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 applicati on of an anti-CD4 antibody. For this purpose, (i) full-length CD4 and a truncated CD4 mutant, lacking a 32-residue-long C-terminal intracell ularly exposed domain, were expressed in Spodoptera frugiperda (Sf9) i nsect cells, (ii) a monoclonal antibody, C6, with specificity for the C-terminal domain was generated, and (iii) a versatile apparatus for f luorescence microphotolysis (FM) studies was constructed. By these mea ns it was found that the commercial anti-CD4 antibody Leu3a-PE, in con trast with several other anti-CD4 antibodies, could be used as a fluor escent label of CD4 without interfering greatly with CD4 mobility. Lab elled by Leu3a-PE, full-length CD4 had a lateral diffusion coefficient of D = (4.7 +/- 1.9) x 10(-10) cm(2)/s and a mobile fraction of f(m) = 80 +/- 16 % (room temperature). Within experimental accuracy the tru ncated CD4 had the same mobility as full-length CD4. Introduction of t he C6 antibody into Sf9 cells by microinjection or by fusion with C6-l oaded liposomes decreased the mobility of full-length CD4 (f(m) = 40 % ) but not of truncated CD4 (f(m) = 80 %). Treatment of Sf9 cells with phorbol ester also reduced the mobility of full-length CD4 (f(m) = 50 %) but not truncated CD4 (f(m) = 90 %). A calmodulin inhibitor but not a protein kinase C (PKC) inhibitor abolished the phorbol ester effect .