The Reorientation of T-Cell Polarity and Inhibition of Immunological Synapse Formation by CD46 Involves Its Recruitment to Lipid Rafts
Figure 1
Palmitoylation regulates CD46 recruitment to DRM. (a) Schematic of CD46, with cytoplasmic sequences for the Cyt1 and Cyt2 alternatively spliced isoforms. Sequence begins within the transmembrane domain at residue 326 [28] and ends at the C-terminus. The transmembrane cysteine that we explore here is in bold and underlined. (b) MD45 cells expressing CD46 variants were either untreated (i)–(iii) or ligated with a CD46-specific antibody (iv)–(vii), lysed with TTX-100, fractionated on sucrose gradient, electrophoresed and probed with antibodies specific for Tfr, Flotillin, or CD46. Fractions 3–8 contain DRM proteins, and Fraction 10 contains the detergent-soluble protein. The lower, faint band visible in panels (iv)–(vii) represents the cross-linking antibody. (c) Cells were incubated with radiolabeled palmitoic acid, lysed and immunoprecipitated with antibodies to CD46, electrophoresed, and autoradiographed. Duplicate gels (lower panels) were immunoblotted with antibodies to CD46 to assess CD46 expression levels. (i) HeLa cells, CHO cells, and CHO cells transfected with CD46Cyt1. (ii) MD45 cells transduced with CD46Cyt1 or CD46Cyt1.C-A as shown. These data are representative of 3 independent experiments.
