title: Protein kinase C inhibits caveolae-mediated endocytosis of TRPV5.
abstract: Transient receptor potential vanilloid 5 (TRPV5) constitutes the apical entry pathway for transepithelial Ca(2+) reabsorption in kidney. Many hormones alter renal Ca(2+) reabsorption at least partly by regulating TRPV5. The mechanism for acute regulation of TRPV5 by phospholipase C-coupled hormones is largely unknown. Here, we found that protein kinase C (PKC) activator 1-oleoyl-acetyl-sn-glycerol (OAG) increased TRPV5 current density and surface abundance in cultured cells. The OAG-mediated increase of TRPV5 was prevented by preincubation with specific PKC inhibitors. Coexpression with a dominant-negative dynamin increased the basal TRPV5 current density and prevented the increase by OAG. Knockdown of caveolin-1 by small interference RNA (siRNA) prevented the increase of TRPV5 by OAG. In contrast, knockdown of clathrin heavy chain had no effects. OAG had no effect on TRPV5 expressed in caveolin-1 null cells derived from caveolin-1 knockout mice. Forced expression of recombinant caveolin-1 restored the regulation of TRPV5 by OAG in caveolin-1 knockout cells. Mutations of serine-299 and/or serine-654 of TRPV5 (consensus residues for phosphorylation by PKC) abolished the regulation by OAG. Parathyroid hormone (PTH) increased TRPV5 current density in cells coexpressing TRPV5 and type 1 PTH receptor. The increase caused by PTH was prevented by PKC inhibitor, mutation of serine-299/serine-654, or by knockdown of caveolin-1. Thus, TRPV5 undergoes constitutive caveolae-mediated endocytosis. Activation of PKC increases cell surface abundance of TRPV5 by inhibiting the endocytosis. This mechanism of regulation by PKC may contribute to the acute stimulation of TRPV5 and renal Ca(2+) reabsorption by PTH.
authors.affiliation: Department of Medicine (Division of Nephrology), UT Southwestern Medical Center, Dallas, Texas 75390-8856, USA.
authors.forename: Seung-Kuy
authors.initials: SK
authors.lastname: Cha
authors.forename: Tao
authors.initials: T
authors.lastname: Wu
authors.forename: Chou-Long
authors.initials: CL
authors.lastname: Huang
mesh_terms: D000818:Animals; D019921:Biotinylation; D021941:Caveolae / Q000187:drug effects / Q000502:physiology*; D002460:Cell Line; D004247:DNA / Q000096:biosynthesis / Q000235:genetics; D004285:Dogs; D004594:Electrophysiology; D004705:Endocytosis / Q000502:physiology*; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology; D010281:Parathyroid Hormone / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D010766:Phosphorylation; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D011817:Rabbits; D011956:Receptors, Cell Surface / Q000187:drug effects / Q000378:metabolism; D050916:TRPV Cation Channels / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*
chemical_list: D007527:Isoenzymes; D010281:Parathyroid Hormone; D011956:Receptors, Cell Surface; D050916:TRPV Cation Channels; C492239:TRPV5 protein, human; D049452:Green Fluorescent Proteins; D004247:DNA; D011493:Protein Kinase C