title: Downregulation of the vasopressin type 2 receptor after vasopressin-induced internalization: involvement of a lysosomal degradation pathway.
abstract: Vasopressin (VP) increases urinary concentration by signaling through the vasopressin receptor (V2R) in collecting duct principal cells. After downregulation, V2R reappears at the cell surface via an unusually slow (several hours) "recycling" pathway. To examine this pathway, we expressed V2R-green fluorescent protein (GFP) in LLC-PK1a cells. V2R-GFP showed characteristics similar to those of wild-type V2R, including high affinity for VP and adenylyl cyclase stimulation. V2R-GFP was located mainly in the plasma membrane in unstimulated cells, but it colocalized with the lysosomal marker Lysotracker after VP-induced internalization. Western blot analysis of V2R-GFP showed a broad 57- to 68-kDa band and a doublet at 46 and 52 kDa before VP treatment. After 4-h VP exposure, the 57- to 68-kDa band lost 50% of its intensity, whereas the lower 46-kDa band increased by 200%. The lysosomal inhibitor chloroquine abolished this VP effect, whereas lactacystin, a proteasome inhibitor, had no effect. Incubating cells at 20 degrees C to block trafficking from the trans-Golgi network reduced V2R membrane fluorescence, and a perinuclear patch developed. Cycloheximide reduced the intensity of this patch, showing that newly synthesized V2R-GFP contributed significantly to its appearance. Cycloheximide also inhibited the reappearance of cell surface V2R after downregulation. We conclude that after downregulation, V2R-GFP is delivered to lysosomes and degraded. Reappearance of V2R at the cell surface depends on new protein synthesis, partially explaining the long time lag needed to fully reestablish V2R at the cell surface after downregulation. This degradative pathway may be an adaptive response to allow receptor-ligand association in the hypertonic and acidic environment of the renal medulla.
authors.affiliation: Program in Membrane Biology and Renal Unit, Department of Medicine, Massachusetts General Hospital East, 149 13th St., Charlestown, MA 02129, USA. bouley@receptor.mgh.harvard.edu
authors.forename: Richard
authors.initials: R
authors.lastname: Bouley
authors.forename: Herbert Y
authors.initials: HY
authors.lastname: Lin
authors.forename: Malay K
authors.initials: MK
authors.lastname: Raychowdhury
authors.forename: Vladimir
authors.initials: V
authors.lastname: Marshansky
authors.forename: Dennis
authors.initials: D
authors.lastname: Brown
authors.forename: Dennis A
authors.initials: DA
authors.lastname: Ausiello
mesh_terms: D000818:Animals; D002460:Cell Line; D015536:Down-Regulation; D004847:Epithelial Cells / Q000378:metabolism / Q000648:ultrastructure; D005786:Gene Expression Regulation / Q000502:physiology; D008247:Lysosomes / Q000378:metabolism*; D017483:Receptors, Vasopressin / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014667:Vasopressins / Q000494:pharmacology / Q000502:physiology*
chemical_list: D017483:Receptors, Vasopressin; D011993:Recombinant Fusion Proteins; D014667:Vasopressins