Modulation of CD4 lateral mobility in intact cells by an intracellularly applied antibody.
This study shows that the lateral mobility of CD4, an important plasma-membrane immune receptor, can be modulated by intracellular application of an anti-CD4 antibody. For this purpose, (i) full-length CD4 and a truncated CD4 mutant, lacking a 32-residue-long C-terminal intracellularly exposed domain, were expressed in Spodoptera frugiperda (Sf9) insect cells, (ii) a monoclonal antibody, C6, with specificity for the C-terminal domain was generated, and (iii) a versatile apparatus for fluorescence microphotolysis (FM) studies was constructed. By these means it was found that the commercial anti-CD4 antibody Leu3a-PE, in contrast with several other anti-CD4 antibodies, could be used as a fluorescent label of CD4 without interfering greatly with CD4 mobility. Labelled by Leu3a-PE, full-length CD4 had a lateral diffusion coefficient of D = (4.7 +/- 1.9) x 10(-10) cm2/s and a mobile fraction of fm = 80 +/- 16% (room temperature). Within experimental accuracy the truncated CD4 had the same mobility as full-length CD4. Introduction of the C6 antibody into Sf9 cells by microinjection or by fusion with C6-loaded liposomes decreased the mobility of full-length CD4 (fm = 40%) but not of truncated CD4 (fm = 80%). Treatment of Sf9 cells with phorbol ester also reduced the mobility of full-length CD4 (fm = 50%) but not truncated CD4 (fm = 90%). A calmodulin inhibitor but not a protein kinase C (PKC) inhibitor abolished the phorbol ester effect.
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D016367:Baculoviridae / Q000235:genetics; D015704:CD4 Antigens / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D002147:Calmodulin / Q000037:antagonists & inhibitors; D002460:Cell Line; D002462:Cell Membrane / Q000276:immunology*; D006801:Humans; D008081:Liposomes; D008845:Microinjections; D008856:Microscopy, Fluorescence; D008858:Microscopy, Phase-Contrast; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000235:genetics; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D018411:Spodoptera; D013449:Sulfonamides / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D015704:CD4 Antigens; D002147:Calmodulin; D008081:Liposomes; D013449:Sulfonamides; C017967:W 7; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate