CD2-mediated signaling requires its cytoplasmic domain. (A) A schematic of two CD2 constructs tested for localization and recruitment of signaling proteins is shown. One is the control wild-type protein (CD2WT), and the second is a truncated CD2 molecule that lacks the entire cytoplasmic domain (CD2TM). Both constructs were tagged with mCherry at the C terminus and transfected into a J.CaM2 cell line, which is deficient in both CD2 and LAT. N, N terminus; C, C terminus, TM, transmembrane domain. (B–E) J.CaM2 cells were cotransfected with either CD2WT-mCherry or CD2TM-mCherry and either GFP-LckY505F or GFP-LAT and were imaged by TIRF microscopy ∼2 min after contact with a CD58-containing bilayer. The white boxes show regions that are magnified in the panels on the right. (F) Normal Jurkat cells GFP-tagged SH3 domains of CD2AP interacting with CD58 bilayers were fixed and stained with an antibody against phosphotyrosine (pY) of active Src kinase. The merge of differential interference contrast image and GFP fluorescence (left) shows that the cell overexpressing the dominant-negative GFP-tagged SH3 domain has little phosph-Src staining compared with the untransfected cells (immunofluorescence image on the right). Bars, 5 µm.