Signal transduction in SF9 insect cells: endocytosis of recombinant CD4 after phorbol ester treatment.
In Spodoptera frugiperda (Sf9) insect cells infected with a recombinant baculovirus carrying a gene construct encoding human CD4 endocytosis of CD4 is induced after stimulation with phorbol-12-myristate-13-acetate (PMA). Stimulation of endocytosis with PMA reduced the amount of full-length CD4 on the plasma membrane of Sf9 cells by 50% after 2 hours. Endocytosis of CD4 is blocked after intracellular delivery by cationic liposomes of a monoclonal antibody directed against a cytoplasmic sequence of CD4. Endocytosis is also blocked by the calmodulin inhibitor W7. The PKC inhibitor H7 does not inhibit PMA-induced endocytosis. A truncated CD4, in which the last 32 C-terminal amino acids were deleted did, not respond to PMA. Our results show that PMA can stimulate the calmodulin-dependent signal transduction for endocytosis of full-length CD4 in Sf9 cells. Phosphorylation of CD4 in Sf9 cells was not detectable after PMA treatment and PKC is not required for endocytosis.
Center for Blood Research Laboratories, Boston, MA.
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Grebenkämper
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Nicolau
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000818:Animals; D015703:Antigens, CD / Q000096:biosynthesis / Q000378:metabolism*; D015704:CD4 Antigens / Q000096:biosynthesis / Q000378:metabolism*; D002147:Calmodulin / Q000037:antagonists & inhibitors; D002460:Cell Line; D002462:Cell Membrane / Q000276:immunology / Q000378:metabolism; D004705:Endocytosis / Q000187:drug effects*; D006801:Humans; D007546:Isoquinolines / Q000494:pharmacology; D007700:Kinetics; D010879:Piperazines / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D015398:Signal Transduction; D018411:Spodoptera; D013449:Sulfonamides / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D013997:Time Factors; D014162:Transfection
D015703:Antigens, CD; D015704:CD4 Antigens; D002147:Calmodulin; D007546:Isoquinolines; D010879:Piperazines; D011994:Recombinant Proteins; D013449:Sulfonamides; C017967:W 7; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate