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.
Chen
Li-ei
LE
Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 294 Taiyuan Road, Shanghai 200031, China. lwchen28@yahoo.com
Gao
Can
C
Chen
Jie
J
Xu
Xue-Jun
XJ
Zhou
De-He
DH
Chi
Zhi-Qiang
ZQ
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