Internalization and recycling of human mu opioid receptors expressed in Sf9 insect cells.
Internalization and recycling of G protein-coupled receptors (GPCRs), such as the mu-opioid receptor, largely depend on agonist stimulation. Agonist-promoted internalization of some GPCRs has been shown to mediate receptor desensitization, resensitization, and down-regulation. In this study, we investigated whether different mu opioid agonists displayed different effects in receptor internalization and recycling, the potential mechanisms involved in ohmefentanyl-induced internalization process. In transfected Sf9 insect cells expressing 6His-tagged wild type mu opioid receptor, exposure to 100 nM ohmefentanyl caused a maximum internalization of the receptor at 30 min and receptors seemed to reappear at the cell membrane after 60 min as determined by radioligand binding assay. Ohmefentanyl-induced human mu opioid receptor internalization was concentration-dependent, with about 40% of the receptors internalized following a 30-min exposure to 1 microM ohmefentanyl. 10 microM morphine and 1 microM DAMGO could also induce about 40% internalization. The antagonist naloxone and pretreatment with pertussis toxin both blocked ohmefentanyl-induced internalization without affecting internalization themselves. Incubation with sucrose 0.45 M significantly inhibited ohmefentanyl-induced internalization of the mu receptor. The removal of agonists ohmefentanyl and morphine resulted in the receptors gradually returning to the cell surface over a 60 min period, while the removal of agonist DAMGO only partly resulted in the receptor recycling. The results of this study suggest that ohmefentanyl-induced internalization of human mu opioid receptor in Sf9 insect cells occurs via Gi/o protein-dependent process that likely involves clathrin-coated pits. In addition, the recycling process displays the differential modes of action of different agonists.
Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 294 Taiyuan Road, Shanghai 200031, China. lwchen28@yahoo.com
Li-ei
LE
Chen
Can
C
Gao
Jie
J
Chen
Xue-Jun
XJ
Xu
De-He
DH
Zhou
Zhi-Qiang
ZQ
Chi
D000701:Analgesics, Opioid / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation / Q000187:drug effects; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)- / Q000494:pharmacology; D005283:Fentanyl / Q000494:pharmacology; D006801:Humans; D006982:Hypertonic Solutions; D007313:Insecta / Q000378:metabolism*; D018613:Microscopy, Confocal; D009020:Morphine / Q000494:pharmacology; D009270:Naloxone / Q000494:pharmacology; D009292:Narcotic Antagonists / Q000494:pharmacology; D037342:Pertussis Toxin / Q000494:pharmacology; D011956:Receptors, Cell Surface / Q000187:drug effects*; D017450:Receptors, Opioid, mu / Q000819:agonists / Q000378:metabolism*; D014162:Transfection
D000701:Analgesics, Opioid; D006982:Hypertonic Solutions; D009292:Narcotic Antagonists; D011956:Receptors, Cell Surface; D017450:Receptors, Opioid, mu; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D009270:Naloxone; D009020:Morphine; D000242:Cyclic AMP; D037342:Pertussis Toxin; D005283:Fentanyl