T‐Cell Antigen Receptor‐Induced Signaling Complexes: Internalization Via a Cholesterol‐Dependent Endocytic Pathway

T‐cell antigen receptor engagement causes the rapid assembly of signaling complexes. The adapter protein SLP‐76, detected as SLP‐yellow fluorescent protein, initially clustered with the TCR and other proteins, then translocated medially on microtubules. As shown by total internal reflection fluorescence microscopy and the inhibition of SLP‐76 movement at 16°C, this movement required endocytosis. Immunoelectron microscopy showed SLP‐76 staining of smooth pits and tubules. Cholesterol depletion decreased the movement of SLP‐76 clusters, as did coexpression of the ubiquitin‐interacting motif domain from eps15. These data are consistent with the internalization of SLP‐76 via a lipid raft‐dependent pathway that requires interaction of the endocytic machinery with ubiquitinylated proteins. The endocytosed SLP‐76 clusters contained phosphorylated SLP‐76 and phosphorylated LAT. The raft‐associated, transmembrane protein LAT likely targets SLP‐76 to endocytic vesicles. The endocytosis of active SLP‐76 and LAT complexes suggests a possible mechanism for downregulation of signaling complexes induced by TCR activation.