Schematic of human CAR constructs. Full-length human CAR (amino acids 1 to 365) contains an N-terminal exoplasmic region, a single transmembrane (TM) domain, and a C-terminal cytoplasmic tail that includes tandem cysteines at positions 259 and 260 (shaded area) next to the transmembrane domain. C₂₅₉A, C₂₆₀A-CAR is a full-length CAR construct in which cysteines 259 and 260 are replaced with alanines. In tailless-CAR (amino acids 1 to 260), the cytoplasmic tail is deleted, with the exception of cysteines 259 and 260, whereas in the C₂₅₉Stop-CAR construct (amino acids 1 to 258), the entire cytoplasmic tail is removed.

Immunolocalization of CAR in epithelial cells. The intracellular membrane distribution of CAR in NHBE cells (A) or of human CAR (B and D) or nonpalmitylated C₂₅₉A, C₂₆₀A-CAR (C and E) expressed in MDCK cells by Ad-mediated gene transfer was analyzed by fixation in 4% paraformaldehyde for 30 min at room temperature, lysis for 20 min at room temperature with 1% Triton X-100 in phosphate-buffered saline containing 1 mM CaCl₂ and 5 mM MgCl₂, and staining with anti-CAR monoclonal antibody (RmcB) in phosphate-buffered saline containing 1 mM CaCl₂ and 5 mM MgCl₂ containing 0.2% albumin, followed by a fluorescein isothiocyanate-conjugated goat-anti-mouse secondary antibody. The distribution of CAR protein is visualized in white, and the 4′,6′-diamidino-2-phenylindole-stained nuclei in panel E are grey. Cells were monitored by laser scanning confocal microscopy en face (A to C) or in the x-z direction (D and E). Bars, 10 μm.

Palmitylation of human CAR at cysteines 259 and 260. (A) Control CHO cells (lanes 1 and 2) and CHO-CAR cells (lanes 3 and 4) were starved for 1 h at 37°C in Dulbecco modified Eagle medium containing 2% dialyzed fetal bovine serum and radiolabeled for 4 h at 37°C with ¹²⁵I-IC16, an iodinated palmitate analog, at 20 μCi/ml (top). In parallel, cells were incubated for 1 h at 37°C in Dulbecco modified Eagle medium minus methionine and cysteine containing 2% dialyzed fetal bovine serum and radiolabeled for 4 h at 37°C with Expre³⁵S³⁵S protein labeling mix at 25 μCi/ml (³⁵S-Met; bottom). Lysates from duplicate radiolabeled dishes were subjected to immunoprecipitation with anti-CAR monoclonal antibody (RmcB) and then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Radiolabeled CAR migrated with an apparent molecular mass of 46 kDa, as indicated by the arrow. (B) COS-1 cells were transfected with Lipofectamine (Invitrogen, Carlsbad, Calif.) and empty pcDNA3.1 vector (Mock; lanes 1 and 2) or plasmids containing full-length CAR (lanes 3 and 4), C₂₅₉A, C₂₆₀A- CAR (lanes 5 and 6), tailless-CAR (lanes 7 and 8), or C₂₅₉Stop-CAR (lanes 9 and 10) and radiolabeled with ¹²⁵I-IC16 (top) and Expre³⁵S³⁵S (³⁵S-Met; bottom). The tailless-CAR and C₂₅₉Stop-CAR constructs migrated at 33 kDa, as indicated by the arrow.

Adβgal infection of CHO cells expressing CAR and nonpalmitylated CAR. CHO cells were infected with an Ad vector carrying no transgene (AdNull [▴]) or an Ad vector expressing wild-type CAR (AdCAR [○]) or C₂₅₉A, C₂₆₀A-CAR (AdCA, CA [•]) for 1 h at 37°C at 1 (solid lines) or 20 (broken lines) PFU per cell. After 2 days, triplicate monolayers of infected CHO cells were subsequently exposed to Adβgal for 1 h at 37°C at 0, 1, 10, or 50 PFU per cell and β-galactosidase activity was measured 24 h after Adβgal infection. Results are presented as the mean ± the standard deviation of triplicate monolayers of cells and are representative of two experiments. MOI, multiplicity of infection; RLU, relative light units.