title: Vasopressin regulated trafficking of a green fluorescent protein-aquaporin 2 chimera in LLC-PK1 cells.
abstract: Aquaporin 2 (AQP2) transfected into LLC-PK1 cells functions as a vasopressin-regulated water channel that recycles between intracellular vesicles and the plasma membrane upon vasopressin stimulation. The green fluorescent protein (GFP) of the jellyfish, Aequorea victoria, was used as an autofluorescent tag to monitor AQP2 trafficking in transfected LLC-PK1 cells. Two chimeras were constructed, one in which GFP was fused to the amino-terminus of AQP2 [GFP-AQP2(NT)] and the second in which it was fused to the carboxyl-terminus [AQP2-GFP(CT)]. The GFP-AQP2(NT) chimera trafficked in a regulated pathway from intracellular vesicles to the basolateral plasma membrane in response to vasopressin or forskolin stimulation of cells. In contrast, the AQP2-GFP(CT) chimera expressed in LLC-PK1 cells was localized constitutively on both apical and basolateral plasma membranes. The cellular location of this chimera was not modified by vasopressin or forskolin. Thus, while the GFP-AQP2(NT) chimera will be useful to study AQP2 trafficking in vitro, the abnormal, constitutive membrane localization of the AQP2-GFP(CT) chimera suggests that one or more trafficking signals exist on the carboxyl-terminus of the AQP2 protein.
authors.affiliation: Massachusetts General Hospital, and Department of Pathology, Harvard Medical School, Boston 02129, USA.
authors.forename: C E
authors.initials: CE
authors.lastname: Gustafson
authors.forename: S
authors.initials: S
authors.lastname: Levine
authors.forename: T
authors.initials: T
authors.lastname: Katsura
authors.forename: M
authors.initials: M
authors.lastname: McLaughlin
authors.forename: M D
authors.initials: MD
authors.lastname: Aleixo
authors.forename: B K
authors.initials: BK
authors.lastname: Tamarappoo
authors.forename: A S
authors.initials: AS
authors.lastname: Verkman
authors.forename: D
authors.initials: D
authors.lastname: Brown
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D051399:Aquaporin 2; D051403:Aquaporin 6; D020346:Aquaporins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D005576:Colforsin / Q000494:pharmacology; D004705:Endocytosis / Q000187:drug effects; D005089:Exocytosis / Q000187:drug effects; D019084:Fluorescent Antibody Technique, Indirect; D049452:Green Fluorescent Proteins; D018374:LLC-PK1 Cells; D008164:Luminescent Proteins; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism; D010539:Permeability; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D013552:Swine; D014162:Transfection; D014667:Vasopressins / Q000494:pharmacology*; D014867:Water / Q000378:metabolism; D014981:Xenopus
chemical_list: C496025:Aqp2 protein, rat; D051399:Aquaporin 2; D051403:Aquaporin 6; D020346:Aquaporins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D014867:Water; D014667:Vasopressins; D049452:Green Fluorescent Proteins; D005576:Colforsin