Ubiquitin/proteasome‐dependent down‐regulation following clathrin‐mediated internalization of histamine H 1 ‐receptors in Chinese hamster ovary cells

J. Neurochem. (2010) 113 , 990–1001. 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°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.