Internalization of the opioid growth factor, [Met5]-enkephalin, is dependent on clathrin-mediated endocytosis for downregulation of cell proliferation.
The opioid growth factor (OGF; [Met(5)]-enkephalin), a constitutively expressed and tonically active inhibitory peptide, interacts with the OGF receptor (OGFr) to form an endogenous growth-regulating pathway in homeostasis. Amplification of OGF-OGFr interfacing in animal and clinical studies depresses development, neoplasia, angiogenesis, and immunity. Disruption of the OGF-OGFr axis accelerates cell proliferation and has been particularly important in wound repair. To investigate how OGF enters cells, OGF was labeled with 5,6-tetramethylrhodamine OGF (RhoOGF) to study its uptake in live cells. African green monkey kidney cells (COS-7) incubated with RhoOGF exhibited a temperature-dependent course of entry, being internalized at 37 degrees C but not at 4 degrees C. RhoOGF was detected in the cytoplasm 15 min after initial exposure, observed in both cytoplasm and nucleus within 30 min, and remained in the cells for as long as 5 h. A 100-fold excess of OGF or the opioid antagonist naltrexone, but not other opioid ligands (some selective for classic opioid receptors), markedly reduced entry of RhoOGF into cells. RhoOGF was functional because DNA synthesis in cells incubated with RhoOGF (10(-5) to 10(-8) M) was decreased 24-36%, and was comparable to cells treated with unlabeled OGF (reductions of 26-39%). OGF internalization was dependent on clathrin-mediated endocytosis, with addition of clathrin siRNA diminishing the uptake of RhoOGF and upregulating DNA synthesis. RhoOGF clathrin-mediated endocytosis was unrelated to endosomal or Golgi pathways. Taken together, these results suggest that OGF enters cells by active transport in a saturable manner that requires clathrin-mediated endocytosis.
Cheng
Fan
F
Department of Neural and Behavioral Sciences, The Milton S. Hershey Medical Center, Hershey, PA 17033, USA.
McLaughlin
Patricia J
PJ
Banks
William A
WA
Zagon
Ian S
IS
D000818:Animals; D001692:Biological Transport; D019556:COS Cells; D002103:Cadaverine / Q000031:analogs & derivatives; D049109:Cell Proliferation; D002522:Chlorocebus aethiops; D002966:Clathrin / Q000378:metabolism*; D016895:Culture Media, Serum-Free; D015536:Down-Regulation; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology*; D011992:Endosomes; D004744:Enkephalin, Methionine / Q000378:metabolism / Q000494:pharmacology*; D005260:Female; D006056:Golgi Apparatus; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000502:physiology*; D010051:Ovarian Neoplasms / Q000378:metabolism*; D034741:RNA, Small Interfering; D012235:Rhodamines / Q000737:chemistry / Q000494:pharmacology; D013194:Staining and Labeling; D047392:beta-Cyclodextrins
D002966:Clathrin; D016895:Culture Media, Serum-Free; D034741:RNA, Small Interfering; D012235:Rhodamines; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D004744:Enkephalin, Methionine; C008542:monodansylcadaverine; D002103:Cadaverine