title: Alix (AIP1) is a vasopressin receptor (V2R)-interacting protein that increases lysosomal degradation of the V2R.
abstract: The vasopressin type 2 receptor (V2R) is a G protein-coupled receptor that plays a central role in renal water reabsorption. Termination of ligand (vasopressin) stimulation is an important physiological regulatory event, but few proteins that interact with the V2R during downregulation after vasopressin (VP) binding have been identified. Using yeast two-hybrid screening of a human kidney cDNA library, we show that a 100-kDa protein called ALG-2-interacting protein X (Alix) interacts with the last 29 amino acids of the V2R COOH terminus. This was confirmed by pull-down assays using a GST-V2R-COOH-tail fusion protein. Alix was immunolocalized in principal cells of the kidney, which also express the V2R. The function of the Alix-V2R interaction was studied by transfecting Alix into LLC-PK(1) epithelial cells expressing V2R-green fluorescent protein (GFP). Under basal conditions, V2R-GFP localized mainly at the plasma membrane. On VP treatment, V2R-GFP was internalized into perinuclear vesicles in the nontransfected cells. In contrast, V2R-GFP fluorescence was virtually undetectable 2 h after exposure to VP in cells that coexpressed Alix. Western blotting using an anti-GFP antibody showed marked degradation of the V2R after 2 h in the presence of VP and Alix, a time point at which little or no degradation was detected in the absence of Alix. In contrast, little or no degradation of the parathyroid hormone receptor was detectable in the presence or absence of Alix and/or the PTH ligand. The VP-induced disappearance of V2R-GFP was abolished by chloroquine, a lysosomal degradation inhibitor, but not by MG132, a proteosome inhibitor. These data suggest that Alix increases the rate of lysosomal degradation of V2R and may play an important regulatory role in the VP response by modulating V2R downregulation.
authors.affiliation: Cellular Biophysics Laboratory, Department of Anatomy and Physiology, Kansas State University, Manhattan, Kansas, USA.
authors.forename: Xianhua
authors.initials: X
authors.lastname: Yi
authors.forename: Richard
authors.initials: R
authors.lastname: Bouley
authors.forename: Herbert Y
authors.initials: HY
authors.lastname: Lin
authors.forename: Shaliha
authors.initials: S
authors.lastname: Bechoua
authors.forename: Tian-Xiao
authors.initials: TX
authors.lastname: Sun
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Del Re
authors.forename: Toshi
authors.initials: T
authors.lastname: Shioda
authors.forename: Malay K
authors.initials: MK
authors.lastname: Raychowdhury
authors.forename: Hua A J
authors.initials: HA
authors.lastname: Lu
authors.forename: Abdul B
authors.initials: AB
authors.lastname: Abou-Samra
authors.forename: Dennis
authors.initials: D
authors.lastname: Brown
authors.forename: Dennis A
authors.initials: DA
authors.lastname: Ausiello
mesh_terms: D000818:Animals; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D056827:Endosomal Sorting Complexes Required for Transport; D005455:Fluorescent Antibody Technique; D005982:Glutathione Transferase / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D007668:Kidney / Q000166:cytology / Q000378:metabolism*; D008247:Lysosomes / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D017483:Receptors, Vasopressin / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013194:Staining and Labeling; D013552:Swine; D014162:Transfection; D020798:Two-Hybrid System Techniques; D015003:Yeasts
chemical_list: D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D056827:Endosomal Sorting Complexes Required for Transport; C117020:PDCD6IP protein, human; D017483:Receptors, Vasopressin; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase