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.
Grebenkämper
K
K
Center for Blood Research Laboratories, Boston, MA.
Nicolau
C
C
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