Ubiquitin/proteasome-dependent down-regulation following clathrin-mediated internalization of histamine H1-receptors in Chinese hamster ovary cells.
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.
Hishinuma
Shigeru
S
Department of Pharmacodynamics, Meiji Pharmaceutical University, Tokyo, Japan. hishi@my-pharm.ac.jp
Komazaki
Hiroshi
H
Fukui
Hiroyuki
H
Shoji
Masaru
M
D000818:Animals; D016466:CHO Cells; D002712:Chlorides / Q000494:pharmacology; D002966:Clathrin / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D015536:Down-Regulation / Q000187:drug effects / Q000502:physiology; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005372:Filipin / Q000494:pharmacology; D051606:G-Protein-Coupled Receptor Kinase 1 / Q000037:antagonists & inhibitors / Q000378:metabolism; D006632:Histamine / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D061988:Proteasome Inhibitors; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D011969:Receptors, Histamine H1 / Q000187:drug effects / Q000378:metabolism*; D022161:Transport Vesicles / Q000187:drug effects / Q000378:metabolism*; D025801:Ubiquitin / Q000037:antagonists & inhibitors / Q000378:metabolism*; D017967:Zinc Compounds / Q000494:pharmacology
D002712:Chlorides; D002966:Clathrin; D004791:Enzyme Inhibitors; D061988:Proteasome Inhibitors; D011969:Receptors, Histamine H1; D025801:Ubiquitin; D017967:Zinc Compounds; D006632:Histamine; C016837:zinc chloride; D005372:Filipin; D011493:Protein Kinase C; D051606:G-Protein-Coupled Receptor Kinase 1; D046988:Proteasome Endopeptidase Complex