Ubiquitin/proteasome-dependent down-regulation following clathrin-mediated internalization of histamine H1-receptors in Chinese hamster ovary cells

J Neurochem. 2010 May;113(4):990-1001. doi: 10.1111/j.1471-4159.2010.06669.x. Epub 2010 Mar 4.

Abstract

We investigated the regulatory pathways responsible for agonist-induced internalization and down-regulation of G(q) protein-coupled histamine H(1)-receptors in Chinese hamster ovary cells. Histamine-induced internalization and down-regulation of H(1)-receptors were detected as the loss of [(3)H]mepyramine binding sites on intact cells accessible to hydrophilic and hydrophobic H(1)-receptor antagonists, pirdonium and mepyramine, respectively. Pretreatment of cells with 0.1 mM histamine for 30 min at 37 degrees C induced internalization as well as down-regulation of H(1)-receptors, both of which were inhibited either in the presence of an inhibitor against G protein-coupled receptor kinases (ZnCl(2)) or under hypertonic conditions where clathrin-dependent endocytosis is known to be inhibited, but were not affected by inhibitors against caveolae/raft-dependent endocytosis (filipin and nystatin). Down-regulation of H(1)-receptors, but not their internalization, was inhibited by protein kinase C inhibitors (chelerythrin or GF109203X), a ubiquitin E1 inhibitor (UBEI-41) and proteasome inhibitors (lactacystin and MG-132). Neither a Ca(2+)/calmodulin-dependent protein kinase II inhibitor (KN-62) nor lysosomal protease inhibitors (E-64, leupeptin, chloroquine and NH(4)Cl) affected the internalization and down-regulation of H(1)-receptors. These results suggest that H(1)-receptors internalize upon agonist stimulation via G protein-coupled receptor kinase/clathrin-dependent but caveolae/raft-independent mechanisms and are delivered to proteasomes, preferentially to lysosomes, for their prompt down-regulation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CHO Cells
  • Chlorides / pharmacology
  • Clathrin / metabolism*
  • Cricetinae
  • Cricetulus
  • Down-Regulation / drug effects
  • Down-Regulation / physiology
  • Endocytosis / drug effects
  • Endocytosis / physiology*
  • Enzyme Inhibitors / pharmacology
  • Filipin / pharmacology
  • G-Protein-Coupled Receptor Kinase 1 / antagonists & inhibitors
  • G-Protein-Coupled Receptor Kinase 1 / metabolism
  • Histamine / pharmacology
  • Proteasome Endopeptidase Complex / metabolism*
  • Proteasome Inhibitors
  • Protein Kinase C / antagonists & inhibitors
  • Protein Kinase C / metabolism
  • Protein Transport / drug effects
  • Protein Transport / physiology
  • Receptors, Histamine H1 / drug effects
  • Receptors, Histamine H1 / metabolism*
  • Transport Vesicles / drug effects
  • Transport Vesicles / metabolism*
  • Ubiquitin / antagonists & inhibitors
  • Ubiquitin / metabolism*
  • Zinc Compounds / pharmacology

Substances

  • Chlorides
  • Clathrin
  • Enzyme Inhibitors
  • Proteasome Inhibitors
  • Receptors, Histamine H1
  • Ubiquitin
  • Zinc Compounds
  • Histamine
  • zinc chloride
  • Filipin
  • Protein Kinase C
  • G-Protein-Coupled Receptor Kinase 1
  • Proteasome Endopeptidase Complex