ interacting domain reduced the half-life of CFTR in the apical membrane by decreasing CFTR endocytic recycling. Our results identify a new role for PDZ proteins in regulating the endocytic recycling of CFTR in polarized epithelial cells.
authors.affiliation: Dartmouth Medical School, Department of Physiology, Hanover, New Hampshire 03755, USA.
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Swiatecka-Urban
authors.forename: Marc
authors.initials: M
authors.lastname: Duhaime
authors.forename: Bonita
authors.initials: B
authors.lastname: Coutermarsh
authors.forename: Katherine H
authors.initials: KH
authors.lastname: Karlson
authors.forename: James
authors.initials: J
authors.lastname: Collawn
authors.forename: Michal
authors.initials: M
authors.lastname: Milewski
authors.forename: Garry R
authors.initials: GR
authors.lastname: Cutting
authors.forename: William B
authors.initials: WB
authors.lastname: Guggino
authors.forename: George
authors.initials: G
authors.lastname: Langford
authors.forename: Bruce A
authors.initials: BA
authors.lastname: Stanton
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D019921:Biotinylation; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry* / Q000378:metabolism; D004285:Dogs; D004705:Endocytosis; D004847:Epithelial Cells / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013997:Time Factors
chemical_list: D011993:Recombinant Fusion Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator