CD150/TCR colocalization, CD3 triggering-mediated CD150 tyrosine phosphorylation, and identification of c Fyn/Lck phosphorylation sites in CD150.

(A) Cross-linking CD3 on normal human peripheral blood T cells with OKT3 results in colocalization of CD150 and CD3 on the T-cell plasma membrane. The top left panel shows FITC staining with capping of CD3 on PBMCs. Top middle panel is the same field as top left panel showing CD150 localization with Texas red following CD3 capping. Top right panel shows the top panels merged to reveal colocalization of CD150 and CD3 following CD3 cross-linking. White arrowheads highlight selected areas of colocalization. Lower right panel is FITC staining showing capping of CD150 on PBMCs following CD150 capping; bottom middle panel is same field as bottom left panel showing localization of CD3 following CD150 capping. Bottom right panel is the bottom panels merged to show colocalization of CD150 and CD3. (B) Cross-linking of CD3 in the human T-cell line Jurkat, stably transfected with CD150 results in rapid, reversible tyrosine phosphorylation of CD150; results are representative of 3 separate experiments. (C-D) Phosphorylation of chimeric CD8-truncated CD150 (CD8-SLAM4 del1, CD8-SLAM3) constructs by the Src kinases Fyn and Lck in COS-7 cells.