FIGURE 4.

Cytoplasmic sequence necessary for CAR internalization. A, CAR intracellular domain sequence. AP binding site is highlighted in blue, PDZ-binding domain in magenta, and minimum sequence needed for internalization in green. B, expression of CAR constructs in Neu2A cells. Cells were transfected, collected 24 h later and total extracts subjected to SDS-PAGE analyses using an anti-CAR raised against the extracellular domain. C, the AP binding site is not necessary for CAR internalization. Neu2A cells were transfected with CAR-RFP or its mutants. 24 h later, FK^CAV was added for 20 min on ice, the medium was replaced and internalization was allowed for 1 h at 37 °C. Cells were fixed and stained using an antibody anti-FK. Only the construct CAR-261 did not allow FK^CAV internalization. D, quantification of FK^CAV internalized cells (n = three independent experiments, ≥50 cells counted for each condition; results are expressed as mean ± S.E. ***, p value <0.001 versus control). E, hippocampal neurons were transfected with CAR-RFP or CAR-261 and similar procedures were applied. As above, CAR-261 did not internalize FK^CAV. Insets show CAR-FL and FK^CAV in vesicular structures (asterisks), whereas in CAR-261-transfected cells, FK^CAV was mainly at the plasma membrane. F, quantification of FK^CAV-internalized cells (n = 3 independent experiments, ≥21 cells counted for each condition; results are expressed as mean ± S.E. **, p value <0.01 versus control). Scale bars, 10 μm.